The main difference between food proteins and bug proteins in that this hypothesis is concerned, is that to achieve being replicated, bug proteins need a genetic code, while food proteins do not need it, as they have an idiot who “replicate” them (the idiot introduces voluntarily these proteins in its body by eating them again and again over a lifetime).


In the broadest sense, a pathogen can be defined as any substance capable of causing disease. Under this definition, pathogens need not be replicative, and could include toxins, food allergens, and dietary antigens responsible for chronic inflammation, such as gluten peptides in the context of celiac sprue (Bethune 2008).

Only in the context of celiac disease?



This hypothesis is intended to demonstrate by means of looking for evidences that an indefinite but potentially enormous amount of human diseases are directly or indirectly due to the crosslinking (forever covalently binding) of certain food proteins with certain endogenous (own) proteins by a human endogenous (own) enzyme called transglutaminase.

The main question is simple, is it really possible that one of our own enzymes bind multiple food proteins to multiple of our own proteins in any part of our body forever in vivo?

What would be the consequences if that happened?

More interestingly, what would be the consequences if that happened during at least three meals a day for a whole life? Why are some bugs using bug proteins similar to these certain food proteins chosen for the human species as food to attach themselves to us by using our own enzyme transglutaminase?

Are they also using our transglutaminase to invade us?

Have certain food proteins chosen for our species to feed themselves the intrinsic ability to invade us by using our own transglutaminase?

Finally and most importantly , are we sure we are eating the right foods?



Polymerization, transamidation and crosslinking are the same here.


Tissue transglutaminase (TG2 or tTG) is an ubiquitous cellular enzyme (35).


TG2 is a pleiotropic enzyme found both intracellularly and extracellularly in many tissues and organs, including the small intestine (34).


TG2 is upregulated in wound healing, angiogenesis, and apoptosis where its main function is cross linking of proteins via creation of stable isopeptide bonds between a donor glutamine residue and an acceptor lysine residue (35).


TG2 is upregulated during active celiac sprue (34).


In a Ca2+-dependent manner, TG2 catalyzes the transamidation of specific glutamine carboxamide sidechains with amine donors, such as the ε-amino group of lysine, forming isopeptide bond crosslinks between proteins. When water replaces the amine donor as the nucleophile, TG2 instead deamidates these glutamines to glutamates, introducing a negative charge at each modified position (34).


tTG is a calcium dependent ubiquitous intracellular enzyme which catalyses the covalent and irreversible crosslinking of a glutamine residue in glutamine-donor proteins with a lysine residue in glutamine-acceptor proteins which results in the formation of an ε-(γ-glutamyl)-lysine (isopeptidyl) bond. The reaction is a multistep process, in which the active site of the enzyme firstly reacts with the glutamine residue to form the acyl-enzyme intermediate under release of ammonia. In a second step, the complex reacts with a primary amine to form an isopeptide bond and liberate the reactivated enzyme. The final step consists of the formation of covalently crosslinked, often insoluble supramolecular structures (37).

Folk JE. Mechanism and basis for specificity of transglutaminase catalyzed ε-(γ-glutamyl)lysine bond formation. Adv Enzymol Relat Areas Mol Biol 1983;54 :1–56.


Tissue transglutaminase (tTG) exhibits the catalytic activitity of crosslinking certain extracellular proteins during inflammation, mechanical stress and wound healing, and several intracellular proteins in the process of apoptosis (38).


Crosslinking occurs from a limited number of glutamine residues of donor proteins to lysine residues of ubiquitous acceptor proteins (38).



Cell adhesion mediated by specific cell surface molecules plays a pivotal role in life sciences (Alsteens 2009).

Neuronal interactions, cellular communication, tissue development, inflammation, cancer and microbial infection are just a few examples of cellular processes regulated by cell adhesion molecules (Alsteens 2009).


Tissue transglutaminase (TG2) catalyzes the covalent cross-linking of proteins and has been implicated in the modulation of cell adhesion (Lin 2011).



Infectious pathogens adapt to and cause disease in a particular host by evolving virulence traits that provide a context-specific, selective advantage to the pathogen (e.g., by enabling it to breach a specific host epithelial barrier (34).


By contrast, it is difficult to imagine how the ability of (for example) immunotoxic (in celiac sprue) gluten peptides to resist gastrointestinal proteolysis, to exert damaging stress on epithelial cells, to be specifically deamidated by TG2, or to engage in high affinity HLA DQ2 binding affords any increase in fitness to the grains encoding these peptides (34).


Tissue transglutaminase (TG2) catalyzes the covalent cross-linking of proteins and has been implicated in migration, invasion and cancer metastasis (Lin 2011).



Microbial adherence is one of the most important determinants of pathogenesis (9).

Adhesion to host tissues generally is a prerequisite for colonization of the host by infecting microorganisms (Emody 2000).


Attachment of fungi to host cells is a critical part of fungal pathogenesis in animals (12).

During the past decade there has been an increased recognition of the importance of adhesion of fungi to host surfaces–both plant and animal–before penetration (Epstein 2006).

Even if we limit our discussion to fungal-substratum adhesion that occurs on the plant host surface before penetration, adhesion serves multiple functions: most obviously, adhesion keeps a fungus from being blown or rinsed from a potentially suitable environment. Adhesion prevents fungus displacement by water and/or wind (Epstein 2006).

Adhesion-reduced mutants of the plant pathogenic fungus Nectria haematococca were less virulent than the wild-type when deposited on the intact surface of cucurbit fruits, but were equally virulent when deposited into wounded fruits; the results indicated that adhesion is a virulence factor in the “natural” environment and suggested that adhesion prevents displacement by water and assists in efficient localization of secreted enzymes on the host surface (Jones and Epstein 1990).


Hwp1 Hyphal wall protein 1:

Hwp1 is a protein (glyco(manno)protein) located on the surface of a dimorphic fungus called Candida albicans.


Hwp1 is a transglutaminase substrate (N-terminal third of Hwp1 to be precise) (1).


Dimorphic: Candida Albicans fungus has two forms: round-to-oval yeast or blastospore form and filamentous hyphal form.


Hwp1 is expressed exclusively on hyphae surface of adhesive-invasive-competent hyphal filamentous form of Candida Albicans (3).


Candida Albicans (3 - 5 µm in diameter, round-to-oval, yeast or blastospore form)


Candida Albicans (up to 50 μm in lenght, filamentous, hyphal form)


Anatomy of Hwp1: Left: three-dimensional structure (9) and Right: aminoacid sequence (UniProtKB/Swiss-Prot).



During both mucosal colonization and induction of disease, Candida Albicans interact with epithelial cells (14).

The interactions of Candida albicans with epithelial cells include adhesion and invasion (14).

Adhesion: The organism first adheres to epithelial cells. Adherence is mediated by multiple different adhesins that are present on the fungal cell surface. Some adhesins are expressed only by hyphae (like Hwp1 and Als3), whereas others are expressed by both hyphae and yeast-phase organisms (14).

Invasion: Next, the adherent organism can invade both into and between epithelial cells. Invasion into an epithelial cell can occur by induced endocytosis, whereby Als3 and other invasins on the fungal cell surface bind to E-cadherin and other target proteins on the epithelial cell surface. Binding to these epithelial cell proteins induces the epithelial cell to produce pseudopods that engulf the organism and pull it into the cell. C. albicans actively penetrates into epithelial cells by a mechanism that is currently poorly understood. It actively penetrates between epithelial cells by secreting aspartyl proteases that degrade E-cadherin and other interepithelial cell junctional proteins. Invasion into and between epithelial cells is a prerequisite for induction of epithelial cell damage (14):

Two major interactions of Candida Albicans with epithelial cells: Adhesion and Invasion (14).


The outcomes of these interactions are important in determining whether the organism can colonize a mucosal surface and subsequently cause disease (14).


Below: Both Candida Albicans forms; round-to-oval form  and filamentous hyphal form  have adhesion ability but the conversion from the round-to-oval form to the filamentous hyphal form enhances this ability (Höfken 2013):


Below: Possible mechanisms of epithelial or endothelial invasion: Left: Induced endocytosis: the interaction between yeast wall proteins such as als3 and Ssa1 with N-cadherin on the surface of endothelial cell triggers endocytosis. Center: Active penetration into a cell: cells are severely damaged in this process. Right: Active penetration between cells: active penetration between cells by degrading junction proteins and extracellular matrix (Höfken 2013):



In response to various environmental stimuli (in the presence of serum, high temperature, neutral pH, or nutrient-poor media), Candida albicans alters its morphology from a unicellular budding yeast to a multicellular hyphal form.


To stick and invade host tissues, Candida Albicans cells must first switch from the round-to-oval,  yeast or blastospore form to the filamentous, hyphal form, a phenotypic transition that involves dramatic changes both in cellular architecture and gene expression.


Many genes associated with this transition are essential for virulence, including both cell wall proteins and secreted enzymes (6).


The yeast-hypha transition is accompanied by the de novo synthesis of proteins that are targeted to hyphal surfaces (3).


One of these proteins expressed only on hyphae is Hwp1. Other remarkable protein expressed only on hyphae is called Als3.


Yeast-to-hyphal or filamentation (from white cells emerges a projection called hyphae)

Candida Albicans white cells undegoing filamentation by producing cellular projections called hyphaes.

Germ tubes emerge from yeast cells with no septum (wall) formed, then germ tubes elongate and only later a septum is formed in the hyphal tube


Hwp1 (and Als3) appear only on the hyphae during the yeast to hyphae morphological transition.


Several mechanisms have been proposed to contribute to the changes in surface composition of Candida albicans during morphogenesis. Ultrastructural studies support the occurrence of rearrangements or losses of wall components during morphogenesis , whereas unmasking of cryptic antigenic determinants has been suggested using monoclonal antibodies to localize specific antigens (3).




HWP1 encodes for a unique protein with combined mammalian and fungal functional domains (11).

Hwp1 combines features of mammalian transglutaminase substrate proteins with characteristics of fungal cell wall proteins to form an unconventional adhesin at the hyphal wall of Candida albicans (11).

Hwp1 has a half-mammalian- like, half-fungal hybrid primary structure (11).


Hwp1 can be subdivided in two domains: the serine/threonine rich domains I and II  and the antigenic, repetitive or proline/glutamine rich or transglutaminase substrate domain (4).




The central and C-terminal regions of Hwp1 contained a high percentage of serine and threonine residues, serine/threonine-rich regions are predicted to function in extending a ligand-binding domain into the extracellular space (4).

A C-terminal domain rich in hydroxy amino acids serves to extend a binding domain above the cell surface, may help explain the exposure of the antigenic domain of Hwp1 at the cell surface (4).

The features of Hwp1 are consistent with the paradigm that yeast cell surface proteins important for ligand binding have unique N-terminal domains that confer binding specificity but C-terminal domains with common features that permit cell wall anchoring and surface-exposure of N-terminal domains (4).



The N-terminal repetitive domain comprised approximately one-third of the overall amino acid sequence and is located in the N-terminal third of the protein (4).

The N-terminal antigenic domain was more hydrophilic and had an increased concentration of negatively charged residues compared with the remainder of Hwp1 (4).

Transglutaminase substrate Hwp1 domain is a proline- and glutamine-rich protein (3).

Transglutaminase substrate Hwp1 domain is a peptide that is largely composed of an acidic, repeated motif (degenerate amino acid repeat) (series of tandem repeats) 10 amino acids in length that is rich in proline and glutamine residues (3).

Below: Alignment of the repetitive amino acid sequences (repeats starting with amino acid 14) of Hwp1. Gaps have been introduced to maximize the similarities of the repeats (3):

The amino acid composition was notable in having 27% proline, 16% glutamine, and 12% aspartate residues (mole percents) (3).

Common features included proline residues at positions 2, 6, and 9 in all but two of the repeats. Cysteine residues predominated at position 3 and aspartate was found at position 4 (3).

The presence of a cysteine residue in each repeat probably leads to the formation of regularly spaced extracellular disulfide bonds that may be important for the surface conformation of hwp1 and possibly for intermolecular cross-linking of proteins on the cell surface. Given the specificity of hwp1 for hyphal surfaces, the presence of cysteines may be related to the enhanced sensitivity and increased protein release from hyphal forms following treatment with dithiothreitol (3).

The first six repeats had a tyrosine in the fifth position and glutamate in the tenth position, whereas the more carboxy proximal repeats had asparagine and aspartate, at the fifth and tenth positions, respectively (3).

The C terminus of hwp1 was threonine- and serine-rich providing abundant potential sites for O-glycosylation. The lack of agreement between the predicted molecular weight and the SDS-polycrylamide gel-determined molecular weight is typical for proteins with high proline content, although O-glycosylation of serine residues located near the C terminus might also contribute to the discrepancy (3).

The serine and threonines near the C-terminal end of hwp1 are likely to be sites for O-glycosylation and serve as wall spanning domains as has been proposed for other yeast surface proteins (3).


Hwp1 lacks Lys residues that are usually found adjacent to reactive Gln residues in TGase substrates (11).

This absence of Lys residues indicate that Hwp1 participates in cross-linking reactions solely as the Gln donor (11).


Computer modeling predicts that the N-terminal domain of Hwp1 is hydrophilic (11).

Hwp1 was found to be very acidic, having 63 negative charges at neutral pH contributing to the strong negative charge of surfaces of Candida albicans hyphae (4).

The presence of 31 acidic residues characterizes rHwp1N13 as a very soluble protein (11).

An additional feature of the repeats is the presence of acidic amino acids that would confer a negative charge to hyphal surfaces at physiological pH. The presence of anionic proteins on hyphal surfaces has been demonstrated by others (3).

The deduced amino acid composition (of antigenic Hwp1 domain) was hydrophilic throughout the entire sequence ( (3).


Computer modeling predicts that the N-terminal domain of Hwp1 is a predominantly coiled structure (11).

Chou-Fasman predictions of secondary structure showed the sequence to be primarily composed of turns with two helical sections in locations containing five and six consecutive glutamine residues (3).

The changes in ellipticity in the case of SPR3 were suggested to reflect β-turns that exist between the repeating amino acid units. Although there is evidence for similar organized structure in Hwp1, the amount is much lower than that found in SPR3 (11).

Biophysical analysis of the N-terminal domain of Hwp1 discloses a tight disulphide-bonded coil without α helices or β sheets (9).

The TGase substrate domain itself is a tight, disulfide-linked coil without discernible secondary structure (11).

Secondary Structure Features of the N Terminus of Hwp1: rHwp1N13 is a protein that is comprised predominantly of a coiled structure (11).

rHwp1N13 N-terminal domain exists completely as a coil. The N-terminal domain of Hwp1 is likely a rigid coiled structure (11).


Native Hwp1 is a complex mixture of forms that are cell-free, membrane-bound, as well as cell wall-linked (11).

Common occurrence of both membrane and cell wall anchored forms of cell wall localized proteins (11).

Hwp1 maturation into the cell wall-bound form: Presence of a 325-kDa GPI-anchored membrane species, which may be a precursor of a periplasmic 301-kDa intermediate destined to become covalently attached to glucan. Soluble 301-kDa Hwp1 may represent a precursor population of Hwp1 in transition between a membrane protein and the wall-anchored mature form. Small amounts of the 301-kDa species are present in the culture media of germ tubes, indicating that this form, if not bound to the cell wall, can diffuse out into the medium (11).

Furthermore, expressing a C-terminal truncated form of Hwp1 produced a protein of 301 kDa that was not bound to germ tube walls but instead found in the culture medium (11).

The latter results also showed that the C-terminal 26 amino acids of Hwp1 contain the necessary information to guide and link the protein to the β-glucan (11).

The amino acid sequence-based prediction that Hwp1 is a member of cell wall proteins covalently attached to the cell wall β-Glucan of Candida albicans (11).

The cell wall forms of Hwp1 comprise 75% of the total Hwp1 (11).

Although noncovalently bound forms of Hwp1 exist at the surface of germ tubes, it is likely that TGase associated with host buccal epithelial cell surfaces interacts only with the mature cell wall-attached Hwp1 during adhesion (11).

Below: Cell wall anchorage of Hwp1: The majority of Hwp1 is covalently bound to the cell wall; Hwp1 cross-linked to β(1,3)-glucan via β(1,6)-glucan in the cell wall (9):


Hwp1 appear to have N-terminal domain whose functions require exposure to the extracellular environment (4).

Data supports the existence of an N-terminal domain of Hwp1 that is set apart from the remainder of the protein (9).

Chymotrypsin sensitivity of native Hwp1 on germ tube surfaces further strengthening the argument for the separation of the N-terminal domain from the rest of the protein (11).

The surface probabilities were also highest for the sites with consecutive glutamine residues and for glutamines bounded by prolines (3).

Hwp1 is an outer protein with a cell surface-exposed NH2-terminal domain (1).

Hwp1 interacts with the cornified epithelium through the N-terminal domain (11).



The presence of amino acid repeats on hyphal surfaces is not surprising given that tandem amino acid repeats are widely distributed on surfaces of broad groups of microorganisms including fungi, parasites, viruses, and bacteria. The functions of repetitive surface proteins have frequently been found to involve the host and include attachment sites to host cells, evasion of phagocytosis, invasion of host cells, and neutralization epitopes (3).



Screens for germ tube–specific surface proteins that might function in adherence led to the identification of hyphal wall protein 1 (Hwp1), a protein with expression limited to surfaces of germ tubes and true hyphae (12).

Hwp1 is an important developmentally regulated adhesin (9).

Hwp1 is not found on surfaces of yeast forms or pseudohypahe forms of Candida Albicans (9).

Hwp1 is specifically localized in the walls (surfaces) of germ tubes and (true) hyphae of Candida albicans (3, 4).

Hwp1 is expressed on the surface of hyphae in the pathogen Candida albicans (3).

Hwp1 is exposed on surfaces of hyphae grown in mammalian hosts (3).

Hwp1 is present in hyphal but not yeast forms (3).


Using recombinant Hwp1 it was confirmed that Hwp1 is localized to hyphal surfaces of Candida Albicans (3).

Antiserum raised to a recombinant Hwp1 encoded by HWP1 cDNA should crossreact with hyphal surfaces. Serum from rabbits immunized with rhwp1 was tested for the ability to recognize antigens on C. albicans hyphal surfaces in immunofluorescence assays (3).

Immune serum stained hyphal surfaces but not the parent blastoconidia of C. albicans. Thus the antiserum was specific for rhwp1 and hyphal surfaces of C. albicans (3).

Hwp1 was found to be present on hyphal surfaces by immunofluorescence assays using monospecific antisera raised to the recombinant protein (3).

Antibodies from the screening antiserum that cross-reacted with hyphal surfaces of C. albicans in an immunofluorescence assay that employs germ tube forms as antigens. Yeast surfaces were negative or weakly fluorescent (3).

Below: Detection of hwp1 on hyphal surfaces of Candida Albicans grown in laboratory cultures. Candida Albicans yeasts bearing germ tubes were treated with various primary anisera followed by fluoresceinated goat anti rabbit IgG in indirect immunofluorescence assays; preimmune serum (left), mono-specific antiserum to rhwp1 (right) (3):

Hwp1 is exposed on hyphal surfaces and not on yeast surfaces of C. albicans (3).


Antibodies to Hwp1 were used to detect Hwp1 on Candida Albicans surfaces (11).

Below: Detection of hwp1 on hyphal surfaces of Candida Albicans grown in laboratory cultures. Immunofluorescence assay was performed to detect Hwp1 on the surface of germ tubes of a Candida Albicans without Hwp1 (left) and a Candida Albicans with Hwp1 (right) (11):


To determine if hwp1 was produced by C. albicans growing in mammalian hosts, immunofluorescence assays were performed on paraffinembedded tissues from beige mice that were heavily colonized with C. albicans (3).

Large numbers of organisms were seen in the lumen and keratinized superficial layers of the stomach following indirect immunofluorescence staining with polyvalent rabbit antiserum to C. albicans (3).

Monospecific antiserum to rhwp1 stained the filamentous but not yeast forms of C. albicans in the tissue sections, indicating that hwp1 is specific to hyphal forms during growth in the host as well as during growth in laboratory medium (3).

Below: Detection of hwp1 on hyphal surfaces of C. albicans grown in host tissues. Sections treated with various antisera from an 11-month-old females mouse that had been colonized for 9 months with C. albicans: preimmune serum (left), monospecific anti-serum to rhwp1 (right). The large arrowhead points to positive-staining hyphae, and the small arrowhead points to negative-staining yeast (3):



Anti-Hwp1 antibody staining reveals that Hwp1 is expressed in serum-induced hyphae of Candida Albicans (5).

Below: White cells and daughter buds of Candida Albicans did not stain with anti-Hwp1 antibody (5):

Below: White cells of Candida Albicans induced to form hyphae by suspending white cells in serum, the walls of the resulting germ tubes and hyphae stained selectively with Hwp1 antiserum (5):

Mother cells remained unstained, demonstrating that differentially expressed Hwp1 localized exclusively in the hyphal wall (5).

In an analysis of 500 white budding cells stained with anti-Hwp1 antibody, 0% of mother and 0% of daughter cells stained (5).

In a similar analysis of serum induced hypha-forming cells, 0% of the mother cells and 100% of the hyphae of each strain stained (5).

In an analysis of 500 untreated opaque phase cells 0% of the cells stained (5).

These results demonstrate that Hwp1 is differentially expressed in hyphae, and that budding opaque phase cells do not express Hwp1, even though they have been shown to express other hypha wall antigens (5).


Synthesis of this Hwp1 protein occurred exclusively during hyphal growth, showing that the bud-hypha transition controls the antigenic surface composition of hyphae by production of de novo proteins (3).

The hyphae-specific surface location was also seen on organisms colonizing the gastrointestinal mucosa of mice, indicating that Hwp1 is produced and developmentally regulated during growth in host tissues (3).


To determine if hwp1 was the sole antigen recognized by the screening hyphae-specific antiserum, blocking experiments were performed with this antiserum as well. Recombinant hwp1 did not block the fluorescence exhibited by the hypha-especific antiserum, indicating that antigens other than hwp1 are present on hyphal surfaces (3).




Hwp1 is a substrate for mammalian transglutaminase (1).


Potential transglutaminase Q residues substrate of transglutaminase in the proved transglutaminase substrate N-terminal of Hwp1 (Hwp1: aminoacids 0 to 240, P46593 UNIPROT)


Potential transglutaminase susbtrates in the “only Q” zone of the proved transglutaminase substrate N-terminal of Hwp1 (Hwp1: aminoacids 40 to 164, P46593 UNIPROT)



Employing recombinant Hwp1 (rHwp1) it has been showed that the mature N-terminal third of Hwp1 serves as a substrate for mammalian TGases (11).

To determine if the N-terminal third region of Hwp1 is a substrate for TGase, we examined a recombinant protein, rHwp1∆C37,  a partial protein encoded by the N-terminal repetitive proline- and glutamine-rich domain (aminoacids 40 to 197 of Hwp1) for the ability to incorporate [14C]putrescine in the presence of TGase2 (1).

The recombinant protein was similar to other TGase2 substrates in the generation of multiple species of radiolabeled reaction products including monomers displaying increased migration , species of high molecular weight , and dimers bridged by putrescine. The production of all radiolabeled forms depended on the presence of active Tgase (1).

In addition to [14C]putrescine, TGase2 catalyzed the incorporation of another TGase cosubstrate, monodansylcadaverine, into rHwp1∆C37. The behavior of rHwp1∆C37 in interactions with TGase2 matched that of SPR proteins and other host TGase substrates and implicated the NH2-terminus of Hwp1 (aminoacids 40 to 197 of Hwp1) in cross-linking reactions with primary amines through Nε-(γ-glutamyl)lysine isodipeptide bonds (1).




Transglutaminase activity is produced by mammalian cells and not by Candida albicans (17).

With the Candida albicans adhesin Hwp1, the host transglutaminase covalently links the pathogen to the host epithelial cell (1).

Hwp1 is a substrate for transglutaminase activity derived from a host.

No intrinsic Candida albicans transglutaminase was detected. No endogenous TGase activity of Candida albicans was detected in whole organisms or in broken cell walls (1).

Candida Albicans adhesion represents a paradigm for microbial adhesion because it implicates essential host enzymes (1).


Hwp1 serves as a substrate for mammalian TGases (11).


Hwp1 participates in the formation of covalent bonds to primary amines and to a buccal epithelial cell (BEC) surface protein catalyzed by human transglutaminases (TGs) (13).


BEC TGases and TGase substrates participating in interactions with C. albicans were not identified (1).

The TGase activity on surfaces of buccal epithelial cells, attributed to TGase 1 by others utilizes rHwp1 as a substrate (11).


TG1 (keratinocyte transglutaminase) is active on the surface of buccal epithelial cells (BECs) where it functions in assembly of the cornified cell envelope, a scaffold of cross-linked proteins that gives mucosal cells their barrier properties. We found the participation of Hwp1 in the formation of a stabilized (heat and detergent resistant) TG-dependent adhesion to BECs, and speculated that TG1 on the surface of BECs catalyzed this reaction. In support of this conclusion, Candida Albicans without Hwp1 is unable to cause oropharyngeal candidiasis in mice. However, we were unable to verify the role of TG1 directly as mice without TG1 die of dehydration soon after birth (13).

A mechanism of adhesion based on transglutaminase activity on BECs could explain the requirement for Hwp1 in oral candidiasis (13).


In vitro, both native and recombinant Hwp1 are substrates for guinea pig liver tissue transglutaminase (TG2) (1, 13).

Hwp1 is a substrate for human tissue transglutaminase TG2 (13).

It is plausible that human tissue TG2 catalyzes transamidation reactions involving Hwp1 (13).



Hwp1 is a transglutaminase substrate located on hyphal surfaces of Candida Albicans (1).

The NH2-terminal domain of native Hwp1 appears to be a surface-exposed TGase substrate available for interactions with exogenous mammalian Tgases (1).


To asses presence of Hwp1 as a transglutaminase substrate on germ tube surfaces of Candida Albicans, strains with or without Hwp1 were used for comparison in TGase assays (1).

Below: Detection of TGase substrates of wild-type strain (with Hwp1, left) versus a mutant strain (without Hwp1, right) of Candida Albicans: Candida Albicans germinated cells were incubated with TGase2 (guinea pig liver tissue transglutaminase) and 5-(biotinamido) pentylamine (a transglutaminase substrate used for fluorescence staining of TGase substrates). TGase substrates were stained on germ tube surfaces of the strain with Hwp1 (left) but not on the strain without Hwp1 (right) thus indicating the requirement of Hwp1 for TGase substrate activity (1):


The results showed that Hwp1 functions as a substrate for transglutaminase and is the major substrate on hyphal surfaces.


Expression of Hwp1 on the surface of Candida albicans germ tubes was visualized by cross-linking 5-(biotinamido)pentylamine (5-BPA) to the germ tube surfaces by human recombinant tissue transglutaminase (hTG2) (13).

The primary amine, 5- (biotinamido)pentylamine and human recombinant tissue transglutaminase (hTG2) were used to visualize Hwp1 on the fungal surfaces (13).

Left column, light images; right column stained images (13).

Below: Candida Albicans without Hwp1 (SCH1211) (13):

Below: Candida Albicans with Hwp1 (SC5314) (13):



Searches of computer data bases showed that hwp1 was similar to a wide variety of proteins containing amino acid repeats having periodic proline residues and/or glutamine residues; however, no identical sequences were found (3).

Hwp1 (Hwp1 transglutaminase substrate domain) shares features with surface proteins of other lower eukaryotic microorganisms (3).

Hwp1 mimicks host cell transglutaminase (TGase)1 substrates (11).

In mimicking host ligands, Hwp1 adds an example from the fungal kingdom to known microbial adhesins that imitate mammalian proteins (11).



SPRs are proteins located in terminally differentiating mammalian stratified squamous epithelium and in cornified cell envelope (CE) (Tarcsa 1998).

Stratified squamous epithelium: The outermost layer of the skin and the inner lining of the mouth, esophagus, and vagina. It consists of squamous (flattened) epithelial cells arranged in layers upon a basal membrane. Only one layer is in contact with the basement membrane; the other layers adhere to one another to maintain structural integrity. This type of epithelium is well suited to areas in the body subject to constant abrasion, as it is the thickest and layers can be sequentially sloughed off and replaced before the basement membrane is exposed (Wikipedia).

There are two types of stratified squamous epithelium: Nonkeratinized: Non-keratinized surfaces must be kept moist by bodily secretions to prevent them from drying out. Types of non-keratinized stratified squamous epithelium include cornea (see also corneal epithelium), oral cavity, esophagus, anal canal, vagina, and the internal portion of the lips. Keratinized: Keratinized surfaces are protected from abrasion by keratin and kept hydrated and protected from dehydration by glycolipids produced in the stratum granulosum. Examples of keratinized stratified squamous epithelium include epidermis of the palm of the hand and sole of the foot and the masticatory mucosa (Wikipedia).

Cornified cell envelope: It is a layer of protein assembled at the cell periphery in different epithelia that affords essential barrier function to stratified squamous epithelia. SPRs serve as structural protein precursors of the cornified cell envelope (Tarcsa 1998).

To date, three classes of SPRs have been identified: SPR1 (two members in all species examined so far), SPR2 (8–11 members), and a single member of the SPR3 class (Tarcsa 1998).

The expression of SPR proteins varies widely between different epithelia. For example, the epidermis expresses SPR1a (also known as cornifin α) and a limited number of SPR2 members. Other internal epithelia express more abundant amounts of other SPR2 members together with SPR1a. The SPR3 (also known as cornifin β) protein is most abundantly expressed in oral epithelia, the esophagus and rodent forestomach epithelium. Cultured keratinocytes from several epithelial sources tend to express both SPR1a and 1b, as well as a limited selection of other members. In addition, the amounts expressed vary during differentiation. Furthermore, a much wider selection of SPR members is expressed in epithelia in response to UV damage, chemical treatment and in hyperproliferative or malignant diseases (Tarcsa 1998).

All SPRs are built according to a common plan. They possess (head) amino- and (tail) carboxyl-terminal domains that are enriched in Gln, Lys, and Pro residues of sequences that are characteristic of and highly conserved between members within each class (Tarcsa 1998).

The SPR name in fact is based on a distinctive central domain consisting of a series of peptide repeats of either eight (SPR1 and SPR3) or nine (SPR2) residues that are enriched in Pro residues and of sequences that vary between the three classes, but members within each class are also highly conserved. The numbers of repeats range from as few as three in all human SPR2 proteins and the smallest mouse SPR2 protein to more than 23 in SPR3 proteins (Tarcsa 1998).


SPRs are transglutaminase substrates (1, 8, 11).

SPRs become cross-linked to other proteins by transglutaminases (Tarcsa 1998).

The Cornified cell envelope (CE) is made insoluble by extensive cross-linking of several defined structural proteins including the SPRs by disulfide bonds and Nε-(γ-glutamyl)lysine or N1,N8-bis(γ-glutamyl)spermidine isopeptide bonds formed by the action of transglutaminases (Tarcsa 1998).

TGases catalyze the usually irreversible formation of an intra- or more usually intermolecular isopeptide bond between acyl donor Gln and acceptor Lys residues. Unambiguous evidence that SPRs serve as CE precursors and are in fact good substrates for TGases in vivo comes from direct sequencing analyses. Many peptides recovered from limited proteolysis experiments of CEs isolated from human foreskin epidermis, cultured epidermal keratinocytes and mouse forestomach epithelium contained recognizable SPR sequences cross-linked through one or more isopeptide bonds to other CE proteins. Examination of these peptides revealed that Gln and Lys residues on only the head and tail sequences of the SPRs were involved in the cross-links. Furthermore, the data indicated that the SPRs functioned primarily as cross-bridging proteins, by mediating links between other proteins, often loricrin in the epidermis or loricrin, trichohyalin, and themselves in the forestomach CEs. They also form cross-links between themselves in the CEs of cultured keratinocytes that may serve as a model system for other internal stratified squamous epithelia (Tarcsa 1998).

Individual members of the SPR family can function as both amine donors and acceptors for the TGase 1 enzyme in vitro (Tarcsa 1998).

Although the three TGases commonly expressed in epithelia (TGases 1, 2 and 3) can cross-link SPR2 in vitro, TGase 3 is used almost exclusively for cross-linking SPRs to CE structures in epithelia in vivo (Tarcsa 1998).

Recombinant human SPR2 protein was used as a complete substrate by the TGase 1 and 3 enzymes present in the epidermis and other stratified squamous epithelia, but by the TGase 2 enzyme only very weakly, if at all (Tarcsa 1998).

Keratinocyte Transglutaminases create a host barrier defense by cross-linking SPR proteins and other proteins through covalent Nε-(γ-glutamyl)lysine iso-dipeptide bonds (1).

Cross-linking of epithelial cell proteins is essential. Mice lacking keratinocyte Transglutaminase die within a few hours of birth (1).

Although both SPR proteins and Hwp1 are TGase substrates, Hwp1 lacks the tripartite domain structure that serves to cross-link the head and tail domains of SPR proteins to themselves or to other proteins (11).

Perhaps Hwp1 contributes to proliferation of Candida albicans in keratinized epithelium by interacting with keratinocyte Tgases (1).


Hwp1 (Hwp1 transglutaminase substrate domain) has similarities to mammalian small proline-rich proteins (SPRs) (1).

Similarity in primary structure of rHwp1 to mammalian small proline-rich proteins (SPRs) (11).

Biophysical properties of the Hwp1 transglutaminase substrate domain were explored using a recombinant protein representative of the N-terminal domain of Hwp1 and were similar to other transglutaminase substrates, the small proline-rich proteins (SPRs) of cornified envelopes found in stratified squamous epithelia of mammals (11).

Similiarities of Hwp1 to small proline-rich (SPR) proteins are likely responsible for the TGase substrate properties of Hwp1 (11).

Role for Hwp1 in formation of stable complexes with human buccal epithelial cells (BECs) was suggested by the amino acid sequence of the NH2-terminal domain, which resembles mammalian TGase substrates , particularly the head and central domains of small proline-rich (SPR) proteins (1).


The predicted mature N terminus of Hwp1 resembles those of all three SPR in having Ser (S) as the first amino acid (11).

The presence of Tyr (Y) residues close to the N terminus as well as immediately prior to the first string of glutamine residues is similar to SPR2 and SPR3 (11).

The Hwp1 repeats (10 amino acids) are similar in size to SPR internal domain repeats (8 amino acids) and are also similar in the presence of Cys (C), Pro (P), and Glu (E) residues (11).

The Hwp1 repeats differ from those of the SPR family in the absence of Lys residues indicating that, unlike the SPR family, Hwp1 participates in cross-linking reactions solely as the Gln donor (11).

Below: Similarities between Hwp1 and SPR proteins: Comparison of the sequence of the N-terminal domain of Hwp1 and three members of the SPR family. Residues in bold type become cross-linked in the presence of epithelial cell Tgases in vitro. Glns in Hwp1 that are predicted to become cross-linked are shown underlined in bold type (11):

The similarity of Hwp1 to SPR proteins suggests that interactions of BEC TGases with SPR proteins are indicative of interactions with Hwp1 and that the primary role of Hwp1 is in the formation of stable attachments (1).

The similarity of Hwp1 to small proline-rich proteins that are expressed only in stratified squamous epithelium suggests that Hwp1 may be more important for mucosal than systemic candidiasis (9).


Long incubation periods (18 hours) are required for maximal cross-linking of SPR2 protein by epithelial cell TGase3 (1).


The overall coiled structure (of N-terminal domain of Hwp1) is similar to the human SPR protein family of cornified cell envelope proteins (11).



APRPs are proteins located in saliva in humans as well as other animals (Bennick 1982).

Saliva is a secretion produced by the salivary glands in the oral cavity. The macromolecules in saliva consist almost exclusively of a complex mixture of proteins called salivary proteins. The largest (~70% of the human salivary proteins) protein components of salivary proteins are the salivary proline-rich proteins. Salivary proline rich proteins are a family of proteins that can be divided into three groups acidic (30%), basic (23%) and glycosylated (17%) proteins. Acidic ones are the acidic proline-rich proteins or APRPs (Bennick 1982).

The primary structure of the acidic proline-rich proteins is unique and shows that the proteins do not belong to any known family of proteins (Bennick 1982).

Amino acid analysis of unfractionated human salivary proteins have demonstrated an unusually large amount of proline varying from 16 to 33% of all amino acids. This observation is all the more interesting because amylase which accounts for approximately 30% of the parotid protein only contains 2.3% proline which is similar to that of most proteins (Bennick 1982)

The human salivary proline-rich proteins (PRPs) are characterized by a predominance of the amino acids proline (25-42%), glycine (16-22%) and glutamic acid/glutamine (15-28%), which together make up 70-85% of the proteins (Kim 1993).

It is characteristic that proline accounts for 25 to 42% of the amino acids in salivary proline-rich proteins. In addition there are high contents of glutamine (glutamic acid) and glycine. Together these three residues account for 70 to 88% of all of the aminoacids in the proteins. (Bennick 1982)

In APRPs proline accounted for 24 to 27% of the residues and this amino acid together with glutamic acid (or glutamine) and glycine constituted 70 to 75% of all the amino acids (Bennick 1982).

In APRPs the sequence from residue 31 to the C-terminus consisted mostly of proline, glycine and glutamine. In this part of the protein there was a high degree of sequence repetition. The sequence pro-gln-gly-pro-pro (PQGPP) ocurred for example 5 times. This suggested the presence of recurrent folding of the peptide chain and gene multiplication. (Bennick 1982).

APRPs perform unique functions. The need for these functions arise because of the particular environment of the oral cavity, which is the only place in the organims where mineralized tissue is exposed to an external environment. The oral cavity also provides an environment where certain microorganisms may flourish, and ingested food is a potential source of chemical, mechanical and thermal irritation of the hard and soft tissues. (Bennick 1982)


APRPs are transglutaminase substrates (3).

APRPs are substrates for buccal epithelial cell transglutaminase (3).

A membrane-bound epithelial enzyme, transglutaminase, catalyzes the covalent cross-linking of acidic proline-rich proteins (APRPs) to surface proteins of buccal epithelial cells (BECs) (10).

Buccal epithelial cells transglutaminase (BECs TGase) catalyzes the covalent crosslinking of acidic proline-rich proteins (APRP) to BECs (10).

Some biological properties of human salivary proline-rich proteins have been demonstrated in vitro studies. The acidic salivary proline-rich proteins bind Ca2+ (Kim 1993).

APRPs will bind calcium with a strength which indicates that they may be important in maintaining the concentration of ionic calcium in saliva (Bennick 1982).

Transglutaminases require Ca2+ to act.


The significance of [125I]APRP cross-linking in Candida adhesion has not been determined as of yet (10).

APRPs may interfere with TGase catalyzed mechanisms of adhesion (10).

Because [125I]APRP are also crosslinked to BECs surface proteins, however, it is tempting to speculate that APRPs may compete with cross-linking between Candida and BEC cell surfaces and, thus, interfere with TGase-catalyzed components of Candida/BEC adhesion (10).

Presumably, the reactivity of APRP with TGase is dependent on their high glutamine content (10).

Because other members of the family share this characteristic, it would not be surprising if TGase reacts with all members of the proline-rich family of proteins (10).

This family comprises approximately 70% of the total protein in parotid saliva, suggesting that proline-rich proteins may have a significant effect on TGase-catalyzed components of Candida adhesion (10).


Hwp1 (Hwp1 transglutaminase substrate domain) shares features with host acidic salivary proline-rich proteins (3).

Like hwp1, aprp contain glutamine residues within proline-rich repeats, a conformational arrangement that may be favorable for formation of ε (γ glutamyl) lysine cross-links by transglutaminase, given the properties of other known substrates for epithelial cell transglutaminases (3).

The similarity of hwp1 to aprp could be important for interactions between C. albicans and the oral mucosa. The presence of a polyphenol-binding extracellular proline-rich wall protein of the plant fungal pathogen Colletotrichum graminicola lends support to the idea that the presence of proteins with aprp-like properties might be a common feature of fungal cell walls (3).

The high percentage of proline residues and the length of the repeat (10 amino acids) place hwp1 within a varied group of proline-rich proteins in which the proline residues are proposed to function in maintaining the polypeptide chains in extended conformations and to mediate noncovalent interactions between protein chains or, in the case of salivary proteins, to bind toxic plant polyphenols. Of particular interest is the presence of acidic salivary proline-rich proteins (aprp) in this group of proline-rich proteins (3).

Because Candida albicans expresses TGase reactive proteins and because APRP appears to be a good substrate for TGase, it is not surprising that TGase catalyzes cross-linking of [125I]APRP to Candida surface proteins (10).

An aprp-like, transglutaminase substrate might also be present on surfaces of C. albicans (3).


When C. albicans interacts with the oral epithelium in vivo, the organisms are coated with saliva, which has been reported to either inhibit or enhance the adherence of C. albicans to oral epithelial cells (20).

Umazume M, Ueta E, Osaki T (1995) Reduced inhibition of Candida albicans adhesion by saliva from patients receiving oral cancer therapy. J Clin Microbiol 33: 432–439.

Holmes AR, Bandara BM, Cannon RD (2002) Saliva promotes Candida albicans adherence to human epithelial cells. J Dent Res 81: 28–32

Also, it is possible that salivary proteins could potentially act as bridging molecules between the hyphae and oral epithelial cells and thereby facilitate endocytosis of C. Albicans (20).

To investigate these possibilities, we incubated C. albicans hyphae in the presence and absence of 20% saliva and then measured their interactions with both intact FaDu oral epithelial cells and oral epithelial cell surface proteins. Killed organisms were used in these experiments to obviate potential effects of saliva on the growth of the hyphae. We have previously shown that killed hyphae adhere to and are endocytosed by oral epithelial cells similarly to live hyphae. Incubating hyphae in saliva prior to adding them to the epithelial cells had no effect on the number of endocytosed or cell-associated organisms. As predicted by the endocytosis assay, saliva also had no detectable effect on the binding of epithelial cell surface proteins, including E-cadherin, to C. albicans hyphae. Therefore, salivary components do not act as bridging molecules between the organisms and epithelial cells (20).



[14C]putrescine in the presence of purified TGase, but not [14C]putrescine alone, was shown to be cross-linked into surface proteins of both morphogenetic forms (blastospore > hyphal forms) of Candida albicans (10).

TGase catalyzed cross-linking of [14C]putrescine into proteins was used to detect the expression of TGase reactive substrates on the cell-wall surface of both the blastospore and hyphal forms of Candida albicans what could cause Candida albicans adhesion by catalyzing Tgase cross-linking between Candida and oral epithelial cells (10).

Purified TGase was used to cross-link [14C]putrescine into TGase proteins on the surface of both the blastospore and hyphal forms of C. albicans. Following incubation, [14C]putrescine cross-linking to proteins on the cell walls of blastospores appeared to be constant when maintained in M199 over a 3-h period (BELOW; open bars). When blastospores were induced to germinate in the same medium, however, the amount of [14C]putrescine cross-linked to cell-wall proteins significantly increased over the same time period (BELOW; black bars) (10)

BELOW: Reaction of Candida cell-wall proteins with BEC TGase. lodinated proteins extracted from Candida albicans cell wall and proteins expressed on Candida albicans cell-wall surfaces react with BEC and purified TGase, respectively. Right: lodinated cell-wall extract alone (lane a) was cross-linked when incubated with BEC (lane b) and cross-linking was inhibited with iodoacetamide (lane c). Left: Over time, cross-linking of [14C]putrescine to Candida Albicans blatospores by purified TGase was constant (open bars), but cross-linking increased proportionally with time following germination (black bars). [14C]putrescine was covalently cross-linked into cell wall proteins of blatospore (inset; lane a) and hyphal forms (inset; lanes) (10):

Following incubation of Candida albicans with [14C]putrescine alone, only negligible amounts (<3% of that in experimental blastospores) were associated with the Candida cell-wall digest (10).

Fluorographs of the digested cell-wall material from both the blastospore and hyphal forms of Candida albicans showed a single high molecular weight component at the top of the resolving gel (10).

A representative fluorograph of blastospore and hyphal form digests (inset; lanes a and b, respectively) indicates that [14C]putrescine was indeed cross-linked to proteins in the cell-wall digests (10).

The expression of TGase reactive proteins appears to increase following transformation of Candida from the blastospore to the hyphal form. It is not clear whether this represents increased expression of a blastospore-phase protein in hyphal cell walls or a de novo expression of a hypha-specific TGase reactive protein (10).

Because [14C]putrescine-labeled complexes were too large to be separated by a 10% SDS-PAGE gel, it is not clear whether the complexes contained a high molecular weight protein(s) or TGase protein(s)/carbohydrate complex(es). Given the high content of manno-protein in the Candida cell wall, it seems likely that the latter is most probable (10).


TGase cross-links APRPs to candidal and epithelial surface proteins; APRPs were cross-linked to the Candida surface; When incubated with [125I]APRPs and purified TGase, both morphogenetic forms of Candida albicans bound dramatically more APRP than controls without TGase (10).

Incubation of Candida albicans with TGase and [125I]APRPs suggests that [125I]APRP can be cross-linked to TGase-reactive proteins on the surface of Candida albicans (10).

Both blastospore (open bars) and the hyphal form (closed bars) retained significant amounts of [1251]APRP (10).

Incubation with [125I]APRP alone resulted in the binding of <2% of that retained by the experimental blastospores (10).

Representative autoradiographs of the digested cell-wall material from both the blastospore and hyphal forms (lanes c and d, respectively) show high molecular weight complexes (10).

The molecular weights of these components are significantly higher than that of [125I]APRP incubated in the reaction buffer alone (lane a) or with [125I]APRP incubated with either morphogenetic form of Candida albicans (lane b, representative autoradiograph), suggesting that [125I]APRP is cross-linked with Candida cell-wall proteins into high-molecular-weight complexes (10).

The cross-linking of [125I]APRP to blastospores or hyphal forms was somewhat different than that of [14C]putrescine (10).

In general, less [125I]APRP remained cross-linked to the blastospore (open bar) than the hyphal form (closed bar); however, the difference between the amount of [14C]putrescine cross-linked to blastospores and hyphal forms is greater than [125I]APRP to the same morphogenetic forms under similar conditions (10).

Additionally, the pattern of [125I]APRP cross-linking to each morphogenetic form over time was not the same as for [14C]putrescine. For instance, the amount of [125I]APRP cross-linked to blastospore and hyphal forms decreased between 2 and 3 h, whereas in the same time period, [14C]putrescine cross-linking stayed the same or increased in blastospores and hyphal forms, respectively. It is possible that this is an experimental artifact; however, the same trends were repeated in two similar experiments and these results could not be explained by cell loss during extraction procedures (10).

Comparison between [14C]putrescine and [125I]APRP cross-linking to Candida albicans surface in the presence of TGase suggest that these two TGase substrates may be cross-linked to two different groups of proteins on the Candida cell surface (10).

BELOW: TGase-catalyzed cross-linking of APRPs to Candida Albicans. Purified TGase appeared to cross-link [125I]APRP to proteins on the surface of both blatospore and hyphal forms of Candida Albicans. Left: [125I]APRP could not be completely extracted from either blastospore (open bars) or hyphal forms (closed bars). Right: TGase/[125I]APRP-treated Candida Albicans indicate that cell-wall digests of the blastospore form (lane c) and the hyphal form (lane d) contain [125I]APRP cross-linked into a high-molecular-weight complex (arrow). [125I]APRP alone incubated with reaction buffer (lane a, representative sample) or with both forms of Candida (lane b, representative sample) were not cross-linked into higher-molecular-weight complexes (10):


Epithelial TGase may stabilize Candida adherence by cross-linking Candida and BEC surface proteins (10).

A component of blastospore form Candida/BEC adherence was shown to be resistant to detachment (10).

Iodoacetamide is the simplest irreversible inhibitor of transglutaminase. It inhibits transglutaminase-mediated protein cross-linking by binding to the active site of the enzyme and preventing substrate binding.

Inhibition of BEC TGase (using iodoacetamide) increased detachment of Candida Albicans blastospore forms from buccal epithelial cells under dissociation conditions (10).

Pretreatment of BECs with iodoacetamide was shown to inhibit the cross-linking of salivary components on the surface of BECs (10).

Pretreatment of BECs with iodoacetamide increased detachment of C. albicans blastospore forms from BECs under dissociation conditions (10).

Pretreatment of BECs with iodoacetamide decreased adherence of blastospore form of Candida albicans by approximately 75% (10).

Pretreatment of BECs with iodoacetamide (plot b) increased dissociation of blastospores significantly (~75%) over that of control samples not inhibited by iodoacetamide (plot a) (10).


BELOW: Implication of TGase in Candida Albicans blastospore forms / buccal epithelial cells (BECs) adhesion. Inhibition of BEC TGase increased detachment of Candida Albicans blastospore forms from BECs. Pretreatment of BECs with iodoacetamide increased dissociation of blastospore forms (plot a) over that of control samples not treated with iodoacetamide (plot b) (10):

In the present study, the increased dissociation of blastospores and hyphal forms from iodoacetamide-treated BECs suggests that inhibition of BEC TGase may destabilize Candida adhesion by inhibiting cross-linking between Candida and epithelial cell surfaces (10).

The inability of heating in SDS to increase the dissociation of either experimental or control blastospores suggests that TGase-catalyzed cross-linking may play a significant role in stabilization of C. albicans (ATCC 28366) adhesion to BECs (10).



Studying  attachment of Candida albicans to the oral mucosa it was shown that pretreatment of BECs with rHwp1N13 produced inhibition of germ tube stabilized adhesion by 78% (11).

The reasons that inhibition did not reach 100% are not known (11).

Why didn´t recombinant Hwp1 protein reach 100% of inhibition of Candida Albicans germ tube stabilized adhesion to buccal epithelial cells?

The presence of additional TGase substrates  is unlikely given that null hwp1/hwp1 mutants do not possess surface TGase substrate activity (11 making reference to 1).

One possibility is that the physical interaction of the germ tube surface to a BEC exposes cross-linking sites that are not accessible to masking by soluble rHwp1N13 molecules, thereby leading to incomplete inhibition of stabilized adhesion (11).




Adhesion of Candida albicans to host epithelial cells is a critical, essential first step in the infection process, for both colonization and subsequent induction of mucosal disease (14).

Candida albicans must be adherent enough to persist on mucosal surfaces (3).

The adhesion of different Candida species to host tissue correlates with their virulence, Candida Albicans adhering far better than other Candida species or the apathogenic yeast Saccharomyces cerevisiae to epithelial and endothelial cells as well as to extracellular matrix proteins (Emody 2000).

In addition, differences in relative virulence have also been observed in Candida Albicans strains with differing degrees of adherence, and spontaneous mutants defective in adherence to buccal epithelial cells are less virulent in animal models than the parent strain (Emody 2000).

As Candida Albicans adheres to many different types of host surfaces, it probably possesses a broad panel of adherence factors (Emody 2000).

Because adherence is essential for C. albicans to persist on mucosal surfaces, it is not surprising that this organism expresses multiple different surface structures that mediate adherence to epithelial cells (14).

A large number of specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells (13).

These various adhesins frequently exhibit differential expression on yeast versus hyphae, and mediate adherence by different mechanisms (14).

Although the numerous binding activities that have been attributed to hyphae involve a variety of host cells and host proteins, a frequent finding is that surface proteins of hyphae are involved in binding (3).

Hwp1 is one of these adhesins with an important role in adhesion.


Hwp1 is one of the Candida albicans surface proteins on hyphae.

Hwp1 on hyphae mediates fungal adhesion to epithelial cells (13).

The involvement of Hwp1 in a novel mechanism of adherence was predicted after the discovery that the amino acid sequence of the NH2-terminal domain resembled mammalian proteins that are substrates for transglutaminase (TGase) cross-linking enzymes (12).

Epithelial TGases in rodents and other mammals are essential for formation of a cross-linked network of squamous epithelial cell–specific proteins such as involucrin, loricrin, and the small, proline-rich proteins that make up the primary host defense barrier. The presence of Hwp1 might enable Candida albicans to form tight attachments to the oral mucosa through TGase-catalyzed cross-linking between Hwp1 and epithelial cell proteins. In in vitro experiments, both the amino-terminal domain of Hwp1 and germ tubes expressing Hwp1 became tightly attached to buccal epithelial cell surfaces through epithelial cell TGase activity (12).

Localization of the adhesive domain of Hwp1 is to the N-terminal third of the protein (repetitive proline/glutamine rich tgase substrate region) (9).

Hwp1 forms tight attachments to stratified squamous epithelial cells with cell surface-exposed TGase activity (11).


Hwp1 forms covalent cross-links to buccal epithelial cells in vitro by functioning as a substrate for mammalian transglutaminases (12).

Hwp1 becomes cross-linked to TGase-expressing surface squames of the oral mucosa (9).

Mammalian transglutaminase cross-links Candida albicans to human buccal epithelial cells (BECs) (1).

Hwp1 in hyphae of Candida Albicans is crosslinked to human buccal epithelium by mammalian transglutaminase (1).

TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1) would be correlated with tight binding of C. albicans via Hwp1 to the host cell surfaces (13).

TGase activity of the surfaces of BEC has been attributed to membrane-bound TGase 1 where it functions to cross-link proteins in the formation of the cornified cell envelope (11).

During stabilized adhesion of germ tubes to the surface of BEC, the insoluble TGase 1 probably contacts the most surface-exposed Hwp1 in the cell wall and cross-links the protein (and the organism) to yet unidentified host surface protein(s) (11).

Physical constraints would prevent BEC TGase 1 from penetrating the hyphal wall to interact with the non-wall species of Hwp1 (11).


Tgase covalently link Candida Albicans to the intestinal epithelium and endomysium by means of Hwp1 (2).

Hwp1 is used by Candida albicans to adhere to the intestinal epithelium. Adherence of the yeast to intestinal epithelial cells is mediated by transglutaminase. Mammalian transglutaminasa covalently link Candida Albicans to the intestinal epithelium and endomysium (2).

Tissue transglutaminase and endomysium components could become covalently linked to the yeast (2).

Adherence of the yeast to intestinal epithelial cells is mediated by transglutaminase, and a peptide that represents aminoacids 40–197 of Hwp1 (rHwp1ΔC37), which is a tissue transglutaminase substrate (2).

In immunofluorescence localization of TGase1 in epithelial tissues, diffuse signals were detected with anti-TGase1 antibodies in non-junctional plasma membranes such as microvilli (15).

Candida Albicans adhesion (filamentous, hyphal forms)


TGase stabilizes Candida albicans adhesion by covalently cross-linking Candida albicans Hwp1 and buccal epithelial cells (BECs) surface proteins

The primary role of Hwp1 is in the formation of stable attachments by means of cross-linking of germ tubes to buccal epithelial cells (BECs) (1).

Requirement for Hwp1 in stabilized attachment of germ tubes to buccal epithelial cells (BECs): unlike most pathogens that form various types of weak interactions with host cells, Candida albicans germ tubes form nondissociable complexes with human buccal epithelial cells (BECs) that are characteristic of transglutaminase (TGase)-mediated reactions in stability and in being prevented by TGase inhibitors (1).


HWP1 null mutants have been shown to be defective in forming stable attachments to buccal epithelium: Hwp1 is required for formation of stable complexes between C. albicans germ tubes and BECs: Candida albicans strains lacking Hwp1 were unable to form stable attachments to human buccal epithelial cells (BECs). The stable attachments greatly reduced in strains lacking Hwp1 as seen by comparing strains with or without Hwp1 in stabilized adhesion assays, which involved treatment of germ tube:BEC complexes with heat and the anionic detergent SDS to dissociate weak, noncovalent bonds.  The mutant strain without Hwp1 was greatly impaired in the ability to form stable attachments to BECs in that stabilized adhesion was only 23% of the other strains and was equivalent to values for other strains when TGase inhibitors were added (1).


rHwp1∆C37 forms stable attachments to BECs: rHwp1ΔC37, a recombinant protein that encompasses the NH2-terminal proline- and glutamine-rich domain of Hwp1 (aminoacids 40 to 197 of Hwp1) was radiolabeled and incubated with BECs in the presence or absence of iodoacetamide followed by treatment with heat and SDS. The results showed rHwp1∆C37 associated with BECs was sixfold greater when TGase was not inhibited thus providing further support for the role of Hwp1 in mediating stabilized adhesion (1).

Below: Radiolabeled rHwp1ΔC37 were incubated with BECs under adhesion assay conditions with or without iodoacetamide. Then associated rHwp1ΔC37 with BECs envelopes were determined (1):


Hyphae complexed to human BECs in vivo in specimens of pseudomembranous candidiasis of the buccal mucosa were not dissociated by heat and detergent treatments (up to 30 min) used in the stabilized adhesion assays, indicating predominance of stable attachments (1).

Germ tubes and hyphae of C. albicans exhibit highly polarized, apical growth and require mechanisms for anchoring. In mimicking mammalian TGase substrates, Hwp1 forms stable attachments between germ tubes and mammalian cells (1).

Stabilized adhesion was prevented by monodansylcadaverine, a competitive inhibitor of TGase-mediated protein cross-linking reactions, and by iodoacetamide, supporting the involvement of BEC Tgases (1).

Below: Candida Albicans strains with or without Hwp1 were used for comparison in TGase assays for stabilized adhesion. Transglutaminase inhibitors also were used (1):





The function of the N terminus of Hwp1 in stabilized adhesion to the surface of BECs was investigated by using rHwp1N13, comprising amino acids 1–148 of the mature protein (aminoacids 40 to 187 of Hwp1) as a competitor in adhesion assays (11).

The ability of rHwp1N13 to compete or inhibit stabilized adhesion of germ tubes to BECs was determined by preincubating rHwp1N13 or salivary amylase (negative control) with BECs prior to the addition of germ tubes. The adhesion of germ tubes to BECs in the presence of rHwp1N13 or salivary amylase was set relative to assays in the absence of added proteins (11).

Preincubation with rHwp1N13 prior to the addition of germ tubes reduced relative adhesion by 60% compared with controls without added protein or in the presence of the control protein, salivary amylase (11).

Inhibition was 78% of the maximum expected amount based on adhesion of the hwp1/hwp1 null mutant, which was 23% of control (11).

Below: Inhibition of germ tube adhesion by rHwp1N13. BECs were incubated with rHwp1N13 or salivary amilase prior to adding radiolabeled wild type germ tubes to measure the ability of rHwp1N13 to interfere with germ tube adhesion. Germ tube adhesion to BEC was determined relative to assays performed in the absence of added protein (No protein, set at 100% adhesion). The germ tube adhesion inhibition by rHwp1N13 was significant relative to no protein. Salivary amylase did not have an appreciable effect on germ tube adhesion (11):

The results show that the stabilized adhesion function of Hwp1 maps to the N-terminal portion of the protein (11).

The 78% inhibition of germ tube stabilized adhesion by pretreatment of BECs with rHwp1N13 is consistent with the importance of the TGase substrate domain of native Hwp1 in attachment of Candida albicans to the oral mucosa (11).

The reasons that inhibition did not reach 100% are not known (11).


Adhesive mechanism = covalently cross-linking of Candida albicans Hwp1 to BEC surface proteins by transglutaminase.

The host protein(s) participating in cross-links to Hwp1 remain unknown (9).

The presence of host innate and specific immune responses to adhesins and their potential role in preventing candidiasis have not been approached experimentally (9).


NOBILE 2006 (17):

Whether Hwp1 functions as an adhesin in the absence of host transglutaminase activity is less certain, though the possibility has never been ruled out. Indeed, a possible function for Hwp1 in C. albicans cell-cell adherence comes from the finding that it is induced by mating factor and is deposited on the surface of the bridge between mating partners. This localization might be expected for a cell-cell adhesin (17).


PHAN 2007 (20):

In our assays, the mutant without Als3 had significantly reduced adherence to endothelial cells, but normal adherence to oral epithelial cells (20).

It has been reported by others that a mutant without Als3 had reduced adherence to endothelial cells, buccal epithelial cells, and FaDu epithelial cells (20).

Also, we found that S. cerevisiae expressing Als3 bound to both endothelial cells and FaDu oral epithelial cells (20).

The discrepancy between the previous and current results is likely due to differences in the methodology of the assays. Specifically, the longer incubation time and the use of a 24-well tissue culture plate (rather than a 6-well plate) in the current endocytosis assay make it less sensitive to differences in adherence among strains (20).

In the affinity purification experiments, hyphaeof the  mutant without Als3 failed to bind to multiple host proteins, including N-cadherin and E-cadherin. Thus, these results are consistent with the model that Als3 binds to a broad range of host substrates. However, our data also indicate that there is some specificity in the binding of Als proteins to host constituents (20).


Coating the latex beads with rAls3-N resulted in a much greater increase in endocytosis than adherence. Also, coating beads with rAls1-N resulted in little to no increase in adherence, even though previous studies have clearly demonstrated that Als1 is an adhesin (20).

The likely explanation for the relative lack of adherence of the beads is that they were coated with fragments of Als1 or Als3 that lacked the tandem repeats present in the central portion of the full-length proteins (20).

Previously, we have found that the adhesive function of Als1 is dramatically reduced when some or all of the tandem repeats are eliminated (20).

Similarly, Oh et al. found that versions of Als3 with fewer tandem repeats mediated less adherence than did versions of Als3 with more tandem repeats. (20).

In addition, Rauceo et al. reported that the absence of tandem repeats reduced the binding affinity of Als5 (20).


S. cerevisiae expressing Als1 or Als3 binds to a variety of host constituents, including endothelial cells, epithelial cells, laminin, and fibronectin (20).




Als3 is another Candida Albicans surface protein.

Als3 is a Candida Albicans protein belonging to the The Candida albicans ALS (agglutinin-like sequence) gene family (14).

Als proteins have adherence properties (20).

The different Als proteins mediate adherence to broad variety of host substrates, and this adherence is likely critical for Candida albicans to infect host surfaces (20).

Each Als protein has three domains. The N-terminal domain contains the substrate-binding region. The central portion of the protein consists of a variable number of 36–amino acid tandem repeats. The C-terminal domain is rich in serine and threonine, and contains a GPI anchorage sequence that is predicted to be cleaved as the protein is exported to the cell surface (20).

Below: Als3 protein (Klotz 2010):

Als3 & Invasion:

Transmission electron microscopic imaging of biopsy specimens from humans with oropharyngeal, vaginal and cutaneous candidiasis shows the presence of intraepithelial cell organisms, demonstrating that epithelial invasion occurs during these diseases (14).

Candida albicans is unique among oral pathogens in its ability to invade cornified layers of stratified squamous epithelium of the tongue, buccal surfaces, hard and soft palate, and esophagous (11).

In mice infected with C albicans, invasion and lysis of the villi in the intestine has been reported (2).

The switch from yeast to filamentous growth facilitates tissue invasion and is associated with the transition of Candida albicans from a harmless colonizer to a pathogen that causes symptomatic infections.

Hyphae appear to be the invasive form of the organisms, as the majority of intracellular organisms are hyphae, whereas yeast are typically located either between or on the surface of epithelial cells (14).

Epithelial cell invasion is important for the pathogenesis of mucosal candidiasis, because mutants of Candida albicans with reduced capacity to invade epithelial cells in vitro usually have reduced virulence in experimental animal models of mucosal candidiasis (14).

Candida Albicans (filamentous, hyphal forms invasion)


Candida Albicans (filamentous, hyphal forms invasion)


Adherent Candida albicans cells can invade epithelial surfaces both by penetrating into individual epithelial cells, and by degrading interepithelial cell junctions and passing between epithelial cells (14).

Invasion into epithelial cells is mediated by both induced endocytosis and active penetration, whereas degradation of epithelial cell junction proteins, such as E-cadherin, occurs mainly via proteolysis by secreted aspartyl proteinases (14).

The mechanisms by which Candida albicans invades epithelial cells have been investigated using in vitro models. The results of these studies suggest that Candida albicans can invade epithelial cells by two distinct mechanisms (14):


One mechanism is the induction of epithelial cell endocytosis by the organism. Endocytosis is induced by invasin-like proteins that are expressed on the surface of a C. albicans hypha. These proteins bind to epithelial cell surface proteins and induce the epithelial cell to produce pseudopods that engulf the organism and pull it inside the cell (14).

Candida albicans induces its own endocytosis by multiple epithelial cell lines, including HeLa cells, HET1-A esophageal cells, FaDu pharyngeal cells, OKF6/TERT-2 oral epithelial cells, and reconstituted human epithelia, which is a three-dimensional model of oral or vaginal epithelium (14).


Another mechanism of invasion is the active penetration of a hypha either into or between epithelial cells. This process requires fungal viability (14).

Candida albicans likely invades epithelial cells from different anatomic sites via different mechanisms. For example, the organism invades oral epithelial cell lines by both induced endocytosis and active penetration, whereas it invades a gastrointestinal epithelial cell line only by active penetration (14).


Below: High-power view of lingual longitudinal section of immunodeficient infected mouse being invaded by Candida Albicans showing that damage resulted from microabscesses formed in response to hyphal invasion of the keratinized layer (stained pink). Polymorphonuclear leukocytes (PMNL) form giant aggregates (pink arrow) that sever the keratin layer containing hyphae (red arrow) from the underlying epithelium. Although polymorphonuclear leukocytes (PMNL) occasionally appeared to be directed toward hyphae, PMNL more commonly formed intraepithelial microabscesses that fused and separated hyphae-laden keratin from the underlying stratum spinosum (12):

Below: Histopathologic sections of depapillated areas on the dorsal tongue surface of a rat, showing epithelial invasion by C. Albicans hyphal elements.  Candida albicans produce fungal hyphae that penetrated the lingual epithelium and stopped short of the prickle cell layer. C. albicans hyphae penetrating the lingual mucosa were 5.00 to 17.71 mm (Samaranayake 2001):


Als3 not only mediates epithelial cell adherence, but also functions as an invasin that induces epithelial cell endocytosis (14).

Als3 has been made responsible of invasion.

Als3 can act as an invasin protein and induce endocytosis by normally nonphagocytic host cells (20).

Candida albicans hyphae invade endothelial cells and oral epithelial cells in vitro by inducing their own endocytosis (20).

Als3, like Hwp1 appears exclusively on hyphae and is the fungal surface protein that mediate this process (20).

Candida albicans mutant without Als3 has markedly impaired capacity to invade epithelial cells (14).

Als3-mediated adhesion has not been related to transglutaminase but what about invasion?

Hwp1 has not been related to invasion however some transglutaminase interaction could exist in the invasion process on the basis described in section more below  “E-CADHERIN: THE LINK BETWEEN ENDOCYTOSIS AND ACTIVE PENETRATION?” . If this transglutaminase interactions really exist, a protein substrate of transglutaminase different from Hwp1 (since Hwp1 has not been implicated in invasion) must exist on the surface of Candida Albicans. Will it be Als3 protein?

Hwp1 versus Als3:

Below: Comparisson of Hwp1 (Sundstrom 2002) and Als3 (Klotz 2010):


- Cell surface proteins (like all ALS family) (14)

- Adhesins; mediate binding to diverse host substrates (like all ALS family) (14)

- Mannoproteins (like all ALS)

- N-terminal domain contains the substrate binding region (like all ALS) (14)

- C-terminal domain is rich in serine and threonine (like all ALS) (14)

- Als1, Als3 and Als5 mediate adherence to multiple host constituents including oral epithelial cells (Hwp1 also mediates adherence to oral epithelial cells), whereas Als6 and Als9 bind to a much more limited range of host substrates and do not mediate adherence to epithelial cells. Als7 does not bind to any host substrates tested to date. The adherence function of Als2 and Als4 has not been investigated by this approach (heterologous expression of a C. albicans ALS gene in the normally non-adherent yeast, Saccharomyces cerevisiae) (14)

- Studies of C. albicans deletion mutants suggest that Als2, Als3 and Als4 mediate adherence to epithelial cells. Als1 has been found to mediate adherence to mouse tongues in an ex vivo assay, but not to exfoliated human buccal epithelial cells (14).

- Expressed only on the surface of Candida albicans hyphae. Some adhesins are preferentially expressed by specific morphological forms of C. albicans. For example, ALS3 and HWP1 are expressed by hyphae, but not yeast-phase organisms. In contrast, ALS1 is expressed by yeast cells under some conditions and for only a short time after hyphal formation is initiated (14).


- Repeats in central domain (variables number of tandem repeat sequences) (like all ALS) (14)

- N-termini of Als proteins predicts the presence of antiparallel β sheets, indicating that these proteins are members of the immunoglobulin superfamily (all ALS) Interestingly, the three-dimensional structures of the N-termini of most Als proteins are predicted to be similar to the three-dimensional structure of bacterial adhesins, including invasin of Yersina pseudotuberculosis, and collagen-binding protein and clumping factor A of Staphylococcus aureus (14)

- The N-terminal region of Hwp1 functions as a substrate for epithelial cell-associated transglutaminases that covalently link it to other proteins on the epithelial cell surface (14).



The interactions of Candida Albicans mutants without Als1 and without Als3 with human umbilical vein endothelial cells and the FaDu oral epithelial cell line were investigated. Using our standard differential fluorescence assay, we determined the capacity of each strain of C. albicans to adhere to these host cells and induce its own endocytosis (20).

Below: Hyphae of the mutant without Als1 interacted with both host cell types similarly to the wild-type strain, in contrast, there was a 90% reduction in the number of hyphae of the mutant without Als3 that were endocytosed by either endothelial or oral epithelial cells compared to the wild-type control strain (20):


Previously, we have reported that N-cadherin is one of the endothelial cell surface proteins that mediate endocytosis of C. Albicans (20).



Endocytosis of Candida albicans by endothelial cells is induced when the organism binds to N-cadherin on the endothelial cell surface (20).

Below: We used an affinity purification approach to determine whether Als1 or Als3 was required for Candida albicans hyphae to bind to N-cadherin in endothelial cell membrane extracts (20).

As predicted by the results of the endocytosis assay, mutant without Als1 bound to the same endothelial cell surface proteins, including N-cadherin, as did the wild-type strain. In contrast, the mutant without Als3 did not bind to N-cadherin, and it bound very weakly to the other endothelial cell surface proteins (20).

Als3 is required for C. albicans to bind to N-cadherin and other proteins on the endothelial cell surface (20).


The surface proteins on oral epithelial cells that are bound by Candida albicans hyphae have not been identified previously (20).

Below: We found that the wild-type strain of Candida albicans bound to at least four different proteins on the surface of FaDu oral epithelial cells. Many of these proteins appeared to be different from the endothelial cell proteins that were bound by wild-type C. albicans. FaDu oral epithelial cells express very low levels of N-cadherin (unpublished data), but high levels of E-cadherin. Therefore, we investigated whether E-cadherin was one of the epithelial cell proteins that was bound by C. albicans. Using an anti-E-cadherin monoclonal antibody, we detected a significant amount of this protein in immunoblots of epithelial cell membrane proteins that were bound by hyphae of wild-type C. Albicans. Furthermore, although the mutant without Als1 still bound to E-cadherin, the mutant without Als3 did not (20):

Candida albicans hyphae bind to epithelial cell E-cadherin in an Als3-dependent manner (20).

Invasion by endocytosis occurs as a result of the N-terminal region of Als3 binding to either N-cadherin or E- cadherin (20).


Binding of Candida albicans hyphae to N-cadherin and E-cadherin on the surface of intact host cells required Als3 and was associated with induction of endocytosis (20).


Monolayers of endothelial cells or FaDu oral epithelial cells were infected with the various strains of C. albicans, after which N-cadherin and E-cadherin were detected by indirect immunofluorescence. To determine if the organisms were in the process of being endocytosed, the host cells were also stained for f-actin, which accumulates around such organisms. We observed that endothelial cell N-cadherin colocalized with hyphae of the wild-type strain and the mutant without Als1 that were being endocytosed (20).

In contrast, N-cadherin did not colocalize with hyphae of the mutant without Als3, and almost none of these hyphae were endocytosed. As expected, N-cadherin accumulated around hyphae of the als3D/als3D::ALS3 complemented strain similarly to the wild-type control strain (20).

Below: N-Cadherin from Endothelial Cells Colocalizes with Wild-type C. Albicans, but Not a mutant without Als3. Confocal micrographs of uninfected endothelial cells (A-C), or endothelial cells infected with the wild type strain (D-G), the mutant without Als3 (H-K) or the mutant without Als3 complemented with Als3 (L-O). The cells were stained for N-cadherin (A), (D), (H) and (L), actin microfilaments (B), (E), (I) and (M), and C. Albicans (F), (J) and (N). The merged images are shown in (C), (G), (K) and (O). Arrows indicate the accumulation of N-cadherin and microfilaments around the organisms (20):


A comparable pattern was observed with E-cadherin and oral epithelial cells (20).

E-cadherin colocalized with the endocytosed hyphae of all strains except for the mutant without Als3. Also, very few hyphae of the mutant without Als3 were endocytosed. The anti-N-cadherin and anti-E-cadherin monoclonal antibodies did not bind to C. albicans hyphae in the absence of endothelial or epithelial cells, indicating that these antibodies did not recognize any cross-reacting C. albicans antigens (20).

Below: E-Cadherin from Oral Epithelial Cells Colocalizes with Wild-type C. Albicans, but Not a mutant without Als3. Confocal micrographs of uninfected FaDu oral epithelial cells (A-C), or epithelial cells infected with the wild type strain (D-G), the mutant without Als3 (H-K) or the mutant without Als3 complemented with Als3 (L-O). The cells were stained for E-cadherin (A), (D), (H) and (L), actin microfilaments (B), (E), (I) and (M), and C. Albicans (F), (J) and (N). The merged images are shown in (C), (G), (K) and (O). Arrows indicate the accumulation of E-cadherin and microfilaments around the organisms (20):


Collectively, these findings support a model in which C. albicans Als3 binds to endothelial cell N-cadherin and oral epithelial cell E-cadherin, thereby stimulating actin-mediated endocytosis of the organism (20).


A striking finding was that the 3-D structures of the N-terminal regions of Als1 and Als3 are predicted to have recurrent β barrel domains and a global negative surface potential similar to the N-terminal regions of N-cadherin and E-cadherin (20).

The N-terminal regions of Als1 and Als3 bear a striking resemblance to the N-terminal ectodomains of N- and E-cadherin, however, the predicted N-Terminal Structures of Als1 and Als3 are different (20).


The predicted parameters of Als3 binding to N-cadherin were very similar to those of one molecule of N-cadherin binding to another (N-cadherin self-association), but different regions of Als1 and Als3 bind to N-cadherin. The N1 and N3 domains of Als1 bind to domains N1 and N2 of N-cadherin, whereas the N2 and N3 domains of Als3 bind to domains N1 and N2 (20).


Als1 and Als3 interact with N- and E-cadherin via different domains, and the geometry of Als3 binding to E-cadherin is similar to that of E-cadherin binding to itself (20).

The N-terminal regions of Als1 and Als3 are also predicted to interact differently with E-cadherin. The energetics of Als1 binding to E-cadherin are considerably weaker than either Als3 binding to E-cadherin or E-cadherin binding to itself (20).

The Als3-E-cadherin interaction is somewhat different from the Als3–N-cadherin interaction. The geometry of this interaction assumes a parallel orientation, such as that which occurs when E-cadherin binds to itself (20).


Als3 Is Predicted to Bind to Cadherins Differently than Als1 (20).

Als3 functions as a molecular mimic of mammalian cadherins, thereby facilitating C. albicans invasion of endothelial and oral epithelial cells (20).



Relatively few Candida albicans adhesins have been evaluated for invasin function (has Hwp1 be?); analysing additional adhesins will help clarify the relationship between adherence and invasion (14).

Als1 and Als3 act as invasins.

Als1 has lower invasive (endocytosis) capacity than Als3.

It is also virtually certain that C. albicans proteins other than Als1 and Als3 can induce epithelial cell endocytosis. For example, Candida albicans mutant without Als3 and without Als1 has only modestly reduced virulence in the mouse model of oropharyngeal candidiasis, indicating that other invasin-like proteins or other mechanisms of invasion can compensate for the absence of Als1 and Als3. Determining how C. albicans invades epithelial cells independently of Als1 and Als3 is an important future challenge (14).

Another Als Candida Albicans protein working as invasin is Als5.

Als5 induces weak endothelial cell endocytosis similar to that induced by Als1 (20).

Als3, Als1 and Als5 are Candida Albicans proteins belonging to the The C. albicans ALS (agglutinin-like sequence) gene family (14).

Computer-assisted modeling of the N-termini of Als1, Als3, and Als5 predicts the presence of antiparallel β sheets that define the immunoglobulin superfamily (20).


Saccharomyces cerevisiae expressing Als3 were avidly endocytosed by endothelial cells (20).

Als1 and Als3 have very similar binding specificities in vitro, and the N-terminal domain of either protein is sufficient to promote N- or E-cadherin-dependent endocytosis (18).

The fact that Als1 and Als3 have a shared function is consistent with their similarity in sequence (88% amino acid identity of their N-terminal 772 residues) and in their predicted N-terminal-domain structures (18).

Even though N-terminal regions of Als1 and Als3 share general features, they are predicted to differ both in their 3-D structure and their interactions with N- and E-cadherin. While Als1 appears to favor an extended conformation, Als3 tends toward a more folded structure that forms a more distinct molecular cleft (20).

Our molecular modeling results also suggest that Als1 binds more weakly to N- and E-cadherin than does Als3 (20).

Als3 binds with greater affinity to N- and E-cadherin than does Als1 (20).

Although the N-termini of Als1 and Als3 share considerable homology at the amino acid level, Als3 induced endocytosis much more efficiently than did Als1 (20).

Similar studies suggest that Als1 can also induce epithelial cell endocytosis, although with lower efficiency than Als3 (14).

The Als family of proteins contains six members in addition to Als1 and Als3. Most of these proteins are known to have adhesive function. When expressed in S. cerevisiae, Als5 induces weak endothelial cell endocytosis similar to that induced by Als1 (20).

Similar experiments suggested that Als1 and Als5 could also induce endocytosis, although with considerably less efficiency than Als3 (20).

Als6, Als7, and Als9 do not appear to mediate significant endothelial cell endocytosis, and Als2 and Als4 have not yet been tested in this assay (20).

Below: sequence comparisson between Als proteins related to its invasive ability and presence of potential transglutaminase substrates:


Als protein















Weak invasion







Weak invasion







No invasion







No invasion







No invasion







Als protein













Weak invasion






Weak invasion






No invasion






No invasion






No invasion






Als protein













Weak invasion






Weak invasion






No invasion






No invasion






No invasion







Als3 Is Required for C. albicans to Damage Endothelial Cells and Oral Epithelial Cells (20).

Infection of endothelial cells or oral epithelial cells with wild-type C. albicans hyphae in vitro causes significant damage to these cells, and endocytosis of the organisms is required to induce host cell damage (20).

Therefore, we investigated the extent of damage to endothelial cells and oral epithelial cells caused by the mutants without Als1 and Als3. Consistent with the results of the endocytosis assay, the mutant without Als1 caused the same amount of damage to both types of host cell as the wild-type strain. In contrast, the mutant without Als3 caused essentially no damage to either cell type. Complementing the als3D/als3D null mutant with a wild-type copy of ALS3 restored its capacity to damage these host cells. These data indicate that the inability of the mutant without Als3 to invade endothelial cells and oral epithelial cells is associated with a significantly reduced capacity to damage these cells (20).

A mutant without Asl3 caused virtually no damage to either endothelial cells or FaDu oral epithelial cells (20).

Below: A Candida Albicans mutant without Als3  has reduced capacity to damage Endothelial Cells and Oral Epithelial cells. The extent of host cell (endothelial and epithelial cells) damage by  Candida Albicans strains was determined by a 51Cr Release Assay (20):

A Candida Albicans mutant without Als3 was endocytosed poorly by endothelial cells and two different oral epithelial cell lines (20).


These current results are also consistent with our previous data that S. cerevisiae expressing Als3 are endocytosed by endothelial cells (20).

Similarly, Zhao et al. reported that a mutant without Als3 had markedly reduced capacity to damage reconstituted human epithelium. Our results suggest that the reduced capacity of the mutant without Als3 to damage endothelial cells and oral epithelial cells in vitro is due to its defect in invading these cells (20).

The invasion and damage defects of the mutant without Als3 suggest that Als3 is important for virulence during hematogenously disseminated and oropharyngeal candidiasis (20).


This process is passive on the part of the organism because killed hyphae are endocytosed similarly to live hyphae (14).

Studies with killed Candida Albicans indicate that induction of endocytosis is passive on the part of Candida Albicans because killed hyphae are endocytosed as avidly as are live hyphae (20).


Latex beads coated with the recombinant N-terminal portion of Als3 are avidly endocytosed by epithelial cells (14).


To determine if either Als1 or Als3 alone was able to induce endocytosis, we coated latex beads with the purified, recombinant N-terminal portion of Als1 (rAls1-N) or Als3 (rAls3-N), which consisted of amino acids 17 to 432 of either protein (20).

Latex beads coated with rAls3-N were efficiently and specifically endocytosed by both endothelial cells and oral epithelial cells (20).

Latex beads coated with just the N-terminus of Als3 were able to induce endocytosis. However, it was notable that coating the latex beads with rAls3-N resulted in a much greater increase in endocytosis than adherence (20).

The rAls3-N–coated beads were avidly endocytosed by CHO cells expressing either N-cadherin or E-cadherin, but not by CHO cells that did not express these cadherins (20).

The mutant without Als1 was endocytosed normally by oral epithelial cells, whereas latex beads coated with rAls1-N induced some endocytosis by these cells. The probable explanation for these apparently conflicting results is that mutant without Als1 still expressed Als3 on its surface. Thus, the presence of Als3 masked the effects of the absence of Als1 when the endocytosis assay was performed using whole organisms. Studies performed with latex beads coated with rAls1-N indicated that Als1 is capable of inducing epithelial cell endocytosis by itself (20).

Als1 and Als3 by Themselves Are Sufficient to Induce Endocytosis. However, beads coated with rAls3-N were endocytosed more avidly by CHO cells expressing N- or E-cadherin than were beads coated with rAls1-N (20).

Beads coated with rAls3-N were endocytosed much more efficiently than were beads coated with rAls1-N (20).


Als3, and to a lesser extent Als1, bind directly to N-cadherin and E-cadherin, and that this binding is sufficient to induce endocytosis. These data also indicate that the cadherin-binding domains of Als1 and Als3 are located in the N-termini of these proteins (20).


Tandem repeats influence the conformation and/or the substrate accessibility of the N-terminal region of Als3 and thereby enhance its binding affinity for host substrates. However, even the weak binding of the N-terminal portion of Als3 to N-cadherin or E-cadherin is sufficient to induce endocytosis (20).


Cadherins on the surface of human cells normally bind other cadherins for adhesion and signaling; however, we found that Als3 also binds to cadherins on endothelial cells and oral epithelial cells, and this binding induces these host cells to take up the fungus (20).

The results with the mutant without Als3 and the latex beads coated with rAls3-N demonstrate that endothelial cell N-cadherin and epithelial cell E-cadherin are two host cell ligands for Als3. Furthermore, binding of the N-terminus of Als3 to either of these cadherins is sufficient to induce endocytosis (20).

Als3 induces endocytosis by binding to E-cadherin and other proteins on the epithelial cell surface (14).

Als3 was required for Candida albicans to bind to multiple host cell surface proteins, including N-cadherin on endothelial cells and E-cadherin on oral epithelial cells (20).

The structure of Als3 is predicted to be quite similar to that of the two cadherins studied, and the parameters of the binding of Als3 to either cadherin are similar to those of cadherin–cadherin binding (20).

Molecular modeling of the interactions of the N-terminal region of Als3 with the ectodomains of N-cadherin and E-cadherin indicated that the binding parameters of Als3 to either cadherin are similar to those of cadherin–cadherin binding. Therefore, C. Albicans. Als3 is a functional and structural mimic of human cadherins, and provide new insights into how C. albicans invades host cells (20).

Computer-assisted molecular modeling of the binding of Als3 with N- and E-cadherin suggests that Als3 is a structural and functional mimic of these cadherins (20).

Als3 is a fungal invasin that mimics host cell cadherins and induces endocytosis by binding to N-cadherin on endothelial cells and E-cadherin on oral epithelial cells (20).

Binding of Als3 to E-cadherin is sufficient to induce endocytosis because latex beads coated with recombinant Als3 are internalized efficiently by Chinese hamster ovary cells expressing human E-cadherin (14).

Latex beads coated with the recombinant N-terminal portion of Als3 were endocytosed by Chinese hamster ovary cells expressing human N-cadherin or E-cadherin, whereas control beads coated with bovine serum albumin were not (20).


Endothelial cell endocytosis of Candida albicans is induced when the organism binds to N-cadherin and other endothelial cell surface proteins. This binding induces microfilament rearrangement, which results in the formation of pseudopods that engulf the organism and draw it into the cell (20).

Endothelial cell endocytosis of Candida albicans is dependent on extracellular calcium (transglutaminase is a calcium dependent enzyme) and is governed at least in part by the tyrosine phosphorylation of endothelial cell proteins (see more below transglutaminase type 1 (TGase1) was identified as a tyrosine-phosphorylated form colocalized with E-cadherin by Hiiragi 1999) (20).


The interaction of Als3 with E-cadherin activates the clathrin-dependent endocytosis pathway (14).

siRNA knockdown of components of this pathway, including clathrin, dynamin and cortactin, inhibits the endocytosis of Candida albicans by approximately 60% (14).

The fact that siRNA knockdown of the clathrin pathway results in incomplete inhibition of endocytosis suggests that additional signalling pathways also govern this process. These alternative signalling pathways are likely activated by receptors other than E-cadherin (14).

In support of this hypothesis, wild-type C. albicans hyphae bind to multiple epithelial cell surface proteins in addition to E-cadherin. Furthermore, Candida albicans mutant without Als3 fails to bind to several of these surface proteins, suggesting that Als3 has more than one epithelial cell target protein (14).

It is highly likely that additional host cell ligands, other than N- or E-cadherin, also contribute to the endocytosis of C. Albicans (20).

For many microbial pathogens, invasion of host cells is critical for the initiation and maintenance of infection, and many of these organisms have more than one mechanism for inducing their own uptake by host cells. Thus, it is logical to speculate that C. albicans has also evolved at least one Als3-independent mechanism to invade host cells (20).



Candida albicans can also invade epithelial cells by an active process that is independent of endocytosis called active penetration (14)

Active penetration seems to require hyphal formation and is not inhibited by when the epithelial cells are treated with a microfilament inhibitor, such as cytochalasin D (14).

During active penetration, the organism can either invade into an epithelial cell without inducing the formation of epithelial cell pseudopods or pass through the intercellular junction between epithelial cells (14).


The mechanism by which C. albicans actively penetrates into an epithelial cell is incompletely understood. It is possible that Saps contribute to this process (14).

Saps are Secreted Aspartyl proteases, one of the other Candida Albicans adhesins (14).


Pepstatin A, an inhibitor of aspartyl proteinases (Saps) has been reported to inhibit the invasion of C. albicans into corneocytes in mice with cutaneous candidiasis (14).

Some, but not all investigators have found that pepstatin A reduces C. albicans-induced damage to reconstituted human epithelia (14).

This reduced damage may be due in part to decreased epithelial cell invasion (14).

However, a limitation of the pepstatin A studies is that this protease inhibitor not only blocks C. albicans Sap activity, but it also inhibits the function of aspartyl proteases produced by the epithelial cell. Therefore, pepstatin A has effects on both the organism and the epithelial cell (14).

C. albicans mutants containing disruptions of various SAP genes have reduced capacity to damage vaginal and oral epithelial cells in some in vitro models, but not others (14).

It is possible that Sap activity is required for active penetration into epithelial cells because it alters the surface characteristics of C. albicans rather than degrading host cell proteins. Dalle et al. (2009) recently found that pepstatin A inhibits C. albicans invasion into epithelial cells only when the hyphae are pre-incubated with this inhibitor, prior to being added to the epithelial cells. They also found that triple mutants lacking either Sap1-3 or Sap4-6 have reduced capacity to invade epithelial cells. Importantly, these defects in epithelial cell invasion persist even when the mutants are killed (14).

 Taken together, these results suggest the possibility that Saps may activate by proteolysis some C. albicans surface proteins that are required for the organism to invade into an epithelial cell (14).


Saps may be more important for C. albicans to invade epithelial surfaces by passing between epithelial cells rather than penetrating into them. Several groups have observed that infection of epithelial cells by C. albicans in vitro results in proteolytic degradation of E-cadherin (14).

E-cadherin is concentrated at the intercellular junctions between epithelial cells, and its degradation is associated with loss of integrity of the epithelium (14).

In support of these in vitro findings, E-cadherin antigen is reduced in the oral epithelium of HIV-infected patients who have oropharyngeal candidiasis compared with those without this disease (14).

Degradation of E-cadherin is likely mediated at least in part by Saps because it can be blocked by pepstatin A (14).

In addition, a rim101Δ/Δ mutant, which has reduced expression of SAP4, SAP5 and SAP6, has reduced capacity to degrade E-cadherin and disrupt epithelium in a three-dimensional model. These defects are rescued by overexpression of SAP5 in this mutant (14).


Collectively, these results suggest that the major mechanism of C. albicans invasion between epithelial cells is by proteolysis of intercellular junctions, whereas invasion into individual epithelial cells occurs by both induction of epithelial cell endocytosis and active penetration via a mechanism that is yet to be defined (14).

A characteristic finding during oropharyngeal candidiasis is the destruction and loss of the superficial oral epithelium due to fungal invasion (14).

Similarly, when live C. albicans is incubated with epithelial cells in vitro, significant epithelial cell damage occurs (14).

Candida albicans must be at least partially endocytosed to cause epithelial cell damage because mutants with defects in inducing epithelial cell endocytosis cause less damage to these cells (14).

Similarly, inhibiting epithelial cell endocytosis of wild-type C. albicans with the microfilament inhibitor, cytochalasin D protects epithelial cells from damage (14).

However, epithelial cell damage is not a direct consequence of the endocytic process, because killed hyphae are avidly endocytosed, but cause no detectable damage (14).

Also, altering the capacity of a C. albicans mutant to induce endocytosis does not necessarily alter the amount of epithelial cell damage that it causes. For example, a rim101Δ/Δ mutant has impaired capacity to induce epithelial cell endocytosis and cause epithelial cell damage. Overexpression of PGA7 in the rim101Δ/Δ mutant results in increased epithelial cell endocytosis, but has no effect on the extent of epithelial damage that is induced. Thus, epithelial cell endocytosis can be dissociated from epithelial damage (14).

The mechanism by which C. albicans induces epithelial cell damage is incompletely understood. As mentioned above, Saps likely contribute to this process. However, C. albicans must cause epithelial cell damage by additional mechanisms because in some systems pepstatin A does not protect epithelial cells from damage, and sap mutant strains of C. albicans cause wild-type levels of epithelial cell damage (14).

It is possible that phospholipases secreted by C. albicans may also contribute to epithelial cell damage, although this hypothesis has not yet been rigorously tested (14).


Below the relationships found between transglutaminase and the two modes of invasion of Candida albicans (FOTGCREN):

E-cadherin & ENDOCYTOSIS:

Candida Albicans Als3 induces endocytosis by binding to E-cadherin and other proteins on the epithelial cell surface (14).

The interaction of Als3 with E-cadherin activates the clathrin-dependent endocytosis pathway (14).

It was found that clathrin is able to act as a Gln-donor in transglutaminase catalyzed reactions (Orru 2003).

The interaction between the Gln-donor clathrin and a Lys-donor cytoskeletal protein could be mediated by tissue transglutaminase (tTG) activity (Orru 2003).

It has been reported that tissue transglutaminase (tTG) is involved in the receptor-mediated endocytosis process in different cellular systems (Orru 2003).

Monodansylcadaverine, a synthetic amine substrate of tTG widely used to inhibit tTG activity in vivo, was shown to inhibit the internalization of ligands via the clathrin-mediated endocytotic pathway (Orru 2003).


Several groups have observed that infection of epithelial cells by Candida albicans in vitro results in proteolytic degradation of E-cadherin (14).

E-cadherin is concentrated at the intercellular junctions between epithelial cells, and its degradation is associated with loss of integrity of the epithelium (14).

In support of these in vitro findings, E-cadherin antigen is reduced in the oral epithelium of HIV-infected patients who have oropharyngeal candidiasis compared with those without this disease (14).

Degradation of E-cadherin is likely mediated at least in part by aspartyl proteinases (Saps) because it can be blocked by pepstatin A (14).

Degradation of E-cadherin can be blocked by pepstatin A (14).

Pepstatin A, an inhibitor of aspartyl proteinases (Saps), is also a transglutaminase inhibitor. Pepstatin A suppressed the activity of transglutaminase 1 (Egberts 2004).

Pepstatin A has been reported to inhibit the invasion of Candida albicans into corneocytes in mice with cutaneous candidiasis (14).

Some, but not all investigators have found that pepstatin A reduces Candida albicans-induced damage to reconstituted human epithelia. This reduced damage may be due in part to decreased epithelial cell invasion (14).


HIIRAGI 1999 (15):


Cell-cell adhesion is essential for the formation and maintenance of the integrity of animal body as a community of a wide variety of cells (15).

During development, the intercellular junctional complex, composed of tight junctions, adherens junctions (AJs), and desmosomes, is repeatedly destroyed and formed (15).

Below: Intercellular junctions in simple epithelia: Left: Schematic representation of intercellular junction complexes in polarized simple epithelia. Right: Electron micrograph showing intercellular ultrastructures. Mv: Microvilli; TJ:tight junction; AJ: adherens junction; DS: desmosome. Taken from (Michels 2010)

AJs are electron microscopically characterized by their electron-dense plasmalemmal undercoats through which actin filaments are densely associated with plasma membranes (15).

E-cadherin, a transmembrane protein responsible for Ca2+-dependent cell-cell adhesion, is concentrated and functions as a major adhesion molecule at AJs (15).

Below: Principal interactions of structural proteins at cadherin-based adherens junction. Actin filaments are linked to α-actinin and to membrane through vinculin. The head domain of vinculin associates to E-cadherin via α-, β-, and γ-catenins. The tail domain of vinculin binds to membrane lipids and to actin filaments (Wikipedia):

Furthermore, several constituents of AJ undercoats, which may connect E-cadherin to the actin-based cytoskeleton or regulate some AJ functions, have been identified, including α-, β-, and γ-catenins, vinculin, and p120 (15).

The molecular mechanism of the formation and destruction of AJs can thus be rephrased as the mechanism responsible for the assembly and disassembly of the multimolecular complexes consisting of E-cadherin and these undercoat-constitutive proteins (15).

Tyrosine phosphorylation of proteins has been shown to be directly involved in the assembly and disassembly of AJs (15).

Unexpectedly , when we attempted to identify heavily tyrosine-phosphorylated proteins in the isolated junctional fraction from the mouse liver, an enzyme with protein cross-linking activity, transglutaminase type 1 (TGase1), was identified as a tyrosine-phosphorylated form (15).

Below: Identification of heavily tyrosine-phosphorylated proteins in the junctional fraction from mouse liver: among the various isolated proteins,  three bands which were heavily tyrosine phosphorylated in the junctional fraction yielded several sequences that were identical to partial amino acid sequences of mouse N-cadherin, β-catenin, radixin, and human TGase1. Because the former three proteins are known to be directly involved in the functions of the junctional complex, some important function in junctions was also expected for TGase1 (15):


Transglutaminase type 1 was reported to be expressed only in keratinocytes (skin epidermis, involved in the covalent cross-linking of proteins in keratinocytes playing a central role by its cross-linking activity in the formation of the cornified cell envelope of terminally differentiated epidermis) but its expression in tissues other than the skin have not been examined (15).

However, transglutaminase type 1 was also expressed in large amounts in tissues containing simple epithelia, such as the lungs, liver, and kidneys (15).

Below: Analysis of TGase1 expression in mouse multiple tissues. TGase1 was detected in the liver, kidneys, lungs, and skin (15):

Fairly large amounts of TGase1 were concentrated in cell-to-cell adherens junctions in simple epithelial cells where they were covalently cross-linked to various proteins to form large multimolecular complexes (15).


Endogenous transglutaminase type 1 was immunofluorescently mostly colocalized with E-cadherin in cultured mouse simple epithelial cells (15).

Below: TGase1, E-cadherin and ZO-1 staining in cultured mouse epithelial cells. TGase1 and E-cadherin were diffusely co-distributed on lateral membranes (d´–f´ and arrowheads) with significant concentration at junctional regions (AJs) (arrows). In contrast, ZO-1 was concentrated more apically than TGase1 and E-cadherin. TGase1 was concentrated more basally than ZO-1 in junctional regions (a´–c´ and arrows). (15):


In the liver and kidney, immunoelectron microscopy revealed that endogenous transglutaminase type 1 was concentrated, although this localization was not exclusive, at cadherin-based adherens junctions in simple epithelial cells (15).

Below: Immunoelectron microscopy of ultrathin cryosections of liver and kidney epithelial cells revealed that TGase1 was concentrated at adherens junctions, although this was not exclusive, and not at tight junctions. When ultrathin cryosections of mouse liver (c– e) and kidney (f and g) were labeled with anti-TGase1 mAb, immunogold particles were detected in adherens junctions (AJ) but not in tight junctions (TJ) (15):

It should be emphasized here that even in simple epithelial cells TGase1 was not exclusively distributed at Ajs (15).


Transglutaminase cross-linking activity was also shown to be concentrated at intercellular junctions of simple epithelial cells (in vitro and in vivo labeling) (15).

Using a primary amine fot labeling TGase-specific substrates in liver fractions from mouse, it was shown that the endogenous TGase activity, i.e. the amount of amine-labeled proteins, was significantly concentrated in the junctional fraction. This reaction was actually dependent on the endogenous TGase activity, because it was completely suppressed by 10 mM EDTA or 10 mM cystamine (data not shown), potent inhibitors of Tgases (15).

Below: Mouse liver fractions transglutaminase labelled with an amine. The cross-linked proteins were enriched at the junctional fraction in the presence of CaCl2, but not in the presence of EDTA (a potent inhibitor of TGase activity), indicating that the endogenous TGase activity itself was concentrated in the junctional fraction. Analysis of the junctional fraction revealed that TGase1 was concentrated in the junctions not only as a full-length 97-kDa form but also as higher molecular mass forms. Because TGase itself was known to be cross-linked to various proteins through its own enzymatic activity, in the junctions this “auto-cross-linking” also would produce higher molecular mass forms of TGase1. The numerous biotin-labeled bands indicated that in the junctions TGase1 used various proteins as substrates, in amine cross-linking experiments with the isolated junctional fraction, various proteins appeared to be cross-linked by endogenous TGase1 activity (the identities of the major substrates for TGase1 in AJs remains to be elucidated). Thus, in the intercellular junctions, especially in AJs, we would expect the presence of highly complicated multimolecular complexes, in which constituents including TGase1 itself were covalently cross-linked through the TGase1 enzymatic activity. (15).


Below: Cultured mouse simple epithelial cells transglutaminase labelled with an amine to visualize endogenous transglutaminase (mTGase1) activity. The localization of the cross-linked proteins overlapped with endogenous TGase1 at cell-cell borders (a–c). In the absence of the amine (B-amine) (d´) or in the presence of cystamine (f´) in the culture medium there was no stained protein, indicating that this is a way to represent the cross-linking activity (e´) of endogenous TGase (d–f) (15):

The TGase activity was also enriched in the isolated junctional fraction from the liver and co-concentrated with E-cadherin at AJs in cultured simple epithelial cells (15).


The amine used in this study (5-(biotinamido)pentylamine) is not a specific substrate for TGase1 (15).

Among the four types of intracellular TGases (types 1–4), only type 2 (TGase2) was reported to be expressed in various types of cells. In agreement with previous reports, Western blotting with anti-TGase2 mAb revealed that TGase2 was abundant in the cytoplasm of the liver and was not concentrated in the junctional fraction in contrast to TGase1 (15).

Furthermore, in cultured simple epithelial cells, TGase2 was not detected by Western blotting or immunofluorescence staining with anti-Gase2 mAb (data not shown) (15)

Therefore, the TGase activity detected with 5-(biotinamido)pentylamine in this study was mostly attributed to TGase1, although the possibility cannot be ruled out that as yet unidentified type of TGase gives rise to part of observed activity, and the TGase1 concentrated at E-cadherin-based cell adhesion sites appeared to be active as a transglutaminase within cells. The “membrane-associated TGase activity,” which was previously reported in the liver, would be the same as the activity described in this study (15).


Because it is widely thought that the plasmalemmal undercoat structures in AJs are involved in the stabilization and/or up-regulation of cadherin-based cell adhesion, it is reasonable to speculate that TGase1-mediated cross-linking of proteins plays a role in further stabilization and up-regulation of cell-cell adhesion (15).

However, this cross-linking is not necessarily irreversible in vivo. The enzymatic activity that catalyzes the breakdown of g-glutamylamines to free amines and 5-oxo-proline, i.e. the breakdown of the TGase-mediated covalent bonds, was identified in rabbit tissues such as the kidneys, liver, and intestine. Recently, TGase2 and factor XIIIa themselves were reported to possess such hydrolytic activities (56). Therefore, it is possible that the TGase1-mediated covalent cross-linking is dynamically regulated in simple epithelial cells (15).

The formation of covalently cross-linked multimolecular complexes by transglutaminase type 1 is an important mechanism for maintenance of the structural integrity of simple epithelial cells, especially at cadherin-based adherens junctions (15).

TGase-dependent cross-linking of proteins plays some important role in the regulation of AJ assembly and disassembly (15).

The TGase1-mediated covalent cross-linking of proteins is directly involved in the formation and maintenance of intercellular junctions, especially adherens junctions Ajs (15).

Further studies are required to determine whether the TGase1-mediated cross-linking of proteins is one of the important and general mechanisms involved in formation, maintenance, and regulation of the structural integrity of simple epithelial cells, especially at Ajs (15).



A systemic infection is so named because the pathogen that causes it, and often the symptoms that it causes, are spread throughout the systems of the body, instead of being localized in one area.


Candida albicans is an opportunistic pathogen which causes both superficial and systemic infections in mammals (39).


The main characteristic of such an infection is that it affects the bloodstream of an individual, with the result that the symptoms spread to the whole of the body.


Microbial translocation can be defined as the passage of both viable and non-viable microbes and microbial products across the intact intestinal barrier (Alexander 1998).


The human gastrointestinal tract is colonized by a dense population of microorganisms, referred to as the bacterial flora. Although the gut provides a functional barrier between these organisms and the host, bacterial translocation is a common event in the healthy person. However, in critically ill patients, with various underlying diseases, this bacterial translocation may lead to infections and consequently to a further reduction in general health status (Wiest 2003).


From the historical observations of Berg and Garlington who defined bacterial translocation as “the passage of viable bacteria through the epithelial mucosa into the lamina propria and then to the mesenteric lymph nodes, and possibly other tissues,” this concept has been re defined several times. Currently, this definition has become broader and includes the passage of both viable and nonviable microbes and microbial products such as lipopolysaccharide (LPS) across ananatomically intact intestinal barrier (G D'Ettorre 2012).


In mucosal candidiasis It exists the risk of that mucosally derived organisms translocate and cause systemic candidiasis. This would happen by translocation of Candida albicans to internal organs (12).


SUNDSTROM 2002 MICE MUCOSAL CANDIDIASIS ASSAY (12):                                          

Candida Albicans (with or without Hwp1 gene) were orally administrated to “abnormal” immunodeficient mice (12).

In murine systemic candidiasis, Candida albicans accumulates in the kidneys (12).

Quantitative kidney cultures were done to determine whether C. albicans (orally administrated) translocated across the GI tract (12).

Candida albicans was rarely found in the kidneys (12).

Kidneys of 1 tgε26 mouse that was given Candida Albicans with Hwp1 and 1 bg/bg-nu/nu mouse that received Candida Albicans without Hwp1 yielded positive cultures (12).

Only 2 of 25 ill orally colonized mice had Candida Albicans in its kidneys because of translocation of C. albicans from GI tract to internal organs (12).

These results are consistent with other reports, which showed that translocation across the GI tract occurs ≥3 weeks after monoassociation in bg/bg-nu/un and tge26 mice (12).


STAAB 2013 MICE MODEL OF TRANSLOCATION (13):                                          


A common route of infection in hospitalized patients is the translocation of endogenous Candida albicans from the intestinal tract into the blood stream associated with antibiotic use and hematological immunosuppression or chemotherapy that result in the loss of intestinal barrier function (13).

We examined the role of Hwp1 in gut translocation of Candida albicans to the bloodstream using a murine model (C57BL/6 mice) that mimics this route of infection (13).

C57BL/6 mice were first treated with antibiotics and fluconazole to reduce  the GI bacterial and fungal normal gut flora and to allow the colonization of Candida albicans in their GI tracts and later were fed Candida Albicans in their drinking water to establish the colonization (13).

We found that all three Candida albicans strains, Candida Albicans with Hwp1 (SC5315), Candida Albicans without Hwp1 (SCH1211) and Candida Albicans with Hwp1 re-introduced (reconstituted strain HR615), colonized the intestinal track of C57BL/6 mice to similar levels (13).

Candida Albicans mutant without Hwp1 was capable of colonizing the mouse gut to equal levels as Candida Albicans mutants with Hwp1 (13).

Hwp1 was not influential in gut colonization of mice. Gut colonization levels are independent upon HWP1 expression (13).


The gut-colonized mice were treated with intraperitoneal injections of cyclophosphamide to induce immunosuppression / intestinal mucosa damage, and allow translocation of C. albicans into the blood stream and dissemination to the liver (13).

All of the mice colonized with strains expressing at least one allele of HWP1 (n=14) died by day 8 post-immunosuppression while 2 of the 7 mice colonized with Candida Albicans without Hwp1 (SCH1211) strain survived to the end of the study (13).

The survival rate between Candida Albicans with Hwp1 (SC5314) and Candida Albicans without Hwp1 (SCH1211)-colonized mice was statistically different; however the survival rates of mice harboring Candida Albicans without Hwp1 (SCH1211) or Candida Albicans with Hwp1 re-introduced (reconstituted strain HR615) were not different from each other (13).

Candida albicans was recovered (at similar levels) from the livers of all the mice at the time of death demonstrating translocation from the GI tract to the organ independent of HWP1 expression (13).

However, Candida Albicans mutant without Hwp1 was less virulent relative to the wild type or the reconstituted strain in this model, thus defining a new phenotype for Hwp1 in murine candidiasis (13).

Below: Survival of mice post immunosuppression with cyclophosphamide. Mice colonized with the Candida Albicans without Hwp1 (SCH1211) strain were less virulent relative to Candida Albicans with Hwp1 (SC5314). Single expression of HWP1 -Candida Albicans with Hwp1 re-introduced (reconstituted strain HR615)- did not restore wild type survival kinetics in mice, although none of the mice colonized with HR615 survived to the end of the observation period. Hwp1 is required for full virulence in an animal model of gut translocation canididasis (13):

Expression of a single allele of HWP1 in HR615 did not fully correct the hwp1 null phenotype. The results observed with HR615 suggested that Hwp1 may participate in yet undefined functions that aid dissemination via the blood stream that require wild type Hwp1 expression levels (13).

Lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice (13).

These results suggested that wild type hyphal surface levels of Hwp1 were needed for rapid translocation of C. albicans from the mouse gut into the blood stream. However, once translocation had occurred, the lack of Hwp1 did not prevent dissemination to and establishment of C. albicans in the liver (13).

Wild type levels of Hwp1 were needed for the rapid translocation of C. albicans from the gut into the blood stream and infection of murine livers (13).

C. albicans cells unable to form filaments and express HWP1 (cph1Δ/Δ efg1Δ/Δ) do not translocate from the mouse gut to the liver as well as SC514; in contrast, cells locked in the filamentous morphology (tup1Δ/Δ) appear more virulent relative to wild type cells in this animal model of candidiasis (13).

Koh AY, Köhler JR, Coggshall KT, Van Rooijen N, Pier GB (2008) Mucosal damage and neutropenia are required for Candida albicans dissemination. PLOS Pathog 4: e35.

These results did not distinguish between the ability of C. albicans to undergo morphological transitions or the expression of adhesins (e.g. Hwp1, Als3p) associated with the hyphal morphology as factors affecting gut translocation (13).

Because hwp1 null strains are not deficient in filamentation, we were able to consider hyphae formation and HWP1 expression as separate variables in our studies (13).

The results here implied that expression of adhesins and perhaps other proteins associated with the hyphal form are the key variables essential for C. albicans translocation (13).

The mechanism by which Hwp1 aids translocation is not yet understood but we propose two models that are not mutually exclusive:


A critical amount of C. albicans self-aggregation is necessary to achieve a threshold fungal burden traversing the damaged intestinal mucosa from the lumen into the blood stream (13).

Dissemination to the liver of the hwp1 null strain was delayed as a consequence of decreased cell numbers trafficking from the GI tract to the blood stream (13).

In support of this hypothesis, the flocculent tup1Δ/Δ cells (cells locked in the filamentous morphology (tup1Δ/Δ)) are more virulent in this gut translocation candidiasis model relative to wild type even when gut colonization levels are two logs below wild type (13).


Alternatively, Hwp1 contributed to interactions with epithelial cells lining the intestinal mucosa and the lack of Hwp1 hampered the initial binding and subsequent translocation of the fungus into the blood stream (13).

Hwp1 also participates in adhesion of C. albicans to oral epithelial cells in a non-TG dependent manner, therefore it is plausible that the lack of such fungal-host interactions may affect the kinetics of GI translocation (13).


Hwp1 & NICHE:

STAAB 2013:


HWP1 gene expression differs from other niche-specific genes in that HWP1 is normally expressed in hyphal cells regardless of host niche (13).


Hwp1 engenders C. albicans with niche-specific capabilities to inhabit the oral cavity (13).

Hwp1 as a niche-specific virulence attribute required for colonization and local invasion of oral tissue (13).


Hwp1 & Casein:




Below the sequence alignment between Hwp1 and Bovine and Human Caseins (UniProtKB/Swiss-Prot):

P46593 HWP1_CANAL Hyphal wall protein 1 Candida albicans (strain SC5314 / ATCC MYA-2876) (Yeast) versus

P02666 CASB_BOVIN Beta-casein Bos taurus (Bovine)

P05814 CASB_HUMAN Beta-casein Homo sapiens (Human)


                  ::  :.   ::***   : 
                       *  * *        *          
                  ***   *  *      *     **   *
Compare Beta casein Human and Bovine with Hwp1 here:
                  : *   * *   **
                        *     *
P46593|HWP1_CANAL EPCDYPQQ 59-66; 71-78; 82-89; 92-99; 102-109
                   * . **   
                    *   *



STAAB 1999 (1):

Using a Candida Albicans Hwp1 recombinant protein, rHwp1∆C37, that en-compasses the NH2-terminal proline- and glutamine-rich domain, we examined its ability to incorporate [14C]putrescine in the presence of TGase2 (from guinea pig livers), levels of radioactivity associated with the recombinant protein were equivalent to those of casein, a known Tgase substrate, and were fourfold greater than for bovine serum albumin (BSA), a negative control (1).

Below: Incorporation of [14C]putrescine by rHwp1∆C37, casein, and BSA mediated by TGase (1):

Examination of proteins after TGase reactions showed no radioactivity associated with BSA; however, casein was similar to Candidas Albicans recombinant Hwp1 protein in the generation of multiple species of radiolabeled reaction products including monomers displaying increased migration , species of high molecular weight , and dimers bridged by putrescine. The production of all radiolabeled forms depended on the presence of active TGase. Casein was implicated in cross-linking reactions with primary amines through Nε-(γ-glutamyl)lysine isodipeptide bonds (1).


A number of small intestinal disorders may have a similar small intestinal biopsy appearance than those caused by gluten in celiac disease. Between them: specific lesions caused by Candida and nonspecific lesions caused by soy protein (and/or milk protein) (Freeman 2001).


Hwp1 & Gluten:

Candida colonization is hypothesized to be a trigger for celiac disease, which has inexplicably increased in prevalence over the past decades in tandem with increased antibiotic use and Candida infections.




Below the sequence alignment between Hwp1 and different Alpha/beta-gliadins (UniProtKB/Swiss-Prot):

P46593 HWP1_CANAL Hyphal wall protein 1 Candida albicans (strain SC5314 / ATCC MYA-2876) (Yeast) versus

P04727 (GDA7_WHEAT) Alpha/beta-gliadin clone PW8142 – Prolamin - Triticum aestivum (Wheat)

P04725 (GDA5_WHEAT) Alpha/beta-gliadin A-V Triticum aestivum (Wheat)

P02863 (GDA0_WHEAT) Alpha/beta-gliadin Triticum aestivum (Wheat)

P18573 (GDA9_WHEAT) Alpha/beta-gliadin MM1 Triticum aestivum (Wheat)


                  ***:: :  ::
                  *:* :: *  ****

Below: Peptides #8 (LQPQNPSQQQPQEQVPL) and #9 (VPVPQLQPQNPSQQQPQEQVPL) of the well-investigated α2-gliadin protein; Q residues targeted by TG2 in bold; For peptide #8 (LQPQNPSQQQPQEQVPL), Q14 was found to be transamidated whereas Q9 was deamidated. These modifications were observed for the same glutamine residues in peptide #9 which represents an N-terminally extended derivative of peptide #8 (Dorum 2010):


                   * **    *   * ***        ***        **      * 


P46593|HWP1_CANAL QPDVPCDN 119-126; 129-136
P04727|GDA7_WHEAT QPQQPISQ 111-118
P04725|GDA5_WHEAT QPQQPISQ 114-121
P02863|GDA0_WHEAT QPQQPISQ 109-116
P18573|GDA9_WHEAT QPQQPISQ 123-130
                  **: * .:


                    * :*  :   *  ***


P46593|HWP1_CANAL PQEPCD 80-85; 90-95; 100-105; 110-115 (5 between them)
P04727|GDA7_WHEAT PQLPYP 86-91
P04725|GDA5_WHEAT PQLPYP 89-94
P02863|GDA0_WHEAT PQLPYS 84-89
P18573|GDA9_WHEAT PQLPYP 84-89; 91-96; 98-103 (2 between them)
                          ** *

Below: Peptides #1, #2, #3, #4 and #5 of the well-investigated α2-gliadin protein; Q residues targeted by TG2 in bold; The DQ2-α-II epitope was present in all of the five peptides in one or two copies (peptides #1–5). The α-gliadin derived 33mer peptide that is known to be a superior TG2 substrate was not identified. However, several isoforms (peptides #3 and #5) and truncated versions (peptides #1, #2, and #4) of this peptide were observed (Dorum 2010):

The immunostimulatory α-gliadin derived 33mer fragment, which is a very good TG2 substrate and which is resistant to proteolysis, was not among the identified TG2 gluten substrates. Recently, the occurrence of T-cell epitopes in α-gliadin proteins of different cultivars was reported. This study found, as was indicated in an earlier study, that the complete α33mer fragment only exists in α-gliadin proteins encoded in the D-genome of Triticum aestivum . Thus, the α33mer peptide was likely present in a low amount in the PTCEC gluten digest we used which could explain its absence among the identified TG2 substrates. We observed, however, several variants of the a33mer peptide (peptides #3 and #5) and as well as truncated versions of these peptides (peptides #1, #2, and #4). Notably, it has been demonstrated that such shorter versions can be as potent as the a 33mer peptide in stimulating intestinal T-cell responses. Among the identified TG2 peptide substrates a few peptides were observed as transamidated and deamidated at the same time (Dorum 2010).



The virulence factor of C albicans—hyphal wall protein 1 (Hwp1)—contains aminoacid sequences that are identical or highly homologous to known coeliac disease-related α-gliadin and γ-gliadin T-cell epitopes (2).

Below: Comparison of the aminoacid sequences of wheat gliadins with those of the cell-wall component hyphal wall protein 1 (HWP1) of Candida albicans, in particular with HWP1 aminoacids 40–197, shows many identical and homologous sequences in the proteins (2):

Below: Hwp1 contains sequences of known coeliac disease-related T-cell epitopes from α gliadins and γ gliadins (2):

Hwp1 and γ gliadin have three and five identical PQQPQ repeats, respectively, that are also present in most T-cell stimulating epitopes (2).

The sequence YPQQPQ is present in Hwp1 and in the DQ2 γ-gliadin epitopes DQ2-γ-V, T-cell γ-III plus γ-IV, and an unspecified DQ2 T-cell epitope (2).

The homologous sequence FPQQPQ is present in epitope DQ2-γ-III (2).

Sequence PQQQ is present in HWP1 and in epitope DQ2-γ-IV (2).

The immunodominant sequence PQPQLPY from α gliadin is selectively deamidated by tissue transglutaminase to give PQPELPY. This sequence is highly homologous to sequences PQPDIPC and PQPDVPC that both arise twice in HWP1 (2).

CROSSLINKING Hwp1 versus Gluten:

Transglutaminases have a pivotal role in blood clotting and wound healing (ie, factor XIIIa), and usually form isopeptide bonds between glutamines and lysines in proteins, thus forming cross-linked protein networks (2).

The direct interaction between C albicans and transglutaminase was noted in an immunological study. When the yeast has been in contact with human tissue (including skin) or human cell lines it binds a component on its cell wall that is immunoreactive with rabbit polyclonal antibody against human plasma transglutaminase factor XIIIa (2).

Arrese JE, Pierard GE. Factor XIIIa-related antigen immunoreactivity of fungal cell wall: a biologically relevant feature? Dermatology 1995; 190: 119–23.

Factor XIIIa might, thus, be covalently linked to C albicans (2).

Factor XIIIa and tissue transglutaminase have large stretches of identical aminoacid sequences (2).

The gliadin T-cell epitopes and aminoacid sequences in HWP1 contain identical and highly homologous sequences, and transglutaminase binds to C albicans (2).

IMMUNOLOGY Hwp1 versus Gluten:


After an injury extracellular concentrations of tissue transglutaminase will raise and become available (2).

If a foreign protein (like Hwp1 or gliadin) were covalently linked (crosslinked) by transglutaminase to the intestinal epithelium, endomysium or any other human tissue or protein conjugates Hwp1-endomysium or tgase and Gliadin-endomysium or tgase would be exposed to the immune system and autoreactive antibodies to endomysium or tgase would be developed, since peptides from these proteins have become part of a foreign immunoreactive adjuvant (related to 2 but gliadin is not quoted).

Hypothetical gluten-tissue transglutaminase hybrids have, by contrast to the C albicans-transglutaminase species, never been found (2).


Chronic mucocutaneous candidiasis (CMC) is a rare disorder characterised by chronic and recurrent infections, predominantly caused by Candida albicans affecting the mucous membranes, nails and skin (Ee 2005).

Chronic mucocutaneous candidiasis is an immune deficiency disease (Kirkpatrick 1989).

In virtually all cases the etiologic agent of CMC is Candida Albicans (Kirkpatrick 1989).

BRINKERT 2009 (21):

Chronic mucocutaneous candidiasis may cause elevated gliadin antibodies (21).

We present a 4-year-old boy admitted to the hospital due to the typical symptoms of celiac disease with severe dystrophy, anaemia and elevated gliadin IgG antibodies. Upper endoscopy ruled out celiac disease but showed severe Candida esophagitis. Due to an impaired T-cell function especially following Candida antigen stimulation in vitro, plus recurrent Candida infections of the skin, the diagnosis of chronic mucocutaneous candidasis (CMC) was made. Under the treatment with fluconazol, trimethoprim/sulfmethoxazole and IVIG, the child improved impressively. Gliadin antibodies declined steadily (21).

The common symptoms growth retardation, anaemia and elevated gliadin antibodies are suggestive for celiac disease but very unspecific. The rare immunodeficiency CMC may cause elevated gliadin antibodies (21).

PALLER 2011:

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent infections of the skin, nails, and mucosae with Candida species, usually Candida Albicans.

This disorder appears to be a common phenotype for a variety of detfects in the immune response, most notably in the cellular branch of the immune system, an mainly the specific responses to antigens of Candida species.

Chronic mucocutaneous candidiasis (CMC) has been reported in association with elevated gliadin antibodies and a celiac disease-like presentation, with weaning of the antibodies during anti-candidal therapy.


Candida:  In rare cases,  an immunodeficiency state called chronic mucocutaneous candidasis (CMC) may cause elevated gliadin antibodies. A 4-year-old boy admitted to hospital with typical symptoms of celiac disease:  severe dystrophy, anaemia  and elevated gliadin IgG antibodies, was found on upper endoscopy to have Candida esophagitis, but no evidence of  celiac disease. Presence of impaired T-cell function along with recurrent Candida infections of the skin led to a diagnosis of  CMC. Treatment with systemic antifungals produced excellent  results and a steady decline in gliadin antibody levels.


A number of small intestinal disorders may have a similar small intestinal biopsy appearance than those caused by gluten in celiac disease. Between them: specific lesions caused by Candida (Freeman 2001).

Below: Upper line: Rat tongues dealing with Candida albicans: Left: tongue papillae almost intact. Right: tissue damaged, loss of filiform papillae. The normal peaked papillary tissue architecture is flattened. Flattening of tongue papillae (Campos 2009) Lower line: Intestinal mucosa of celiac disease patient intestine dealing with gluten: Left: Healthy small intestinal mucosa. Right: Mucosal surface damaged; “flat” mucosal surface with absence of villi, striking hyperplasia of the crypts, and extensive infiltration of the lamina propria by lymphocytes and plasma cells:

Below: Upper line: Rat tongues dealing with Candida albicans (Samaranayake 2001): Left: tongue papillae intact (uninfected epithelium). Normal filiform papillae. Right: tissue damaged; loss of filiform papillae and flat-surfaced lingual epithelium. Lower lines: Intestinal mucosa of celiac disease patient intestine dealing with gluten: Left: Healthy small intestinal mucosa. Right: Mucosal surface damaged; “flat” mucosal surface with absence of villi, striking hyperplasia of the crypts, and extensive infiltration of the lamina propria by lymphocytes and plasma cells:.


Adf1 Adherence factor 1:

Adf1 is a protein located ¿on the surface (40)? of a dimorphic fungus called Candida albicans.


Adf1 has not been tested as transglutaminase substrate yet.


Adf1 is expressed on both forms (round-to-oval yeast or blastospore form and filamentous hyphal form) of Candida Albicans (40).


Anatomy of Adf1: aminoacid sequence (P46589 UNIPROT)



ADF1 (also called AAF1) is a Candida Albicans gene that encodes a protein called: Adherence factor Adf1 (recommended name) or Adhesion and aggregation mediating surface antigen Aaf1 (alternative name) (Uniprot).



Biological process: 1 adhesion of symbiont to host, 2 cell adhesion, 3 filamentous growth (Uniprot)




Adf1 has not been tested to date as a transglutaminase substrate.

Below: Comparisson between Hwp1 (with a proved N-terminal substrate of transglutaminase) and Adf1 (FOTGCREN 2015):


Adf1 versus Hwp1 (P46589 versus P46593 UNIPROT)




It is generally recognized that adhesion of C. Albicans directly to host surfaces is a crucial initial step in colonization and pathogenesis (39).

Various investigations have been carried out to determine the nature of the adhesins which mediate the initial attachment of C. Albicans to host cells (39).

New genetic techniques have been developed for elucidating the nature of the adhesin(s) involved in binding of C. Albicans like the expression of C albicans genes cloned in Saccharomyces cerevisiae that complement functionally known mutations or confer new attributes to the recipient. In the present study, we followed such an approach in an attempt to identify C albicans genes related to adhesion (39).

C. albicans adhesion was studied by expression of C. albicans DNA sequences encoding adhesion functions in a non adherent strain of Saccharomyces cerevisiae (39).

Adherent transformants were selected from a population of non-adherent S. cerevisiae cells harboring a C. albicans genomic library. An adherent transformant which exhibited enhanced adhesion to polystyrene and epithelial cells as well as autoaggregation properties was isolated. The DNA fragment responsible for these properties was partially characterized (39).

One of these transformants, bearing a reduced insert of 3.3kb retained the ability to autoaggregate, to bind to treated and untreated polystyrene, and to adhere to buccal epithelial cells, unlike appropriate controls (39).

Hybridization of a smaller 2.7-kb segment with C.albicans and S.cerevisiae DNA confirmed its origin as a single-copy sequence in the C.albicans genome as well as the absence of a homologous sequence in the genome of S.cerevisiae (39).

The data suggest that the adhesion and aggregation phenomena of the transformant cells are related to expression of a C.albicans surface antigen encoded by the cloned DNA fragment (39).

In the present study, we have shown that a unique DNA segment from C. albicans can transform nonadherent S. cerevisiae cells to adherent and autoaggregative phenotypes. Although adhesion to polystyrene was initially used for selection, it was subsequently found that the DNA segment concomitantly confers autoaggregation as well as adhesion to buccal epithelial cells. To the best of our knowledge, this is the first report of a candidal DNA segment which mediates adhesion to plastic as well as to host surfaces (39).

Preliminary results (not shown) with rabbit anti-serum prepared against the transformant and adsorbed with S. cerevisiae cells suggest the presence of a specific surface antigen(s) that is present on both transformant TY21-1 and C. albicans cells but absent from the S. cerevisiae control (39).

These findings raise the possibility that the C. albicans DNA segment encodes a surface component which is not expressed in host S. cerevisiae cells and which may be related to adhesion in the donor strain (39).

Alternatively, the sequence may encode a protein with a regulatory function related to adhesion. Work is under way to elucidate the DNA sequence of the segment studied here, to identify the gene product, and to study its expression in both the yeast and mycelial phases of C. Albicans (39).

The finding that transformant cells adhere to buccal epithelial cells raises the possibility that the gene product is involved in adhesion to host tissue. Further work is under way to test this hypothesis. The highest levels of adhesion to buccal epithelial cells were observed for the donor C. albicans cells, which contrasted with their relatively poor

adhesion to plastic surfaces (39).



An adherent transformant Saccharomyces cerevisiae clone, harboring a 3.3-kb DNA fragment from a C.albicans genomic library, was isolated (40).

This transformant exhibited enhanced adhesion to buccal epithelial cells (40).

We have provided evidence for a component on the outer surface of transformant S. cerevisiae cells which (40):

(i) is not observed in S. cerevisiae cells lacking the DNA fragment,

(ii) may be related to a specific candidal surface antigen.

We identified a DNA fragment from C. albicans that confers adhesion properties in S. cerevisiae cells (40).

A 3.3 kb DNA fragment from Candida albicans which confers adhesion in Saccharomyces cerevisiae was isolated and partially characterized (40).

Evidence is presented that the adhesion phenotype observed in S.cerevisiae cells transformed with the candidal DNA fragment is due to expression of a C.albicans surface antigen (40).

Transformant cells bearing this fragment exhibited pronounced adhesion to buccal epithelial cells (40).


In the present study, a specific antibody preparation was employed to probe the outer surface of transformant and donor strains (40).

The antibody preparation (40):

(ii) bound to the surface of transformant cells in a specific manner

(iii) recognized a specific component of ca. 30 kDa in candidal cell surface extracts

Rabbit antiserum, prepared against transformant S.cerevisiae cells was prepared (40).

Immunofluorescence micrography showed that the adsorbed antiserum bound to the surface of transformant S.cerevisiae cells as well as to C.albicans cells, but only marginally to the S.cerevisiae control (40).

Microscopic observation indicated strong specific immunofluorescence of donor C. albicans and transformant cells, as compared with barely observable immunofluorescence of control S. cerevisiae cells (40).

Westernblot (immunoblot) analysis of candidal extracts revealed that the absorbed antiserum recognized a major candidal antigen of ca. 30 kDa which was present on both yeast-phase and germ tube cells (40).

Preliminary data (not shown) suggest that the antigen may be associated with morphogenesis since immunofluorescence of the donor C. albicans cells is heavily concentrated along the developing germ tubes (40).

Antiserum, raised against transformant cells and adsorbed exhaustively by using control cells, recognized a specific antigen of ca. 30 kDa which was extracted from both yeast-phase and germ tube candidal cells. Interestingly, the same extraction procedure failed to yield an antigen of similar molecular weight from Saccharomyces cells, possibly as the result of strong anchorage of the component in the cell wall (40).

The data suggest that the observed adhesion phenotype is due to the presence of a specific candidal antigen on the outer surface of the transformant cells (40).

Taken together, the data suggest that the TY21-1-specific antibodies (40):

(i) recognize a trypsin- and proteinase K-sensitive outer surface component which is present on transformant but not on control S. cerevisiae cells,

(ii) inhibit autoaggregation through specific binding to this component on the transformant cell surface

(iii) recognize a specific antigen of ca. 30 kDa which is extractable from donor candidal cells.

Work is under way to further determine whether the candidal antigen recognized by the specific antiserum is identical or similar to that appearing on transformant cells. This antigen may be related to the cysteine-rich ca. 30-kDa hydrophobins which have been isolated from the outer surface of Schizophyllum commune and Aspergillus nidulans

 but have yet to be identified or characterized in C. Albicans (40).

Work is under way to determine whether the transformed DNA fragment directly encodes this surface component and whether it acts as an adhesin in C. albicans cells (40).

STURTEVANT 1997 (41):

While adhesion molecules in C. albicans have been biochemically characterized, the genes encoding these proteins have not. Diverse approaches have been used to identify these genes including screening libraries (41).

Screening Saccharomyces transformed with Candida DNA to identify adhesin genes in Candida, a C. albicans DNA sequence has been isolated which confers an adherence phenotype to S. cerevisiae (41).

BARKI 1993:

Barki et al.described a sequence from Candida that induces adhesion in S. cerevisiae. S. cerevisiae, normally a non-adherent species, was transformed with a Candida genomic library.  One clone was identified by its ability to adhere avidly to polystyrene and autoaggregate. A second round of transformation confirmed the ability of the DNA sequence to confer adhesion of the transformed yeast to buccal epithelial cells (41).

Barki M, Koltin Y, Yanko M, Tamarkin A, Rosenberg M. Isolation of a Candida albicansDNA sequence conferring adhesion and aggregation on Saccharomyces cerevisiae. J Bacteriol 1993; 175:5683-5689.

BARKI 1994:

An antibody was produced against the Saccharomyces transformant. Immunofluoresence studies showed reactivity on the surface of transformants and the antibody retarded autoaggregation. Immunoblotting recognized a major antigen around 30 kD. However, the gene has been fully sequenced, AAF1, and shares significant sequence homology with genes involved in transcription repression or activation due to glutamine and proline rich regions. In light of this, the antibody data is suspect since the expression of one or more Saccharomyces genes in the transformant may be altered. The function of this gene in C. albicans has not been reported. It is possible that AAF1 activates a structural adhesin gene(s) in Candida (41).

On the other hand, the ensuing transcriptional changes induced by AAF1 may be different between the two yeast species. Nevertheless it is of interest that Saccharomyces could be induced to adhere and aggregrate. Either AAF1 induces a cryptic adhesin in S. cerevisiae or the transcriptional activation of genes may alter the surface in such a way that so that proteins normally expressed in yeast are overexpressed or rearranged on the surface (41).

Barki M, Koltin Y, van Wetter M, Rosenberg M. A Candida albicanssurface antigen mediating adherence and autoaggregation in Saccharomyces cerevisiae. Infect Immun 1994; 62:4107-4111.

GAUR 1997:

Two C. albicans adherence genes, AAF1 and aINT1, have been cloned and partially characterized. Barki and coworkers described the rst C. albicans gene, AAF1, which was cloned based upon its ability to confer upon Saccharomyces cerevisiae the ability to adhere to polystyrene. This gene was also found to be responsible for autoaggregation and adherence to buccal epithelial cells. The AAF1 protein was localized to the C. albicans cell surface by immunouorescence (Barki 1994).

Barki, M., Y. Koltin, M. Van Wetter, and M. Rosenberg. 1994. A Candida albicans surface antigen mediating adhesion and autoaggregation in Saccharomyces cerevisiae. Infect. Immun. 62:4107–4111.

BROWN 2006:

Candida Albicans facing epithelial and endothelial barriers: Adhesion of Candida Albicans to epithelial cells is the first important step of colonisation. Molecular methods have been used to identify genes involved in adhesion to epithelial cells. Non-adherent S. Cerevisiae were transformed with a Candida Albicans genomic library, and adherent transformants were found to contain AAF1, the first Candida Albicans adherence gene described (Barki et al. 1993). These proteins were further characterised and shown to promote adhesion of Candida Albicans to oral epithelial or kidney cells (Brown 2006).


Adf1 & Gluten:


A search in Uniprot for a fragment of the celiac toxic peptide QQQPF in non-Triticeae proteins (bugs):

α-gliadin(31–43) LGQQQPFPPQQPY



Gave this:


Adhesion and aggregation mediating surface antigen [Candida albicans SC5314]

RecName: Full=Adherence factor; AltName: Full=Adhesion and aggregation mediating surface antigen [Candida albicans SC5314]

Sequence ID: P46589.2 ADF1_CANAL Length: 612 Number of Matches: 1

Query  2   QQQPF  6


Sbjct  67  QQQPF  71


Adf1 (P46589) versus gliadin (P04121) fixing the QQQPF motif:









Adf1 (P46589) versus different gliadins around the QQQPF motif:


CMPARISSON BETWEEN Adf1 and a Saccharomyces cerevisiae protein that also has the QQQPF motif:
Adf1 (P46589) a Candida Albicans with the QQQPF motif versus gliadin:
Psp1 (P50896) a Saccharomyces cerevisiae with the QQQPF motif versus gliadin:


P50896= RecName: Full=Protein PSP1; AltName: Full=Growth inhibitory protein 5; AltName: Full=Polymerase suppressor protein 1 [Saccharomyces cerevisiae S288c]


Adf1 & Gluten & Casein:


An alignment with blast directed the gliadin sequence alignment to the N terminal half of Adf1 (left) and the casein sequence to half C terminal sequence of Adf1 (right). So Adf1 is “the half gliadin half casein” protein:






As obligate intracellular parasites, all viruses must have ways of entering target cells to initiate replication and infection (Sieczkarski 2002).

Cellular membranes present a barrier between the viral particle and intracellular site(s) of replication in the cytosol or nucleus (24).

In animal cells, viruses can enter target cells in two principal ways: by a direct mechanism at the cell surface (plasma membrane) or by following their internalization into cellular compartments (for example, endosomes) (Sieczkarski 2002).

Non-enveloped and enveloped viruses enter the cytosol directly at the plasma membrane or via host cell endocytic pathways (24).

While enveloped viruses are bound by a lipid bilayer, non-enveloped viruses are surrounded by a proteinaceous capsid. Both enveloped viruses and non-enveloped viruses have evolved complex mechanisms to enter cells (24).

The mechanisms employed by non-enveloped and enveloped viruses to cross membrane barriers differ significantly, most likely as a consequence of the biophysical constraints imposed by the viral envelope (24).

Non-enveloped viruses can enter the cytosol by directly penetrating the plasma membrane, as well as through a variety of endocytic mechanisms leading to penetration of internal membrane(s). Internal membranes crossed by non-enveloped viruses include the endosomal membrane (e.g. adenovirus), the Golg i ( e.g. papillomavirus;) and the endoplasmic reticulum (e.g. SV40). Strategies to disrupt or traverse host cell membranes must be included in the mechanisms of non-enveloped virus entry. However, the precise molecular and biophysical means by which non-enveloped viruses gain entry to the cytosol have not been clearly defined in all cases (24).


Infection with an enveloped virus requires the fusion of the viral envelope with a cellular membrane. In some cases, this can occur at the plasma membrane, as reported for HIV, where binding to plasma membrane-expressed forms of CD4 and chemokine receptors induce changes in the viral envelope glycoprotein that are thought to mediate membrane fusion under neutral pH conditions. Fusion of other enveloped viruses occurs within the low-pH environment of an acidic endosomal compartment. Enveloped viruses typically reach the endosomal compartment via trafficking in clathrin-coated vesicles, although a caveolar route of entry has been reported for human coronavirus 229E (24).

The entry of both enveloped and non-enveloped viral particles requires specific interactions between host cell molecules, or receptors, and viral encoded envelope or capsid proteins. One key result of this is to bring the virus into close contact with the plasma membrane (24).

In addition to primary receptors critical for virus attachment to the cell surface (e.g. CD4 for human immunodeficiency virus (HIV) ), important co-receptors have been identified (e.g. chemokine receptors CXCR4 or CCR5 for HIV) (24).

It is now becoming apparent that a wide variety of host cell molecules are important for virus internalisation in the absence of any direct association with the virus particle. This is leading to the notion of “entry factors ” , for example the tight junction proteins claudin-1 and occludin appear to have an indirect role in HCV entry (24).


Following attachment to the host cell surface, virus entry at the plasma membrane has been described for many viruses such as HIV and Poliovirus. Enveloped viruses can fuse directly with the plasma membrane, releasing the capsid directly into the cytosol, whilst non-enveloped viruses disrupt or form pore(s) in the plasma membrane to gain entry (24).

In contrast to fusion at the plasma membrane, many viruses (such as SV40 and Influenza A) utilise intracellular trafficking pathways to fuse with internal membranes in order to release their genomic material into the cytosol (24).

Receptor trafficking pathways often define particle internalisation routes and viruses typically enter the cell by a single defined pathway, although examples have been reported where viruses utilise multiple pathways in diverse cell types (24).

The majority of host cell membrane proteins internalise through clathrin-mediated endocytosis. The clathrin-mediated pathway is ubiquitous (24).

The majority of virus families utilize endocytosis as a means of entry into cells (Sieczkarski 2002).

This is not surprising, considering the many benefits that endocytosis oers. Many viruses have a low-pH-dependent conformational change that triggers fusion, penetration and/or uncoating and endocytosis is crucial to these viruses due to the acidification occurring within the endosomal pathway. It is also becoming appreciated that viruses without a strict low-pH step for entry also enter cells via endocytosis, as endosomes offer a convenient and often rapid transit system across the plasma membrane and through a crowded cytoplasm. For nuclear replicating viruses especially, the endosome can deliver its viral cargo to the vicinity of the nuclear pore, ready for translocation into the nucleoplasm (Sieczkarski 2002).

The first molecule shown to be required for endocytosis was clathrin, which mediates the primary route of endocytic internalization into cells. In response to an internalization signal (involving typically either a YXXΦ or di-leucine sequence in the cytoplasmic tail of a receptor), clathrin is assembled on the inside face of the plasma membrane to form a characteristic invagination or clathrin-coated pit (Sieczkarski 2002).

As befits its major role in endocytosis, clathrin has been shown to play a major role in the internalization of many viruses (Sieczkarski 2002).

Internalization from the plasma membrane is made by sorting and tracking events within intracellular vesicles (endocytic compartments).

Endocytic compartments are pleiomorphic structures that fuse with one another to promote ligand tracking. Subsequent to internalization, endosomes often undergo complex

tracking and sorting events. Two principal post-internalization endocytic tracking routes exist in the cell, which can be termed recycling or lysosome-targeted. Regulation of sorting and tracking is determined by inherent signals on the internalized receptor and by signalling events within the cell (Sieczkarski 2002).

The internalized vesicle acquires properties that are defined temporally and are thus termed `early' and `late' endosomes. The early endosome is an often pleiomorphic tubulo-vesicular structure and is a major sorting station where internalized cargo can be delivered back to the plasma membrane (the recycling pathway) or can progress to the late endosome. Late endosomes, comparatively, have a mostly juxtanuclear distribution, are more spherical and contain internal vesicles - leading to the term multi-vesicular bodies. They also dier from early endosomes in that they have a significantly lower pH (approximately pH 5.5 versus pH 6.2-6.5 in the early / recycling endosome). Late endosomes subsequently progress to lysosomes, which are characterized by the presence of degradative proteases and hydrolases, delivered by communication of endosomes with the transGolgi network (Sieczkarski 2002).

In the case of endocytic entry, internalization itself is generally not sucient for productive infection, as incoming viruses are still part of the extracellular space while in endosomes. Therefore, endocytosed viruses must penetrate or fuse with the endosomal membrane to be released into the cytoplasm. In addition, the endocytic pathway is often used by viruses requiring a specific localization within the cell for a successful infection (Sieczkarski 2002).

Viruses not only depend on the machinery of the cell for internalization but also for tracking within the cytoplasm and the ability to find the correct site for replication (Sieczkarski 2002).


gp120 & gp41 glycoproteins 120 & 41:

gp120 & gp41 are two proteins (glycoproteins) located on the surface of a virus called Human immunodeficiency virus (HIV).


gp120 & gp41 are both transglutaminase substrates (22,23)


HIV (100 - 120 nm in diameter - Russell Kightley)




Anatomy of gp160 and constituent parts: gp120 & gp41 Left: three-dimensional structure and Right: aminoacid sequence.


gp160; the source of gp120 and gp41:

The HIV evelope (env) protein is initially synthesized as a single polypeptide precursor termed gp160. Prior to delivery to the plasma membrane, gp160 undergoes a single post-translational cleavage, which creates an exterior env glycoprotein (gp120) and a transmembrane env glycoprotein (gp41) noncovalently associated (22).

The native and functional HIV-1 envelope glycoprotein (Env) complex is present on the virus surface as a trimer, each of the monomers made of noncovalently loosely associated gp120 surface and gp41 transmembrane glycoproteins (Visciano 2013).

Like all retroviruses, HIV displays a heterodimeric env protein (gp120-gp41 complex) which is synthesized as a polyprotein (gp160) and intracellularly cleaved (23).

Via a host-cell mediated process, gp160 is cleaved to form gp120 and the integral membrane protein gp41. As there is no covalent attachment between gp120 and gp41, free gp120 is released from the surface of virions and infected cells (Berman 1994).

Human immunodeficiency virus (HIV) env glycoproteins, like  those of other retroviruses, are synthesized as a precursor (gp160) that is cleaved to generate the surface (gp120) and transmembrane (gp41) env proteins, which are non-covalently associated to each other (23).

The HIV envelope protein is a glycoprotein of about 160 kd (gp160) which is anchored in the membrane bilayer at its carboxyl terminal region. The N-terminal segment, gp120, protrudes into the aqueous environment surrounding the virion and the C-terminal segment, gp41, spans the membrane (Berman 1994).



gp120 contains the CD4-binding domains, while gp41 anchors the gp120-gp41 complex in the viral env or host-cell membrane (23).


Unlike other chronic viral infections, which are rarely fatal, human immunodeficiency virus (HIV) infection eventually ends with severe immunodeficiency and death. A plausible explanation is that the virus, by virtue of its high mutation rate, produces antigenic variations in its envelope (env) which let it evade the immune response of the host and kill the helper T-cells essential in maintaining an effective immune state (22).

HIV-1 diversity facilitates evolution of resistance to antiretroviral therapy and escape from host immune responses (Abigail 2013).


gp120 is the portion of the HIV envelope protein which is on the surface of the virus (Berman 1994).

gp120 is known to possess the CD4 binding domain by which HIV attaches to its target cells (Berman 1994).

The amino acid sequence of gp120 contains five relatively conserved domains (C1, C2, C3, C4 and C5) interspersed with five hypervariable domains (V1, V2, V3, V4 and V5). The positions of the 18 cysteine residues in the gp120 primary sequence, and the positions of 13 of the approximately 24 N-linked glycosylation sites in the gp120 sequence are common to all gp120 sequences (Berman 1994).

gp120 associates with gp41 by the regions located in the amino-terminal half (22).

gp120 determines the tissue selectivity of viral infection by binding with its carboxy-terminal region ¿C4? to CD4 receptor, present on the surface of helper T-lymphocytes, macrophages and other cells (22).


Among the products of the HIV genome, gp120 shows the greatest sequence variation. Since the gp120 heterogencity has been observed even in serial isolates from the same infected individuals, it was suggested that ongoing molecular evolution of the virus may occur during the course of infection and interfere with the host responses to HIV (22).

The hypervariable domains (V domains) contain extensive amino acid substitutions, insertions and deletions. Sequence variations in these domains result in up to 30% overall sequence variability between gp120 molecules from the various viral isolates. Despite this variation, all gp120 sequences preserve the virus's ability to bind to the viral receptor CD4 and to interact with gp41 to induce fusion of the viral and host cell membranes (Berman 1994).


HIV-1 gp120: Schematic of gp120 with the 5 conserved domains (C1–C5 and five variable domains (V1–V5). Sites for N-linked glycosylation are shown (Sanders 2008)


Diagram of sequence elements in gp120 and definition of its core. The branched symbols mark glycosylation sites (Chen 2005)



HIV-1 gp120: Schematic of gp120 with the 5 conserved domains (C1–C5 and five variable domains (V1–V5) (Briz 2006)


gp120 contains 9 conserved disulfide bridges. Also relevant is the so-called V3-loop. This is a surface-exposed highly immunogenic antibody-binding and hypervariable (immunological escape) region of gp120, which has been extensively sequenced.

gp120 domains (the location of the V3 loop in the gp120 molecule can be seen in this schematic visualization and the 9 conserved disulfide bridges in red)


gp120 from HIV BH10


Below: The C2 terminus region of gp120. Sequence comparison of the gp120 molecules from different isolates. The sequences corresponding to the peptides that inhibit gp120 Sag binding are boxed in grey. Peptides that are potential or proved transglutaminase substrates are boxed in black with glutamine targeted boxed in red. Gaps are introduced to maximize homology and dots indicate homology (edited from Karray 1997):



Sequence alignment of the C2 region of gp120 molecules from different HIV/SIV isolates (Uniprot):




The C-terminus of the second conserved region (C2) of the envelope glycoprotein gp120 encompassing peptide 254-274 CTHGIRPVVSTQLLLNGSLAE (in P03377) has been reported to be critical in the postbinding event during virus penetration (22).



The C-terminus of the second conserved region (C2) of the envelope glycoprotein gp120 encompassing peptide 273-295 RSANFTDNAKTIIVQLNESVEIN is called peptide NTM by Veljkovic (25).

This region is crucial for several important functional and immunological properties of HIV-1 (25).

This region is important for infectivity and neutralization of the human immunodeficiency virus type 1 (HIV-1) (25).

The conserved area of HIV-1 gp120; C-terminus of the C2 region, has immune tolerance: the human immune system is unresponsive, or tolerant, to epitope(s) within this part of the molecule (25).



gp120 HIV 273-299 Q255 (Q287 in gp160)

C-terminus of gp120 HIV BH-10

Shielding of the highly conserved region IRSANFTDNAKTIIVQLNQS, participating in gp120/CD4 interaction, with variable V3 loop provides an important viral defense against immune survivalence (25).

D’Costa, S. et al. Aids Res. Hum. Retrovir. 2001; 17: 1205

The third variable region, V3, of the gp120 surface envelope glycoprotein is an approximately 35-residue-long, frequently glycosylated, highly variable, disulfide-bonded structure that has a major influence on HIV-1 tropism (Hartley 2005).

The HIV-1 gp120 V3 loop is encountered in a large sequence variability (Tamamis 2014).



VIP is a human protein function as a neuromodulator and neurotransmitter very widely distributed in the peripheral and central nervous systems called "intestinal" because it was originally isolated from intestinal extracts. A huge number of biological effects have been attributed to VIP. With respect to the digestive system, VIP seems to induce smooth muscle relaxation (lower esophageal sphincter, stomach, gallbladder), stimulate secretion of water into pancreatic juice and bile, and cause inhibition of gastric acid secretion and absorption from the intestinal lumen. It is a potent vasodilator, regulates smooth muscle activity, epithelial cell secretion, and blood flow in the gastrointestinal tract . As a chemical messenger, it functions as a neurohormone and paracrine mediator, being released from nerve terminals and acting locally on receptor bearing cells.


There is a sequence similarity between VIP and the C-terminus of the second conserved region (C2) of HIV envelope glycoprotein gp120 (25):


P01282 (VIP_HUMAN) UniProtKB/Swiss-Prot Vasoactive intestinal peptide VIP Homo sapiens (Human) versus

P03375 (ENV_HV1B1) UniProtKB/Swiss-Prot Surface protein gp120 Human immunodeficiency virus type 1 group M subtype B (isolate BH10) (HIV-1)

RSANFTDNAKTIIVQ255LNESVEINCTRP (gp120 HIV 273-299 Q255 (Q287 in gp160))

SDAVFTDNYTRLRKQ16  MAVKKYLNSILN (Vasoactive intestinal peptide VIP Human Q16)


Human natural anti-vasoactive intestinal peptide (VIP) antibodies reactive with this gp120 region play an important role in control of HIV disease progression (25).

The bioinformatic analysis based on the time-frequency signal processing revealed non-obvious similarities between NTM and VIP (25).



Antibodies in sera of HIV-infected patients reacting with the C-terminus of the C2 region strongly correlate with disease progression (25)

Neurath, A.R., Strick, N., Tajlor, P., Rubinstain, P. & Stevans, C.E. (1990) Search for epitope-specific antibody responses to the human immunodeficiency virus (HIV-1) envelope glycoproteins signifying resistance to disease development. AIDS Res. Hum. Retroviruses 6, 1183–1192.

Human immunodeficiency virus (HIV) disease progression varies greatly between individuals and it appears that host factors play an important role in determining the clinical outcome in HIV infection (25).

In order to define these host factors, Neurath and co-workers have investigated antibody profiles in two groups of HIV-infected patients: those who remained healthy for at least 10 years and those who developed AIDS within 5 years of the onset of infection (25).

They demonstrated that antibodies recognizing the peptide RSANFTDNAKTIIVQLNESVEINCTRP (amino acids 280–306 within the C2 region of the envelope glycoprotein gp120 from the BH-10 isolate of HIV-1) are significantly more prevalent in asymptomatic carriers than in patients who progressed to AIDS (6/9 in asymptomatic vs. 0/9 in AIDS patients) (25).

Based on these results, it appears that the absence or disappearance of these antibodies my represent a possible factor contributing to disease progression (25).

For this reason, it has been proposed that maintenance of a high level of these antibodies by immunotherapy, based on active immunization with antigens containing this peptide and/or administration of the corresponding antibodies, should be considered as a modality for therapy of HIV-1 infection. This assumption was strongly confirmed by recently reported results of therapy performed by passive immunization with human HIV-negative plasma enriched with antibodies reactive with the C2-derived peptide encompassing amino acids 280–302 (25).

Despite the presence of the strongest T-cell epitope of gp120, which is active in vitro and an exposed B-cell epitope, the C-terminus of the C2 region encompassing amino acids 280–306 is not immunogenic in humans. Absence of the active B-cell epitope within this peptide indicates that antibodies in sera of HIV patients recognizing this region of HIV-1 gp120 represent autoreactive antibodies elicited by some human antigen. Vasoactive intestinal peptide (VIP) was identified as the human antigen likely inducing these natural antibodies, which are cross-reactive with peptide RSANFTDNAKTIIVQLNQSVEIN (denoted as peptide NTM) derived from the C2 region of HIV-1 gp120 (25).



The transmembrane protein gp41 contains several glutamine residues, 75% of which are located on the outer surface of the virus (23).


Schematic Representation of gp41

Important functional regions include the fusion peptide (FP, purple box), the N- and C-terminal heptad repeat regions (NHR, green box, and CHR, red box, respectively), and the transmembrane region (TM, yellow box). The various domains are not drawn to scale

(Cardoso 2005)



The initial event in the HIV-infection process involves the binding of gp120, the coat glycoprotein of the virus, with a subset of peripheral T cells expressing the cell surface glycoprotein CD4 (23).

The CD4 molecule functions as a receptor for gp120, being essential for HIV entry into the host cell and for membrane fusion, which contributes to cell-to-cell transmission of  the virus and to its cytopathic effects characterized by syncytia formation and cell death (23).

CD4 may interact not only with gp120 but also with gp41, determining significant conformational changes in its own structure and in that of the transmembrane protein (23).




The process of HIV infection involves the binding of glycoprotein (gp) 120, a surface component of the viral envelope glycoprotein, to CD4 and CXCR4 receptors expressed on the target cell surface (Takano 2007).

gp120 role:

gp120 first binds to the CD4 receptor (Takano 2007).


HIV entry begins with the high affinity binding of gp120 to the host cell CD4 (Anastassopoulou 2012).


gp120-CD4 binding results in exposure of novel binding sites in gp120 by which it binds to the co-receptor CXCR4 (Takano 2007).


gp120-CD4 binding induces a major conformational change in Env that exposes or creates a binding site on gp120 for the coreceptor, typically either CCR5 or CXCR4 (Anastassopoulou 2012).


gp41 role:

Sequential binding of gp120 to CD4 and the CCR5 or CXCR4 co-receptor lead to the release of gp41 with subsequent fusion of the viral and the host membrane.

The chemokine receptor, typically CCR5 or CXCR4, triggers molecular rearrangements in the gp41.



Despite being extensively studied, the entry process of HIV, the causative agent of AIDS remains a debated issue (24).

The events that follow the interaction of gp120 with the cell membrane and which precede the internalization of the viral contents in the cell have not yet been established (23).

In this respect, it has been postulated that, after gp120 binding, HIV enters the cell either by direct fusion of the virus envelope with the plasma membrane or by receptor-mediated endocytosis of a CD4-HIV complex (23).


Below: Two main virus entry pathways: a | Clathrin-mediated endocytosis, b | Fusion at the cell membrane (Dimitrov 2004):

Below: Schematic model of cell entry of enveloped viruses (Teissier 2010):

Below: Entry of enveloped viruses into cells. The virus particle bears viral attachment  proteins VAPs embedded in its plasma membrane, which interact with cell surface molecules (virus receptors) attaching the  virion to  the  cell surface. The membrane of the virion may then fuse directly with the plasma membrane releasing the genome into the cytoplasm. Alternatively the  virus particle is  internalized by  adsorptive or  receptor-mediated endocytosis and delivered to an endosome. The acidic pH triggers fusion of the viral membrane with the endosome membrane, liberating the genome (Lentz 1990):


It has long been thought that productive entry of HIV occurs via direct fusion of the viral envelope with the host cell plasma membrane (24).


Coreceptor engagement of CD4-bound gp120 induces additional reconfigurations, leading to the insertion of the gp41 fusion peptide (FP) into the host cell membrane and the formation of a pre-fusion complex. This pre-fusion intermediate is then refolded into an energetically favorable six-helix bundle that brings the two membranes in close proximity so that fusion can occur; the viral core is thereby released into the cytoplasm (Anastassopoulou 2012).



Fusion of the HIV cell to the host cell surface (NIAID)



HIV gp120 binds to CD4 on TT-cells and then to a coreceptor. Causes gp41 attachment to the cell membrane = virus-cell fusion and HIV infection (Immunotech Laboratories)



- One key piece of evidence for an endocytic-independent entry is the observation that infection is insensitive to neutralisation of endosomal pH. In fact, blocking endosome acidification was in some cases seen to augment viral infection, perhaps by sparing particles from lysosomal degredation (24).

- CD4 endocytosis was not required for HIV entry, suggesting that endocytosed particles were degraded by host cell lysosomes (24).



Whilst the clathrin-mediated entry pathway has been documented for almost as long as the classical plasma membrane entry mechanism, it has only recently been confirmed that this route offers a productive entry pathway for HIV (Daecke 2005) (24).

Recent data may even suggest that clathrin-mediated HIV entry is the only productive pathway for infection (Miyauchi et al. 2009) (24).


- EM studies have shown internalised virions in membrane-bound vesicles. Moreover, the rates of entry and uncoating of radio labelled virions in the human T-lymphoid cell line CEM were consistent with a receptor-mediated mechanism of entry (24).

- Live cell imaging has demonstrated that HIV fusion at the plasma membrane did not proceed further than mixing of lipids and could therefore not lead to productive infection (24).

Below: Steps of virus entry via clathrin-mediated endocytosis. ( A) Virus approaches the cell surface. (B ) Biochemical interactions between ligands and receptors attract virus to the cell surface. ( C) Virus attaches to the cell surface and signals the cell. (D) A clathrin-coated pit is formed around the bound virus. (E ) A clathrin-coated vesicle is formed, and the dynamin at the neck region facilitate vesicle scission. (F ) The vesicle travels to cell interior (Barrow 2013):






The molecular events, underlying both virus-cell and cell-cell fusion, are poorly understood. In particular, little is known both about the molecular mechanisms which follow the interaction of gp120 with CD4, or other HIV receptors occurring on CD4- cells, and the molecular mechanisms of gp41 anchorage to the cell membrane. Since several proteins capable of interacting with cell surfaces (i.e. fibrinogen, fibrin, fibronectin, vitronectin and von-Willebrand factor) have been reported to be substrates for TGase, our studies have examined whether HIV env proteins possess this feature (23).



gp160 is completely ineffective as transglutaminase substrate (23).



Conversion by proteolysis, of gp160 into gp120 and gp41, exposes amino-acceptor (acyl donor) glutaminyl residues not only in gp120 but also in gp41 (23).

gp120 & gp141 are both transglutaminase substrates (22,23)



MARINIELLO 1993 (gp120) (22):

It was tested TGase-dependent incorporation of [14C]Spd into gp160, gp 120 and various gp120 fragments at 37ºC, pH 8.0 (22).


In the attempt to investigate if the acyl donor sites occur in biologically important determinants of gp120, several glutamine-containing fragments of the protein were tested as acyl donor TGase substrates (22):

gp120 fragments 105-117, 201-216, 302-324, 346-359, 414-434, 419-430 and 428-445, including the principal neutralizing determinant and the CD4-binding domain of gp120, were ineffective as acyl donor (amino-acceptor) substrates of the enzyme when tested at a concentration five times higher than that used to assay the 254-274 fragment (22).









Only the fragment 254-274, occurring in a highly conserved domain of gp120 and supposed to be involved in the mechanisms of HIV penetration into the cells, was able covalently to bind radioactive Spd in the presence of TGase and Ca2+ (22).

gp120 fragment 254-274 is a TGase amino-acceptor substrate (22).

Gln265 (as amino-acceptor) is suggested as the reactive site in gp120 recognized by transglutaminase (22).

Q265 could be a candidate as reactive site responsible for the substrate activity of gp120, even though the structures of oligopeptides derived from a protein may not be strictly considered the same as a segment of intact protein from which the peptide is derived. In fact, since a whole protein may be a substrate of TGase, whereas the constituent oligopeptides may not be, it was suggested that conformational factors are often predominant in determining substrate features (22).




Recombinant gp120, but not its precursor gp160, is an amino-acceptor substrate for TGase in vitro (23).

Human immunodeficiency virus envelope glycoprotein gp120, but not its precursor gp160, covalently incorporates both spermidine and glycine ethyl ester in the presence of Ca2+ and transglutaminase purified from guinea pig liver (22).

The examined ability to act as enzyme substrate of various glutamine-containing gp120 fragments, including the principal neutralizing determinant, the CD4 binding domain, and the sequence 254-274 (222-242 without peptide signal), suggested to be involved in post-binding events and in virus entry in the host cell, indicated the glutamine-265 (glutamine 232 without peptide signal) as possible reactive acyl donor site of the protein (22).



Mariniello 1993 proved transglutaminase substrate in the HIV gp120 protein. Q231 (gp120: aminoacids 220 to 240, P03377 UNIPROT)



A mistake took me to propose this Q residue as potential transglutaminase substrate:


FOTGCREN 2014 proposed transglutaminase substrate in the HIV gp120 protein. Q255 (gp120: aminoacids 234 to 272, P03377 UNIPROT)


Currently the only evidence that could prove that this peptide is a substrate for transglutaminase is its homology with VIP (see more below).



In the C-terminus of C2 region of HIV / SIV there are two conserved residues Q and K which could be potential transglutaminase substrates: KTIIVQLN


36 HIV-1 15 group M complete KTIIVQLN: 13 subtype B, 1 subtype U and 1 subtype D
2 SIV Chimpanzee 
P04578|ENV_HV1H2              VNFTDNAKTIIVQLNTSVEINC     HIV-1 group M subtype B HXB2
P03375|ENV_HV1B1              ANFTDNAKTIIVQLNQSVEINC     HIV-1 group M subtype B BH10
P12490|ENV_HV1N5              ENFTNNAKTIIVQLNKSVEINC     HIV-1 group M subtype B NY5
P31819|ENV_HV1KB              ENFTDNVKTIIVQLNETVKINC     HIV-1 group M subtype B KB-1/ETR
P05879|ENV_HV1C4              ENFTNNAKTIIVQLNVSVEINC     HIV-1 group M subtype B CDC-451
Q73372|ENV_HV1B9              ENFTDNAKTIIVQLNESVVINC     HIV-1 group M subtype B strain 89.6
P12491|ENV_HV1Z3              ENITNNAKTIIVQLNETVKINC     HIV-1 group M subtype U Z3
P03378|ENV_HV1A2              DNFTNNAKTIIVQLNESVAINC     HIV-1 group M subtype B ARV2/SF2
P12488|ENV_HV1BN              ENFTNNVKTIIVQLNESVEINC     HIV-1 group M subtype B BRVA
P19551|ENV_HV1MF              ANFTDNAKTIIVQLNTSVEINC     HIV-1 group M subtype B MFA
P03377|ENV_HV1BR              ANFTDNAKTIIVQLNQSVEINC     HIV-1 group M subtype B BRU/LAI
P04579|ENV_HV1RH              ENFTDNVKTIIVQLNASVQINC     HIV-1 group M subtype B RF/HAT3
P18799|ENV_HV1ND              ENLTNNVKTIIVQLNASIVINC     HIV-1 group M subtype D NDK
P20871|ENV_HV1JR              DNFTDNAKTIIVQLNESVKINC     HIV-1 group M subtype B JRCSF
P35961|ENV_HV1Y2              ENFTNNAKTIIVQLNESVVINC     HIV-1 group M subtype B YU-2
P19549|ENV_HV1S3              DNFTNNAKTILVQLNVSVEINC     HIV-1 group M subtype B SF33
O41803|ENV_HV19N              ENFTDNTKVIIVQLNNSIEINC     HIV-1 group M subtype G 92NG083
Q9WC69|ENV_HV1S9              ENISDNAKNIIVQLNDTVEIVC     HIV-1 group M subtype J SE9173
Q9WC60|ENV_HV1S2              ENISDNAKNIIVQLNKTVEIVC     HIV-1 group M subtype J SE9280
Q9Q714|ENV_HV1V9              KNITDNTKNIIVQLNEPVQINC     HIV-1 group M subtype H VI991
P04583|ENV_HV1MA              ENLTDNTKNIIVQLNETVTINC     HIV-1 group M subtype A MAL
Q9QBY2|ENV_HV196              ENITDNTKNIIVQLNETVQINC     HIV-1 group M subtype K 96CM-MP535
Q9QBZ8|ENV_HV197              EDITKNTKNIIVQLNEAVEINC     HIV-1 group M subtype K 97ZR-EQTB11
Q75008|ENV_HV1ET              ENLTNNAKIIIVQLNESVEITC     HIV-1 group M subtype C ETH2220
P20888|ENV_HV1OY              SNFTNNAKIIIVQLNKSVEINC     HIV-1 group M subtype B OYI
P12487|ENV_HV1Z2              ENLTNNAKIIIVQLNESVAINC     HIV-1 group M subtype D Z2/CDC-Z34
P04580|ENV_HV1Z6              ENLTNNAKIIIVQLNESVAINC     HIV-1 group M subtype D Z6
P05878|ENV_HV1SC              ENFTDNAKTIIVQLKEAVEINC     HIV-1 group M subtype B SC
P19550|ENV_HV1S1              ENFTDNAKTIIVQLKESVEINC     HIV-1 group M subtype B SF162
P12489|ENV_HV1J3              ENFTDNAKTIIVQLKEPVVINC     HIV-1 group M subtype B JH32
P05881|ENV_HV1ZH              ENFTDNAKIIIVQLVKPVNITC     HIV-1 group M subtype A Z321
Q9QBZ4|ENV_HV1MP              KNITDNTKNIIVQFNRSVIIDC     HIV-1 group M subtype F2 MP255
Q9QBZ0|ENV_HV1M2              ENISDNTKTIIVQFKNPVKINC     HIV-1 group M subtype F2 MP257
O70902|ENV_HV190              KNISDNTKNIIVQLKTPVNITC     HIV-1 group M subtype H 90CF056
O91086|ENV_HV1YF              VIRNDSHSNLLVQWNETVPINC     HIV-1 group N YBF30
P17281|ENV_SIVCZ              ENKSKNTDVWIVQLVEAVSLNC     SIV-cpz CPZ GAB1 (Chimpanzee)
Q1A243|ENV_SIVEK              VIRNNSKDTLLVQLNESIPINC     SIV-cpz EK505    (Chimpanzee)
                                               : : *   
10 HIV-1
12 HIV-2

2 SIV Chimpanzee

2 SIV Rhesus monkey

2 SIV Sootey mangabey

4 SIV African Green monkey

1 SIV Mandrill

O89292|ENV_HV193              QNISDNAKTIIVHLNESVQINC     HIV-1 group M subtype F1 93BR020
Q9QSQ7|ENV_HV1VI              QNISNNAKTIIVHLNESVQINC     HIV-1 group M subtype F1 VI850
O12164|ENV_HV192              KNLTDNVKTIIVHLNESVEINC     HIV-1 group M subtype C  92BR025
P31872|ENV_HV1W1              ENFTDNAKTIIVHLNESVEINC     HIV-1 group M subtype B  WMJ1
P05880|ENV_HV1W2              ENFTDNAKTIIVHLNESVEINC     HIV-1 group M subtype B  WMJ22
P05877|ENV_HV1MN              ENFTDNAKTIIVHLNESVQINC     HIV-1 group M subtype B  MN
P05882|ENV_HV1Z8              ENLTNNVKTIIVHLNESVEINC     HIV-1 group M subtype D  Z84
P04581|ENV_HV1EL              ENLTNNAKNIIAHLNESVKITC     HIV-1 group M subtype D  ELI
Q79670|ENV_HV1MV              KNITESAKNIIVTLNTPINMTC     HIV-1 group O MVP5180
Q77377|ENV_HV1AN              KDILEGGKNIIVTLNSTLNMTC     HIV-1 group O ANT70
Q1A261|ENV_SIVMB              RNVSNDMDTIIVKLNETVRLNC     SIV-cpz MB66 (Chimpanzee)
Q89607|ENV_HV2EH              WHGKDNRTIISLNSYYNLTMHC     HIV-2 subtype B EHO
Q76638|ENV_HV2UC              WHSKDNRTIISLNKYYNLTMHC     HIV-2 subtype B UC1
Q74126|ENV_HV2KR              WHGRDNRTIISLNTHYNLTMHC     HIV-2 subtype A KR
P24105|ENV_HV2CA              WHGKDNRTIISLNKHYNLSMYC     HIV-2 subtype A CAM2
P05883|ENV_HV2NZ              WHGKDNRTIISLNNFYNLTMHC     HIV-2 subtype A NIH-Z
P18094|ENV_HV2BE              WHGRDNRTIISLNKYYNLTMRC     HIV-2 subtype A BEN
P17755|ENV_HV2D1              WHGKDNRTIISLNKYYNLTMHC     HIV-2 subtype A D194
P04577|ENV_HV2RO              WHGRDNRTIISLNKYYNLSLHC     HIV-2 subtype A ROD
P18040|ENV_HV2G1              WHGRDNRTIISLNKYYNLSIHC     HIV-2 subtype A Ghana-1
P20872|ENV_HV2ST              WHGRDNRTIISLNKFYNLTVHC     HIV-2 subtype A ST
P15831|ENV_HV2D2              WHEKDNRTIISLNTYYNLSIHC     HIV-2 subtype B D205
P11267|ENV_SIVML              WHGRDNRTIISLNKYYNLTMKC     SIV-mac K78  (Rhesus monkey)
P05885|ENV_SIVM1              WHGRDNRTIISLNKHYNLTMKC     SIV-mac Mm142-83 (Rhesus monkey)
P19503|ENV_SIVSP              WHGRSNRTIISLNKYYNLTMRC     SIV-sm PBj14/BCL-3 (Sooty mangabey)
P12492|ENV_SIVS4              WHGKSNRTIISLNKYYNLTMRC     SIV-sm F236/smH4 (Sooty mangabey)
Q02837|ENV_SIVG1              GGNDNDTVIIKLNKFYNLTVRC     SIV-agm.gri AGM gr-1 (Green m grivet)
P05886|ENV_SIVVT              HRVNNNTVLILFNKHYNLSVTC     SIV-agm.ver AGM TYO-1 (Green m vervet)
P27977|ENV_SIVVG              HRVSNDSVLVLFNKHYNLTVTC     SIV-agm.ver AGM3 (Green m vervet)
P27757|ENV_SIVV1              HGVSNDSVLILLNKHYNLTVTC     SIV-agm.ver AGM155 (Green m vervet)
Q8AIH5|ENV_SIVTN              FVNTSVNTPLLVKFNVSINLTC     SIV-cpz TAN1 (Chimpanzee)
P22380|ENV_SIVGB              EEFHQRKFVYKVPGKYGLKIEC     SIV-mnd GB1 (Mandrill)
                                               : : *   


There are a Q an d a K residue located at the C terminus of the conserved C2 region of HIV gp120, near the beginning  of the variable V3 loop region.


Sequence FTDN represent an exposed B-cell epitope (25)

There are aminoacids preceding this Q255 which are contact residues for CD4 based on crystal structure analyses (Decker 2005):

Envelope gp120 alignments for HIV-2 (7312A and UC1), SIV (Mac239 and Ver-Tyo1), and HIV-1 (YU2 and HXB2).

Red dots indicate HIV-1 contact residues for CD4 based on crystal structure analyses

Asterisks below the sequence indicate conservation of amino acid identity across all five virus strains

(Decker 2005)

Residues of gp120 preceding the zone are in direct contact with CD4 (Kwong 1998):

gp120 structure-based sequence alignment.

The sequences are shown of HIV-1 B, C, O , HIV-2 , and SIV

The secondary-structure assignments are shown as arrows.

Solvent accessibility is indicated for each residue by an open circle if the fractional solvent accessibility is greater than 0.4, a half-filled circle if it is 0.1 to 0.4, and a filled circle if it is less than 0.1.

N-linked glycosylation is indicated by ‘m’ for the high-mannose additions

Residues of gp120 in direct contact with CD4 are indicated by an asterisk. Direct contact is a more restrictive criterion of interaction than the often-used loss of solvent accessible surface; residues of gp120 that have lost solvent-accessible surface but are not in direct contact include 278, 282.

(Kwong 1998)

Residues of gp120 preceding the zone are in direct contact with CD4 (LIAO 2013):

Sequence alignment of outer domain of the crystallized gp120, and diverse HIV-1 Env proteins recognized by CH103 (an antibody)

Secondary structure elements are labelled above the alignment.

Symbols in yellow or green denote gp120 outer domain contacts for CD4 and CH103, respectively, with open circles with rays representing side-chain contacts, and filled circles representing both main-chain and side-chain contacts.

(Liao 2013)


Interestingly, in the lethal HIV-1 group M , it seems to be better conserved lysine (K) residues that glutamine (Q) residues:

gp120 alignment (Bunning 2009)



Location of transglutaminase substrate in gp120 from envelope protein of HIV


gp120 from HIV BH10 (in this case the Q position would be Q257)



Identification of Q and K residues sensitive to tTG activity in Vasoactive Intestinal Peptide (VIP): Q16,K20, and K21 were found to be reactive within the VIP sequence. Published data reported Q16 and K21 as tTG substrates (Esposito et al. 1999), while no indications were available about K20. The exploitation of state-of-the-art technology in conjunction with classical biochemical methods led the authors to identify K20 as a new NH2-donor substrate for tTG within the VIP sequence (26).

The analysis allowed the identification of peptide 15–20 of the VIP sequence, revealing that the only Q residue, Q16, is a tTG substrate (26).

The analysis showed the presence of VIP peptides 16–21 and 21–23, carrying modified K20 and K21, respectively (26).


P01282 (VIP_HUMAN) UniProtKB/Swiss-Prot Vasoactive intestinal peptide VIP Homo sapiens (Human) versus

P03375 (ENV_HV1B1) UniProtKB/Swiss-Prot Surface protein gp120 Human immunodeficiency virus type 1 group M subtype B (isolate BH10) (HIV-1)

RSANFTDNAKTIIVQ255LNESVEINCTRP (gp120 HIV 273-299 Q255 (Q287 in gp160))

SDAVFTDNYTRLRKQ16  MAVKKYLNSILN (Vasoactive intestinal peptide VIP Human Q16)

Below: Identification of Q and K residues in VIP sensitive to tTG activity (26):



MARINIELLO 1993 (gp41) (23):

We initially investigated whether one or more of gp41 glutamine residues could incorporate radioactive spermidine, in the presence of purified TGase and Ca2+ in vitro (23)

Since the transmembrane protein gp41 contains several glutamine residues, 75% of which are located on the outer surface of the virus, we initially investigated whether one or more of these residues could incorporate radioactive spermidine, in the presence of purified TGase and Ca2+ in vitro (23).

TGase-catalyzed incorporation of  spermidine into gp41 (23).

Recombinant gp41, the transmembrane glycoprotein of the human-immunodeficiency-virus (HIV) envelope, is an amino acceptor and donor substrate for transglutaminase in vitro (23).

gp41 is not only able to act as a TGase amino acceptor but also as an amino-donor substrate of transglutaminase (this presence of amine-donor site(s) in the gp41 was verified by the tgase-catalyzed incorporation of substance P (a peptide known to contain a reactive glutamine residue) into gp41, confirming the existence of one or more cross-linking lysine residues in the glycoprotein sequence (23).

Ability of TGase to produce gp41 homodimer and homopolymers. In vitro cross-linking of gp41 into both a homodimer and homopolymer(s) (23).

To explore and identify the specific gp41 site(s) involved in the reaction, we tested several overlapping synthetic peptides which covered all the glutamine residues present in the gp41 sequence located on the outer surface of the virus (23).

These synthetic peptides were tested as TGase substrates by TGase-catalyzed incorporation of  spermidine (23).

The gp41 peptides which were assayed contained all the glutamines present  in the external part of the transmembrane protein (23).

Below: Primary structure of HIV transmembrane gp41. The underlined fragments are the peptides assayed as TGase amino-acceptor substrates; the glutamine residues in white boxes represent  those found to be reactive sites for the enzyme. Outer (OUT) and inner (IN) viral environments (23):


gp41 fragments 27-42, 104-116, 128-144 and 143-154 are completely ineffective as TGase amino-acceptor substrates (23).






Gln51, Gln52, Gln66 (as amino-acceptor) and Lys77 (as amino-donor) residues were suggested as reactive sites in gp41 recognized by transglutaminase (23).



Mariniello 1993 (gp41) (23) proved transglutaminase substrates in the HIV gp41 protein (gp41: aminoacids 42 to 86, P04578 UNIPROT)



A specific HIV-1 gp41 sequence, denoted CS3, inhibits T-cell activation in vitro and antibody specific to CS3 has been linked to the absence of disease (28).



CS3 region (underlined) in the HIV gp41 protein (gp41: aminoacids 42 to 86, P04578 UNIPROT)


A binding domain for a Gln66-containing and Lys77-containing region of transmembrane protein (amino acids 65-81) has been reported to be expressed on the surface of CD4+ cells. gp41 interaction with such a receptor is probably required for HIV fusion and internalization (23).




MARINIELLO 1993 (gp41) (23):

CD4 is unable to function as amino-acceptor substrate of transglutaminase when incubated alone with Tgase (23).

CD4 acquired amino-acceptor substrate property in the presence of similar amounts of gp41 (23).

There is an effect of CD4 on the ability of gp41 to act as TGase amino-acceptor substrate (23).

CD4 seems to acquire a TGase amino-acceptor substrate ability in the presence of  similar concentrations of gp41 (23).

Possible effect on the TGase-catalyzed  structural modification of gp41 by soluble CD4 (23).

The presence of CD4 in the reaction mixture negatively influenced spermidine incorporation into transmembrane protein monomer and dimer, apparently leading to an increased production of either homopolymers or heteropolymers. These phenomena were less evident when lower concentrations of CD4 were used (23).

These findings could be a consequence of non-covalent interaction between gp41 and CD4, affecting the molecular features of both proteins as amino acceptors for TGase. This hypothesis is supported by  the experiment showing an increase in CD4 immunoprecipitation, specifically observed when the protein was incubated with gp41 both in the presence and absence of either gp120 or bovine serum albumin (used as a control) (23).

Soluble CD4, even though unable to function as an amino-acceptor transglutaminase substrate, becomes active in the presence of gp41, negatively influencing the enzyme-catalyzed incorporation of the polyamine spermidine into the transmembrane protein (23).

The existence of a putative gp41-binding site for a cell surface protein exposed after HIV interaction with CD4, might help to understand how CD4 activates the fusion potential of the gp120-gp41 complex (23).


AMENDOLA 1994 (30):

We analyse whether the interaction between the HIV protein gp120 and the CD4 receptor can cause the tTG (tissue transglutaminase) expression (30).

tTG (tissue transglutaminase) is a Ca2+ dependent enzyme that is not active at the Ca2+ levels normally detected in viable cells (30).

The binding of gp120 to the CD4 molecules is able to induce the expression and the subsequent activation of tTG (30).

It is interesting to note that untreated cells do not express tissue transglutaminase (tTG), while the enzyme is markedly induced upon the binding of gp120 to the CD4 receptor and/or after stimulation with the antigen (30).


Tissue transglutaminase (tTG) is not detectable in untreated cells (30).

Below: The untreated T cells grown in presence of rIL-2; note the absence of positive reaction to the tTG antibody (bar = 12 µm) (30):


Tissue transglutaminase (tTG) is induced in the human T cells (clone HC4) during the first 24 h exposure to gp120. The number of tTG-positive cells reached a value close to 30% following 24 h treatment with gp120 (30).

Below: ‘Tissue’ transglutaminase expression in gp120 treated human T cells. T cells (clone HC4) immunostaining with the monospecific anti-tTG antibody. The clone T cell HC4 pretreated with gp120 for 24 h; about 30% of the T cells present an intense tTG expression in the cytoplasm (arrow heads; bar ≈ 12 µm) (30):

This tTG protein expression in the absence of the extreme apoptotic phenotype could highlight a pre-apoptotic stage during which the T cells are ‘primed’ for apoptosis which is then affected if the primed cell receives additional CD3-transduced signals (30).

AMENDOLA 1996 (31):

This study indicates that tTG gene expression is induced in immune system of seropositive individuals (31).

It is largely accepted that the pathological activation of T cells might represent the principal cause of the progressive CD4+ T-cell loss typical of AIDS (31).

In AIDS it occurs a selective depletion of CD4+ T cell lymphocytes in vivo (31)

The perturbation of the CD4 T-cell receptor due to gp120-CD4 interaction might be responsible for CD4+ T-cell anergy and/or the induction of apoptosis (31).

The enzyme transglutaminase is not necessarily active at Ca2+ levels found in normal cells (31).

Resting T cells do not express tTG; however, upon crosslinking of the CD4 receptor by gp120, human T cell clones start to synthesize the protein (31).

Peripheral blood mononuclear cells and lymphoid tissues from HIV-infected individuals display high levels of "tissue" transglutaminase (tTG) with respect to seronegative persons (31).

In asymptomatic individuals, > 80% of the circulating CD4+ T cells synthesize tTG protein (31).

In HIV-infected lymph nodes tTG protein is localized in large number of cells (macrophages, follicular dendritic cells, and endothelial cells), showing distinctive morphological and biochemical features of apoptosis as well as in lymphocytes and syncytia (31).

These findings suggest that the presence of the tTG protein in the cytoplasm of a T cell may highlight a preapoptotic stage (31),

We investigated tTG expression in peripheral blood mononuclear cells (PBMCs) and in lymph nodes of a large cohort of HIV-infected individuals (31).

We investigated tTG expression in the PBMCs from 11 healthy and 49 HIV-infected subjects. With respect to seronegative donors, the HIV-infected individuals analyzed showed a drastic increase (7- to 8-fold) in the number of circulating cells expressing the tTG gene (31).

Below: Characterization of tTG expression in PBMCs from HIV-infected individuals as compared with seronegative donors: Immunocytochemical detection of tTG in PBMCs obtained from a seronegative (A) versus a HIV-seropositive individual (stage IV) (B). (A and B) Arrowheads indicate the presence of tTG-positive monocytes. (Bars: A and B, 30 μm) (31):

We investigated the phenotype of those PBMCs which synthesize tTG protein in asymptomatic individuals and AIDS patients: Both CD4+ and CD8+ T cells from HIV-infected individuals expressed tTG protein. Approximately 80% of the total CD4+ T cells were stained by the tTG antibody, representing about 90% of the total tTG-positive PBMCs population in asymptomatic individuals; the remaining 10% was composed by CD8+ T cells and monocyte/macrophages (31).

tTG Localization in Lymphoid Tissues of HIV-Infected Individuals: To verify whether the tTG expression observed in PBMCs reflects a similar pattern in lymphoid tissues, tTG localization in situ in lymph nodes from both HIV-infected and seronegative individuals was investigated (31).

The paracortical and interfollicular regions as well as the apical light zone of the follicle of HIV-1 lymph nodes were markedly stained by the tTG antibody (31).

Below: Immunohistochemical localization of tTG protein in lymph nodes sections from uninfected (A) and HIV-1 infected (B) individuals. Note the large increase in the tTG specific staining observed in the cortex/paracortex area and in interfollicular zone of the lymph nodes of the seropositive individuals (B) when compared with a seronegative control (A) where only endothelial cells are stained by the tTG antibody (A, arrowheads) Bars: 80 μm (31):

Below: D and E show a higher magnification of the cortex/paracortex and interfollicular zones, respectively, of a lymph node from HIV-1 and HIV-2 coinfected individuals. Note the dramatic fragmentation of accessory cells (macrophages and follicular dendritic cells) into tTG-positive apoptotic bodies (arrowheads) and the staining of polynucleated syncytia (arrows). Bars: 5 μm It is interesting to note that the accumulation of tTG protein was not limited to T cells, but the enzyme was also localized in a large number of polymorphic cells and in the syncytia (31):

Correlation Between HIV-1 Infection and Plasma Concentrations of Free ε(-γ-Glutamyl)Lysine: The increased concentration of epsilon(gamma-glutamyl)lysine isodipeptide, the degradation product of tTG cross-linked proteins, observed in the blood of HIV-infected individuals demonstrates that the enzyme accumulated in the dying cells actively cross-links intracellular proteins (31).

Results indicate that in HIV-infected individuals there is a significant and progressive enhancement in the plasma concentration of the free ε(γ-glutamyl)lysine cross-link, reaching its maximum in stage IV patients (31).

The enhanced levels of epsilon(gamma-glutamyl)lysine in the blood parallels the progression of HIV disease (31).

This study demonstrates that induction of tTG occurs in the immune system of HIV-infected individuals and that the enzyme is also activated, as indicated by the increase in the plasma concentrations of ε(y-glutamyl)lysine isodipeptide (31).

The number of tTG-positive PBMCs reaches a plateau in the asymptomatic phase whereas the plasma concentration of ε(γ-glutamyl)lysine continues to increase, reaching its maximal concentration in parallel with the massive disruption of tTG-positive cells observed in late stages of the disease in the lymphoid tissues (31).

The tTG-positive PBMCs of asymptomatic individuals are predominantly CD4+ cells and an increase of tTG in CD8+ cells and other cell types undergoing apoptosis is observed in AIDS patients (31).


tTG-dependent cross-linking of proteins might play an important role by reducing viral spreading in the course of HIV infection. This hypothesis is supported by the fact that apoptosis of HIV-infected monocyte/macrophages (which express high level of tTG) is characterized by a reduced viral release and that the viral glycoproteins gp41 and gp120 act as substrates for tTG (31).



MARINIELLO 1993 (gp41) (23):

gp41 could cross-link to receptor(s) occurring on HIV-target cells and/or gp120 with both glutaminyl and lysyl residues (23).

Transglutaminase reactive sites in gp41 have been proposed for possible cross-linking reactions with gp120, CD4 or other receptor(s) occurring on the surface of HIV-target cells (23).

An extracellular or membrane-associated molecular form of transglutaminase could cross-link the virus env glycoproteins to each other and/or to CD4 or some different protein receptor occurring on the surface of the HIV-target cell (23).



MARINIELLO 1993 (gp41) (23):

Several functions have been ascribed to transglutaminases including receptor-mediated endocytosis (23).

The existence of membrane-associated TGase on the surface of human alveolar macrophages, human peripheral blood mononuclear cell and rabbit hepatocytes have been reported; the occurrence, in blood plasma, of the coagulation factor XIII, a zymogenic form of  the enzyme, is also well known (23).

It  has been demonstrated that rabbit hepatocyte surface-expressed TGase can serve as a crucial component of a binding site for exogenous fibrinogen or fibronectin, covalently incorporating these glycoproteins into high-molecular-mass complexes on the outside of the cell (23).

For both endothelial cells and  melanoma cells, the TGase-mediated covalent cross-linking of  the  cell-bound fibrinogen suggests the possibility that the surface-expressed enzyme may also serve as a binding site for glycoproteins in these cells (23).

These data emphasize the possibility that TGase catalyzes the formation of a covalent bond between protein(s) of the HIV envelope (env) and protein(s) present on the membrane of HIV-target cells (23).

Possible role for human-immunodeficiency-virus (HIV) internalization, by some molecular forms of  transglutaminase either occurring extracellularly or associated to the membrane of  the HIV-target cells (23).

Possible role for transglutaminase in virus entry into host cells, via receptor-mediated endocytosis, and/or in HIV-induced CD4+ T-cell depletion via apoptosis (23).

In conclusion, the experimental evidence that soluble gp120 and gp41 can act as TGase substrates in vitro supports the notion that the enzyme could participate in the process of HIV entry into the host cells (23).

Primary amines and several peptides which share the property of being competitive inhibitors or substrates of TGase have been shown to inhibit internalization of ligands through coated pits and vesicles, we believe that  this potential new target for anti-HIV therapy deserves to be investigated first of all by testing  TGase inhibitors and alternative substrates as potential drugs blocking  virus entry (23).

Since it has been shown that the interaction of gp120 with CD4, as well as with other receptor(s) occurring in ganglion cells and hippocampal neurons, results in an increased intracellular Ca2+ concentration and that TGase, a strictly Ca2+-dependent enzyme, could be involved in receptor-mediated endocytosis, we hypothesized that transglutaminase may play a role in the process of virus internalization by cross-linking HIV env glycoproteins to specific receptor(s) occurring on the target-cell su rface (23).


MARINIELLO 1993 (gp120) (22):

These data should stimulate further experiments on the possible changes of the properties of HIV following the preliminary treatment of the virus with active TGase and various acyl acceptor substrates. In fact, the TGase-catalyzed covalent modification of gpI20 could influence the ability of HIV both to penetrate inside the cells and to infect them (22).

In this respect, the existence has been previously reported of a membrane-associated TGase both in intact resting human peripheral blood mononuclear cells and on the surface of alveolar macrophages possibly related to receptor-mediated functions of lymphocytes and/or macrophages (22).

Therefore, a role played in HIV internalization by some molecular forms of the enzyme, either membrane-associated or occurring outside of the cell (such as blood plasma Factor XIII), cannot be ruled out and deserves further investigation (22).



MARINIELLO 1993 (gp41) (23):

Programmed cell death (apoptosis) may be responsible for the deletion of reactive T cells that contributes to HIV-induced immunodeficiency. However, some of the stimuli inducing apoptosis were capable of both inducing TGase expression and increasing ε(γ-glutamyl)lysine-cross-link concentration. Therefore, the ability of HIV env glycoprotein to act as TGase substrates could be consistent also with a putative role played by the enzyme in virus-induced CD4+ T-cell depletion via apoptosis during the progression of HIV infection (23).


It has been proposed that transglutaminase TGE is involved in the biochemical pathway of programmed cell death (29).



A syncytium (plural syncytia) is a multinucleated cell that can result from multiple cell fusions of uninuclear cells (i.e., cells with a single nucleus) (Wikipedia).

Syncytia can form when cells are infected with certain types of viruses, notably HIV. During infection, viral fusion proteins used by the virus to enter the cell are transported to the cell surface, where they can cause the host cell membrane to fuse with neighboring cells (Wikipedia).

HIV infects CD4+ T cells and makes the cell produce viral proteins, including fusion proteins. Then, the cell begins to display surface HIV glycoproteins, which are antigenic. Normally, a cytotoxic T cell will immediately come to "inject" lymphotoxins, such as perforin or granzyme, that will kill the infected T helper cell. However, if T helper cells are nearby, the gp41 HIV receptors displayed on the surface of the T helper cell will bind to other similar lymphocytes. This makes dozens of T helper cells fuse cell membranes into a giant, nonfunctional syncytium, which allows the HIV virion to kill many T helper cells by infecting only one (Wikipedia).

MARINIELLO 1993 (gp120) (22):

Several functions have been ascribed to transglutaminases including cell-cell adhesion (22).

AMENDOLA 1996 – FERRI 2000:

In vivo, in lymphoid tissues from AIDS patients, syncytium formation is accompanied by over-expression of tissue transglutaminase, a marker of apoptosis.


TGE (transglutaminase) has been implicated in apoptosis of the cell (demonstrated in the liver cell), altering the cell structure and rendering it "insoluble"; these apoptotic cells undergo phagocytosis, when phagocytosis of several of the "insoluble" cells must temporarily render the "host cell" multi-nucleated. This may be the more logical explanation for the mechanism of syncytia formation seen in AIDS, and explained as a virus induced phenomenon (29).



It has been advocated by the main view a role of the HIV virus in immunosuppression in AIDS - and only in the "at-risk" groups.


The primary role of the virus in suppres­sion of the immune system in AIDS is being openly and clearly questioned. Particularly as the limited number of T cells affected by the retrovirus could not explain the extreme immunosuppression (29).


One very important issue in AIDS research seems to be the small number of lymphocytes that are infected by the retrovirus at any one time (29).

Although the trigger mechanism for virus replication is not understood, it seems to be associated with the direct activation of the infected cell; whereas, the virus by itself does not seem to activate the cell into virion production (29).

It is naturally understood that the lymphocytic activation is tightly coupled to the immune system activation. Therefore, virion production is also coupled to a generalized immune system activation. Since one of the most important components of the immune system build-up is the simultaneous development of an immune system memory-bank, logically, the immune system education for the newly manufactured cells must also be a part and parcel of the process. The mechanism and the process of virion production by a comparatively small number of lymphocytes seems to find better explanation as a natural component of the mechanisms involved in the immune system memory-bank build-up than a source of immune suppression in AIDS. Extending this explanation, when the immune system becomes gradually suppressed in the group of conditions associated with AIDS, what should be an "educational" antigenemia becomes exaggerated and exposed. Although a tunnel vision concentration on HIV seems to be the vogue, the above logic for the possession of a mechanism of virion production to maintain the memory-bank of the immune systems of the body could also apply to other viral disease recurrences seen in AIDS (29).


There are a main view that only acknowledge a long-term role for the virus in establishment of AIDS (29).

This main view claims that the antibody to HIV becomes a marker of disease progression (29).

However, it seems that anti-p24 antibody from healthy individuals with high antibody titre and antigen neutralizing capacity, given to very sick patients, for the duration of its presence in the serum, does clear the antigenemia (29).

This reported fact indicates that the presence of antibody to the virus is not a sign of disease progression (29).

The presence of HIV antibody as the marker of the progress of AIDS is inaccurate, all seropositive cases do not develop the clinical disease (29).


For evaluation of any disease condition, a detailed all-encompassing attention to the role of the milieu interieur is still important, more so in chronic viral infections. From this angle of view, it may even be possible to rationalize the diversity of disease conditions for which HIV seems to be made responsible, including the forms seen in hotter climates and poorer countries (29).

Since in the West, AIDS is primarily a disease of an "at-risk" section of the population, with clearly established and continuously participating "life-style" patterns, the physiological influence of the perpetuation of the "life-style" should also become a factor for consideration in this disease (29).

The body has normal physiological shut-down mechanisms for the immune system, for the establishment of which humoral and enzyme factors are manufactured. Otherwise, how will the body survive in its stressful environment of antigens? Within this normal physiological activity of the body, immunosuppressive or immune permissive factors are produced (29).

The ongoing "stressful" life-style of the at-risk group may be an important factor in immunosuppression (29).

To continue to allocate a dominant role to HIV in all pathological conditions that, at some time or another, in some cases, have also demonstrated markers of having come across the virus is also a marker of bankruptcy of scientific reasoning, particularly when the majority of the "at-risk" group so openly belong to a "life-style" participating sector of the society. How can it be justified that the disparity of the chemistry of the constantly determined "life-style" practitioners should not be investigated and yet the investigation of a viral causative factor, in so many different conditions, be so conclusively advocated? After all, if the camel had a back-breaking point to the weight of the last straw, surely the human body must also have a breaking point to being "life-stylishly" loaded. Do we measure the straw or the inherent structurallphysiological limitations? (29).

The following are some proposed physiologically induced permissive common factors that need to be considered when the etiology of AIDS is being researched (29):



The semen contains uteroglobin (UG)-like protein (SV-IV - 4) and transglutaminase (TGE) (29).


TGE is highly immunosuppressive and anti-inflammatory (29).

Lymphocyte proliferation is arrested by TGE (29).

The semen seem to render the allograft sperm non-antigenic to the recipient uterus, in all stages of its intrauterine life, could possibly also render the antigenic virus non-antigenic to the same immune system that would otherwise have established an immune response to the sperm (29).

TGE seems to transform a non-species specific protein from the seminal vesicle (SV-IV - demonstrated in the rat) that is highly suppressive to the lymphocyte proliferation (4).

There is a Transglutaminase-catalized Crosslinking Of An Immunesuppressive And Anti-inflammatory Protein Secreted From The Rat Seminal Vesicles (Porta 1988).


Semen introduced into the mouth or the lower part of the intestine will probably have the same immunosuppressive impact on the lymphatic system as it is naturally designed to do in the reproductive organs of the male and the female of the species. Thus, if the use of a condom seems prudent, the reason for its use should clearly identify the immunosuppressive property of the semen! (29).


In hemophiliacs who receive factor VIII concentrate, containing factor XIII, which is a TGE (transglutaminase), the same TGE-induced immunosuppression seems to be indicated (29).


Even the glucocorticoid's induced thymic involution seems to be mediated through the activation of the TGE in this gland (29).

Since glucocorticoids seem to promote TGE production for their role in thymic involution, the common factor between the permissive state of physiology of AIDS in homosexuals, drug addicts and hemophiliacs seems logically to be the immunosuppressive role of transglutaminase, rather than the direct action of HIV in AIDS (29).


The vertical transfer of the virus from the mother to the immune-naive fetus - that should inherit immunity from the already immunosuppressed mother, with the same amino acid pool available to the fetus as the composition of the pool in the mother - will establish without any form of resistance in a similar manner, not because of the aggressive properties of the virus but, because of the permissive state of the naturally suppressed immune system in the mother and the child (29).


gp120 & gp41 & Casein:



TGase activity was assayed by a radiometric method, based on the Ca2+-dependent (active TGase) incorporation of [14C]spermidine into protein amino-acceptor substrates (23).

It was measured tgase-catalyzed incorporation of spermidine into casein, gp160, gp120 and gp41 (which was showed by incorporation of radioactivity into the protein) (23).

Below: Transglutaminase amino-acceptor substrate activity of casein (N,N-dimethylated casein) compared to gp41, gp120 and gp160. Proteins were incubated with purified guinea pig liver transglutaminase in the presence of [14C]spermidine at 37ºC, pH 8.0 (23):

The ability of gp41 to incorporate radioactive spermidine was approximately 50% of that exhibited by N,N-dimethylated casein, a well known and very effective amino-acceptor substrate of the enzyme (23).

gp41 effectiveness to incorporate spermidine in the presence of active TGase was found to be more than twice as high as that exhibited by gp120 and only half of  that of N,N-dimethylated casein, a well known and very effective amino-acceptor substrate (23).


TGase activity was assayed by a radiometric method, based on the Ca2+-dependent (active TGase) incorporation of labelled amines into receptive acyl donor substrates (22).

It was measured tgase-catalyzed incorporation of spermidine and glycine ethylester into casein, gp160 and gp120 (which was showed by incorporation of radioactivity into the protein) (22).

Below: Transglutaminase amino-acceptor substrate activity of casein (N,N-dimethylated casein) compared to gp120 and gp160. Proteins were incubated with purified guinea pig liver transglutaminase in the presence of [14C]spermidine at 37ºC, pH 8.0 (22):

The ability of gp120 to incorporate covalently both Spd and glycine ethylester when incubated in the presence of Tgase and Ca2+ (acyl donor substrate activity exhibited by gp120 with the two acyl acceptors used) was about 18% of that exhibited by N,N-dimethylated casein, a well known and very effective amino-acceptor substrate of the enzyme (22).


gp120 & gp41 & Gluten:




Below the sequence alignment between gp120 and different Alpha/beta-gliadins (UniProtKB/Swiss-Prot):

P03375 (ENV_HV1B1) Surface protein gp120 Human immunodeficiency virus type 1 group M subtype B (isolate BH10) (HIV-1) versus

P04727 (GDA7_WHEAT) Alpha/beta-gliadin clone PW8142 – Prolamin - Triticum aestivum (Wheat)

P04725 (GDA5_WHEAT) Alpha/beta-gliadin A-V Triticum aestivum (Wheat)

P02863 (GDA0_WHEAT) Alpha/beta-gliadin Triticum aestivum (Wheat)

P18573 (GDA9_WHEAT) Alpha/beta-gliadin MM1 Triticum aestivum (Wheat)

SANFTDNAKTIIVQLNESVEINC (gp120 HIV-1 group M subtype B (BH10) 273-299 Q255 (Q287 in gp160))

QQNPQAQGSVQPQQLPQFAEIRN (Alpha/beta-gliadin PW8142 Triticum aestivum (Wheat) Q253 (Q273 w s peptide))

QLNPQAQGSVQPQQLPQFAEIRN (Alpha/beta-gliadin A-V Triticum aestivum (Wheat) Q259 (Q279 w s peptide))

QQNPQAQGSVQPQQLPQFEEIRN (Alpha/beta-gliadin Triticum aestivum (Wheat) Q228 (Q248 w s peptide))

QQNPQAQGSVQPQQLPQFEEIRN (Alpha/beta-gliadin MM1 Triticum aestivum (Wheat) Q249 (Q269 w s peptide))


Below: Transglutaminase Q residues substrate of transglutaminase in the amino acid sequence of the recombinant α-gliadin. The QXP sequence deamidated by tTGase are underlined (Mazzeo 2003):


KTIIVQLNESVEINCTR (gp120 HIV-1 group M subtype B (BH10) 273-299 Q255 (Q287 in gp160))

QQILQQILQQQLIPCRD (Alpha/beta-gliadin PW8142 Triticum aestivum (Wheat) Q122 (Q142 w s peptide))

QQILQQILQQQLIPCRD (Alpha/beta-gliadin A-V Triticum aestivum (Wheat) Q123 (Q143 w s peptide))

QQILQQILQQQLIPCMD (Alpha/beta-gliadin Triticum aestivum (Wheat) Q117 (Q137 w s peptide))

QQILQQILQQQLIPCRD (Alpha/beta-gliadin MM1 Triticum aestivum (Wheat) Q134 (Q154 with signal peptide))


P03375 (ENV_HV1B1) UniProtKB/Swiss-Prot Surface protein gp120 Human immunodeficiency virus type 1 group M subtype B (isolate BH10) (HIV-1) versus

P18573 (GDA9_WHEAT) UniProtKB/Swiss-Prot Alpha/beta-gliadin MM1 protease resistant 31-mer α2(58–88; LQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) (Dieterich 2005).

   CTHGIRPVVSTQLLLNGSLAE (gp120 HIV 215-235 Q226 (Q258 in gp160) proved transglutaminase substrate)

RSANFTDNAKTIIVQLNESVEINCTRP (gp120 HIV 241-267 Q255 (Q287 in gp160) proposed transglutaminase substrate)

LQPFPQPQLPYPQPQLPYPQPQLPYPQPQP (protease resistant 31-mer α2(58–88) Triticum aestivum (Wheat))






In order to infect a target cell, the HIV envelope glycoprotein gp120 has to interact with the cellular receptor CD-4 and co-receptor, CC or CXC chemokine receptors (Suresh 2006).

In addition to CD4, the human immunodeficiency virus (HIV) requires a coreceptor for entry into target cells. The chemokine receptors CXCR4 and CCR5, members of the G protein-coupled receptor superfamily, have been identified as the principal coreceptors for T cell line-tropic and macrophage-tropic HIV-1 isolates, respectively (Berger 1999).


No specific coeliac disease-related receptors for gluten peptides have been reliably identified on the epithelial cells, although some preliminary studies have indicated that gliadin is recognised by a G protein-coupled receptor in the chemokine family CXCR3 on intestinal epithelial cells (Lammers et al., 2008).

Gliadin Induces an Increase in Intestinal Permeability and Zonulin Release by Binding to the Chemokine Receptor CXCR3 (Lammers 2008).



HIV recruitment of CD4+ T cells to infect them and increase their number by replication:

HIV preferentially infects HIV-specic CD4+ T cells in vivo. Simply by virtue of their specicity, HIV-specic CD4+ T cells are more likely to be in prolonged close proximity to actively replicating HIV—their cognate antigen—in the lymph node (Douek 2002).

The recruitment of HIV-specic T cells into infected lymphoid sites, enhanced by inammatory chemoattractants, may affect the rate of viral clearance but may also provide cellular substrates for viral replication. During acute infection in vivo, rapidly proliferating HIV-specic CD4+ T cells are highly susceptible to HIV infection (Douek 2002).

As CD4 cells multiply to fight infection, they also make more copies of HIV.


Activation of small intestinal gluten-reactive CD4+ T cells is a critical event in celiac disease. Such cells predominantly recognise gluten peptides in which specific glutamines are deamidated. Deamidation may be catalysed by intestinal tissue transglutaminase (TG2), a protein which is also the main autoantigen in celiac disease (36).

Gliadin activation of CD4+ T cells to modify the intestinal barrier and let more gluten to cross:

Once gliadin peptides have crossed the epithelial barrier and entered the lamina propria, gliadin is thought to be deamidated by TG2. After deamidation, gliadin peptides are easily recognised by HLA DQ2 or DQ8 molecules on antigen-presenting cells, which in turn activate a specific population of CD4+ T cells. T cells start to proliferate and accumulate in the mucosal lamina propria. Coeliac disease-specific T cells raised against gluten have been obtained both from the small-intestinal mucosa and from the peripheral blood of patients suffering from coeliac disease (Stenman 2011).

By producing proinflammatory cytokines such as interferon-gamma (IFN-γ) the activated CD4+ T cells further provoke intraepithelial cytotoxic T cells to damage intestinal epithelial cells. Activated T cells also induce B cells to differentiate into plasma cells, producing disease-specific IgA-class antibodies. It has been demonstrated that coeliac autoantibodies may contribute to intestinal barrier modulation by increasing the transcellular transport of gliadin through the epithelial barrier increasing epithelial cell permeability (Stenman 2011).



Induction of tTG (tissue transglutaminase) occurs in the immune system of HIV-infected individuals and that the enzyme is also activated (31).


TG2 (tissue transglutaminase) overexpression and activation is also induced by toxic gliadin peptides (Paolella 2013).


Elafin is a potent endogenous human serine protease inhibitor (Galipeau 2014).

Elafin has been identified as a substrate for the cross-linking activity of tissue transglutaminase (Galipeau 2014).


In Nairobi, Kenya, a small group of highly exposed but HIV-uninfected sex workers show no signs of infection, despite repeated exposure to HIV for as long as 20 years and are epidemiologically defined as relatively resistant to HIV infection (Iqbal 2009).

Elafin/trappin-2, a small molecular weight protein, was elevated in secretions from HIV-resistant women. The association of elafin/trappin-2 with reduced HIV susceptibility was then prospectively confirmed in an independent cohort of high-risk female sex workers (FSWs) (Iqbal 2009).

Mucosal levels of elafin/trappin-2 were elevated in the genital tract of HIV-resistant sex workers, and the association of elafin/trappin-2 with HIV protection was confirmed in an independent, prospective study. This suggests that elafin/trappin-2 is playing an important role in mediating natural protection against HIV infection, either alone or in conjunction with other adaptive/innate protective immune mechanisms (Iqbal 2009)..


CD is one of the most common gastrointestinal diseases. The only treatment is a life-long adherence to a gluten-free diet (GFD) (Galipeau 2014).

Elafin expression is significantly decreased in patients with active CD, compared with patients without CD (Galipeau 2014).

Patients with active CD have reduced elafin expression within the small intestinal epithelium compared with patients in whom CD was excluded (Galipeau 2014).

In vitro elafin reduces the deamidation of an immunogenic gliadin peptide, an important step in the autoimmune process of CD (Galipeau 2014).

Our in vitro results demonstrate that the addition of elafin inhibited the deamidation of the digestion-resistant 33-mer gliadin peptide, which is one of the potential triggers of the adaptive immune response in CD. The comparison of elafin n to tridegin, a known transglutaminase inhibitor, revealed that elafin n moderately inhibits TG-2. These data support that elafin interacts with TG-2, raising the possibility that in addition to correlating with mucosal damage a reduction of elafin in active CD could have a pathogenic role (Galipeau 2014).

In conclusion, the reduced small intestinal elafin expression in patients with active CD, coupled to the finding that elafin may compete with gliadin peptides and reduce their deamidation, raise the possibility that loss of this protease inhibitor contributes to CD pathogenesis. The results in the animal model of gluten sensitivity suggest that mucosal delivery of elafin restores small intestinal barrier function and inflammation, likely through preservation of tight-junction function and antiinflammatory effects in the small intestine. Owing to the combination of specific and nonspecific beneficial effects of elafin in our study, we propose that its replacement could have potential as adjuvant therapy in gluten-related disorders (Galipeau 2014). 



Bacteria can enter the host through several pathways, like the respiratory tract, the orogastric way or by accessing the blood stream through skin wounds (Tosi 2010).



Once inside, bacteria can stably occupy a niche in the target organism, mainly by adhering to the epithelia in different cellular districts (Tosi 2010).

The adhesion process is thus essential for many bacteria in order to allow both survival and colonization (Tosi 2010).

Adhesion is mediated by diverse bacterium-specific adhesion molecules, called adhesins, which are located at the bacterial surface  and recognize either specific host cellular receptors or proteins of the extracellular matrix (Tosi 2010).

Adhesins can be present on elongated appendices on the surface of the bacterial cells (pili or fimbriae), or be directly tethered to the outer membrane. In both cases, these proteins mediate the first steps of the host-microbe interactions (Tosi 2010).

Bacterial adhesins are very diverse and are generally specific for certain receptors. For instance, the human-infecting Gram-negative pathogen Bartonella quintana, which causes a wide spectrum of pathologies in humans, produces the protein BadA (Bartonella adhesin A), a protein of 328 kDa that allows these bacteria to self-aggregate as well as to attach to the extracellular matrix proteins fibronectin and collagen. Streptococcus pneumoniae cbpA, a 75 kDa protein, binds to sialic acid present on cytokine-activated epithelial cells and to interact with the polymeric immunoglobulin receptor (pIgR) present on the human mucosa (Tosi 2010).



The invasion of non-phagocytic host cells (such as epithelial cells) by bacteria has been reported throughout the literature, particularly for gastrointestinal bacteria (Pinnock 2012).

Two main mechanisms of cellular invasion have been described; these are the trigger and zipper mechanisms (Pinnock 2012).

The trigger mechanism involves the delivery of bacterial virulence factors, upon host cell contact, delivered into the host cell via bacterial type III secretion systems. This results in the  direct activation of cytoskeletal proteins that can cause ruffling of the host cell membrane and lead to bacterial entry. Salmonella typhimurium and Shigella flexneri utilise this mechanism for cellular invasion (Pinnock 2012).

The zipper mechanism is initiated via bacterial ligands binding to host-cell surface receptors. This results in receptor clustering and the formation of a ‘phagocytic cup’. Invasion occurs following intracellular signalling and actin remodelling resulting in the engulfment of bacteria. Listeria monocytogenes and Yersinia pseudotuberculosis are thought to invade host cells via this mechanism (Pinnock 2012).



Bacteria commonly express proteinaceous appendages on their outer surfaces (Enersen 2013).

A pilus (Latin for 'hair'; plural : pili) is a hairlike appendage found on the surface of many bacteria (Wikipedia Pilus).

The terms pilus and fimbria (Latin for 'fringe'; plural: fimbriae) can be used interchangeably, although some researchers reserve the term pilus for the appendage required for bacterial conjugation (Wikipedia Pilus).

All pili are primarily composed of oligomeric pilin proteins (Wikipedia Pilus).


Thread-like adhesive organs known as pili enable many pathogens to attach themselves to the infected host’s target cells (Peter Rüegg 2008).

One class of extracellular polymers, known as pili or fimbriae (non-flagellar appendages), is used in attachment to and invasion of host cells. Pili and fimbriae are synonymous terms, with both commonly used, and are derived from Latin; pili for ‘hair’ or ‘fur’ and fimbriae for ‘fringe’. Since the first observations of these non- flagellar peritrichous appendages in the early 1950s, several distinct types of structures have been identified and characterized in Gram-negative bacteria, and later in Gram-positive bacteria (Enersen 2013).

Adhesins are a group of extracytoplasmic proteins found in pathogenic bacteria as well as environmental species, and they can be divided into two major classes. Fimbrial adhesins are composed of heteropolymers of pili subunits, while non-fimbrial adhesins consist of homo- trimers or a single protein (Enersen 2013).

The well-known bacterium Escherichia coli, for example, is responsible for more than 80 percent of all infections of the human urinary tract, and uses the numerous pili on its surface to attach itself to the epithelial cells of the urinary passages (Peter Rüegg 2008).

The adhesive threads also help the bacteria to penetrate into host cells, where the pathogens can evade attack by antibiotics (Peter Rüegg 2008)


UPGP Unidentified PG Protein:

UPGP is a protein located on the surface of a bacterium called Porphyromonas gingivalis.


UPGP is a transglutaminase substrate (32).


Porphyromonas gingivalis (Tsute Chen)

Electron micrograph showing the budding vesicles and fimbriae of the strain ATCC 33277




Fimbriae are thin, filamentous structures that protrude from the surface of the cell (Dashper 1998).

Fimbriae are short hair-like projections that protrude from the outer membrane of this organism (Pinnock 2012).

Fimbriae are fine and numerous appendices that protrude from the outer cell membrane (Moreno 2013).

Porphyromonas gingivalis (Wang 2007)

Electron micrograph of wild-type P. gingivalis (strain 33277) surface structures were visualized by transmission electron microscopy.

P. gingivalis (strain 33277) expresses long fimbriae.


These proteinaceous filaments play important roles in host adhesion and in cellular invasion of the bacterium (Pinnock 2012).


Escherichia coli Type 1 pili (Crespo 2012)

E Coli Pili are filamentous, noncovalent protein complexes mediating bacterial adhesion to the host tissue.

All structural pilus subunits are homologous proteins sharing an invariant disulfide bridge.

E Coli Pili consists of the disulfide bond formation in each of the up to 3,000 subunits of the pilus.

E Coli FimH is the protein that is always situated at the tip of a pilus and is responsible for the actual adhesive attachment.

Although presently, the tertiary structure of Porphyromonas Gingivalis FimA is unknown, and an experimental structure resembling a protein with high homology to FimA has not been found, it is possible to speculate that Porphyromonas Gingivalis fimbrillin (FimA) generates multimers employing a ‘donor strand exchange’ in a manner resembling E. Coli type I pili (Enersen 2013).


Although Porphyromonas gingivalis is a microorganism that has been isolated in gingivitis, it has also been isolated in healthy patients in low proportions (Moreno 2013).

Porphyromonas gingivalis is a microorganism with considerable genotypic diversity; hence, we can find clones more pathogenic than others and this could be the reason that explains the presence of the bacteria in healthy patients who have no signs of periodontal disease and in patients with severe periodontal disease, where there are signs of marked destruction of supporting tissue (Moreno 2013).

In the different studies reported in literature a distribution may be noted, where the fimA II genotype is the most frequently found in patients with periodontitis, while in healthy patients the most frequent genotype is fimA I. This could explain the existence of strains more virulent than others and because of this some patients can be positive for P. gingivalis and not develop periodontal disease (Moreno 2013).


P. gingivalis shown to express on the cell surface two distinct fimbria-molecules, long and short fimbriae (Enersen 2013).

P. gingivalis possesses two fimbrial types, a major (FimA) type and a minor (Mfa1) type (Pinnock 2012).

P. gingivalis expresses two distinct fimbria-molecules on its cell surface, one of which is composed of a subunit protein (named FimA or fimbrillin) termed long or long fimbriae, while the other consists of a subunit Mfa protein termed short, minor, or Mfa fimbriae (Enersen 2013).

MAJOR OR LONG FIMBRIAE (repeated subunit FimA or fimbrillin + subunits FimC, FimD and FimE):

Long fimbriae were suggested to comprise polymerized FimA and accessory proteins (FimCDE) (Enersen 2013).

The major or long fimbriae (FimA) are composed of filaments of a repeating 43 kDa protein, fimbrillin or FimA (Pinnock 2012).

The major or long fimbriae (FimA) is comprised of subunits of a protein called fimbriline, which is encoded by a gene denominated fimA (Moreno 2013).

The major fimbriae is confgured by long appendices measuring approximately 0.3 to 1.6 micra long (Moreno 2013).

Major or long fimbriae are classified into six types (I, II, III, IV, V and Ib) based on the diversity of fimA genes encoding FimA or fimbrillin (the main subunit of long fimbriae) (Enersen 2013).


The primary protein sequences of FimA share no significant homology with other described fimbrial proteins, indicating that P. gingivalis may possess a unique class of fimbria subunits (Enersen 2013).

Much of the amino-terminal sequence of fimbrillin is conserved, however, molecular cloning and sequencing of the gene has shown little homology with other Gram-negative bacteria, suggesting the major fimbriae is specific for P. gingivalis (Pinnock 2012).

The presence of an extremely long signal peptide, and requirements for Arg- and Lys-specific proteases (gingipains) for extracellular maturation, indicates that FimA is a novel group of fimbriae different from the type I and IV families (Enersen 2013).

The hypothesis and presented data supporting the variable virulence potential among different fimA genotypes of P. gingivalis imply a possible role for the tertiary structure in the function of FimA. However, the level of transcription of the gene is also an important factor (Enersen 2013).

Translation of fimA nucleotide sequences performed in a study by Enersen 2008 resulted in the same number of primary protein structures as sequence variants. Although the fimA gene was conserved, there were some minor variations between isolates belonging to the same genotype, resulting in corresponding variations in the primary protein sequence of the FimA monomer, which was also shown by Fujiwara 1993. Whether these mutations result in a FimA monomer with a changed structure that influences the pathogenicity of the isolate may partly depend on how the secondary structure of the molecule folds into a tertiary structure (Enersen 2013).

Presently, the tertiary structure of FimA is unknown, and an experimental structure resembling a protein with high homology to FimA has not been found. Advanced bioinformatic data yet unpublished, based on results presented by Shoji and co-workers as well as other bioinformatic sources, indicate that the structure of FimA may resemble the structure of protein NP_809975 of Bacteroides thetaiotaomicron found in the RCSB Protein Data Bank (structure 3GF8). From this structure, it is possible to speculate that fimbrillin generates multimers employing a ‘donor strand exchange’ in a manner resembling E. coli type I pili (Enersen 2013).

FimA also appears to be hypervariable, which is consistent with its importance as a virulence factor for the species (Enersen 2013).

FimA of TYPE II:

P. gingivalis expressing type II fimbriae have been shown to be the most prevalent strains isolated from patients exhibiting periodontitis, have an increased ability to adhere to and invade oral epithelial cells (Pinnock 2012).

Studies of clones with type II fimA have revealed their significantly greater adhesive and invasive capabilities as compared to other fimA type clones (Enersen 2013).

It has been found, in experimental studies that fimA II can adhere to epithelial cells and invade the cell more eficiently than the other genotypes and it does it through specifc receptors in the host, including Integrin α5β1 (Moreno 2013).

Nakagawa 2002 demonstrated that recombinant FimA protein corresponding to fimA genotype II has a greater ability to adhere to and invade human epithelial cells than FimA corresponding to the protein from other genotypes. The pathogenicity of the various fimA genotypes has also been evaluated in animal models, with fimA genotypes II, Ib, and IV shown to cause stronger infectious symptoms and inflammatory changes as compared to strains harboring fimA genotypes I and III (Enersen 2013).

In addition, mutants in which the fimA type I gene was substituted with type II showed enhanced bacterial adhesion/invasion. In contrast, substitution of type II with type I resulted in diminished efficiency, supporting the notion that type II fimbriae are a critical determinant of virulence (Enersen 2013).

Results of several clinical studies also support findings that nucleotide variation of the gene is likely related to virulence. In chronic marginal periodontitis, P. gingivalis isolates with fimA genotypes II, IV, and Ib have been shown to be significantly more prevalent than isolates with other genotypes (Enersen 2013).

Also, studies of the pathogenic potential of distinct fimA genotypes in patients suffering from aggressive periodontitis have indicated that genotype II strains are more prevalent (Enersen 2013).

In contrast, isolates with fimA genotype I are the most prevalent among P. gingivalis-positive healthy adults, followed by genotype V (Enersen 2013).

In addition, fimA genotyping of cultured clinical strains of P. gingivalis sampled from individuals with periodontitis support previous findings that genotypes II, IV, and Ib are related to virulence (Enersen 2013).

Genotypes II and IV are associated with periodontal disease, while genotype I is related to gingival health (Moreno 2013).

Type II fimbriae from strain OMZ314 were shown to significantly induce cytokine expressions, thus the immune subversion potential may vary among fimA types, though definitive findings have not been presented (Enersen 2013).


FimC, FimD, and FimE are accessory or minor components associated with the main FimA protein (Enersen 2013).

FimC, FimD, and FimE are adhesive tip components (Enersen 2013).

FimC, FimD and FimE mutants

Loss of function experiments have confirmed that P. gingivalis mutants deficient for Fim C, D, or E have drastically attenuated virulence (Wikipedia).

FimC-, fimD-, and fimE- deficient mutants lost their auto-aggregation ability (Enersen 2013).

FimC-, fimD-, and fimE- deficient mutants showed diminished efficiencies of their long fimbriae to bind to GAPDH of S. oralis as well as fibronectin and type I collagen (Enersen 2013).

Fimbriae of the fimCDE mutant lost their ability to down-regulate IL-12, a key cytokine involved in intracellular bacterial clearance (Enersen 2013).

The fimCDE mutant failed to exploit CXCR4 in vivo for immune subversion (Enersen 2013).


FimE is known to be required for assembly of FimC and FimD onto FimA fibers (Enersen 2013).


FimC and FimD bound to fibronectin and type 1 collagen, whereas FimE failed to interact with these matrix proteins (Enersen 2013).

FimC and FimD (but not FimE) were shown to interact with CXC-chemokine receptor 4 (CXCR4) (Enersen 2013).

Together, these reports indicate the importance of FimCDE in the virulence of P. gingivalis and assembly of fully functional fimbriae (Enersen 2013).



Long fimbriae interact with CXCR4, a TLR2- associated receptor (Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system), which limits TLR2 activation in human monocytes and mouse macrophages. Furthermore, long fimbriae induce CXCR4-mediated activation of cAMP-dependent protein kinase A, which in turn inhibits TLR2-induced NF-kB activation in response to P. gingivalis. These results suggest that long fimbriae enable P. gingivalis to resist clearance in vitro and in vivo, promoting its adaptive fitness (Enersen 2013).


MINOR OR SHORT FIMBRIAE (subunit or monomer Mfa1):

Minor fimbriae are comprised of minor fimbria protein subunits (Mfa1) encoded by the Mfa1 gene (Moreno 2013).

Short fimbriae are homopolymers of a subunit protein mfa1 with an apparent molecular mass of 75 kDa and antigenicity distinct from long fimbriae. Short fimbriae are shorter than long fimbriae and can only be visualized when the latter are absent (Enersen 2013).

Minor fimbriae measure from 3.5 to 6.5 nanometers long, signifcantly shorter than the major fmbriae (Moreno 2013).

The minor fimbriae (Mfa1) are shorter, and as such are hidden beneath the longer and more abundant FimA fimbriae. These minor fimbriae are important in co-adhesion with early colonisers of the oral cavity such as Streptococcus gordonii, therefore establishing colonisation, and auto-aggregation, contributing to the stability of this bacterium within the microbial biofilm (Pinnock 2012).



Among the virulence factors that develop P. gingivalis, there are fimbriae, which have been considered the main virulence factor of this microorganism, given that it confers it the capacity to adhere and invade tissues, which characterizes its high pathogenicity over periodontal tissue (Moreno 2013).

Fimbriae are considered to be critical factors that mediate bacterial interactions with and invasion of host tissues (Enersen 2013).

P. gingivalis fimbriae seem to participate in nearly all interactions between the bacterium and the host, as well as with other bacteria (Enersen 2013).

Fimbriae are a critical factor for colonization of P. gingivalis in subgingival regions, as they promote both bacterial adhesion to and invasion of targeted sites (Enersen 2013).

Porphyromonas gingivalis is found on and within oral and gingival epithelial cells following binding to surface components of host cells, which serve as receptors for the bacterium (32).


Adherence is an essential first step in the colonisation of the oral cavity by P. Gingivalis (Pinnock 2012).

Adhesion of microorganisms to oral structures and/or other microorganisms prevents the ever present risk of being ‘washed away’ by gingival crevicular fluid GCF (Pinnock 2012). 

A first step in colonization is attachment to host epithelial cells and P. gingivalis can bind to several host proteins and putative host cell receptors for the organism have been described (32).


Adherence to oral structures is facilitated by numerous bacterial cell surface-associated components, including  fimbriae (Pinnock 2012).

The major feature of Porphyromonas gingivalis which is important in adhesion to host cells is thought to be fimbriae (Pinnock 2012).

Fimbriae main function is adhesion to periodontal tissue, endothelial cells, and other tissues, given that they have isolated from ovarian and lung abscesses (Moreno 2013).

To survive within the oral cavity, P. gingivalis must first adhere to oral structures,  i.e. epithelium,  tooth surfaces, or most commonly primary bacterial colonisers. It does this via adhesins present on its cell surface, which include fimbriae (Pinnock 2012).

Adhesion is thought to be via the association of fimbriae with epithelial cells, which is a two-way  process, i.e., to adhere, bacterial fimbriae must bind to some receptor(s) located on the epithelial cell surface (Pinnock 2012).

This initial adherence of P. gingivalis to oral surfaces is a pre-requisite to, but the major rate-limiting step in, host cell internalisation (Pinnock 2012).

Long fimbriae extend a significant distance from the bacterial cell wall, which suggests that they are the first bacterial components to interact with other bacteria as well as host cells (Enersen 2013).

Long fimbriae are considered to be directly responsible for many of the adhesive properties of the organism, binding specifically to and activating various host cells, such as human epithelial, endothelial, and spleen cells, as well as peripheral blood monocytes, resulting in the release of inflammatory cytokines and several different adhesion molecules (Enersen 2013).

Various investigators have used purified fimbriae, recombinant fimbrillin, and antibodies to show that P. gingivalis fimbriae mediate bacterial adherence to a wide variety of molecules and oral substrates. These include salivary molecules, such as proline-rich proteins, proline-rich glycoproteins, statherins, oral epithelial cells, fibrinogen, fibronectin, and lactoferrin, and bacteria, such as oral streptococci and actinomyces species (Enersen 2013).

FimA fimbriae adhere to diferent proteins of eukaryotic cells like fbronectin, collagen, laminin, the proline-rich protein derived from saliva and statherin, as well as to prokaryotic proteins like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Streptococcus oralis (Moreno 2013).

Indeed, P. gingivalis long fimbriae have been reported to mediate coadhesion with Actinomyces viscosus, Treponema denticola, Streptococcus gordonii, and Streptococcus oralis via specific interactions with their receptors, including dentilisin of T. denticola, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of S. gordonii and S. oralis. GAPDH is a well-characterized glycolytic protein involved in energy production and has been suggested to be a multi-functional house-keeping protein that is conserved by eubacteria and eukaryotic cells. The interaction between long fimbriae and GAPDH is the initial contact event that allows for localization of P. gingivalis on the streptococcal surface. The binding domains of the subunit protein FimA that mediate attachment to streptococci are localized to a C-terminal region spanning amino acid residues 266-337. Human GAPDH has also been shown to bind to long fimbriae (Enersen 2013).

Attaching Porphyromonas gingivalis fimbriae on its epithelial cells

Université de Rennes 1



Invasion of non-phagocytic cells is a key virulence property of P. Gingivalis (Dolgilevich 2011).

Scanning electron micrographs show that P. gingivalis invades primary cultures of gingival epithelial cells and epithelial cell lines (Pinnock 2012).

Porphyromonas gingivalis is found inside oral and gingival epithelial cells in vivo (32).

P. gingivalis has the ability to adhere to and invade a variety of eukaryotic cells in vivo and in vitro, including various oral cell  lines, fibroblasts, endothelial  cells, and oral epithelial cells (Pinnock 2012).

Invasion is a rapid process, shown by the internalisation of fluorescently labelled P. gingivalis within primary cultures of gingival epithelial cells (Pinnock 2012).

P. gingivalis cells located within the perinuclear region of the epithelial cells, after approximately 10-15 minutes (Pinnock 2012).

Invasion of host cells could provide protection against the host immune response and other local defences (Pinnock 2012).

P. gingivalis invasion of epithelial cells aids in P. Gingivalis viability and it has been reported that  P. gingivalis has the ability to replicate within epithelial cells (Pinnock 2012).

In addition, it has been reported that P. gingivalis can spread inter- and intra-cellularly (Pinnock 2012).


The epithelial receptor-mediated entry of P. gingivalis and the rearrangement of the filamentous actin and microtubule networks, suggests a ‘zipping’ mechanism of invasion (Pinnock 2012).

However, there may be more than one mechanism of invasion operating due to the variety of virulence features expressed and different P. gingivalis strains may preferentially utilise a particular mechanism (Pinnock 2012).

These mechanisms may include clathrin-mediated endocytosis (Boisvert  and  Duncan,  2008) or lipid rafts (Tsuda et al., 2008), both of which have been implicated in P. gingivalis invasion (Pinnock 2012).


The invasion of oral epithelial cells by P. gingivalis has been shown previously to be dependent on bacterial fimbriae, with type II fimbriae rendering the bacterium most highly invasive (Nakagawa et al., 2002; Kato  et al., 2007).


Below: Mechanism of host cell internalisation by invasive bacteria. The zipper mechanism involves the binding of bacteria to host cell receptors and formation of a phagocytic cup resulting in bacterial invasion (Pinnock 2012):



Bacterial internalisation may occur via vesicular trafficking (Pinnock 2012).

This involves a dynamic system where vesicles bind at the cell membrane resulting in the carriage of extracellular molecules into the intracellular environment (Pinnock 2012).

Clathrin-coated vesicles have been implicated in the epithelial internalisation of P. gingivalis (Pinnock 2012).

Clathrin-coated vesicles consist of the protein, clathrin, which forms a ‘basket-like’ structure, stabilised by proteins called adaptins. Dynamin, which is a GTP-binding protein, forms a ring around the neck of the vesicle resulting in scission of a ligand-bound vesicle, in a GTPase dependent manner, which may then be transported intracellularly with help from the actin cytoskeleton (Pinnock 2012).

These vesicles may then be delivered to early or late endosomes. Early endosomes are involved in ligand dissociation and vesicular recycling, whereas late endosomes are involved in the delivery of vesicles to lysosomes, where they are hydrolysed and breakdown products delivered into the cell cytoplasm for recycling (Pinnock 2012).

It has been proposed that the trafficking of P. gingivalis within early endosomes may present a mechanism of bacterial exit from the cell via endosomal recycling to the cell membrane (Pinnock 2012).

P. gingivalis cells have been observed free within the cytoplasm and also surrounded by endosomal membranes (Pinnock 2012).

It has been suggested that shortly after invasion, P. gingivalis traffics to autophagosomes, which are multi-membranous vacuoles important in the recycling of cellular organelles (Pinnock 2012).

Autophagosomes mature into autolysosomes where degradation of the vacuole load occurs (Pinnock 2012).

Dorn et al. (2001) showed the localisation of P. gingivalis within autophagosomes,  which lack hydrolytic enzymes.The authors proposed that the bacteria are able to replicate within this vacuole, until released to cause re-infection. In addition, this vacuole may provide a niche in which this organism can increase the concentration of free amino acids required for survival (Pinnock 2012).

Endocytosis and cell invasion of Porphyromonas gingivalis

Atsuo Amano



FimA fimbriae bind eukaryotic proteins such as collagen type I, laminin and fibronectin, and prokaryotic proteins such as glyceraldehyde-3-phosphate dehydrogenase, playing a role in the initial colonisation of the oral cavity (Pinnock 2012).

FimA fimbriae bind transglutaminase substrate proteins (FOTGREN 2014):



We studied the potential involvement of tissue transglutaminase in the binding of P. gingivalis to epithelial cells (32).

Transglutaminase 2 (TG2) plays a critical role in the adherence of P. gingivalis to host cells (32).

Studies of confocal microscopy indicate colocalization of P. gingivalis with TG2 on the surface of HEp-2 epithelial cells, with clusters of TG2 seen at bacterial attachment sites (32).

By silencing the expression of TG2 with siRNA in HEp-2 cells, P. gingivalis association was greatly diminished (32).

The bacterium does not bind well to a mouse fibroblast cell line that produces low amounts of surface TG2, but binding can be restored by introduction of TG2 expressed on a plasmid (32).

TG2 can form very tight complexes with fibronectin (FN), and the complementary binding sites of the two proteins are known (32).

A synthetic peptide that mimics the main FN-binding sequence of TG2 blocks the formation of TG2-FN complexes and is highly effective in inhibiting adherence of P. gingivalis to host cells (32).

These findings provide evidence of a role for cell-surface TG2 in bacterial attachment and subsequent internalization (32).


In response to adhesion of P. gingivalis to oral epithelial cells, numerous host cell changes occur that may aid bacterial internalisation. These include: intracellular Ca2+ fluxes (Pinnock 2012).

Calcium ion fluxes have been reported to occur following contact of P. gingivalis with epithelial cells. These transient increases in calcium ion concentrations may contribute to intracellular signalling pathways resulting in cytoskeletal rearrangement (Pinock 2012).

Invasion of primary gingival epithelial cells by P. gingivalis can be inhibited in the presence of cytochalasin D, which inhibits actin polymerisation, and nocodazole, which depolymerises microtubules, suggesting a significant role for cytoskeleton rearrangement in host cell internalisation (Lamont et al., 1995).

Transglutaminase enzymatic activity is calcium-dependent, and we found that P. gingivalis association with host cells was dramatically increased in the presence of calcium ions (32).

In addition, inhibitors of transglutaminase block the binding of P. gingivalis to epithelial cells (32).




Porphyromonas gingivalis is the major causative agent of periodontitis, and it may also be involved in the development of systemic diseases (atherosclerosis, rheumatoid arthritis) (32).

Porphyromonas gingivalis has been implied in diverse systemic complications like cardiovascular disease, preeclampsia, and low birth weight, given its capacity to colonize other tissues, which has been evidenced by its presence in atheromatous plaques (Moreno 2013).

Several studies have reported the relationship between periodontal disease and cardiovascular disease, while at the same time the presence of P. gingivalis was found in specimens of atheromatous plaques (Moreno 2013).

The fimA genotypes that have been associated to periodontitis are also frequently found in cardiovascular specimens, which suggests the possible role of the type II and IV clones at the start and progression of cardiovascular disease (Moreno 2013).



In research on mice, it has been found that fimbriae stimulate the production of interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8), and tumor necrosis factor (TNF) by peritoneal macrophages and that in humans it triggers TNF secretion per monocyte. These cytokines are potent infammatory mediators, which can lead to the activation and destruction of bone tissue and periodontal tissue. Hence, fimbriae, besides being considered important virulence factors in colonization and invasion of P. gingivalis in oral tissues, can also collaborate with the infammatory response dependent on the immune response upon stimulating secretion of the cytokines mentioned (Moreno 2013).



Few studies on Porphyromonas gingivalis fimA genotypes can be related to systemic complications attributed to the spread of bacteria via bacteremia (Moreno 2013).

fimA A II and IV genotypes were more aggressive and generated greater damage to the tissue than fmA I genotypes in a model of systemic infection disseminated in mice (Moreno 2013).

Findings from a study of 2009 confirm this pathogen´s capacity to enter the circulatory system (Moreno 2013).


Fimbriae & Casein & Gluten:



Below the sequence alignment between Fimbriae (FimA, Mfa1, FimC, FimD and FimE) and Alpha/beta-gliadin and Beta-casein (UniProtKB/Swiss-Prot):



B2RH54|FIMA1_PORG3 Major fimbrial subunit protein type-1 OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimA

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum



                   .*:  ** .  . :     *: :*.* :..:  ::* :     :*  :          :*

B2RH54|FIMA1_PORG3 Major fimbrial subunit protein type-1 OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimA

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
                     :   ::: *: : .:.:             :: :*:*:                    

G1UBU7|G1UBU7_PORGT FimA type II fimbrilin OS=Porphyromonas gingivalis (strain TDC60) GN=fimA

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

                         . .*:* :    .*:  ** .  . :     *: :*.* :..:  ::* :   
                    :*  :          :*  ::*:        :*  :*::   :    * .***. * **:
P04727|GDA7_WHEAT   NPQAQGSVQPQQLPQFAEIRNLALQ----------------------------------- 286
                    .*     *: :    *:**:    *                       

Below: Extracts from Amano 1996 and Nagano 2012:

Genotypes II and IV are associated with periodontal disease, while genotype I is related to gingival health (Moreno 2013).


The sequence similar to gliadin is in types II and III, not in type IV ¿?


No apparent difference between type II and III in the zone: YDGSQGGNQISQDTPLEIKRV:


G1UBU7|G1UBU7_PORGT FimA type II fimbrilin OS=Porphyromonas gingivalis (strain TDC60) GN=fimA

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2

                     :*:*: *   *  . .*.     : .  * : *    .  .:  **  . *        
                      *..:::*  *:     *::  *   * :   .**:    **:          **:.. 

Below: Sequence homologies among type I, II, III, and IV fimbrillin of P. gingivalis. The deduced amino acid sequences of type I fimbrillin (6), type II fimbrillin of P. gingivalis HW24DI, type III fimbrillin of P. gingivalis ATCC 49417, and type IV fimbrillin of P. gingivalis HG564 (7) are aligned. Spaces (–) are inserted to maximize the sequence homologies. Amino acids identical to those in the type I sequence are represented by colons. Arrows show putative binding domains defined by the peptide inhibition study. Symbols: •———–, synthetic peptide inhibiting fimbrial binding of FimA to both PRP1 and statherin (Amano 1996)





B2RHG1|B2RHG1_PORG3 Mfa1 fimbrilin OS=Porphyromonas gingivalis (strain ATCC 33277) GN=mfa1

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

                        * **: **::   ::. :      :*.* ::.      .:*: . :   **:*   
                    *:  *     : .::   :  :  .**  * *     :* :    . *     *  :   
                    .* :  * * *:.:: ::  :* : :   * *    *              * :*::  .
P04727|GDA7_WHEAT   IHNVVHAII---------------------------------MHQQEQQQQLQQQQQQQL 223
                     :.*.:*::                                 : * *::* *.:::   :
                     ::      .    :. :*:    :* .  * ::  : *      :.  *:* :*: :  

F5X8I7|F5X8I7_PORGT Mfa1 fimbrilin OS=Porphyromonas gingivalis (strain TDC60) GN=mfa1

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

                        * **: **::   ::. :      :*.* ::.  .  : :   .   **:*:  **
                      *     : .::   :  :  .**  * *     :* :    . * *:    ..   *:
P04727|GDA7_WHEAT   --------NVVHAIIMHQQEQ------------------------QQQLQQQ-QQQQLQQ 225
                            .*.:*::  : *.                        :  : *  ::* *. 
P04727|GDA7_WHEAT   QQLPQFAEIRNLALQTL-----PAMCNVYIPPHCS-TTI--------------------- 304
                     ::* :*   *  ** :       :.  :: *.   : *                                                                                         


B2RHG1|B2RHG1_PORG3 Mfa1 fimbrilin OS=Porphyromonas gingivalis (strain ATCC 33277) GN=mfa1

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
P02666|CASB_BOVIN   SSSE--------------ESITR-----------INKKIEKFQSEEQ------------- 53
                    *..               .. .*           :. * * * .. :             
                                          :.      * * :.: : * * :  *  .*  .     
                     ::***   : : .: .:.*:                         *..   *.*. * :

F5X8I7|F5X8I7_PORGT Mfa1 fimbrilin OS=Porphyromonas gingivalis (strain TDC60) GN=mfa1

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
P02666|CASB_BOVIN   INKKIEKFQSEEQQQ-------------------------------------------TE 57
                    :..* *.:: :   *                                             
                            *  * :  . :*:* **: :*     * * : :  :  :* * * :*     
P02666|CASB_BOVIN   LTLTDVENLHLPL--PLLQSWM---------------------------------HQPHQ 164
                    :. .:: .:*:     *  .*                                   :*.*
F5X8I7|F5X8I7_PORGT PLPDQDTFMSVEVTVLPWKVHSYEVDL--------------------------------- 565
                    ***    *    *  *  

Fim C

B2RH57|B2RH57_PORG3 Minor component FimC OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimC

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

P04727|GDA7_WHEAT   -PQEQVPLVQQQQFPG------------------------------QQQQFPPQQPYPQP 63
                       ::* :   : : *                               *.:*   :*:   
                         :* *:      :*: :     :: : :   :   *:   *  **          *
                    :**  :. :               . ::*  :*    :*: *  **:   : :*  .* .
                    *::*    : : *    : *   *    ::. .*:* :.   : ::::     

Below: Identification of tTGase-modified peptides by MALDI-MS and definition of the deamidated glutamine residues present in the recombinant α-gliadin by nanoESI-MS/MS experiments. It was not possible t o define which of the underlined Q residues is deamidated in peptide 118–128 (Mazzeo 2003):

Below: Identification of deamidation sites in the 26 synthetic peptides overlapping the entire sequence of the recombinant α-gliadin by nanoESI-MS/MS experiments. It was not possible to define which of the underlined Q residues is deamidated in peptide 120–139 (Mazzeo 2003):

INCREDIBLE ALIGNMENT 1 FimC Alpha/beta-gliadin
B2RH57|B2RH57_PORG3 FimC (85-345)
P04727|GDA7_WHEAT   Alpha/beta-gliadin (41-301)




F5XAW1|F5XAW1_PORGT Minor component FimC OS=Porphyromonas gingivalis (strain TDC60) GN=fimC

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

                          :. ** *            : .**.*   :*         :* :          
                                         :*: * . *.:*   :*:        :* *:  :   :*
                    : :     :: : :       *: . *  **:         *:**  :. :         
                          . ::*  :  :  :*: *  **:   : :*     .*::*: ** :        
                    :  ::*   :. .*:* :.   : ::::     :.. :  *   : * :  : :      

B2RH57|B2RH57_PORG3 Minor component FimC OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimC

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
                    :::: *    .* *.*: *    **:. *  .*  :    : ** : **. ** :  ** 
                         **                    *        *:* *  *:::* .*     . ::
P02666|CASB_BOVIN   LLQSWMHQPHQPLPPTVMFPPQSVLSLSQS------------------------------ 183
                        *       ** *     **    *                                             

F5XAW1|F5XAW1_PORGT Minor component FimC OS=Porphyromonas gingivalis (strain TDC60) GN=fimC

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2



                      :  :**                   .*::::: *     * *.*: *    **:. * 
                     .*  :    : *: : **. ** :  **      **         *::*  ::      
                          *:* *  *::** .*    .  :: *:              :: :    * * :
                                                      *        * *     **::. *  

Fim D

B2RH58|B2RH58_PORG3 Minor component FimD OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimD

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum



                    *:             **::.::   .   :.         . *:** *  .:        
                     .::  :     ::* .: ::: :  .  :   .  :*:..*.* :  *           
                            :::.   ::.:  : :: : :*:**. :          .::.*:**.     
INCREDIBLE ALIGNMENT 2 FimD Alpha/beta-gliadin
B2RH58|B2RH58_PORG3 FimD (255-469)Strain ATCC 33277
P04727|GDA7_WHEAT   Alpha/beta-gliadin (85-299) 










O32389|O32389_PORGN FimD protein OS=Porphyromonas gingivalis GN=fimD

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

                    *:             **::.::   .   :.         . *:** *  .:        
                     .::  :     ::* .: ::: :  .  :   .  :*:..*.* :  *           
                            :::.   ::.:  : :: : :*:**. :          .::.*:**.     

F5XAW0|F5XAW0_PORGT Minor component FimD OS=Porphyromonas gingivalis (strain TDC60) GN=fimD

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum



                    : ::: :  .  .   .  :*:..*.* :  *                 *: :..:.** 
                    .:  : :: : :*:**. : :     :  .::.*::** : *:   ..          * 

B2RH58|B2RH58_PORG3 Minor component FimD OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimD

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2                                                                              
                           *    :          :**.: : *:*:: : ...  * : : **:   :   
P02666|CASB_BOVIN   YQEPVLGPVRGPFPIIV-------------------------------------- 224
                    **  **.*      **                                         
O32389|O32389_PORGN FimD protein OS=Porphyromonas gingivalis GN=fimD
P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
                           *    :          :**.: : *:*:: : ...  * : : **:   :   
P02666|CASB_BOVIN   YQEPVLGPVRG-----PFPIIV-------------------------------------- 224
                    **  **.*         **                                         


F5XAW0|F5XAW0_PORGT Minor component FimD OS=Porphyromonas gingivalis (strain TDC60) GN=fimD

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
                    *..::**  ::*       :.:          : .::**: *          **  *:* 
                        * . ** .         **:. :      *:: *       * :. .:  .  : :

Fim E

B2RH59|B2RH59_PORG3 Minor component FimE OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimE

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

                                       .*:  *.: *::        *: *:::  :*    .  ** 
                    :   *     : ..     * **             :  ***  *        : :* . 
                     : ::*:  :     :::*.:      *   **:    :*  * .  : ::..:     :
INCREDIBLE ALIGNMENT 3 FimE Alpha/beta-gliadin
B2RH59|B2RH59_PORG3 FimE (154-294)
P04727|GDA7_WHEAT   Alpha/beta-gliadin (47-187) 


                    : ::  *::: :                        . ::  :*  *  ..: .* *: :
                    :. * * .  :*      : * *   :: :  .  **.. *  *  :       .:*.: 
P04727|GDA7_WHEAT   ----------------QPSQQNPQAQ---------------------------------- 266
                                       * **: :                                  

F5XAV9|F5XAV9_PORGT Minor component FimE OS=Porphyromonas gingivalis (strain TDC60) GN=fimE

P04727|GDA7_WHEAT Alpha/beta-gliadin clone PW8142 OS=Triticum aestivum

                                       .*:  *.: *::        *: *:::  :*    .  ** 
                    :   *     : ..     * **             :  ***  *        : :* . 
                     : ::*:  :     :::*.:      *   **:    :*  * .  : ::..:     :
                    : ::  *::: :                        . ::  :*  *  ..: .* *: :
                    :. * * .  :*      : * *   :: :  .  **.. *                   
P04727|GDA7_WHEAT   -------QYPSSQGSFQPSQQNPQA----------------------------------- 265
                           :: . .  .   * **:                                    

B2RH59|B2RH59_PORG3 Minor component FimE OS=Porphyromonas gingivalis (strain ATCC 33277) GN=fimE

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
                       **::   :: :  .    * :** :    *: *..   **. ::   . *:  * . 

F5XAV9|F5XAV9_PORGT Minor component FimE OS=Porphyromonas gingivalis (strain TDC60) GN=fimE

P02666|CASB_BOVIN Beta-casein OS=Bos taurus GN=CSN2
                       **::   :: :  .    * :** :    *: *..   **. ::   . *:  * . 






BETHUNE 2008 (34):

Exposure and susceptibility to the pathogen:

The first step in any pathogen-initiated disease is exposure of the host to the pathogen. Whether this exposure results in disease depends both on the virulence of the pathogen and on the susceptibility of the host. Highly virulent infectious agents, such as human immunodeficiency virus (HIV), cause disease in virtually all exposed individuals, such that the primary determinant of disease incidence is the frequency of new exposures. However, exposure to most pathogens is necessary but not sufficient to cause disease, and the genetic and conditional susceptibility of the host are additional determinants of disease progression. Indeed, less virulent pathogens may be in frequent contact with potential hosts but cause symptomatic disease in only a small fraction of those exposed. For example, persistent H. pylori infection is present in roughly half of the world’s population, and in up to 80% of populations in developing areas, yet only 10%–20% of those infected experience peptic ulcer disease, and only 1% develop gastric cancers (34).

Gluten peptides are similar to H. pylori and other high prevalence, low virulence pathogens in that they are ubiquitous but cause disease only in susceptible individuals (34).

Gluten-containing grains such as wheat, rye, and barley are extremely common dietary components in modern agricultural societies. Additionally, many ostensibly gluten-free products contain gluten contaminants due to the use of these proteins in food processing, as well as in certain non-food items such as cosmetics and household cleaning products (34).
Despite the nearly universal presence of gluten as a dietary protein source, the prevalence of celiac sprue is established by serological screening to be 1:100–1:200, and many of these cases are asymptomatic and undiagnosed (34). (Is Celiac sprue the only way of damage caused by gluten?)



BETHUNE 2008 (34):

Invasion across intestinal epithelium:

Pathogens that survive the harsh conditions in the stomach and proximal gut must overcome yet another barrier to pathogenicity in the form of the intestinal epithelium. The epithelial layer of the small intestine is a villous structure in which polarized enterocytes are joined together at their apical surface by transmembrane protein complexes called tight junctions and adherens junctions (34).

Tight junctions are located most apically and are composed of junctional adhesion molecules (JAM), claudins and occludin, while adherens junctions lie just below the tight junctions and are formed by homotypic interactions of E-cadherin. The tight and adherens junctions are connected to the actin cytoskeleton by associated proteins zonula occludins 1 (ZO-1) and α/β-catenins, respectively. These junctional complexes allow diffusion of small molecules between contiguous cells via the paracellular pathway, while preventing the entry of microbes and potentially antigenic macromolecules. Absorption across enterocytes, via the transcellular pathway, is likewise selective toward dietary protein-derived amino acids, di-, and tri- peptides, which are taken up by specic transporters on the apical membrane. Larger proteins and microbes are deterred from being transported via this pathway by secretory immunoglobulin (Ig)A and by the mucus layer coating the apical membranes of these cells (34).

The mucosal epithelium is thus selectively permeable to nutrients while acting as a barrier to pathogens and potentially antigenic macromolecules (34).

The ability to invade across the epithelium to access a privileged niche is a key determinant of whether a gastrointestinal microbe is pathogenic or commensal. Transepithelial invasion by microbes commonly involves disruption of the apical-junctional complex (34).

For example, Vibrio cholerae and Bacteroides fragilis secrete proteases that cleave the extracellular domains of occludin and E-cadherin, respectively. Adenovirus and rotavirus, among many other viral pathogens, also directly target junctional proteins, disturbing their proper function (34).

Still other pathogenic microbes that cannot overcome the apical junctional complex bypass it altogether by availing themselves of preexisting points of entry. For instance, Listeria monocytogenes exploits the transient luminal exposure of its host receptor, E-cadherin, at sites of epithelial cell extrusion at the tips of intestinal villi. Shigella flexneri, Salmonella typhimurium, and Yersinia pseudotuberculosis all cross the epithelium by being captured by specialized antigen-sampling epithelial cells, called M cells, and subsequently escaping macrophage-mediated destruction once translocated. Infectious prions are also observed to invade across enterocyte layers, and may do so using M cells as a portal (34).


GARDNER 1988 (27):

We know enough to conclude that macromolecular absorption is not a large-scale process in adults but it is not possible yet to state with reliable accuracy what fraction of the protein in a nutritionally complete meal will enter the circulation in macromolecular form. Yet this quantification is a most important question (27).


It is commonly assumed either

(a) that dietary proteins are digested completely to free amino acids within the lumen of the gastrointestinal tract before absorption occurs, or

(b) that only trace amounts of macromolecular fragments enter the circulation and that these are of absolutely no nutritional, physiological, or clinical relevance.

The first of these assumptions is blatantly untrue. It is now known that intestinal peptide transport is a major process, with the terminal stages of protein digestion occurring intracellularly after transport of peptides into the mucosal absorptive cells. Also, there now is irrefutable evidence that small amounts of intact peptides and proteins do enter the circulation under normal circumstances (27).

The second assumption is a gross simplification, but it does highlight two areas in which current knowledge is seriously deficient, namely the actual quantitative significance of intact protein absorption and the biological and medical relevance of this process and of abnormalities in it (27).


There is now no reasonable doubt that small quantities of intact proteins do cross the gastrointestinal tract in animals and adult humans, and that this is a physiologically normal process required for antigen sampling by subepithelial immune tissue in the gut (27).


It is too small to be nutritionally significant in terms of gross acquisition of amino-nitrogen, but since it has important implications relating to dietary composition it must receive consideration from nutritionists (27).


The process of intact protein absorption occurs without eliciting harmful consequences for most individuals, but it appears likely that a small number of people absorbing these "normal" amounts may react idiosyncratically; also, some individuals may absorb excessive amounts, and they may suffer clinically significant consequences. Likewise, individuals with diminished absorption of intact protein may be at risk (27).


Normal absorption probably occurs predominantly by transcellular endocytosis with some possible contribution by a route between cells; increased net entry of protein to the circulation may reflect:

(a) increased paracellular (intercellular) passage,

(b) increased transcellular passage, and/or

(c) decreased lysosomal proteolysis.

Tests to distinguish among these possibilities are strongly desirable (27).


Intact protein absorption may be involved in the pathogenesis of inflammatory bowel disease, "food allergies," and other diseases, including even major psychiatric disorders, but the current evidence is mainly indirect and suggestive (27).


Great caution and careful objective studies are needed to establish whether such relationships with disease do exist and to unravel the underlying basic physiological mechanisms (27).


Intact protein absorption must now be regarded as a normal physiological process in humans and animals. There is a good deal of indirect evidence suggesting that augmented absorption of intact proteins into the circulation may be pathologically significant, and there are numerous diseases for which the pathophysiology is poorly understood and for which hypotheses implicating enhanced permeability of the gastrointestinal tract to macromolecules have been postulated (27).

But, while there is no doubt that intestinal permeability is increased in a wide range of diseases, it must be stressed that there is not yet enough evidence to prove a casual link between enhanced intact protein absorption and disease. There also are grounds for believing that decreased absorption of intact protein may be disadvantageous but this too is not proven (27).



It is no longer tenable to regard the gut as an impenetrable physical "barrier", since many particles and large molecules previously regarded as "nonabsorbable" are in fact absorbed; indeed, some of these are now used diagnostically to measure intestinal permeability or "leakiness". Information about gut leakiness un various diseases, both gastrointestinal and systemic, is accruing and is proving helpful in understanding factors that reduce the barrier function of the gastrointestinal tract (27).



Peptide transport systems, distinct from free aminoacid carriers, have been characterized in intestinal brush-border membranes and are now thought to play a major role in protein assimilation (27).

Intracellular hydrolysis of small peptides thus becomes an important terminal stage of protein digestion. The concept that significant ammounts of small peptides can escape total digestion to amino acids and enter the circulation in intact form is a new one, but it is gaining acceptance; one potentially important consequence of this is that biologically or pharmacologically active peptides arising during protein digestion may reach peripheral tissues (including central nervous system) in active form and the effects may be profound. In general, the effects of intact biologically active peptides entering the circulation are likely to be deleterious (27).



The small and large intestines are now regarded as important immune organs; macromolecular antigen transport appears to be a normal and essential part of immune function, and specific cells adapted for this function are now recognized. Also, it is now clear that there is a highly developed enteric nervous system ("a brain in the bowel"), and recent evidence shows that electrolyte and fluid movements are under local neural control. Hence, the possibility that similar interrelationships exist to control or modulate macromolecular transport should not be neglected (27).



Probably the most compelling single item of evidence showing that intact proteins or macromolecular fragments of them are absorbed is provided by the demonstration, repeatedly made by numerous independent workers, that antibodies to many food proteins and their immune complexes occur in the circulation of healthy individuals -probably all individuals. Furthermore, increased levels of antibodies are found in individuals with various diseases likely to promote intestinal "leakiness", such as celiac disease, although Pitcher-Wilmott et al disagreed (27).

While it is theoretically possible that such antibodies might arise through the intestinal immune system responding to luminal proteins rather than absorbed ones, analyses of plasma by radioimmunoassay now show the presence of orally administered proteins, such as ovalbumin in blood: these show a time-course or tolerance curve generally similar to that for absorbed amino acids or peptides. Hence, it is impossible to escape the conclusion that immunologically significant amounts of intact protein (or immunologically identifiable large fragments thereof) have been absorbed (27).

This conclusion is reinforced by numerous animal and isolated-tissue experiments. For example, McLean & As have reported the time course of appearance of intact (or largely intact) horseadish peroxidase in blood and several peripheral tissues in fish in vivo: approximately 0,001% (rainbow trout)  or 0,7% (carp) of the oral dose of 20 mg  was detected in intact form in the tissues examined, which did not include muscle or brain. Several studies, notably those by Walker and his associates and by Desjeux and his coworkers have demonstrated passage of high molecular-weight fragments of protein across isolated animal jejunum (27).

There also have been numerous demonstrations that inert particles [e. g. carbon particles in Indican ink] and viruses can cross healthy intestine. The cells responsible have been identified as M cells. Additionally, we have rather dramatic evidence provided by the drastic consequences of botulism, in which a high-molecular-weight fragment (~106 daltons) of protein has been shown to cross the intestine (27).

While all  these techniques have limitations, the concordance between results obtained by independent workers using different experimental approaches is now so strong that we cannot fail to accept that intact proteins and high-molecular-fragments thereof do cross the gastrointestinal tract in humans and animals (both neonates and adults) (27).



These, by virtue of their simplicity, have commonly been made. While they can provide qualitative evidence that intact protein has been absorbed and can show the time-course of the event (though few studies provide this information), they, alone, are unable to provide information about the total amount absorbed in macromolecular form. Studies on distribution of absorbed macromolecular fragments in various organs as well as in blood can be particularly iluminating (with horseadish peroxidase), but these are not practicable in humans! The rapidity of the uptake by petipheral tissues and the amounts that may be sequestered by, for instance, the liver are striking (with horseadish peroxidase). Further, the presence of active proteases and peptidases with broad specificity in plasma is generally neglected: this has certainly accounted for some failures to detect the appearance of peptides in blood after protein or peptide meals, and it is possible that rapid proteolysis in the circulation has often resulted in erroneous estimates of the quantity of the intact protein crossing the gastrointestinal tract (27).



For obvious reasons, much works has been performed on animal species. However a sight must not be lost of one particular difference between humans and other mammals. Most species acquire the majority of their passive immunity via the gastrointestinal tract postpartum, and their gastrointestinal tract thus has to be able to transport in (selectively IgG in many especies) for the first few days (21-22 days from the mat) weeks of life: then  "closure" occurs and this process ceases. Thus, in this initial neonatal period, absorption of intact proteins plays a vital role. In contrast, however, humans acquire passive immunity via the placenta,  and "closure" (i.e. cessation of transmission of IgG) occurs apparently abruptly, or lose to, birth. The monograph by Baintneam deals in valuable detail with the macromolecular absorption in the context of immune transmission (27).

Hence, the mechanisms and routes of intact protein transport in neonatal animals may be fundamentally different from those operating in humans (27).

However, observations on neonatal animals can provide information about the physiology of  "closure" that cannot be obtained in humans.

A striking example of interspecific differences is provided by Mc Lean & Ash who are report an 1000-fold greater absorption of intact horseadish peroxidase by carp rainbow trut. They suggest that agastric species may have special requirements for maintenance of their immunocompetence, and that specially adapted enterocytes may be responsible of augmented intact protein absorption (27).



The gastrointestinal tract is a major site of immunologically competent tissue -hence the expresion gut- associated lymphoid tissue. Throughout the intestine, the lamina propia contains a substantial population of lymphocytes and macrophages, furthermore, the small intestine contains many Peyer's Patches, clearly discernible nodules of lymphoid tissue. Two distinct functions seem to operate immunization against antigens that have crossed the epithelium  and inhibition of antigen uptake by promotion of intraluminal (or brush-border face) digestion (27).

A major step in understanding the gut's function as an immunological organ and the significance and mechanism of macromolecular transport was taken in 1973 when Bockman & Cooper, followed by Owen & Jones in 1974, showed that Peyer's Patches were covered by a special type of cell, hitherto unrecognized. This is the M cell or membranous cell [or lymphoepithelial cell]. These can be identified by electron microscopy, and they are significantly different from columnar epithelial absorptive cells. It was initially thought that their apical surface had microfolds rather than microvilli, but it is now clear that they do possess irregular short and wide microvilli, although there are fewer than on columnar absorptive cells. Vesicles are particularly abundant in the cytoplasm, a reflection of their endocytotic activity, and there appear to be fewer lysosomes in the cytoplasm; this is consistent with a diminished rate of intracellular protein degradation as observed by Desjeux's group (27).

Transport by endocytosis into and across these M cells has now been shown for a number of proteins, viruses, and inert particles. It is hypothesized that the function of M cells is to permite direct access of luminal antigens to the subepithelial lymphocytes, which now can approach close to the intestinal lumen. Hence an immune response is elicited (27).

One important question is that of the relative contributions made by M cells and by "ordinary" columnar epithelial absorptive cells to macromolecular absorption. Owen concluded that horseadish peroxidase entered M cells much more rapidly than columnar cells, but similar rates of entry were reported by Ducroc et al. However, the latter group observed less intracelular degradation in tissue containing Peyer's Patches, so that the net transepithelial passage of macromolecules would be greater for the M cells (27).

Keljo & Hamilton also found a 3-fold increase in the passage of peroxidase across regions of piglet intestine containing Peyer's Patches, which supports the quantitative importance of this route. Walker, taking an overview, suggests that the M-cell route is used preferentially at low (or physiological) loads of luminal antigen, but that all absorptive cells may participate at increased antigen levels (27).



Having established that intact proteins do cross the gastrointestinal tract, it is pertinent to consider what region(s) are involved. Little is known about the possible involvement of regions other than the small intestine, which is certainly a major site of such absorption. Stomach and large intestine are unlikely to be important sites, but they should not be neglected. Rectal entry of intact protein has been shown in some fish species; while this is not likely to be of physiological relevance, it is a potential route for therapeutic administration of polypeptides and vaccines (27).

One route that has been neglected is the buccal mucosa, especially sublingual tissue. The rapid response in allegedly food-allergic patients to food in the mouth and the apparent efficacy of sublingual neutralization or desensitization merit investigation of the mechanisms involved and of the quantities that may enter the body vía this route (27).



Both (a) the paracellular pathway through the "tight" junctions -arguably inaptly named because of their permeability and their major role in fluid  and ion transport -and through cell extrusion zones and areas of damaged mucosa, and (b) the transcellular pathway may be involved in intact protein absorption. However, most evidence favors the latter route as dominant, especially in healthy intestine, although the process is a complex one involving metabolic energy expenditure, cytoplasmic tubule formation, and lysosomal processing. Bockman & Winborn observed ferritin passing through hámster intestinal cells by pinocytosis, with none passing between the cells. Likewise, the corroboration found in more recent work by Desjeux and colleagues suggests that only a small fraction of absorbed horseadish peroxidase crossed by the paracellular route in their rabbit ileal experiments in vitro. Hence, the transcellular route seems to be more important than the paracellular route, although increases in it caused by disease or with excessive exfoliation may even make it a predominant route. Their observations on biopsy material from malnourished infants suggested that decreases in intracellular processing were the basis for the increased transepithelial passage of the peroxidase marker (27).

The permeability probes discussed above for use in humans appear to reflect paracellular leakiness , which undoubtedly is increased at least to small molecules in many diseases. This route also can be used by particles and macromolecules: Volkheimer, who considered that motility was a driving force for particulate absorption, coined the term "persorption" for this process (27).



The scheme described by Walker & Isselbacher meets most of the histochemical and electron microscopic observatíons on macromolecule transport (27).

Protein molecules bind to receptors on the surface of the apical (brush-border) membrane.

The membrane invaginates to form phagosomes or vesicles encapsulating the protein.

The phagosomes migrate in the cytoplasm to lysosomes via a system of cytoplasmic microtubules.

Most fuse to form phagolysosomes or secondary lysosomes in which proteolysis occurs by a series of cathepsins and other acid proteases.

Some apparently fail to fuse or use a separate pathway and leave the cells by exocytosis at the basolateral membrane.

All these steps are energy dependent. A similar process occurs in neonatal animals before "closure" but large numbers of vacuoles are formed; at that stage IgG receptors exist on the brush-border membrane and it is thought that binding to them (and their inclusion in the vesicles) specifically protects the engulfed IgG from proteolysis in the phagolysosomes. In the experiments of Heyman et al, 97% of the peroxidase entering the cells was degraded to fragments of 2000-4000 daltons (27).

Hence it appears that lysosomal proteolysis is a major factor in minimizing entry of intact protein to the circulation, although the mechanism of this process has been less intensively studied in intestinal cells than in, for example, hepatocytes (27).



Although the name hints at a physical process of sealing the epithelial barrier, the events of "closure" appear to relate wholly to intracellular developments associated with intestinal maturation, rather than paracellular events, which lead to the cessation of (or substantial reduction in) intestinal transmission of large amounts of IgG in animals. After closure, brush-border receptors for IgG disappear (27).

In humans, where the intestinal route is regarded as unimportant for transmission of passive immunity, closure is thought to occur suddently at birth. However, the evidence on this point is confussing and suggests that closure has largely occurred by the time of birth but that some further closure does take place in the early days of extrauterine life. Also, there is some legitimate speculation that the intestine at this stage may be particularly vulnerable to damage by some exogenous, some of which may precipitate long-term gastrointestinal disease or "allergy". Objective evidence on this would be welcome since manipulation of infant-feeding practices potentially offers a powerful means of reducing the incidence of disease: for example, it is suggested that the reduction in infant celiac disease observed since the 1970s was associated with an increase in breast-feeding and later introduction of cereals (27).

Evidence shows that, while permeability to macromolecules is greater in pre-term infants than in full-term ones, it is also significantly present during the first few weeks of life of full-term infants and gradually reduces thereafter. Hence, closure may not be as abrupt and complete at birth as is generally presumed, and some passive immunity may also be gained by the gastrointestinal route. Further, it is suggested that exposure to human milk and to dietary antigens does affect this early postuterine maturation process (27).

In rabbits, Udall et al showed that absorption of immunoreactive bovine serum albumin by rabbits decreased markedly after the first week postpartum. Further, the absorption in the first week was greater if the animals were fed on a synthetic "milk" than when they were allowed to breast-feed. Thus factors in the natural milk were thought to be involved in hastening (but not essential for) the intestinal maturation. This is also consistent with the concept that exposure to specific antigens in early life may be relevant in subsequent events including, possibly, the pathogenesis of various gastrointestinal diseases; and with the idea that sensitization to allergens may arise, especially in children, during gastrointestinal inflammation or infection (27).

Leary & Lecce showed that surgically bypassed segments of piglet intestine "closed" at the same time as the neighboring segments that were exposed to food. Hence, hormones (rather than luminal contents) were concluded to trigger closure. While, Svendsen et al thought that insulin played a key role, there is confusing evidence for involvement of various other hormones, notably cortisol: much of the evidence has been discussed by Baintner (27).



The possibility that various ailments lacking other established pathophysiological explanations may be associated with diet frequently is expressed in lay and "fringe" medical circles but also, increasingly, in professional fora (27).

Unfortunately, much evidence has been anecdotal and subjective, and the need to provide sound analyses of the underlying pathophysiological mechanisms has too often been neglected. However, recently there has been some movement to redress this problem; see, for example, the tome by Brostoff & Challacombe. An understanding of intact protein absorption is central to this subject; the conclusion that some intact protein absorption does occur in health and that it may be augmented in disease inevitably provokes enquiry into the clinically relevant consequences. The possibility that, for example, inflammatory bowel diseases, are caused by dietary proteins and can be cured/treated by dietary manipulation has a plausible hypothetical background and some (but not enough) supportive evidence (27).

Dannaeus et al reported increased absorption of ovalbumin in egg sensitive children, and this was reduced by sodium chromoglycate: this suggests that there may have been elevated intestinal permeability (or decreased lysosomal hydrolysis) secondary to a mast cell response (27).

Dohan has advanced the theory that schizophrenia is associated with gluten ingestion in genetically susceptible individuals - note that gluten is the wheat protein known to be causal in celiac disease. Elevated plasma levels of gliadin antibodies have been reported in schizophrenia, but only in a small number of patients. However, gluten exclusion has also been reported to be beneficial in a small subset of schizophrenics and, of particular interest in the present context, intestinal permeability has been found to be increased in some schizophrenics (27). Interpretation of the causal relationships underlying these findings is difficult, but objective tests of  gastrointestinal function and of intact protein absorption may make new advances possible (27).

Other diseases in which food allergy and suggested enhanced macromolecular absorption have been discussed include eczema and rheumatoid arthritis, but there is no general acceptance of gastrointestinal mechanisms in the etiologies of these conditions. Also, only subsets of the populations studied have had increased intestinal permeability, but these may of course reflect true subgroups of etiologies (27).

Andre's work provides a new stimulus and suggestions for an objective assessment of potential adverse effects of dietary proteins on intestinal function but, as always, caution is needed in drawing causal conclusions (27).





Casein is a protein (phosphoprotein, phosphoric acid bound to the protein) that is grouped roughly spherical particles called casein micelles that are in colloidal dispersion in the animal milk. Naturally found only in the milk of mammals.


Casein is a substrate for transglutaminases for the inter-protein cross-linking (transamidation) reaction (33).




Gluten is a protein (a complex mixture of storage proteins) found in various food grains (2).

Wheat gluten contains about equal parts of alcohol soluble gliadins and alcohol insoluble glutenins (36).

The main, toxic, wheat-gluten components are a family of closely related proline-rich and glutamine-rich alcohol-soluble proteins called gliadins (2).

The gliadins consist of protein subtypes A, α, β, γ, and ω (2).

Gliadins have been classified into the major fractions α, γ, and w gliadins, and subdivided into their subcomponents α1–α11 γ1–γ6, w1–3, and w5 (35).


Gluten is a substrate for transglutaminases for the inter-protein cross-linking (transamidation) reaction (33).


Gliadin toxicity (in coeliac disease) was confirmed in several studies, with the main focus on the amino terminal region of A-gliadin, a major component of α-gliadin (35).


Transglutaminase Q residues substrate of transglutaminase in the amino acid sequence of the recombinant α-gliadin (Mazzeo 2003)

The QXP sequence deamidated by tTGase are underlined



BETHUNE 2008 (34):

To cause disease in a susceptible host, infectious pathogens must encounter that host (exposure), overcome barriers to infectivity, access a privileged niche, colonize, and ultimately cause damage to the host either directly, through toxin secretion, or indirectly, through activation of a self-injurious host immune response. In many cases, additional steps, such as activation of the infectious pathogen to a more virulent form and subversion of host processes toward a virulent end, are prerequisite to disease as well (34):


The gluten-induced pathogenesis of celiac sprue proceeds through a remarkably similar trajectory (34).

Below: Each Stage of Celiac Disease Progression; 1 replication via grain cultivation, 2 exposure to gluten peptides, 3 gluten peptides’ proteolytic resistance, 4 Invasion of gluten peptides across the intestinal epithelium, 5 activation of native gluten peptides to their deamidated forms and 6 deleterious response to gluten peptides (34):


Gluten Peptides as Non-Replicative Pathogens:

Gluten peptides in the small intestine cannot replicate but otherwise have many hallmarks of classical pathogens in the context of celiac sprue (34).

Gluten peptides can thus be thought of as non-replicative pathogens, bearing many similarities to infectious pathogens, with the exception of their inability to replicate or colonize an afflicted individual (34).

As non-replicative immunotoxins, ingested gluten peptides possess no capacity to colonize the gut. Instead, chronic inflammation persists in the celiac gut due to the continual dietary reintroduction of immunotoxic peptides from an exogenously replicating pool of cultivated gluten-containing grains (34).

Below: Infectious pathogens (green rounded rectangle; left panel) replicate within a privileged niche in an infected individual (white box; left panel). In contrast to infectious pathogens, gluten peptides (right panel), have no replicative capacity within afflicted individuals. Instead, these immunotoxic peptides are propagated by grain cultivation (34):

Gluten peptides persist as human pathogens uniquely due to our purposeful cultivation of the grains that produce them, and our quite intentional exposure to them by way of diet. Gluten peptides, then, are quintessentially accidental pathogens that cause disease in the most obliging of hosts (34).


Gluten peptides enter the body as components of common dietary grains (34). (Healthy and celiacs (34)).

Below: Infectious pathogens (green rounded rectangle; left panel) are then transmitted , either directly or via a pathogen-bearing vector, to another susceptible host (white box; left panel). In contrast to infectious pathogens, gluten peptides (right panel) are transmitted to celiac sprue patients via intentional or accidental ingestion in the course of their diet (34):


3 SURVIVAL: Evasion of proteolytic digestion:

Gluten peptides evade destruction by gastrointestinal proteases (34). (Healthy and celiacs (34)).

Below: To infect this new host, the pathogen (green rounded rectangle; left panel) must evade host defenses. Similarly, immunotoxic gluten peptides, clustered in proline/glutamine-rich regions of gluten proteins (protein depicted as yellow rectangle containing immunotoxic peptide in green, right panel), cause celiac sprue in susceptible individuals (white box; right panel) by evading gastrointestinal proteolysis. Most dietary proteins (yellow rectangle) are proteolyzed by gastrointestinal proteases and do not enact pathogenic effects (34):


Gluten peptides invade across the intestinal epithelium intact (34). (Healthy and celiacs?)

Below: The pathogen (green rounded rectangle; left panel) (green rounded rectangle; left panel) must invade across host barriers into a privileged niche (pink box), and in some cases become activated to a virulent form. Damage is caused to the host by pathogen- and/or host-mediated processes, while replication within the infected host enables further transmission. Commensal microbes (yellow rounded rectangle) cannot access privileged niches and do not cause disease. In contrast gluten peptides invade across the intestinal epithelium by unknown mechanisms, and, in some peptides, becoming activated by TG2 (represented by Q (glutamine) → E (glutamate) modification), resulting in a deleterious immune response (34):


Immunogenic gluten peptides access the lamina propria by unknown mechanisms (34).


TG2 plays a role in enhancing the immunotoxicity of many gluten peptides (34).

Gluten peptides become activated to a more immunotoxic form via enzymatic deamidation (34). (Healthy and celiacs?))

Immunogenic gluten peptides are deamidated by TG2 (34).

It is TG2 deamidation activity that enables TG2 to activate gluten peptide immunoreactivity (34).

Binding of certain short gluten peptides to HLA-DQ2 or -DQ8 and the resultant T cell stimulation is potentiated when distinct glutamine residues are deamidated by tissue transglutaminase (35).


Gluten peptides exert both innate and immunogenic effects in susceptible individuals, leading to disease (34). (only in celiacs or only the immunogenic response in celiacs and the innate response also in healthy?)

An inflammatory response to these metastable peptides is triggered in genetically susceptible individuals (in celiac patients) that is initially localized to the small intestine but that eventually leads to a systemic humoral response against gluten (34). (only in celiacs?)

Adaptive (deamidated) immunogenic peptides are loaded onto HLA DQ2 (or DQ8) and presented on the surface of antigen-presenting cells (APC) to gluten-specific, DQ2-restricted CD4+ T cells in the lamina propria, causing their activation and clonal expansion. Activated T cells mediate the humoral response, by giving help to both gluten-specific and TG2-specific B cells, as well as the cell-mediated Th1 response, which, through the secretion of proinflammatory cytokines such as IFN- γ and TNF-α ,disrupts tight junction integrity (34).

Innate peptides (non-deamidated) peptides act through unknown mechanisms as a stress signal toward enterocytes, inducing expression of MIC and IL-15. IL-15 promotes the infiltration of CD8+ IEL into the epithelium, and arms them with the NK receptor NKG2D. IL-15 may also influence the Th1 response. Intraepithelial lymphocytes bearing NKG2D target MIC-expressing enterocytes for killing via apoptosis, causing destruction of the epithelial layer, and villous flattening.

The combination of enterocyte apoptosis and tight junction disruption renders the epithelium more permeable, thereby facilitating access of gluten and propagation of the disease. In the continued presence of dietary gluten, chronic inflammation persists, and, in a small percentage of patients, results in enteropathy-associated T cell lymphomas (34).


At two stages in this process, the immunotoxicity of gluten peptides is increased through the actions of endogenous enzymes (proteases before invasion and transglutaminases before immune response enhanced) (34).



Availability of undegraded gliadin sequences in the intestinal mucosa:

For decades, it was hypothesized that celiac patients were missing a critical peptidase, accounting for their inability to properly digest gluten. It has since been established that gluten is proteolyzed to a similar extent by celiac patients and healthy individuals, leaving certain proteolytically resistant peptides intact (34).

Shan L, Molberg O, Parrot I, Hausch F, Filiz F, et al. (2002) Structural basis for gluten intolerance in celiac sprue. Science 297: 2275–2279.

Bruce G, Woodley JF, Swan CH (1984) Breakdown of gliadin peptides by intestinal brush borders from coeliac patients. Gut 25: 919–924.


Evasion of ‘‘host’’ defenses: Due to their route of entry, gluten peptides are most readily compared to pathogens of the gastrointestinal tract. Such pathogens encounter an extremely hostile environment that destroys any exogenous agents not uniquely suited to survive. In the stomach, gastric juices containing a mixture of hydrochloric acid, lysozyme, and pepsin prevent infection by ingested bacteria, and may attenuate the infectivity of low doses of prions (34).

Immunotoxic (for celiacs) gluten peptides have certain unusual structural features that allow them to survive the harsh proteolytic conditions of the gastrointestinal tract and thereby interact extensively with the mucosal lining of the small intestine (34).

Gastrointestinal proteases are the primary defense against potentially toxic dietary proteins. Gastric pepsin, pancreatic proteases trypsin, chymotrypsin, elastase, and carboxypeptidase, as well as exopeptidases anchored to the mucosal surface, cooperatively and rapidly digest most dietary proteins into single amino acids, di-, and tri-peptides. These digestion products are too small to elicit an immune response, and are absorbed across the mucosa for their nutritional value. By contrast, gluten proteins are incompletely digested by gastrointestinal proteases (34).

Hausch F, Shan L, Santiago NA, Gray GM, Khosla C (2002) Intestinal digestive resistance of immunodominant gliadin peptides. Am J Physiol Gastrointest Liver Physiol 283: G996–G1003.

Shan L, Molberg O, Parrot I, Hausch F, Filiz F, et al. (2002) Structural basis for gluten intolerance in celiac sprue. Science 297: 2275–2279.

Piper JL, Gray GM, Khosla C (2004) Effect of prolyl endopeptidase on digestive-resistant gliadin peptides in vivo. J Pharmacol Exp Ther 311: 213–219.

In sharp contrast with virtually all other dietary proteins, gluten proteins are minimally digested by the normal complement of gastrointestinal proteases, yielding proteolytically resistant peptides that accumulate in the proximal small intestine upon gastric emptying of a gluten-containing meal (34).

Hausch F, Shan L, Santiago NA, Gray GM, Khosla C (2002) Intestinal digestive resistance of immunodominant gliadin peptides. Am J Physiol Gastrointest Liver Physiol 283: G996–G1003.

Shan L, Molberg O, Parrot I, Hausch F, Filiz F, et al. (2002) Structural basis for gluten intolerance in celiac sprue. Science 297: 2275–2279.

Ingested gluten is digested to innocuous amino acids and proteolytically resistant, immunotoxic gluten peptides in the small intestine (34).

The structural basis for this proteolytic resistance has been elucidated. Gluten proteins are unusually rich in proline (~15%) and glutamine (~35%) residues, particularly in those regions identied as immunotoxic in celiac sprue (34).

Wieser H (1995) The precipitating factor in coeliac disease. Baillieres Clin Gastroenterol 9: 191–207.

Gluten proteins have a special structure, being rich in glutamine (about 30%) and proline (about 15%), clustered either as polyglutamine stretches or in repeating structures mainly containing glutamine and proline (36).

It is well known that peptide bonds, where proline participates with the imino group, are only susceptible to digestion with few proteolytic enzymes (36).

Cleavage adjacent to proline is highly disfavored for most proteases, and glutamine is not a preferred residue for any of the endoproteases found in the gut. Consequently, peptides of sufficient length to precipitate an immune response evade gastrointestinal digestion to reach the intestinal epithelium unscathed (e.g., a 33-residue peptide) (34).

Shan L, Molberg O, Parrot I, Hausch F, Filiz F, et al. (2002) Structural basis for gluten intolerance in celiac sprue. Science 297: 2275–2279.

While the majority of immunotoxic gluten epitopes identified to date derive from monomeric gliadins, immunotoxic sequences are also present in glutenins. Accordingly, the aggregation of these proteins is likely to contribute to disease by protectively shuttling immunotoxic epitopes through the alimentary tract until their eventual release (34).

The proteolytic resistance of gluten proteins may be further enhanced by their assembly into insoluble aggregates, a property imparted by their primary sequence. Wheat gluten comprises two protein groups, the monomeric gliadins, and the low and high molecular weight (LMW and HMW) glutenins (34).

Both gliadins and glutenins contain intrachain disulfide bonds and exhibit poor aqueous solubility, both of which are likely to reduce their proteolytic susceptibility in the gut. In contrast to gliadins, however, glutenins are also extensively cross-linked by interchain disulfide bonds, resulting in the formation of 500 kDa to10 MDa aggregated protein complexes. These huge gluten networks are further stabilized by hydrogen bonding between the glutamine-rich hexapeptide and nonapeptide repeats that compose ~80% of each ~100 kDa HMW glutenin subunit. Glutamine-rich repeats, such as those present in gluten, are predictive of aggregation propensity, and have been used to identify novel prion-forming proteins (34).

Interestingly, it is via the action of endogenous gastrointestinal proteases that immunotoxic peptides are released to enact their harmful effects. Insofar as the intact dietary gluten proteins harboring these peptides would be less efficiently transported across the intestinal epithelium to be presented to the immune system than their immunotoxic fragments, it can be said that gastrointestinal proteases facilitate the pathogenesis of celiac sprue. This subversion of the normal process of nutrient digestion toward a pathogenic end bears resemblance to the tactic of host subversion commonly employed by infectious pathogens. A relevant example of this is the trypsin-catalyzed cleavage of rotavirus protein VP4 into fragments VP5 and VP8, the latter of which disrupts the barrier function of the epithelium, facilitating viral entry (34).


The highly immunogenic (in celiac disease) 33-mer peptide of α2-gliadin (56–88) is fairly resistant to digestion by intestinal brush border enzymes and likely reaches the lamina propria where T cell activation occurs (in celiac disease) (35).

It has been demonstrated that a 33-mer in a particular alpha-gliadin is resistant to enzymatic hydrolysis (36).

Shan L, Molberg O, Parrot I, Hausch F, Filiz F, et al. (2002) Structural basis for gluten intolerance in celiac sprue. Science 297: 2275–2279.



SKOVBJERG 2004 (36):

In the small intestine, TG2 (tissue transglutaminase) is mainly localized extracellularly in lamina propria and shows increased levels in patients with CD (celiac disease) (36).

CICCOCIOPPO 2003 (37):

Bruce et al. found an increased mucosal activity of tTG in untreated and treated CD patients compared to healthy controls (37).

Bruce SE, Bjarnason I, Peters TJ. Human jejunal transglutaminase: demonstration of activity, enzyme kinetics and substrate specificity with special relation to gliadin and coeliac disease. Clin Sci 1985; 68:573–9.

Signicantly increased level of tTG in untreated CD patients compared to treated CD and healthy controls, such as in treated CD compared to healthy controls (37).

The confocal microscopy experiments designed to assess the localization of tTG and gliadin in mucosal samples showed that in the normal mucosa tTG is expressed only in the subepithelial region from cells that resemble the features of myobroblasts. It has been shown that at this level, tTG is closely associated with the β-integrin functioning as a cell surface adhesion molecule independently from its catalytic activity (37).

Akimov SS, Krylov D, Fleischmann LF et al. Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin. J Cell Biol 2000; 148:825–38.

However, upon mechanical stress, inammation, infection, or during apoptosis, the enzyme is secreted into the extracellular matrix and expresses its catalytic activities crosslinking several proteins, then giving these molecules greater resistance to degradation (37).

Johnson TS, Skill NJ, Meguid El Nahas A et al. Transglutaminase transcription and antigen translocation in experimental renal scarring. J Am Soc Nephrol 1999; 10:2146–57.


In the normal mucosa, tTG positivity was found in close connection to a population of cells situated just beneath the epithelium either in the crypts or in the villi (37).

In untreated coeliac patients the labelling intensity was increased and appears to be like clods at the level of the extracellular matrix, and the epithelium becomes slightly positive at the surface level (37)

The intestine of treated coeliac patients showed at these localizations, a labelling pattern between that of the normal and the active diseased samples (37).

In active CD we observed either an increased labelling intensity of tTG at the level of subepithelial region with a pattern similar to clods that probably reects an extracellular localization, or the presence of positivity in the epithelium that was absent in treated CD and normal mucosa (37).


Our ndings revealed that in CD mucosa of both untreated and treated patients, the levels of tTG are increased in comparison to healthy mucosa, supporting a previous study in which the activity of this enzyme has been found raised in the same conditions (37).

Bruce SE, Bjarnason I, Peters TJ. Human jejunal transglutaminase: demonstration of activity, enzyme kinetics and substrate specificity with special relation to gliadin and coeliac disease. Clin Sci 1985; 68:573–9.


As concern gliadin localization, we observed that in untreated coeliac mucosa there is a strong uorescence both in the epithelium and in the submucosa, whereas in the normal mucosa the labelling is weaker and totally absent in the extracellular matrix at the crypt level (37).

In the biopsied controls we observed the positivity within enterocytes either in the crypts or in the villi, other than in the lamina propria of the villi (37).

In untreated CD the enterocyte staining was highly evident and lamina propria resulted positive either at the crypt or surface level (37).

In treated CD we found only a weak positivity of the brush border of the enterocyte present at the apex of the villi and few spots in the lamina propria of the crypts (37).


These observations allow us to conclude that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, whereas the colocalization is observed only in the lamina propria of the villi in normal mucosa (37).

Therefore, a colocalization of gliadin with tTG is evident in untreated CD patients and healthy controls, even though possible differences in tTG catalytic activity among these groups cannot be excluded (37).

The analysis by confocal microscopy showed that tTG colocalizes with gliadin at the epithelial and subepithelial levels in active CD, and only in the lamina propria of the villi in normal mucosa (37).

Coimmunoprecipitated gliadin (with tTG) result increased in active CD compared with normal mucosa (37)


The increased positivity observed in the submucosa of both untreated and treated CD, together with the immunoprecipitation results, could reect either a clonal proliferation of myobroblasts or the induction of tTG synthesis and secretion by the action of cytokines. The ndings that tTG is regulated by TNF-α  and IL-6, and the demonstration by Nilsen et al. that these two cytokines are up-regulated in the CD mucosa, argues in favour of the latter hypothesis (37).

25 Kuncio GS, Tsyganskaya M, Zhu J et al. TNF-alpha mediates expression of the tissue transglutaminase gene in liver cells. Am J Physiol 1998; 274:G240–5.

26 Ikura K, Shinigawa R, Suto N et al. Increase caused by interleukin-6 in promoter activity of guinea pig liver transglutaminase gene. Biosci Biotechnol Biochem 1994; 58:1540–1.

DIETERICH 2006 (35)


It remains unclear how far an altered expression and distribution pattern of tTG or rather the known increased tTG activity in the duodenal mucosa of patients with coeliac disease, contributes to coeliac disease pathogenesis (35).

Intrinsic tTG colocalises non-covalently with fibronectin in the lamina muscularis mucosae (35).

However, our data proved that excess tTG, as secreted in coeliac disease, will change the binding behaviour and favour association of tTG and tTG-gliadin complexes with collagens, especially in the lamina propria (35).

The resulting increased tTG activity followed by an increase in tTG modified gliadins, gliadin-collagen, or gliadin-tTG complexes in the coeliac lamina propria could favour progression and chronicity of coeliac disease (35).


An immunohistochemical study demonstrated that gliadin colocalised with tTG in the duodenal mucosa of untreated coeliac patients and controls. This colocalisation was mainly found in the epithelial and subepithelial areas in active coeliac disease but was restricted to the lamina propria in controls (35).

Ciccocioppo R, Di Sabatino A, Ara C, et al. Gliadin and tissue transglutaminase complexes in normal and coeliac duodenal mucosa. Clin Exp Immunol 2003;134:516–24.



BETHUNE 2008 (34):

The same proline/ glutamine-rich sequences that render gluten peptides resistant to gastrointestinal proteolysis also make them excellent substrates for TG2 (tissue transglutaminase) (34).

Fleckenstein B, Molberg O, Qiao SW, Schmid DG, von der Mulbe F, et al. (2002) Gliadin T cell epitope selection by tissue transglutaminase in celiac disease. Role of enzyme specificity and pH influence on the transamidation versus deamidation process. J Biol Chem 277: 34109– 34116.

Vader LW, de Ru A, van der Wal Y, Kooy YM, Benckhuijsen W, et al. (2002) Specificity of tissue transglutaminase explains cereal toxicity in celiac disease. J Exp Med 195: 643–649.

TG2 is found in the lamina propria and the brush border of enterocytes (34).

Peptides typied by the 33-mer are excellent substrates for TG2 (34).

DIETERICH 2006 (35):

Confirmation of isolated gliadin fractions as substrates for tTG: using a quantitative assay which is based on tTG catalysed incorporation of monodansyl cadaverine into gliadins followed by measurement of enhanced fluorescence intensity, all of the tested gliadins (α1–11, γ1–6, and ω1, 2, 3, and 5) were found to be good substrates for tTG, showing an increase in fluorescence reaching 50–100% of values obtained with crude gliadin as substrate (data not shown). No incorporation of monodansyl cadaverine was detected with the control substrates bovine serum albumin and lactalbumin (35).

tTG activity was analysed by incorporation of monodansyl cadaverine into gliadins. Fluorescence labelled tTG reactive short gliadin peptides were used to demonstrate their deamidation and explore their cross linking patterns with tTG itself or extracellular matrix proteins. Patient sera and controls were checked for autoantibodies to matrix proteins (35).

All tested gliadins were substrates for tTG. Gliadins α1–α11, γ1–γ6, w1–w3, and w5 were substrates for tTG (35).

The tTG catalysed modifications were not restricted to single gliadin types and epitopes (35).

The glutamine-rich gliadins are excellent donor substrates for the otherwise highly substrate specific tTG (35).

Larre C, Chiarello M, Blanloeil Y, et al. Gliadin modifications catalyzed by guinea pig liver transglutaminase. J Food Biochem 1993;17:267–82.

The amino acid composition around glutamine residues was shown to be critical for the reactivity of tTG with the gliadin peptides (35).

To date, the catalytic activity of tTG for the different gliadins has not been compared. Here we demonstrate that all investigated gliadins are good substrates for tTG (35).

CICCOCIOPPO 2003 (37):

Interestingly enough, Bruce et al. found that gliadin represented a preferential substrate for the enzyme tissue transglutaminase (37).

Bruce SE, Bjarnason I, Peters TJ. Human jejunal transglutaminase: demonstration of activity, enzyme kinetics and substrate specificity with special relation to gliadin and coeliac disease. Clin Sci 1985; 68:573–9.

Growing evidences indicate that gliadin would represent a highly preferred substrate because of its high content of glutamyl residues (37).

SCHUPPAN 2002 (38):

Gliadins are glutamine- and proline-rich proteins of wheat gluten that constitute excellent glutamyl donors for tTG-catalysed crosslinking reactions, giving rise to gliadin- gliadin complexes and even the covalent incorporation of tTG into high molecular-weight aggregates (38).

Dietrich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997;3:797-801.



tTG catalysed modifications are not restricted to single gliadins or gliadin epitopes as a wide panel of gliadins can be modified by tTG. This finding verifies prior reports that applies search algorithms to identify preferred tTG substrates and that yielded matches in the gluten (wheat), hordein (rye), and secalin (barley) databases (35).

Vader WL, de Ru A, van der Wal Y, et al. Specificity of tissue transglutaminase explains cereal toxicity in coeliac disease. J Exp Med 2002;195:643–9.


Substrate specificity of gliadin peptides for tTG:

Site directed deamidation of gliadin peptides by tTG depends on the primary structure of the peptides and some consensus sequences have been identified. Whereas peptides containing the sequence motifs QXP or QXX followed by the hydrophobic residues F, Y, M, W, L, I, or V (with X for any amino acid) are preferred substrates for tTG, the sequences QP or QXXP are not substrates of the enzyme (35).

Vader WL, de Ru A, van der Wal Y, et al. Specificity of tissue transglutaminase explains cereal toxicity in coeliac disease. J Exp Med 2002;195:643–9.






The high selectivity of tTG for glutamine donor substrates is well known and the gliadin derived sequence PQPQLPY was described as high affinity substrate for tTG (35).

Piper JL, Gray GM, Khosla Ch. High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: Implications for coeliac sprue. Biochemistry 2002;41:386–93.

Here we used an alternative approach to verify this consensus sequence in the gliadin peptides α2(56–68) and α2(58–88) as the only recognition site for tTG (35).

Using a fluorometric tTG activity assay, the absolute requirement of Q65 as reactive glutamine in the gliadin peptide α2(56–68; PQLQPFPQPQLPY) was confirmed (35).

This 13 amino acid peptide α2(56–68): LQLQPFPQPQLPY and its deamidated variants E59 and E63 α2(56–68, E59): LQLEPFPQPQLPY; α2(56–68, E63): LQLQPFPEPQLPY; were good substrates for tTG, while substitution of Q65 by E65, α2(56–68, E65): LQLQPFPQPELPY resulted in complete loss of reactivity with tTG, therefore confirming that modification of peptide α2(56–68) by tTG is strictly restricted to Q65 (35).

tTG specifically deamidates glutamine 65 in the immunodominant gliadin peptide α2(56–68) that contains the core sequence PQPQLPY generating PQPELPY (35).

Piper JL, Gray GM, Khosla Ch. High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: Implications for coeliac sprue. Biochemistry 2002;41:386–93.

Below: site specific deamidation of gliadin peptides prevents tissue transglutaminase (tTG) catalysed incorporation of monodansyl cadaverine (MDC). Incorporation of MDC into gliadin peptides is dependent on specific glutamine residues in gliadins and detected by increased fluorescence intensity. (A) Only deamidation of the substrate residue Q65 in the gliadin peptide α2(56–68) resulted in failure of MDC incorporation (35):

Three copies of the preferred recognition motif are found in the protease resistant 31-mer α2(58–88; LQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF) (35).

The multiple presence of this motif suggested that the sole deamidation of Q65 to E65 could not abolish reactivity of this larger peptide with tTG. Accordingly, only replacement of Q65, Q72, and Q79 by glutamic acid in the extended peptide α2(58–88, E65, E72, E79): LQPFPQPELPYPQPELPYPQPELPYPQPQPF; abolished reactivity with tTG, confirming the PQPQLPY motif as the sole tTG recognition sequence in these gliadin peptides (35).

Below: site specific deamidation of gliadin peptides prevents tissue transglutaminase (tTG) catalysed incorporation of monodansyl cadaverine (MDC). Incorporation of MDC into gliadin peptides is dependent on specific glutamine residues in gliadins and detected by increased fluorescence intensity. (B) Deamidation of the three tTG substrate residues Q65, Q72, and Q79 prevented tTG catalysed modification of the protease resistant gliadin peptide α2(56–88) which contains three of the immunodominant substrate sequences (35):



α-gliadin(31–43) LGQQQPFPPQQPY


There are peptides that are described as “toxic”, since they alter the mucosa of celiac patients and induce villous atrophy. A very prominent example  for this is the “toxic” epitope within the sequence 31-49 of the  α-gliadin (LGQQQPFPPQQPYPQPQPF), which is also used in the truncated form 31-43 (Reinke 2009)

Sturgess et al (1994) were among the first to clearly show that a single gliadin peptide could elicit toxicity in vivo (Loponen 2006).

They discovered that the ingestion of an α-gliadin 19-mer polypeptide, consisting of 19 amino acids α-gliadin(31–49) caused atrophy of the villi of celiac patients (Loponen 2006).

A year later, Marsh et al (1995) showed that a 13-residue α-gliadin 13-mer polypeptide peptide of the same protein, but lacking the last six amino acids of the 19-mer, was toxic as well (Loponen 2006).

Kasarda (1997) searched databases to explore for potential celiac-toxic peptides and, based on its presence in wheat, barley, and rye (but not in oats), highlighted the possible importance of the hexapeptide QQQPFP in causing CD (Loponen 2006).


MAMONE 2004:

Identifcation of gliadin Q residues susceptible to crosslinking or to deamidation by tTG is a challenging task (Mamone 2004).

For the first time in a wheat flour, this procedure allowed us to identify, as tTG substrates in vitro, a few gliadin peptides having a specific amino acid composition amongst a myriad of other peptides (Mamone 2004).

In addition, because gliadin proteins are rich in Q and poor in K residues, in the absence of MDC, tTG may act as an acyl acceptor and form complexes with gliadin peptides. Assays to detect gliadin peptide-tTG complexes were unsuccessful owing to the high heterogeneity of the reaction mixture and the very high molecular weight of the protein-peptide

complexes. This heterogeneity would be resolved if, instead of a whole gliadin PT digest (mimicking in vivo digestion), single synthetic peptides amongst those identified were incubated witht TG. To this end, we developed polyclonal antibodies against the 19-mer peptide α /β-gliadin 31-49 (peaks 4 and 9), known to be toxic for CD patients, as a putative substrate in forming peptide-tTG complexes. The choice of this peptide was determined also based on its high Q residue content; that is, a total of seven, with two belonging to the tTG-preferred consensus sequence Q-X-P. SDS-polyacrylamide gel electrophoresis (PAGE) analysis carried out on the reaction mixture containing the gliadin peptide and tTG in the presence of calcium at pH 7.6 allowed us to detect a band of ~80 kDa recognized by the antibodies raised against the synthetic peptide. There was no reactivity in the corresponding tTG positive control (lane 2) with reference to tTG migration. Notably, this clearly indicated that the 19-mer peptide formed relatively stable and

covalent aggregates with tTG in the absence of other binding molecules (Mamone 2004).

Below: Identification by nES-MS/MS of MDC-tagged Q residue gliadin peptides isolated from the fluorescent PT digest by HPLC fractionation. Q* indicates transamidated Q residues. Amino acid residues are indicated with a single letter nomenclature. The peptide chain found under the indicated accession number in the NCBI Data Bank archive is in lower line (Mamone 2004):

DORUM 2010:

The α-gliadin derived peptides #7–9 did not harbor any known T-cell epitopes. Peptide #7 is similar to the p31–44 ‘‘toxic’’ peptide previously reported except a L to P substitution at the N-terminus (Dorum 2010).

The sequences of the remaining peptides (#7, #28, #29, and #31) do not contain obvious binding motifs of HLA-DQ2.5 or HLA-DQ8, and they are thus unlikely to be efficient stimulators of T cells from celiac disease patients (Dorum 2010).

Below : TG2 peptide substrates identified by nano-LC MS/MS. Glutamine residues targete d by TG2 are given in bold. bnot possible to determine which Q residue is transamidated (shown in bold and italic) (Dorum 2010):

MAZZEO 2003:

Below: Identification of tTGase-modified peptides by MALDI-MS and definition of the deamidated glutamine residues present in the recombinant α-gliadin by nanoESI-MS/MS experiments. The deamidation sites are indicated with an asterisk (Mazzeo 2003):

Below: Identification of deamidation sites in the 26 synthetic peptides overlapping the entire sequence of the recombinant α-gliadin by nanoESI-MS/MS experiments. It was not possible to define which of the underlined Q residues is deamidated in peptide 20–39 (Mazzeo 2003):



BETHUNE 2008 (34):


Following TG2-mediated deamidation at select Gln residues (in gluten peptides), these peptides bind with increased affinity to disease-associated HLA DQ2 molecules, and thereby possess increased stimulatory capacity toward DQ2-restricted gluten-reactive T cells. Much of the damage that occurs in celiac sprue is mediated by this disease-specific T cell response. Thus, the deamidation of immunotoxic gluten peptides by endogenous TG2 constitutes the second point at which normal cellular processes are subverted toward a pathogenic end in celiac sprue (34).

Although TG2 is found in the lamina propria and the brush border of enterocytes, the precise location and context in which TG2 encounters and deamidates gluten peptides is not yet known (34).


Notably, the transamidase activity of TG2 is also implicated in celiac sprue. During active disease, celiac patients have circulating antibodies not only against gluten epitopes, but also against TG2 (34)..

Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, et al. (1997) Identication of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 3: 797–801.

TG2 forms covalent complexes with gluten peptides (34).

Fleckenstein B, Qiao SW, Larsen MR, Jung G, Roepstorff P, et al. (2004) Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J Biol Chem 279: 17607–17616.

Since TG2 forms covalent complexes with gluten peptides it has been proposed that intestinal gluten-reactive T cells can provide co-stimulation to B cells expressing TG2-specic antibodies as part of an autoimmune humoral response (34).

Sollid LM, Molberg O, McAdam S, Lundin KE (1997) Autoantibodies in coeliac disease: tissue transglutaminase–guilt by association? Gut 41: 851– 852

Upon treatment with a gluten-free diet, anti-TG2 autoantibody levels decline. Whether anti-TG2 autoantibodies play a role in disease pathogenesis or are simply bystanders is not yet clear. However, these highly disease-specic antibodies do serve an important role in serological screening for celiac sprue (34).



pH dependency of tTG catalysed cross linking:

A low pH favours tTG catalysed deamidation of donor glutamines instead of its incorporation into an isopeptid bond (35).

As low pH values can prevail in inflamed intestinal mucosa tTG catalysed modification of α2(56–68) was checked with pH values adjusted to 6.0, 6.5, 7.0, and 7.5. Spontaneous deamidation of the gliadin peptide was excluded (data not shown). Fluorescence labelled peptide *α2(56–68) was deamidated and cross linked by tTG. Increasing pH values caused a continuous increase in incorporation of the peptide into tTG which was paralleled by the creation of high molecular weight complexes formed by tTG-gliadin multimers. As cross linking also occurred at pH values as low as 6.0, tTG-gliadin complexes should also be formed in inflamed intestine (35).

We observed this incorporation and complex formation at lower pH values also, conditions which were formerly described to favour deamidation over cross linking (35).

Fleckenstein B, Molberg Ø, Qiao SW, et al. Gliadin T cell epitope selection by tissue transglutaminase in coeliac disease. J Biol Chem 2002;277:34109–16.

Therefore, an environment with a pH as low as pH 6 which can prevail in inflammation still leads to tTG catalysed gliadin cross linking (35).


Below: effect of pH on cross linking of gliadin to tissue transglutaminase (tTG). The formation of cross links between tTG and fluorescence labelled peptide *α2(56–68) was investigated after separation by 18% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Increasing pH enhanced cross linking of peptide *α2(56–68) to tTG. The tTG monomer and oligo/multimers with the gliadin peptide attached are visualised as high molecular weight complexes. The two lanes on the left show the *α2(56–68) peptide and its deamidated variant which has a higher mobility than the nondeamidated parent peptide (35):

SKOVBJERG 2004 (36):

Determination of deamidation/transamidation at various lysine concentrations: the relation between deamidation and transamidation was measured by quantifying the amount of released ammonia in the absence (deamidation) or presence (transamidation) of lysine (36).

Solpro 300 (proteolytically digested gluten) was incubated (37 ºC, 60 min) with GPLT in the presence of various lysine concentrations in pH 6.5, 0.5 mM CaCl2. Ammonia released was measured (36).

The relation between deamidation and transamidation was studied in a pure system with gliadin as acyl donor in the presence of either H2O or varying concentrations of lysine, assuming that an increased catalytic rate (measured as production of ammonia) by increasing concentrations of a primary amine is due to transamidation. By this approach, we found a ratio between deamidation and transamidation of 1:4 for TG2 at optimal lysine concentrations. The finding that significant deamidation of PepQ was registered even at high concentrations of lysine strengthens the suggestion that deamidation actually takes place in the intestinal environments, under conditions where the concentration of primary amines, especially peptide-bound lysine, is expected to be high (36).

Interestingly, the studied immunodominant epitope occurs in an oligomerised non-digestible form having more than one deamidation/transamidation site, thus potentially allowing simultaneous cross-linking and deamidation by transglutaminase (36).


The transamidation reaction has been demonstrated to increase with increasing pH (36).

CICCOCIOPPO 2003 (37):


In slightly acidic environment, as occurs in chronic inflammation, tTG-induced gliadin peptide polymerization (transamidation) decreases, whereas deamidation increases (37).

Fleckenstein B, Molberg Ø, Qiao SW et al. Gliadin T cell epitope selection by tissue transglutaminase in celiac disease: role of enzyme specificity and pH influence on the transamidation versus deamidation reactions. J Biol Chem 2002; 277:34109–16.

tTG is best known for its ability to catalyse transamidation reactions in a multistep process, however, at low pH or when no acceptor proteins are available, deamidation of glutamines is favoured over their incorporation into isopeptide bonds (37).

Aeschlimann D. Transglutaminase: Protein cross-linking enzymes in tissues and body fluids. In: Maki M, Tossavaine M, eds. Proceedings of the Workshop on Transglutaminase, Protein Cross-Linking and Coeliac Disease, Tampere, Finland: University of Tampere, 2001:15–22.



CICCOCIOPPO 2003 (37):

Because the formation of tTG-gliadin complexes within the gut mucosal wall, although plays a central role in the pathogenesis of CD, has never been demonstrated, in the present study, we aimed to explore the putative presence of gliadin and tTG complexes in the duodenal mucosa of coeliac patients in comparison to healthy controls (37).

The action of tTG in duodenal mucosa leads to the formation of covalently crosslinked supramolecular structures which, in the subjects following a normal diet, comprise gliadin molecules. The covalent bonds that give rise to these complexes could be dissolved by using strong denaturating and reducing conditions (37).

The puried anti-tTG fraction from sera samples of untreated CD patients were able to recognize and coimmunoprecipitate detectable levels of tTG and gliadin in duodenal mucosa of both active CD and control conditions (37).

To date, it has not been demonstrated whether gliadin is cross-linked to tTG within the gut wall, no direct evidence is available that gliadin is cross-linked to tTG directly in the gut wall, a phenomenon known to occur in vitro (37).

Dieterich W, Ehnis T, Bauer M et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997; 3 :797–801.

Molberg Ø, McAdam SN, Körner R et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat Med 1998; 4


We therefore investigated the putative presence of tTG and gliadin complexes directly in duodenal mucosa (37).

Our study rstly indicated that tTG and gliadin are bound to form supramolecular complexes directly in the duodenal mucosa of both untreated CD patients and biopsied controls (37).

Our ndings rstly demonstrated that gliadin was directly bound to tTG in duodenal mucosa of coeliacs and controls (37).

Gliadin was bound to tTG. A covalent bond links the molecules (37).

tTG is prevalently engaged with gliadin in the formation of supramolecular complexes in the subjects following a gluten-containing diet, as well as with other molecules in the subjects following a gluten-free diet (37).

Taken together, our results suggest that tTG and gliadin form supramolecular complexes directly in normal and diseased duodenal mucosa and that in active CD the levels of both molecules are increased (37).

On the other hand, we cannot exclude the possibility that the tTG coimmunoprecipitates found in our study might include other substrates such as peptides of the extracellular matrix, or some viral, bacterial or other nutritional proteins, whose identity needs further investigations (37).

Schuppan D, Hahn E. Celiac disease and its link to type 1 diabetes mellitus. J Pediatr Endocrinol Metab 2001; 14:597–605.

Finally, our ndings prompt us to hypothesize that, since gliadin is bound to tTG in biopsied controls too, this event is necessary, but not sufcient for the development of enteropathy (37).


Mucosal biopsies obtained from (human) untreated, treated CD patients and biopsied controls. Multiple size-appropriate endoscopic biopsy specimens of the second part of the duodenum were obtained from 13 untreated coeliac patients, 13 coeliac patients on a gluten-free diet for at least 12 months and 13 consenting subjects undergoing upper gastrointestinal endoscopy for functional dyspepsia (37)

SKOVBJERG 2004 (36):

In this paper, it is demonstrated that TG2, when present in the intestinal mucosa, can deamidate an immunodominant peptide epitope. In the presence of primary amines, this reaction is inhibited, although not completely, and cross- linking to intestinal proteins seems to dominate (36).

Incubation of BSA and chicken egg albumin with epitope peptide and active transglutaminase clearly demonstrated that the epitope peptide bound to these proteins, as the binding profile in both cases, follows the protein profile (36).

The cross-linking (between gliadin epitope and transglutaminase) occurs, as demonstrated in this paper and recently by others (Fleckenstein et al 2004), also outside the active site of TG2 (36).

B. Fleckenstein, S.W. Qiao, M.R. Larsen, G. Jung, P. Roepstorff, L.M. Sollid, Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides, J. Biol. Chem. 279 (2004) 17607–17616.

Cross-linking is therefore expected to occur to all proteins having epsilon-amino groups that are reactive with the gliadin epitope (36).

That this really is the case is demonstrated in this paper by its binding to BSA and chicken egg albumin (36).


Synthetic epitope peptides: synthetic alpha-gliadin peptides:

- Synthetic peptide Gliadin 33-mer fragment PepQ =α2(59–72)=QPFPQPQLPYPQPQ

- Synthetic peptide Gliadin 33-mer fragment PepQ in a deamidated (QPFPQPELPYPQPQ- amid) (PepE).

Transglutaminase: Guinea pig liver transglutaminase (GPLT) (36).

Small intestinal mucosa: pigs were anaesthetised, the middle part of duodenum was removed, and the mucosal layer stripped from the underlying layers (36).

Our aim was to study how the two main catalytic activities of transglutaminase (deamidation and transamidation (cross-linking) of an immunodominant gliadin epitope) are influenced by the presence of acceptor amines in the intestinal mucosa (36).



Using synthetic peptides containing the immunodominant gliadin epitope together with monoclonal antibodies specific for the deamidated form of the epitope or for both forms, we characterised both the deamidation and cross-linking to intestinal proteins (36).

We analyse deamidation and cross-linking of these peptides to proteins. Our results show that QPFPQPQLPYPQPQ-amide was deamidated when incubated with purified TG2, with fresh mucosal sheets and with mucosal homogenates. A fraction of the non-deamidated epitope was cross-linked to proteins, including TG2. The results suggest that intestinal TG2 is responsible for generation of the active deamidated epitope. As the epitope often occurs in a repeat structure, the result may be cross-linking of a deamidated, i.e., activated cell epitope (36).

We demonstrated that the epitope was deamidated in an environment similar to the intestinal wall, albeit at a rather low rate. The lower rate was explained by concomitant binding to a multitude of proteins (36).

Cross-linking to mucosal proteins: PepQ was incubated with mucosal homogenate (from pig intestine) at pH 7.3 with 5 mM CaCl2 with or without GPLT (36).

Cross-linking to chicken egg albumin and BSA: PepQ was incubated with GPLT and chicken egg albumin or BSA in 5 mM CaCl2 pH 7.3 (36).

Cross-linking to purified TG2: PepQ was incubated (37 ºC, 60 min) with GPLT at pH 7.3, 5 mM CaCl2. In order to investigate whether other sites besides the active site of TG2 were involved in cross-linking, high concentrations of heat-inactivated GPLT were added to some of the incubations. In these cases we could observe a high molecular weight precipitates.

Transglutaminase assay: The transamidating activity of TG2 in the mucosal homogenate was quantified (36).

Relation between deamidation and cross-linking:

Purified transglutaminases: to study further the relation between deamidation and transamidation with respect to purified transglutaminases, proteolytically digested gluten was incubated with or without various concentrations of lysine. The reaction rate in this experiment was measured by the rate of liberation of ammonia. The results demonstrate that the maximal transamidation rate for TG2 was about four times higher than the deamidation rate (in the absence of lysine) (36).

Cross-linking of PepQ to mucosal proteins:

PepQ  was incubated with mucosal homogenate and GPLT:

Mucosal homogenate: to further characterise the influence of primary amines in the mucosa, we studied the effect of a mucosal homogenate on the deamidation activity of GPLT. PepQ incubated with mucosal homogenate. The result strongly supports the suggestion that the presence of primary amines in the mucosal homogenate inhibits deamidation (36).

We suggest that the inhibition of deamidation by mucosal proteins is due to cross-linking by TG2 (36).

To further study the nature of this cross-linking, a series of experiments was performed. Incubation of PepQ with a mucosal homogenate and GPLT. When the elution fractions were analysed the result fits the assumption that the critical glutamine residue was involved in cross-linking. The possible influence of low molecular weight primary amines was studied by dialysis experiments (below). When the homogenate was dialysed before incubation with PepQ, the fluorescence counts were slightly decreased, indicating that some of the registered high molecular weight compounds were due to the involvement of monoamines and diamines removed during dialysis (36).

Cross-linking of PepQ to chicken egg albumin and BSA:

PepQ was incubated with chicken egg albumin or BSA in the presence of GPLT

Purified proteins:

Chicken egg albumin and BSA: to further study the extent of cross-linking of PepQ to proteins, several purified proteins were studied (36).

The results for chicken egg albumin and BSA clearly show that, in the presence of GPLT, PepQ was cross-linked to these proteins. The high molecular weight peaks seen both in the presence of chicken egg albumin and BSA were only present after incubation with GPLT, and represent the polymerised protein which has also incorporated the epitope peptide (36).


TG2: It has earlier been reported that gliadin peptides bind to TG2 (36).

B. Fleckenstein, S.W. Qiao, M.R. Larsen, G. Jung, P. Roepstorff, L.M. Sollid, Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides, J. Biol. Chem. 279 (2004) 17607–17616.

J.L. Piper, G.M. Gray, C. Khosla, High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: implications for celiac sprue, Biochemistry 41 (2002) 386–393.

R. Ciccocioppo, A. Di Sabatino, C. Ara, F. Biagi, M. Perilli, G. Amicosante, M.G. Cifone, G.R. Corazza, Gliadin and tissue trans- glutaminase complexes in normal and coeliac duodenal mucosa, Clin. Exp. Immunol. 134 (2003) 516 –524.

Corresponding experiments with TG2 show that the epitope peptide is cross-linked to GPLT independent of a native active site conformation, and is in accordance with our demonstration of binding also to other proteins and with recent findings on TG2 of Fleckenstein (36).

To further analyse this reaction, we performed a series of experiments similar to those with BSA and chicken egg albumin.

Cross-linking of PepQ to purified TG2. PepQ was incubated with GPLT with or without addition of heat inactivated GPLT:

The epitope peptide was bound to GPLT (inactive and active) as observed for other proteins. In control experiments without addition of high amounts of inactive GPLT, the antibody reactivity was low, demonstrating that most of the registered epitope peptide was cross-linked to the inactivated GPLT (36).

The results provide a basis for the suggestion that binding of a peptide to a protein, in connection to its modification to a T cell epitope, might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens (36).

DIETERICH 2006 (35):

While tTG mediated deamidation of distinct peptides from α- and γ- gliadins was described in detail, no information on the nature of complexes between gliadins and tTG, and between gliadins and extracellular matrix components in the small intestine is available (35).

Gliadin peptides can react with tTG, resulting in gliadin-tTG cross link and complex formation (35).

Furthermore, tTG catalyses the binding of gliadin peptides to interstitial collagen types I, III, and VI, which suggests the generation of complex neoepitopes and long term immobilisation of pathogenic gliadins in the intestinal extracellular matrix (35).

Haptenisation of gliadins to collagen is associated with increased titres of IgA antibodies to collagens in the sera of coeliac patients and may in part explain the occurrence of autoimmune phenomena in coeliac disease (35).

tTG catalysed the cross linking of gliadin peptides with interstitial collagen types I, III, and VI (35).

tTG formed high molecular weight complexes with all tested gliadins (34).

Haptenisation and long term immobilisation of gliadin peptides by tTG catalysed binding to abundant extracellular matrix proteins could be instrumental in the perpetuation of intestinal inflammation and some associated autoimmune diseases in coeliac disease (35).

Our demonstration of tTG mediated gliadin incorporation into collagen types I, III, and VI, which are predominant interstitial matrix components in the lamina propria, supports the concept that due to immobilisation in the ECM of coeliac patients, local availability of gliadins can reach increased concentrations in vivo (35).

No incorporation of the gliadin peptide *α2(56–68) was noted into collagen type V (35).

We observed no cross linking of the gliadin peptide to collagen type V, thus indicating that this collagen does not serve as a gliadin acceptor substrate. None the less, the telopeptide of collagen type V was described as a glutamine donor substrate for tTG,

Kleman JP, Aeschlimann D, Paulsson M, et al. Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204 rhabdomyosarcoma cells. Biochemistry 1995;34:13768–75.

therefore allowing complex formation with tTG itself (35).


Synthetic epitope peptides: synthetic alpha-gliadin peptides:

Synthetic peptide Gliadin 33-mer fragment: Fluorescence labelled peptide *α2(56–68) LQLQPFPQPQLPY


Collagens: types I, II and VI.

Oesophagus sections (primate)

IN VITRO (gliadin, transglutaminase and collagens type I, III and VI):


tTG catalysed cross linking of gliadin peptides with extracellular matrix molecules (35):

Fluorescence labelled peptide *α2(56–68) was used to investigate tTG mediated cross linking of gliadins to ECM proteins. Both α-chains of collagen type I (with the α1(I)2 α2(I) heterotrimer) and the α1-chain of collagen type III (with the α1(III)3 homotrimer) were cross linked with the gliadin peptide. Incorporation of the gliadin peptide into collagen type VI (with the α1(VI), α2(VI), α3(VI) heterotrimer) seemed to be restricted to the α2>α1 chains (35).


Fluorescence labelled gliadin peptide *α2(56–68) was used to show that tTG promotes the autocatalytic incorporation of this peptide into tTG itself. This again occurs via the substrate glutamine residue at position 65, resulting in the formation of high molecular weight complexes containing autocatalytic tTG multimers, and tTG-gliadin peptide cross links (35).

Six lysine residues of tTG were identified as crosslinking sites with gliadin peptides (35).

Fleckenstein B, Qiao SW, Larsen MR, et al. Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides. J Biol Chem 2004;279:17607–16.

Tissue transglutaminase (tTG) mediated binding of gliadin peptide *α2(56–68) to extracellular matrix molecules. Only α-chains of collagen types I, III, and VI and tTG itself served as gliadin acceptor substrates. There is no spontaneous binding of gliadin to collagen type I in the absence of tTG (35):

EX-VIVO (gliadin, transglutaminase and primate oesophagus sections):

tTG catalysed colocalisation of gliadin peptides with extracellular matrix molecules in tissues (35):


Immunostaining for tTG (fig E) was superimposable on the pattern obtained after incubation of the sections with the *α2(56–68) gliadin peptide, yielding the characteristic honeycomb endomysial pattern with main reactivity in the lamina muscularis mucosae (fig D). Colocalisation of the gliadin peptide with tTG, and the finding that staining with the gliadin peptide was calcium dependent and absent in EDTA containing buffer (data not shown), clearly demonstrates that extracellular tTG can bind gliadin to the matrix. The almost exclusive staining of the lamina muscularis mucosae for tTG (fig E) and fibronectin (fig A) and not for the major interstitial collagen types I and III (fig B), is in line with the reported non-covalent association of tTG with these fibronectin fibres (35).

Below: Localisation of extracellular matrix proteins and gliadin in monkey oesophageal sections by immunofluorescence staining and labelled polyclonal antibodies against transglutaminase and gliadin: Incubation of the sections with the fluorescence labelled gliadin peptide *α2(56–68) (D) or with a TRITC labelled monoclonal antibody against tissue transglutaminase (tTG) (E) demonstrates the honeycomb staining pattern of the lamina muscularis mucosa, characteristic of endomysial autoantibodies in coeliac disease. LMM, lamina muscularis mucosae; LP, lamina propria; TM, tunica muscularis; TS, tela submucosae (35):


In contrast, preincubation of the tissue sections with additional tTG showed a completely different pattern with strong staining of the lamina propria mucosae, thus indicating that an excess of tTG, as is secreted in the coeliac mucosae, will favour binding of tTG to regions with high content of collagens (fig 4F). Accordingly, concurrent incubation of the sections with the *α2(56–68) gliadin peptide and increasing amounts of human recombinant tTG (0.5–2.0 mg/section; fig 4G and H) yielded a diffuse staining pattern with emerging fluorescence in the lamina propria mucosae. This was caused by the gliadin peptide being linked to collagens by extrinsic tTG, in addition to the lamina muscularis mucosae, due to the gliadin peptide being linked to intrinsic tTG by autocatalysis (35).

Below: Localisation of extracellular matrix proteins and gliadin in monkey oesophageal sections by immunofluorescence staining and labelled polyclonal antibodies against transglutaminase and gliadin: (G, H) Addition of *α2(56–68) together with tTG (0.5 or 2.0 μg/section for (G) and (H), respectively) produced a diffuse staining pattern encompassing the lamina muscularis mucosae and the lamina propria mucosae. LMM, lamina muscularis mucosae; LP, lamina propria; TM, tunica muscularis; TS, tela submucosae. (35):



Initiation of deleterious immune response:

The ultimate dening characteristic of pathogens is that they contribute to disease (34).

It has been suggested that pathogens can be classied according to the damage their presence inicts on a host relative to the strength of the host’s immune response (34).

Casadevall A, Pirofski LA (1999) Host-pathogen interactions: redening the basic concepts of virulence and pathogenicity. Infect Immun 67: 3703– 3713.


Those microorganisms classically termed opportunistic cause disease only in the context of compromised immunity. Diseases caused by toxin-producing pathogens comprise damage mediated both by the pathogen and by the host’s immune response, the contributions of each depending on the potency of the toxins produced, as well as on the pathogen’s ability to avoid provoking a strong immune response. At the far end of this continuum, pathogens that produce no toxins of their own precipitate disease in the context of a strong, host-damaging inammatory response. Gluten peptides are examples of this last category (34).


Celiac sprue is a chronic inammatory disease. In infectious disease, chronic inammation occurs when a pathogen continually evades an active immune response, for instance by resisting phagocytic engulfment or by aggressin-mediated killing of macrophages. This inammation persists, resulting in signicant tissue damage, until the colonizing pathogen is cleared (34).


The mechanisms by which gluten peptides precipitate inammation in the celiac gut are only recently becoming clear. Over the past decade, we have begun to appreciate that celiac pathogenesis involves a complex interplay between adaptive and innate responses, each of which is mediated by a distinct class of immunotoxic gluten peptides (34).

Londei M, Ciacci C, Ricciardelli I, Vacca L, Quaratino S, et al. (2005) Gliadin as a stimulator of innate responses in celiac disease. Mol Immunol 42: 913–918.

Jabri B, Kasarda DD, Green PH (2005) Innate and adaptive immunity: the yin and yang of celiac disease. Immunol Rev 206: 219–231.

Gianfrani C, Auricchio S, Troncone R (2005) Adaptive and innate immune responses in celiac disease. Immunol Lett 99: 141–145.



The rst of these classes provokes the T cell–mediated adaptive response. These immunogenic peptides, typied by the 33-mer, are excellent substrates for TG2, and, once deamidated, are potent activators of gluten-specic, DQ2-restricted CD4+ T cells in the lamina propria (34).

Activated CD4+ T cells enact a Th1 response, secreting IFN-γ and other proinammatory cytokines, as well as give help to the B cell–mediated humoral response against both gluten and TG2 (34).


Due to the remarkable concordance between the role that TG2 plays in increasing these immunogenic peptides’ afnity for DQ2, the identity of TG2 as the target of the autoantibody response, and the strong genetic association of DQ2 with disease, research into celiac pathogenesis has largely focused on the adaptive branch of the immune response (34).

However, the gluten-specic adaptive immune response is thought to be insufcient on its own to explain why CD4+ lamina propria T cells trigger an inammatory Th1 response (34)..

91. Nilsen EM, Lundin KE, Krajci P, Scott H, Sollid LM, et al. (1995) Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon gamma. Gut 37: 766–776.

92. Nilsen EM, Jahnsen FL, Lundin KE, Johansen FE, Fausa O, et al. (1998) Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology 115: 551–563.

93. Troncone R, Gianfrani C, Mazzarella G, Greco L, Guardiola J, et al. (1998) Majority of gliadin-specific T-cell clones from celiac small intestinal mucosa produce interferon-gamma and interleukin-4. Dig Dis Sci 43: 156–161.


It also does not provide an explanation for the characteristic expansion of intraepithelial lymphocytes (IEL), the majority of which are CD8+, seen in active celiac intestinal epithelium (34).

Finally, gluten-specic adaptive immunity cannot account for how enterocytes lining the gut are targeted for destruction during active disease (34).



Only recently, it has been demonstrated that T cells within coeliac lesions, other than react toward native gliadin epitopes (Vader 2002), recognize deamidated gliadin epitopes too that are formed in situ by endogenous tTG (Molberg 2001) (37).

14 Vader W, Kooy Y, Van Veelen P et al. The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology 2002; 122:1729–37.

21 Molberg Ø, McAdam S, Lundin KEA et al. T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminase. Eur J Immunol 2001; 31:1317–23.



In recent years, several epitopes activating T cells isolated from the intestinal mucosa of CD patients have been identified. They occur mainly in gliadins of the alpha and gamma-type (36).

Interestingly, the indigestible 33-mer contains several repeats of some of these epitopes, one of which has been characterised as immunodominant (36).

To be active, most of the epitopes need to be deamidated at a specific glutamine residue, a reaction that has been shown to occur in vitro by treatment with purified TG2 (36).

There is also indirect evidence that TG2 is able to deamidate in vivo (36).

Furthermore, by its specificity, TG2 preferentially generates active epitopes on gliadins (36).

Deamidation of the underlined glutamine (Gliadin 33-mer fragment PepQ =α2(59–72)=QPFPQPQLPYPQPQ) by TG2 was shown to be important for recognition by the T cells.



Some non-deamidated gluten peptides can induce T cell responses from celiac patient biopsies (34).

Vader W, Kooy Y, Van Veelen P, De Ru A, Harris D, et al. (2002) The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides. Gastroenterology 122: 1729–1737.

Arentz-Hansen H, McAdam SN, Molberg O, Fleckenstein B, Lundin KE, et al. (2002) Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues. Gastroenterology 123: 803–809.



These outcomes may be explained by the involvement of a non-T cell–mediated innate response, induced by a second class of immunotoxic gluten peptides (34).

The best characterized of these innate peptides, p31–43 (or p31–49), is distinguished from immunogenic gluten peptides in that it does not stimulate gluten-reactive CD4+ T cells (34).

Arentz-Hansen H, Korner R, Molberg O, Quarsten H, Vader W, et al. (2000) The intestinal T cell response to alpha-gliadin in adult celiac disease is focused on a single deamidated glutamine targeted by tissue transglutaminase. J Exp Med 191: 603–612.

Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, et al. (2003) Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet 362: 30–37.

Instead, this peptide acts directly on epithelial cells as a stress signal, causing increased enterocyte expression of both interleukin-15 (IL-15) and the non-classical MHC class I molecules, MIC and HLA-E, when intestinal biopsies derived from treated celiac patients are exposed to it  (Enactment of innate immune responses through direct toxic effects) (34).

Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, et al. (2003) Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet 362: 30–37.

Hue S, Mention JJ, Monteiro RC, Zhang S, Cellier C, et al. (2004) A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease. Immunity 21: 367–377.


IL-15 promotes IEL expansion (in celiac disease) (34).

Di Sabatino A, Ciccocioppo R, Cupelli F, Cinque B, Millimaggi D, et al. (2006) Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease. Gut 55: 469–477.

IL-15 induces the expression of natural killer (NK) receptors NKG2D and CD94 on the surface of effector IEL (34).

These NK receptor-bearing IEL are targeted to kill epithelial cells via NK receptor engagement of MIC stress markers on the surface of enterocytes (34).

The in vivo relevance of these effects is underscored by the presence of upregulated IL-15 (34),

Di Sabatino A, Ciccocioppo R, Cupelli F, Cinque B, Millimaggi D, et al. (2006) Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease. Gut 55: 469–477.

Maiuri L, Ciacci C, Auricchio S, Brown V, Quaratino S, et al. (2000) Interleukin 15 mediates epithelial changes in celiac disease. Gastroenterology 119: 996–1006.

Mention JJ, Ben Ahmed M, Begue B, Barbe U, Verkarre V, et al. (2003) Interleukin 15: a key to disrupted intraepithelial lymphocyte homeostasis and lymphomagenesis in celiac disease. Gastroenterology 125: 730–745.

increased MIC expression on enterocytes, and CD94+ IEL inltration in the intestinal epithelium of active celiac patients (34).

Thus, innate gluten peptides cause damage to the gut by inducing epithelial stress and IL-15 expression, which in turn lead to IEL inltration and targeted killing of MIC-expressing enterocytes by NK receptor+ IEL in a manner independent of T cell receptor specicity (34).


The mechanism by which these peptides induce stress in epithelial cells is still not known. However, inactive TG2 on the surface of enterocytes may mediate this effect, since neutralization of surface TG2 with the monoclonal antibody 6B9 attenuates the innate effects of p31–43 (34).

Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, et al. (2005) Unexpected role of surface transglutaminase type II in celiac disease. Gastroenterology 129: 1400–1413.


Innate immunity may also play a role in directing the gluten-specic adaptive response toward a Th1 cytokine prole. Since IL-12, a major promoter of Th1 differentiation, is absent in celiac sprue, other cytokines must mediate this Th1 differentiation. Two possible candidates are IFN-α and IL-15 (34).

In ex vivo biopsy culture experiments, IL-15 is known to be induced by p31–43, and it also drives secretion of IFN-γ and TNF-α by IEL (34).

Di Sabatino A, Ciccocioppo R, Cupelli F, Cinque B, Millimaggi D, et al. (2006) Epithelium derived interleukin 15 regulates intraepithelial lymphocyte Th1 cytokine production, cytotoxicity, and survival in coeliac disease. Gut 55: 469–477.

Moreover, p31–43 potentiates the activation of lamina propria T cells by immunogenic peptides, and this effect is mitigated by IL-15 inhibition, suggesting both p31–43 and IL-15 inuence the course of the gluten-specic adaptive immune response (34).

Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, et al. (2003) Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet 362: 30–37.


A major outlying question concerns  the provenance of the inammatory immune response. Celiac sprue is widely regarded as a T cell–mediated inammatory disease, but the discovery of an IL-15-mediated innate response to gluten calls into question whether adaptive immunity alone can cause disease (34).

Importantly, both the immunogenic 33-mer and the innate p31–49 peptide induce characteristic villous attening and increased IEL inltration when administered alone to celiac patients (34).

Sturgess R, Day P, Ellis HJ, Lundin KE, Gjertsen HA, et al. (1994) Wheat peptide challenge in coeliac disease. Lancet 343: 758–761.

Fraser JS, Engel W, Ellis HJ, Moodie SJ, Pollock EL, et al. (2003) Coeliac disease: in vivo toxicity of the putative immunodominant epitope. Gut 52: 1698–1702.


DIETERICH 2006 (35):

It is still not clear whether tTG-gliadin cross links are directly involved in the pathogenesis of coeliac disease (35).

However, the presence of serum IgA and IgG antibodies directed against these cross links (own unpublished results) in addition to antibodies against gliadin, tTG, or deamidated gliadins, supports a possible role of these complexes in the immunopathogenesis of coeliac disease (35).

Haptenisation of immunogenic gliadin peptides by collagens I, III, and VI as well as by tTG itself, and viceversa, likely enhances the immune reaction to these gliadin peptides, to tTG, and to collagens (35).

While the occurrence of IgA autoantibodies to tTG is pathognomonic for coeliac disease,

Dieterich W, Ehnis T, Bauer M, et al. Identification of tissue transglutaminase as the autoantigen of coeliac disease. Nat Med 1997;3:797–801.

our finding of significantly increased IgA autoantibodies against collagen types I, III, V, and VI in patients with active coeliac disease compared with non-diseased controls is of particular interest. It is assumed that epitope spreading from gliadins to tTG due to hapten-like gliadin-tTG complexes occurs (35).

Molberg Ø, McAdam SN, Koerner R, et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in coeliac disease. Nat Med 1998;4:713–17.

Accordingly, expansion of the mucosal humoral response from gliadins/deamidated gliadins to cross links of gliadin with collagens (types I, III, and VI), or supposedly of tTG with collagen type V, is likely. This haptenisation of collagens by gliadins combined with a switch from IgA to IgG class antibodies could be responsible for the increased association of coeliac disease with connective tissue diseases (35).

Ventura A, Magazzu G, Greco L. Duration of exposure to gluten and risk for autoimmune disorders in patients with coeliac disease. SIGEP Study Group for Autoimmune Disorders in Coeliac Disease. Gastroenterology 1999;117:297–303.

In this context, it is of interest to follow up our patients with increased IgA autoantibodies against collagens for development of connective tissue diseases (35).

In summary, the confirmation of defined tTG-recognition sites in gliadin peptides in combination with demonstration of cross linking of these preferred peptides to tTG and certain ECM (extracellular matrix) proteins confirms the essential role of tTG in creating immunogenic neoepitopes and enhancing antigenic presentation of gliadins (35).

Therefore, the intestinal immune response of coeliac patients is even more complex due to enhanced tTG mediated binding of gliadins or related immunogenic peptides to matrix components, which in addition creates long term reservoirs of antigenically potentiated gluten components (35).

Furthermore, a mucosal immune reaction against extracellular matrix proteins, as mirrored by IgA autoantibodies to these proteins, may play a role in secondary autoimmune diseases that are associated with coeliac disease (35).

SKOVBJERG 2004 (36):

Cross-linking of a gliadin T cell epitope to transglutaminase may help the production of anti-TG2 by B cells (36).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

Such a binding has been demonstrated using a labelled epitope peptide added to recombinant human TG2, and has been suggested to be due to temporary binding to the active site of the enzyme or to lysines in the protein (36).

B. Fleckenstein, S.W. Qiao, M.R. Larsen, G. Jung, P. Roepstorff, L.M. Sollid, Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides, J. Biol. Chem. 279 (2004) 17607–17616.

J.L. Piper, G.M. Gray, C. Khosla, High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: implications for celiac sprue, Biochemistry 41 (2002) 386–393.

Evidence for cross-linking was also provided by Ciccocioppo (36).

R. Ciccocioppo, A. Di Sabatino, C. Ara, F. Biagi, M. Perilli, G. Amicosante, M.G. Cifone, G.R. Corazza, Gliadin and tissue trans- glutaminase complexes in normal and coeliac duodenal mucosa, Clin. Exp. Immunol. 134 (2003) 516 –524.

If gliadin peptides also bind to other proteins, a similar mechanism may be involved in generation of antibodies against other endogenous proteins (36).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

Our research provides a basis for the suggestion that binding of a peptide to a protein in connection to its modification to a T cell epitope might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens (36).

A short segment, PQPQLPY, was previously identified as a core sequence of the immunodominant alpha-gliadin epitope, which is responsible for most of the stimulatory activity on intestinal and peripheral CD4+ T lymphocytes (36).

Deamidation of the underlined glutamine by TG2 was shown to be important for recognition by the T cells (36).

Patients with CD have circulating antibodies against TG2, which is considered to be the dominating autoantigen, including the reactivity earlier ascribed to endomysium and reticulin. However, not all anti-TG2 activities of patient sera are absorbed by guinea pig or monoclonal TG2, pointing to the existence of other submucosal autoantigens. In addition, some patients have autoantibodies against the cytoskeletal protein actin and possibly other proteins (36).

The binding of the epitope peptide to TG2 supports the hypothesis of Sollid et al. that T cell immune response to gliadin would drive antibody responses towards TG2 that is cross-linked to gliadin T cell epitopes (36).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

The fact that cross-linking is expected to occur to all proteins having epsilon-amino groups that are reactive with the gliadin epitope and that this really is the case is demonstrated in this paper by its binding to BSA and chicken egg albumin strengthens the hypothesis (Sollid 1997) that gliadin (in celiac disease) also drives antibody responses to proteins other than TG2 (36).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

The importance of posttranslational protein modifications in antigen recognition and autoimmunity has recently been reviewed by Doyle et al (2001).

H.A. Doyle, M.J. Mamula, Post-translational protein modifications in antigen recognition and autoimmunity, Trends Immunol. 22 (2001) 443–449.

Sjfstrfm et al. suggested that the intestinal immune system is tolerant to non-deamidated peptides and that a process leading to deamidation of gliadins could contribute to break of tolerance. However, the use of the particular glutamine residue in the gliadin epitope for cross-linking instead of deamidation may argue against this suggestion.(36).

H. Sjfstrfm, K.E. Lundin, a. Molberg, R. Kfrner, S.N. McAdam, D. Anthonsen, H. Quarsten, O. Nore´n, P. Roepstorff, E. Thorsby, L.M. Sollid, Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition, Scand. J. Immunol. 48 (1998) 111 –115.


The importance of the introduction of gluten in relation to breast feeding has also been under investigation. Interestingly, food supplementation with gluten-containing foods before the age of 3 months was shown to be associated with significantly increased islet autoantibody risk (36).

A.G. Ziegler, S. Schmied, D. Huber, M. Hummel, E. Bonifacio, Early infant feeding and risk of developing type 1 diabetes—associated autoantibodies, JAMA 290 (2003) 1721– 1728.

In addition, autoantibodies specific to diabetes mellitus type 1 have in one case been reported to disappear following change to a gluten-free diet (36).

P. Banin, R. Perretta, E. Ravaioli, V. De Sanctis, Regression of autoimmunity and abnormal glucose homeostasis in an adolescent boy with silent coeliac disease, Acta Paediatr. 91 (2002) 1141–1143.

Furthermore, patients with gluten ataxia have been shown to have antibodies against Purkinje cells (36).

M. Hadjivassiliou, S. Boscolo, G.A. Davies-Jones, R.A. Grunewald, T. Not, D.S. Sanders, J.E. Simpson, E. Tongiorgi, C.A. Williamson, N.M. Woodroofe, The humoral response in the pathogenesis of gluten ataxia, Neurology 58 (2002) 1221 –1226.

Cross-linking with TG2 has also been suggested to be involved in autoantigen modifications during apoptosis or cellular injury (36).

P.J. Utz, P. Anderson, Posttranslational protein modifications, apoptosis, and the bypass of tolerance to autoantigens, Arthritis Rheum. 41 (1998) 1152 –1160.

However, in none of these cases the mechanism for generation of neo-epitopes was specified. It can be speculated that, under conditions where undigested gliadin peptides have access to the circulation due to a barrier disturbance, these peptides can potentially be cross- linked to proteins in organs where TG2 is available for the reaction. In this way, gliadin peptides may also be an initiating factor for other autoimmune diseases (36).

It has been suggested that the up-regulation of TG2 in CD may generate additional antigenic neo-epitopes by cross-linking or deamidating viral, nutritional or endogenous proteins, and thereby contribute to initiation of autoimmune diseases (36).

D. Schuppan, R. Ciccocioppo, Coeliac disease and secondary auto- immunity, Dig. Liver Dis. 34 (2002) 13 –15.

CICCOCIOPPO 2003 (37):

The development of autoantibodies to tTG has been suggested to result from the covalent incorporation of tTG into high molecular weight complexes with gliadin, a reaction consequent to the autocatalytic activity of tTG (37).

Aeschlimann D. Transglutaminase: Protein cross-linking enzymes in tissues and body fluids. In: Maki M, Tossavaine M, eds. Proceedings of the Workshop on Transglutaminase, Protein Cross-Linking and Coeliac Disease, Tampere, Finland: University of Tampere, 2001:15–22.


CICCOCIOPPO 2003 (37):

Although the pathogenesis of celiac disease is still obscure, a strong autoimmune component is recognized, as supported by the production of class A immunoglobulin (anti-endomysial antibodies) against reticular components of the extracellular matrix (37).

Mäki M. The humoral immune system in coeliac disease. Baillieres Clin Gastroenterol 1995; 9:231–49.

In 1997, Dieterich et al. showed that immunoprecipitation of human fibrosarcoma cell lysates, using the immunoglobulin A fraction from coeliac sera, resulted in a single protein band of 85 kD, which, after sequence analysis, was assigned to the enzyme tissue transglutaminase (tTG) (37).

Dieterich W, Ehnis T, Bauer M et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat Med 1997; 3 :797–801

The demonstration that tTG was the predominant, if not the sole, endomysial autoantigen in CD was promptly confirmed (37).

Brusco G, Muzi P, Ciccocioppo R et al. Transglutaminase and coeliac disease: endomisial reactivity and small bowel expression. Clin Exp Immunol 1999; 118 :371–5.



CICCOCIOPPO 2003 (37):

The tTG-mediated reactions with polyamines, such as gliadin, may result in protein modification possibly affecting their biological activity, antigenicity, and turnover (37).




KURTH 1984 (33):

A study researching the use of transglutaminase in the food industry for increasing the binding forces between proteins in meat systems to contribute significantly to the tensile strength of proteins in some restructured meat products with the ultimate aim of improving and binding strength in these products (33).

The covalent linkage of nonmeat proteins to myosin at temperatures and pH’s common in meat product processing was investigated (also at 37ºC) (33).


Preparation of substrate proteins (33):

Myosin was prepared from freshly excised sheep M. longissimus dorsi (33).

Soya globulin (mainly 7s) solution was prepared by extracting soya isolate (33).

Hammarsten casein was used as the source of casein in all the experiments reported (33).

Gluten proteins were prepared for iodination by dispersing 1.25 g commercial gluten (33).


Transglutaminase preparation (33):

Transglutaminase was prepared from fresh citrated bovine plasma (33).

Using the enzyme unit definition of Chung and Folk (1972), the specific activity of the enzyme preparation was 37 units/mg, a 1,000-fold increase relative to the initial plasma concentration (33).

In the present study transglutaminase prepared from bovine plasma was employed because this was considered to be a likely source of the enzyme for use in beef products (33).


Iodinated proteins (casein, gluten and soya) were cross-linked to immobilized myosin and then quantitated by gamma counting after noncross-linked material was removed (33).

For assessing transglutaminase action casein, soya protein or gluten solution containing 2.5 mg iodinated protein was added to reaction tubes containing 1.8 mg myosin immobilized on moist compacted Sepharose 4B (33).

A multi-factor experimental design was employed to simultaneously investigate the effect of protein type (i.e. casein, soya and gluten), pH (i.e. 5.5. 6.0 and 7.0) and temperature (4 and 37ºC) on the reaction of the enzyme (33).


The total volume of each reaction mixture was adjusted to 2 ml by the addition of 0.1 M histidine of the appropriate pH to the protein mixture in the reaction tube. Then 20 μl 1M DTT and 20 μl 1M CaCl2 were added to each tube followed by 280 μg of preactivated transglutaminase. A corresponding set of control samples was prepared without transglutaminase. All samples were prepared in triplicate. Samples were incubated at 37°C for 1 hr or at 4-5°C overnight. At the completion of the incubation time the Sepharose beads were washed four times each with approximately 5 ml of 6M guanidine hydrochloride (GuHCl) to remove noncovalently attached iodinated protein (33).


Washing was carried out by vortexing the beads in GuHCl, then pelleting the beads by centrlfugation and aspirating the GuHCl solubilized protein. Covalently attached nonmeat protein was calculated from the disintegrations per minute of 125I in the final pellet (33).

GIutamyI-Iysine analysis: To confirm the presence of glutamyl-lysine in the proteins coupled to the Sepharose beads, both the transglutaminase treated samples and the controls were subjected to amino acid analysis (33).


Below: The data obtained from a typical multi-factor experiment utilizing immobilized myosin are summarized in Table 1. The data in column I are expressed in terms of the weight of nonmeat protein covalently linked to 1.0 g myosin immobilized on Sepharose 4B, corrected for the weight of protein retained in the control samples. Column II lists the weight of nonmeat protein retained in the controls, i.e. incubations without transglutaminase but with the transglutaminase activators. Each value is the mean of triplicate reaction mixtures (33).

It appeared that the type of substrate protein had a greater effect on the reaction rate than the temperature or pH conditions examined. The amount of gluten cross-linked to myosin showed a significant increase with increasing pH. At the higher incubation temperature, there was a significant increase in the amount of casein cross-linked when the pH of the reaction mixture was decreased (33)


Below: Fig. 3 is a portion of a typical chromatogram of the amino acids released following extensive enzymic hydrolysis of the proteins coupled to the Sepharose beads. The arrow in Fig. 3a indicates a peak with the same retention time as authentic glutamyl-lysine. This peak only appeared in samples treated with transglutaminase and was absent in the controls which had no added transglutaminase (Fig. 3b). Thus the presence of this peak coincided with the covalent linking of iodinated protein to myosin (33).

The results reported here show that plasma transglutaminase is able to catalyse covalent cross-links between myosin and several nonmeat proteins (casein, gluten and soya) at temperatures and over a pH range common in meat processing but also at 37ºC. Covalent bonds can be formed by the enzyme at pH values found in both pre-rigor and post-rigor muscle (33).


Bovine plasma transglutaminase catalyzed the formation of ε-(γ-glutamyl) lysine cross-links between myosin and soya protein, casein or gluten at 4-5°C for 16 hr (but also at 37ºC for 1h) and at pH’s from 5.5-7.0 (33).


Casein appeared to be the best substrate for the enzyme and showed the highest degree of cross-linking to myosin. This could be due to some similarities that exist between casein and fibrinogen, an in vivo substrate for plasma transglutaminase (33).

Casein was the best substrate with approximately 0.4 g of casein cross-linked to 1.0 g myosin (33).





GARDNER 1988 (27):

The controversial work of Hemmings leading to his concept of  "distributed digestion" i.e. the proposal that macromolecular fragments of protein were absorbed on a large-scale so that peripheral tissues were a major site for digestion of dietary proteins, was based on the appearance in the tissues of isotopically labelled high-molecular-weight fragments from minute quantities (1-10 mg) of protein introduced intraluminally in suckling and adult rats; the protein was introduced in some experiments with a grossly hypertonic and alkaline solution (2 mol/liter NaHCO3). His results indicating massive scale absorption of 40-70% of bovine IgG or gliadin as high-molecular-weight fragments in adult rats have never been confirmed independently (27).


A long-standing controversial query is whether increased permeability or macromolecular absorption may be associated with (and then possibly causal in) food allergies, skin diseases such as eczema, and schizophrenia -see the section below on food allergy. Although negative findings on schizophrenia were reported, Mindham and Axon and their colleagues have recently reported increased intestinal permeability in a subset (11 of 32) of their psychiatric in-patients; small-intestinal biopsies were normal, so that celiac disease was excluded. It would be of special interest to know whether these same patients had elevated antibody titers against dietary proteins, and whether the leakiness of their intestines (or those of the "negative subjects) was aggravated by the inclusion of potential allergens, including gluten, in the test meal (27).


BETHUNE 2008 (34):

Immunotoxic (in celiacs) gluten peptides invade across epithelial barriers intact to access the underlying gut-associated lymphoid tissue (34). Only in celiacs?


Mechanisms by which gluten is transported:

The pathways and mechanisms by which gluten peptides are transported across the intestinal epithelium are not yet known. During active disease (celiac disease) (i.e., on a gluten-containing diet), the architecture of the celiac epithelium is grossly perturbed. Intestinal biopsies of lesions exhibit villous attening and crypt hyperplasia as well as increased enterocyte apoptosis, suggesting the integrity of the epithelium may be compromised. Moreover, the jejunal tight junction structure is morphologically altered in celiac sprue patients, and molecular analysis of these junctions has recently revealed that both occludin and E- cadherin fail to localize properly. As a result, untreated celiac patients exhibit increased permeability toward small molecules and sugars used as paracellular markers (34).


Transepithelial invasion of gluten peptides:

Transcellular transport is also upregulated during active enteropathy, as evidenced by the increased endocytic uptake of gluten peptides across the apical membrane of celiac jejunal biopsy enterocytes (34).

Zimmer KP, Poremba C, Weber P, Ciclitira PJ, Harms E (1995) Translocation of gliadin into HLA-DR antigen containing lysosomes in coeliac disease enterocytes. Gut 36: 703–709.


Concomitant with this increased uptake, the apical-to-basolateral transcellular flux of specific gluten peptides and their antigenic metabolites across untreated celiac patient jejunal biopsies is also increased relative to controls (34).

Matysiak-Budnik T, Candalh C, Dugave C, Namane A, Cellier C, et al. (2003) Alterations of the intestinal transport and processing of gliadin peptides in celiac disease. Gastroenterology 125: 696–707.

Matysiak-Budnik T, Candalh C, Cellier C, Dugave C, Namane A, et al. (2005) Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac disease. Gastroenterology 129: 786–796.


Results observed about the influence of cytokines in increasing both paracellular and transcellular permeability observed during active celiac sprue leave open the pathways and mechanisms by which gluten peptides first cross the intestinal epithelium to come into contact with the underlying lymphoid tissue and thereby initiate inflammation. This event may depend on genetic predisposition toward impaired gut barrier function, environmental factors that prime the intestine for uptake of gluten, or preexisting routes of luminal antigen uptake that are shared between celiac patients and healthy individuals. Of course, a combination of these factors may be at play (34).


To date, there is minimal evidence for celiac patients possessing genetic defects in gut barrier function.  Defects in epithelial tight junction structure and paracellular permeability persist after treatment with a gluten-free diet, as does the increased transcellular uptake of gliadin into enterocytes (34).

Friis S, Dabelsteen E, Sjostrom H, Noren O, Jarnum S (1992) Gliadin uptake in human enterocytes. Differences between coeliac patients in remission and control individuals. Gut 33: 1487–1492.


However, due to the difficulty of ensuring a completely gluten-free diet in human patients, it is not clear whether these persistent defects reflect genetically encoded traits, incomplete recovery of the gut, or a continued inflammatory reaction to low levels of dietary gluten. To circumvent this issue, longitudinal studies examining permeability in potentially gluten-sensitive individuals prior to dietary intake of gluten and the onset of inflammation are needed. Due to the practical limitations of conducting such studies in humans, the investigation of this question awaits an animal model for celiac sprue, in which gluten intake can be strictly controlled. Of course, a genetically tractable animal model, such as a mouse, will allow for a more sophisticated toolbox to be directed at this question (34).


Alternatively, there may not be any genetic determinants of celiac sprue related to the transepithelial transport of gluten peptides. Instead, other environmental factors, or gluten itself, may contribute to disease onset by attenuating the barrier function of the intestine. Gastrointestinal infections can permeabilize the gut by causing inflammation or by other mechanisms. For example, in a cell culture model of H. pylori infection, the transcellular flux of intact protein is increased due to urease-dependent impairment of lysosomal protein degradation. Physiologically relevant temperature increases, such as may occur in the context of a fever or bacterial infection, may also permeabilize epithelial monolayers by increasing paracellular flux, thereby rendering the intestine conditionally susceptible to opportunistic invasion by gluten peptides (34).


Interestingly, certain gluten peptides may even have an intrinsic ability to directly affect epithelial permeability. Pepsin-trypsin (PT)- gliadin digests induce production of TNF-α in cultured monocytic cell lines (34).

Jelinkova L, Tuckova L, Cinova J, Flegelova Z, Tlaskalova-Hogenova H (2004) Gliadin stimulates human monocytes to production of IL-8 and TNF-alpha through a mechanism involving NF-kappaB. FEBS Lett 571: 81–85.


Proinammatory cytokines, such as TNF-α and IFN-γ have been shown to regulate the permeability of the gut (34).


Moreover, apically administered PT-gliadin causes actin cytoskeletal rearrangement, changes in expression and localization of tight junction proteins, and increased permeability toward paracellular markers in cultured epithelial monolayers (34).

Sander GR, Cummins AG, Henshall T, Powell BC (2005) Rapid disruption of intestinal barrier function by gliadin involves altered expression of apical junctional proteins. FEBS Lett 579: 4851–4855.

Drago S, El Asmar R, Di Pierro M, Grazia Clemente M, Tripathi A, et al. (2006) Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines. Scand J Gastroenterol 41: 408–419.


Finally, there exist multiple pathways by which small amounts of dietary proteins are regularly transported intact across the healthy intestinal epithelium (34).

Gardner ML (1988) Gastrointestinal absorption of intact proteins. Annu Rev Nutr 8: 329–350.

Heyman M (2001) Symposium on ‘dietary influences on mucosal immunity’. How dietary antigens access the mucosal immune system. Proc Nutr Soc 60: 419–426.

Various orally administered proteins are observed to cross the epithelium in healthy individuals while retaining their immunoreactivity and biological activity (34).

Husby S, Foged N, Host A, Svehag SE (1987) Passage of dietary antigens into the blood of children with coeliac disease. Quantification and size distribution of absorbed antigens. Gut 28: 1062–1072.

Peng HJ, Turner MW, Strobel S (1990) The generation of a ‘tolerogen’ after the ingestion of ovalbumin is time-dependent and unrelated to serum levels of immunoreactive antigen. Clin Exp Immunol 81: 510–515.

Ziv E, Bendayan M (2000) Intestinal absorption of peptides through the enterocytes. Microsc Res Tech 49: 346–352.

Gluten is among these, as anti-gliadin antibody-reactive proteins can be detected in the breast milk and sera of healthy human mothers on a gluten-containing diet (34).

Chirdo FG, Rumbo M, Anon MC, Fossati CA (1998) Presence of high levels of non-degraded gliadin in breast milk from healthy mothers. Scand J Gastroenterol 33: 1186–1192.

This low-level intact transport likely operates through a non-degradative transcellular pathway, either through M cells, or following non-specific endocytic uptake at the apical enterocyte membrane (34).

Heyman M (2001) Symposium on ‘dietary influences on mucosal immunity’. How dietary antigens access the mucosal immune system. Proc Nutr Soc 60: 419–426.

Larger gluten peptides, such as the 33-mer, may additionally be transported through enterocytes via the lysosomal pathway, delivering antigenic fragments to the serosa (34).

Matysiak-Budnik T, Candalh C, Dugave C, Namane A, Cellier C, et al. (2003) Alterations of the intestinal transport and processing of gliadin peptides in celiac disease. Gastroenterology 125: 696–707.

Matysiak-Budnik T, Candalh C, Cellier C, Dugave C, Namane A, et al. (2005) Limited efficiency of prolyl-endopeptidase in the detoxification of gliadin peptides in celiac disease. Gastroenterology 129: 786–796.

Receptor-mediated mechanisms for gluten peptide transport have also been proposed, and several proteins implicated in the pathology of celiac sprue have been suggested as candidate receptors. These include gluten-specific IgA and MHC class II molecules, as well as transglutaminase 2 (TG2) (34).

Zimmer KP, Poremba C, Weber P, Ciclitira PJ, Harms E (1995) Translocation of gliadin into HLA-DR antigen containing lysosomes in coeliac disease enterocytes. Gut 36: 703–709.

Raki M, Tollefsen S, Molberg O, Lundin KE, Sollid LM, et al. (2006) A unique dendritic cell subset accumulates in the celiac lesion and efficiently activates gluten-reactive T cells. Gastroenterology 131: 428–438.

The existence of IgA-deficient individuals with celiac sprue suggests that anti-gliadin IgA is not an essential factor for this endocytic gluten uptake (34).

Collin P, Maki M, Keyrilainen O, Hallstrom O, Reunala T, et al. (1992) Selective IgA deficiency and coeliac disease. Scand J Gastroenterol 27: 367–371.

The MHC class II products HLA DR and DP are upregulated in the apical epithelium during active celiac sprue, but expression of the disease-associated HLA DQ products is restricted to the lamina propria (34).

Kelly J, Weir DG, Feighery C (1988) Differential expression of HLA-D gene products in the normal and coeliac small bowel. Tissue Antigens 31: 151– 160.

Dendritic cells present in the lamina propria express both surface TG2 and HLA DQ molecules, and can extend dendrites through the epithelial layer to directly sample luminal antigens. The identification of a subset of mucosal dendritic cells that can activate gluten-reactive T cells raises the intriguing possibility that gluten peptides may invade across the intestinal epithelium via the same cells that present them to the immune system (34).

Raki M, Tollefsen S, Molberg O, Lundin KE, Sollid LM, et al. (2006) A unique dendritic cell subset accumulates in the celiac lesion and efficiently activates gluten-reactive T cells. Gastroenterology 131: 428–438.


SKOVBJERG 2004 (36):

It is generally believed that immunologically active peptide fragments access the subepithelial tissue via a leaky epithelium (36).


CAPUTO 2010:

Although little is known about the processing of gliadin peptides, there is evidence that they enter enterocytes (Caputo 2010):

Caputo I, Barone MV, Lepretti M, Martucciello S, Nista I, et al. (2010) Celiac anti-tissue transglutaminase antibodies interfere with the uptake of alpha gliadin peptide 31–43 but not of peptide 57–68 by epithelial cells. Biochim Biophys Acta 1802(9): 717–727.

Barone MV, Nanayakkara M, Paolella G, Maglio M, Vitale V, et al. (2010) Gliadin peptide P31–43 localises to endocytic vesicles and interferes with their maturation. PLoS One 5(8): e12246.


SILANO 2012:

peptide 31–43 (p31–43): α31–43 PGQQQPFPPQQPY


In recent years, a growing body of evidence has highlighted the ability of some gliadin peptides, such as gliadin-derived peptide 31–43 (p31–43), to specifically elicit a mucosal innate activation in celiac duodenum as well as in intestinal epithelial cell lines (Silano 2012).


Emerging evidences indicate that p31–43 enters intestinal epithelial cells by endocytosis, delays vesicle trafficking, and accumulates in the later endosomal compartments. This leads to prolonged epidermal growth factor receptor inactivation, overexpression of transpresented interleukin (IL) 15/IL15 receptor-α complex as well as increased reactive oxygen species generation, and TG2 activation (Silano 2012).




              1.      STAAB 1999: Staab JF, et al. (1999) Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 283:1535-1538

              2.      NIEUWENHUIZEN 2003: Nieuwenhuizen WF, et al. (2003) Is Candida albicans a trigger in the onset of coeliac disease?. Lancet 361: 2152–54

              3.      STAAB 1996: Staab JF, et al. (1996) Developmental Expression of a Tandemly Repeated, Proline- and Glutamine-rich Amino Acid Motif on Hyphal Surfaces of Candida albicans. The Journal of Biological Chemistry  271:6298-6305

              4.      STAAB 1998: Staab JF, et al. (1998) Genetic Organization and Sequence Analysis of the Hypha-specific Cell Wall Protein Gene HWP1 of Candida albicans. Yeast Vol. 14: 681–686

              5.      DANIELS 2003: Daniels KJ, et al. (2003) The Adhesin Hwp1 and the First Daughter Cell Localize to the a/a Portion of the Conjugation Bridge during Candida albicans Mating. Molecular Biology of the Cell Vol. 14, 4920–4930

              6.      ENE 2009: Ene IV et al. (2009) Hwp1 and Related Adhesins Contribute to both Mating and Biofilm Formation in Candida albicans. Eukaryotic Cell, Vol.8, Nº12, p. 1909–1913

              7.      LACHKE 2003: Lachke SA et al. (2003) Skin Facilitates Candida albicans Mating. Infection and Immunity Vol. 71, No. 9, p. 4970–4976

              8.      HITOMI 2005: Hitomi (2005) Transglutaminases in skin epidermis. Eur J Dermatol 2005; 15 (5): 313-9

              9.      SUNDSTROM 2002: Sundstrom P (2002) Adhesion in Candida spp. Cellular Microbiology 4(8), 461–469

        10.      BRADWAY 1993: Bradway SD et al. (1993) Do proline-rich proteins modulate a transglutaminase catalyzed mechanism of candidal adhesion?. Crit Rev Oral Biol Med 4: 293–99

        11.      STAAB 2004: Staab JF et al. (2004) Expression of Transglutaminase Substrate Activity on Candida albicans Germ Tubes through a Coiled, Disulfide-bonded N-terminal Domain of Hwp1 Requires C-terminal Glycosylphosphatidylinositol Modification. The Journal of  Biological Chemistry 279:40737-40747

        12.      SUNDSTROM 2002: Sundstrom P et al. (2002) Essential Role of the Candida albicans Transglutaminase Substrate, Hyphal Wall Protein 1, in Lethal Oroesophageal Candidiasis in Immunodeficient Mice. The Journal of Infectious Diseases 185:521–30

        13.      STAAB 2013: Staab JF et al. (2013) Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans. PLoS ONE 8(11): e80842

        14.      ZHU 2010: Zhu et al. (2010) Interactions of Candida albicans with epithelial cells. Cellular Microbiology 12(3), 273–282

        15.      HIIRAGI 1999: Hiiragi et al. (1999) Transglutaminase Type 1 and Its Cross-linking Activity Are Concentrated at Adherens Junctions in Simple Epithelial Cells. The Journal of Biological Chemistry 274, Nº 48, pp. 34148-34154

        16.      INCI 2013: Inci et al. (2013) Investigations of ALS1 and HWP1 genes in clinical isolates of Candida albicans. Turkish Journal of Medical Sciences 43: 125-130

        17.      NOBILE 2006: Nobile et al. (2006) Function of Candida albicans Adhesin Hwp1 in Biofilm Formation. Eukaryotic Cell 5(10):1604-1610

        18.      NOBILE 2008: Nobile et al. (2008) Complementary Adhesin Function in C. albicans Biofilm Formation. Current Biology 18, 1017–1024

        19.      BOURTOOM 2009: Bourtoom (2009) Edible protein films: properties enhancement. International Food Research Journal 16: 1-9

        20.      PHAN 2007: Phan et al. (2007) Als3 is a Candida albicans invasin that binds to cadherins and induces endocytosis by host cells. PLoS Biol 5(3): e64.

        21.      BRINKERT 2009: Brinkert et al. (2009) Chronic mucocutaneous candidiasis may cause elevated gliadin antibodies. Acta Paediatr. 98(10):1685-8.

        22.      MARINIELLO 1993 (gp120): Mariniello et al. (1993) Transglutaminase covalently incorporates amines into human immunodeficiency virus envelope glycoprotein gp120 in vitro. International Journal of Peptide & Protein Research 42, 204-206.

        23.      MARINIELLO 1993 (gp41): Mariniello et al. (1993) Human-immunodeficiency-virus transmembrane glycoprotein gp41 is an amino acceptor and donor substrate for transglutaminase in vitro. Eur. J. Biochem. 215, 99-104

        24.      THORLEY 2010: Thorley et al. (2010) Mechanisms of viral entry: sneaking in the front door. Protoplasma 244: 15-24.

        25.      VELJKOVIC 2003: Veljkovic et al. (2003) Design of peptide mimetics of HIV-1 gp120 for prevention and therapy of HIV disease. J. Peptide Res. 62, 158–166.

        26.      RUOPPOLO 2003: Ruoppolo et al. (2003) Analysis of transglutaminase protein substrates by functional proteomics. Protein Sci. 12(6):1290-7.

        27.      GARDNER 1988: Gardner MLG (1988) Gastrointestinal absorption of intact proteins. Annual Review of Nutrition Vol. 8: 329-350.

        28.      QURESHI 1990: Qureshi NM et al. (1990) Characterization of a putative cellular receptor for HIV-1 transmembrane glycoprotein using synthetic peptides. AIDS, 4(6), 553-558.

        29.      BATMANGHELIDJ 1989: Batmanghelidj (1989) Aids: is transglutaminase the primary permissive factor for HIV establishment and spread? Science In Medicine Simplified, Vol. 1. pp. 3-9.

        30.      AMENDOLA 1994: Amendola A et al. (1994) HIV-1 gp120-dependent induction of apoptosis in antigen-specific human T cell clones is characterized by 'tissue' transglutaminase expression and prevented by cyclosporin A. FEBS Letters 339, 258-264.

        31.      AMENDOLA 1996: Amendola A et al. (1996) Induction of "tissue" transglutaminase in HIV pathogenesis: evidence for high rate of apoptosis of CD4+ T lymphocytes and accessory cells in lymphoid tissues. Proc Natl Acad Sci USA Vol. 93, pp.11057-11062

        32.      BOISVERT 2014: Boisvert H et al. (2014) Transglutaminase 2 is essential for adherence of Porphyromonas gingivalis to host cells. Proceedings of the National Academy of Sciences U S A. 111(14):5355-60.

        33.      KURTH 1984: Kurth L et al. (1984) Transglutaminase catalyzed cross-linking of myosin to soya protein, casein and gluten. J. Food Sci. 49, 573–576.

        34.      BETHUNE 2008: Bethune MT et al (2008) Parallels between pathogens and gluten peptides in celiac sprue. PLoS Pathog 4(2): e34

        35.      DIETERICH 2006: Dieterich W et al (2006) Cross linking to tissue transglutaminase and collagen favours gliadin toxicity in coeliac disease. Gut 55:478–484.

        36.      SKOVBJERG  2004: Skovbjerg H et al. (2004) Deamidation and cross-linking of gliadin peptides by transglutaminases and the relation to celiac disease. Biochimica et Biophysica Acta 1690, 220 –230.

        37.      CICCOCIOPPO 2003: Ciccocioppo R et al. (2003) Gliadin and tissue transglutaminase complexes in normal and coeliac duodenal mucosa. Clinical and Experimental Immunology 134:516–24.

        38.      SCHUPPAN 2002: Schuppan D et al. (2002) Coeliac disease and secondary autoimmunity. Digest Liver Dis 34:13-5.

        39.      BARKI 1993: Barki et al. (1993) Isolation of a Candida albicans DNA Sequence Conferring Adhesion and Aggregation on Saccharomyces cerevisiae. Journal of Bacteriology , Vol.175,  Nº17,  p.5683-5689.

        40.      BARKI 1994: Barki et al. (1994) A Candida albicans Surface Antigen Mediating Adhesion and Autoaggregation in Saccharomyces cerevisiae. Infection and Immunity, Vol.62, Nº10, p.4107-4111.

        41.      STURTEVANT 1997: Sturtevant et al. (1997) Candida albicans adhesins: Biochemical aspects and virulence. Rev Iberoam Micol 14: 90-97.



Hippocrates of Cos

Greek physician (460 - 370 BC)


Let food be thy medicine and medicine be thy food

Maybe misquoted citation (Cardenas 2013) but see below:


The importance of food in medicine was recognized in the 5th Century BC by Hippocrates of Cos, who is considered the father of Western medicine. His work was compiled either directly or indirectly through his disciples, so that the existing knowledge on Hippocrates’ medicine consists of more than 60 texts known as The Hippocratic Corpus (Corpus Hippocraticum). This important text in the history of medicine expounds on the theory of diet. Up until Hippocrates, diseases had been seen as a consequence of divine intervention. With him, they became seen as a state caused by natural causes, including diet. There is no doubt about the relevance of food in The Hippocratic Corpus and its role in health and disease states (Cardenas 2013).


In order to fight diseases, Hippocratic doctors used two kinds of interventions. On the one hand, the previously existing therapeutic interventions such as medicines, incisions, and cauterization and on the other hand the new regimen or dietetic interventions. In a hierarchical order, the most important intervention was diet.  Secondly, medicines seemed to be considered as means of evacuation or purgation of impure fluids from the various cavities of the body. The dietetic intervention, which included a food regimen and exercises, was considered revolutionary at the time. The properties of foods were meticulously analyzed in the treatise On Regimen. Physicians were then able to prescribe a detailed food regimen to patients based on their individual nature, activity, age, season, etc. Thus it is considered that medicine in the Hippocratic era was in fact mainly a dietetic medicine, not a pharmacological or surgical medicine (Cardenas 2013).


“Persons in good health quickly lose their strength by taking purgative medicines, or using bad food


“It is a bad thing to give milk to persons having headache, and it is also bad to give it in fevers, and to persons whose hypochondria are swelled up, and troubled with borborygmi, and to thirsty persons; it is bad also, when given to those who have bilious discharges in acute fevers, and to those who have copious discharges of blood”


“Of course I know also that it makes a difference to a man's body whether bread be of bolted or of unbolted flour, whether it be of winnowed or of unwinnowed wheat, whether it be kneaded with much water or witli little, whether it be thoroughly kneaded or unkneaded, whether it be thoroughly baked or underbaked, and there are countless other differences. Barley-cake varies in just the same way. The properties too of each variety are powerful, and no one is like to any other. But how could he who has not considered these truths, or who considers them without learning, know anything about human ailments? For each of these differences produces in a human being an effect and a change of one sort or another, and upon these differences is based all the dieting of a man, whether he be in health, recovering from an illness, or suffering from one. Accordingly there could surely be notfiing more useful or more necessary to know than these things, and how the first discoverers, pursuing their inquiries excellently and with suitable application of reason to the nature of man”


Jean Seignalet

French physician (1936-2003)


"Food is an integral part of medicine and is more than less salt for hypertensives and less sugar for diabetics "


"I have sought to understand scientifically how a inadequate food could lead to a pathology."


“I have no doubt about this: food is both preventive and curative”


“In addition to the genetic predisposition of each one environmental factors are dominant in 90% of diseases


Two out of three cancers depend on food


“The acquired cancers (non-hereditary) (about 95%), even if found predisposed genes, are essentially caused by environmental factors: food, tobacco, asbestos or virus such as in the case of uterine cervical cancer”


“Radiation, chemicals, viruses and non-intestinal bacterias can only explain 40% of acquired cancers. Therefore, for the remaining 60% it seems logical to consider bacterial and food waste resulting intestinal origin of modern food”


"Modern food acts on a key body, the small intestine, providing molecules which can not degrade our enzymes. Large molecules, from food and bacterial origin cross the intestinal barrier and enter the blood. Are deposited in various tissues and clog the body. "


“Intracellular poisoning is the main reason for cell cancerization. Some foreign macromolecules progressively bother blocking the operation of various mechanisms and accumulation of waste breaks certain physiological balance. I am persuaded that this prolonged poisoning by lead cell ends alterations of nuclear DNA and cause genetic abnormalities that lead to cancer


“I have healed from a serious nervous depression by means of a dietary regime that excluded cereals and dairy products, which was rich in raw products”


Cow's milk is a very nutritious food ... for calves in the growing season. Humans digest milk only from our species, and at the the nursing period. The main milk protein, casein, is difficult to digest completely


You get interesting results yet their medical colleagues do not always believe in the benefits of feeding. JS: "Do not believe in this theory and it could be indifferent to them, I can understand. What surprises me most is that they do not want to experience. I have done my duty by exposing my theory. "



T. Colin Campbell

American biochemist



I consider nutrition to be THE premiere science in medicine – end of story.”


“I am most interested in, namely, the comprehensiveness of the nutrition effect on health and disease


“I focused on the role of nutrition on health maintenance and disease occurrence.”


“What we choose to eat also is one of the most emotionally intense topics of human discourse, ranking up there with sex, religion and politics. Yet, properly practiced nutrition, as a dietary lifestyle, can do more to create health and save health care costs than all the contemporary medical interventions put together.”

"We’ve distorted our diet seriously through the ages, and we have all the problems we have because of that distortion."


“The shorthand of the whole thing is we’re eating the wrong foods, basically, animal-­based foods, plus all this processed food, we’re eating the wrong food, and then turning around and relying on this silly notion that we can take a single chemical after we get a disease and hopefully make ourselves well. Ah, we get can get some benefits from that from time to time, but that’s not the long-­term solution to maintain health, it just doesn’t work that way.”


“I want people to talk about and to think about how should we be eating in a very empirical scientific sense, and not with an ideological bent to it.”


“All humans share virtually the same biochemistry and physiology, regardless of ethnicity, race and gender. They differ, both as individuals and as groups of people, in the DEGREE to which they respond to dietary insult. But the direction of the effect is essentially the same.”




"Well, it was very traditionally American, I suppose, rural America. I was raised on a dairy farm, and believed in the good old American diet, so to speak. I milked cows until I went away to school. .I went away to graduate school at Cornell University, and I thought the good old American diet is the best there is. The more dairy, meat and eggs we consumed, the better. When I did my doctoral research, my program was actually focused on the idea that we had to find more productive ways of producing more animal protein, in particular, that is, more meat, milk and eggs. The early part of my career was focused on protein, protein, protein. It was supposed to solve the world’s ills. And I was very much a part of that culture, and believed that that was the ultimate as far as good health is concerned. But as my career began to unfold, especially with all my students, and other colleagues, and a research career that involved experimental research, that is, actually doing the studies, designing studies, doing studies, and publishing the results, I eventually came to the view that I had to seriously question what this good old American diet was all about. When we started doing our research, we found that when we start consuming protein in excess of the amount we need, it elevates blood cholesterol and atherosclerosis and creates other problems. So I obviously made quite a change and quite a shift in my thinking over the last 50 years. And I find that my views are not just based on research that I did, but obviously the research that many, many others have done too. We did some research that I just found very provocative, and just caused me to really begin to question what we really believed as far as diet and health are concerned.”


“I’ve just finally come to the view that nutrition, if it’s properly understood and used, really has enormous potential to create health, maintain health, prevent disease, even cure disease, even cure advanced diseases. And so I just find the whole idea very, very exciting, I think it has a lot of potential, not only to help people be well, if they really understood what all this is about, but in the process, from a more societal point of view, I suppose, it could have a major impact on the runaway health care costs that we’re now experiencing in our country.”


“It's been estimated that the total number of people now living in the world who are going to die prematurely from smoking is of the order of 200 million people, that's a population approaching the size of the United States. When we compare the number of people dying from smoking with the number of people adversely affected by diet, for example, diet is often conceded as causing even great number of premature deaths than smoking. So, just using that very simple comparison, let me suggest that the number of people in the world today who are likely to die prematurely from poor diets, at least in the western sense, could easily be 200 or 300 hundred million people. These are big numbers, these are really big numbers. In fact, I would suggest that's a conservative number. Another way of looking at this is to say 60-70% of the people in the UK and the US and other western countries die prematurely from cancer, heart disease, diabetes and these other western kinds of disease. Since those diseases are preventable by dietary means, might we say that half, or 60-70% of these diseases really can be prevented at least until much older ages through dietary means. This is a large number of people, whatever that number is.”




“My research career started in 1956, so I’ve been around a long time. Most of my career was spent at Cornell University where I had a large research program. Initially, my research focused on diet and cancer. Through my research, I saw some very unusual things that did not appear in textbooks. It challenged my own thinking.”


“My experience and interest over the years has been concerned with the prevention of cancer primarily, particularly the prevention of cancer by dietary means. I happen to now believe very strongly that nutrition has a lot to do with whether or not we get cancer. It is an area that has been unfortunately underplayed and in some cases actually ignored by some of the central authorities who are involved in doing cancer research.”


“It's ironic that traditional cancer research organisations and health organisations will admit that at least a third of all cancers can be prevented by dietary means, but then in the next breath, they'll tell you that they really don't know how. Then you ask them how much money are you spending on this, and what you discover is that they're only spending about 1-2% of their budget at the most. There is some terrible discordance here.”




All that wealth for the few at the expense of health for the many


Money is made when fixing sick people, not in maintaining healthy people. Exceptionally well-endowed and powerful industries that survive on our money are not very serious about converting us to non-customers. Yes, the message is remarkably simple, but history shows us that considerable efforts, intentional or unintentional, have been made to ignore it, misunderstand it or make it complicated.”


“I think that the Food Pyramid is rather trivial and highly political. I have paid little attention to what they say or do, for I know how corrupt is that process.”


“Historically, we have been slaves to a nutrition-less health information system that, in effect, is designed to keep us in mental chains, thus to maintain the status quo.”


Do you feel passionate about this subject? : “Yes I do. I think in part because I was in the other camp in a way, not intentionally, but that's just where I was, from my childhood on through, into science - and I got into science because I thought science was a place where we were supposed to look at things honestly and make our decisions accordingly. I saw evidence that didn't agree with the way I was doing things or the way before us, and so I had to look at that. I thought it was a very simple matter and I got quite excited about this kind of thing, and in fact what I discoved was an enormous hostility and antagonism to the promotion of these ideas. I have to say I became somewhat cynical of the institutions of science because of that. I started thinking a lot about why it is that the institution of science itself behaves in such a way? And, what I'm now discovering is that science is not so ideal, in the way I once thought. I was very naive. The institution of science is closely related to who provides the funds for the science to be done, either directly or indirectly.”


“I think the indirect effect is even greater than the direct effects, and people in science advance their careers by how much research they do, and how much publicity they tend to get. And of course, they are going to advance their careers and get the publicity if they do the research that's generally accepted, in other words supporting the status quo. If a scientist comes along and says something different, they do it at their peril, because they just may not get the publications, they may not get the advancement in their care ers. That's a rather indirect effect, but nonetheless, it's a very serious effect, and they know it. And so, I think the institution of science, which has basically served a very reductionist way of thinking, that is producing little pills and magic bottles to do this that and everything else, that's what medical science has largely been, been fostering, been concerned with, and interested in.”


“And so one can sort of wonder why it is that we tend to focus on one thing at a time? Well, that’s the way things are sold, that’s the way they make money. And that’s the way things get patented in order to protect the intellectual property, in order for it to be marketed. And of course that’s the simplest way to think about things, too, just using one chemical at a time, or many just two or three, or so, working together in pill form. And it’s really quite ludicrous, when really one understands how things work in this very dynamic way in the tissues. On the one hand, one begins to recognize and understand that, and then to turn around and assume that we can take a single chemical, whether it’s a drug, or whether it’s a nutrient supplement, or whether it’s some other kind of thing, to just do one thing and try to correct a whole system by just sort of using one entity, and it just makes no sense. We can only expect to get unintended consequences, I think, by taking that approach. It’s just simply wrong.”

“And of course it serves the free market system and it serves our sense of how to control disease through cure, but, it doesn't serve the public. Prevention is really the way to go, and at the centre of the plate for prevention is nutrition, how we decide to eat and how we decide to behave otherwise, and that's a very comprehensive sort of lifestyle dietary change. That's where we get good health - that's what the public needs to know, and science is not delivering it. When I find I get hounded for my views by some of my colleagues, on these particular points, it makes me angry and in a sense pursue the question even more.”


“I’ve really become, I guess, in my older years, really pretty cynical about the whole medical system, the way we now do it. It’s not really creating health, we know that from the figures, it’s basically sustaining these extraordinary rates of disease that we now have. We’re not really getting anyplace, and we’re spending a heck of a lot of money getting no place. And it seems, the figures show, for example, in the United States, that we spent more per capita on medical care costs than any country in the world, probably somewhere in the neighborhood of about 50% more than the second highest country, I mean, we’re way up there. Yet when our medical system, when our health systems are judged by others in terms of quality of health care, we stand somewhere in the neighborhood of 30th to 40th in the list. So the question arises, why are we spending so much money and getting so little in return? Makes no sense.”


“We have lost respect for nature. We over-name things, we over-quantitate things. We live that way, partly because that’s the way our brains work, maybe we just can’t think in this kind of context – but we have to. That’s the way nature is and until we recognize that this is what life is all about, we’re not gonna make a lot of progress. What we’re going to do is continue to make a lot of money, and let the rich get richer and let the rest of the people serve as slaves. That’s one title I’m considering: The Master and Slave State. We’re focused on money, greed and competition, and the people who really want to go down that route become very hostile when you challenge them


“I say it’s “wealth for the few at the expense of health for the many”. It’s really what it’s all about. To come back to your question of HOW we do this – establishment does not understand nutrition, and whether they know it or not, they are consistently trying to keep this information from the public. So, I say, first of all, it’s about information control – let’s face it. And I show how industries have devised systems to keep things under control. Registered dietitians, for example, are the only ones allowed to practice nutrition professionally. There’s licensing, and the ones who are controlling the licensing is the American Dietetic Association (ADA). And the ADA is a front for the dairy industry, for crying out loud. Two years ago, I was invited to give a keynote there and we were given our registration bags and there on the outside it says “ADA partners” and you see The National Dairy Council, Coca Cola, Pepsi Cola. So I just took a picture and I just showed the others and I said “look at this criminal outfit”. They are the ones that not only control who is allowed to talk about food because of the licenses, but they also control the curriculum in universities as to what courses you have to take to get a Registered dietitian. So, all the Registered Dietitians in this country are working with a corrupt organization and getting trained in an area of nutrition that is controlled. When I tell them this, a lot of dietitians get upset, but all of a sudden they realize that they’ve been had. And the public has really been had.”


The meat and dairy industries have much power and influence in our society. How have these groups affected you personally and in your career: TCC: “They know well who I am, ever since about 1982, and they have tried, at times vigorously, to use less than professional means to silence me and to discredit my reputation. This has only spurred me onwards.”


“It is very frustrating to see, for example, medical professionals not being trained in nutrition, not understanding nutrition, and in many cases as they go out into practice almost denying that the dietary effect is all that important. I mean, that’s a pretty traditional view on the part of many physicians, of course. They have come to rely on drugs, as we all know, and surgery, and so forth, to sort of treat disease once it’s already present, and as I say, and treat it rather ineffectively in many cases.”




“I think the world is changing to some extent in the sense that, one of the biggest innovations in my view that has come along in the last few decades is the vastly improved facilities for communication, I mean virtually anyone in our society can, can get together some equipment and get some ideas and, and basically go out and promote some information and let the public know, and so now the dispersion of information is I think more democratic than it has been, at least I think. Whereas before, 20 or 30 years ago, 40 or 50 years ago, I mean, information was held in the hands of a few, and I think this is going to make a difference.”

“I'm rather optimistic and hopeful because I know it's related to the fact that 'the power and the money' has done tragic things, and they are still very powerful and there is a lot of 'power and money' there, but, thanks to the world of communication, books and the media, it can make a diffence. It really can make a diffence.”




“Every disease begins with genes but this is not a death sentence. For the most part, genes, good and bad, are controlled by nutrition [i.e., the food we consume]. With the right nutrition resources, our marvelous bodies, while always striving for health, manage which genes to express and which ones to keep quiescent. There is also considerable evidence that the initiation and expression of many autoimmune diseases may be influenced by nutrition (and therefore would be repressed by eating whole foods), sometimes initiated early in life, from what mothers eat while pregnant and nursing. We need more awareness of this phenomenon, and then we need to do more research on mother-child interactions.”


“We should not be relying on the idea that genes are determinants of our health. We should not be relying on the idea that nutrient supplementation is the way to get nutrition, because it’s not. I’m talking about whole, plant-based foods. The effect it produces is broad for treatment and prevention of a wide variety of ailments, from cancer to heart disease to diabetes.”


“There are hundreds of studies now showing that people who move from one country to another where the disease risk is very different, take on the risk of the disease of the country to which they move while they keep their genes the same. In other words, diseases don't occur because of a genetic predisposition, it may for individuals be somewhat different, but regardless of our genetic predisposition, we can control whether or not that we get the disease simply through dietary and lifestyle changes.”

“It is true that we have discovered a tremendous amount of information but this does not mean discovering what it all means. Indeed, our focus on details has created an enormous pile of contradictory observations--permitting too many people to construct ideas that please their palates and wallets more than educate their brains.”




“I don't care to pass personal blame or pose conspiracies, for we are all participants in this great war of words of what nutrition really means. Nonetheless, somewhere there is an origin and it is fostered by our professions, my nutrition and medical research community and my clinical colleagues' medical practice community. This is not surprising.The National Institutes of Health (NIH), which is the most influential research funding agency in the world, is comprised of 27 institutes, centers and programs and not one is named the Institute of Nutrition. Research funding is a mere pittance in a couple of the institutes and most of this is dedicated to the study of individual nutrients that I consider pharmacology, not nutrition. Further, there is not a single medical school in the country that teaches nutrition as a basic medical science. At best, a few may have an elective course that treats the subject in a most superficial manner. Public citizens, therefore, are left to fend for themselves against the hyped up claims of the food and drug industries.”


“And there’s nothing that can touch nutrition – if we understand it. We don’t understand nutrition. People in my community of research don’t understand nutrition. People in the medical practitioner community don’t understand it, we talked about that before, how there is no nutrition course requirement in medical school. And NIH is the premiere research funding agency in the world, it has the most money, has a great track record, is highly respected. It’s made up of 27 institutes, centers and programs, on cancer, diabetes, etc…Not one is called the Institute of Nutrition. The head of the NCI and the head of the Heart and Lung would say “oh, we got nutrition built into our fabric, in our system, in our research”, but when they asked them what percent would you actually say is focused on nutrition, they would give a figure of 2% or 3%. And the others don’t have any, so it’s just limited to a couple of the institutes, and only 2%-4%. And most of that is actually spent on clinical trials – where they spend a lot of the little money testing the ability of single nutrients, like “does vitamin C stop colon  polyps?” It’s done with an eye on the corporate sector – that’s what it’s about: What can we put into a pill and see if it works. And it’s just ridiculous. And to add insult to injury, since the director of an institute has to be a medical doctor, that means it has to be someone who is not trained in nutrition, by default. I have served on NIH review committees and it became very clear to me that they were consistently very opposed to nutrition – even though they use the word a lot, they don’t seriously study it.”


“I also have fervent views not to make claims for this dietary lifestyle that are not supported by reliable evidence. Predicting future events for this practice is not an exact science. Forecasting health and disease outcomes is a matter of odds, not a matter of certainty. We cannot say that all ailments will be controlled in all individuals by this nutritional strategy. But, on the basis of probability, it is abundantly clear that this dietary lifestyle has a breadth of effect that is greater than any combination of drugs and procedures ever used, without the accompanying side effects that are common to virtually all drugs.”


“If we are to understand the true value of nutrition. When done right, advanced heart disease can be cured, type 2 diabetes stopped and reversed, cancer can be prevented and, with some newer evidence, controlled after it appears. The range of diseases that can be prevented is more than impressive. The breadth and rapidity of the nutritional effect not only prevents disease but actually treats many of these diseases while restoring and maintaining health. The totality of these health effects are far more than almost anyone knows.”




Casein, a protein found in milk from mammals, is "the most significant carcinogen we consume””


“Let there be no doubt: cows milk protein is an exceptionally potent cancer promoter


“I was actually raised on a farm, and I milked cows until I was well into college, and obviously I ate that kind of diet. When I went away to college I was in pre-vetinary medicine as an undgraduate, but then in my graduate studies I did a PhD dissitation on figuring out ways to produce animal protein more efficiently, so we could eat more animals. That was my background. I was totally a farm boy, totally into that territory.”


You have made some strong statements about the negative effects of casein, a milk protein. Could you give our readers some of the major points you make about the detrimental nature of casein? What led you to those views?: TCC: “In experimental animals (rats and mice) we could turn on and off experimental cancer development by feeding and withdrawing casein at levels above minimum protein requirements. We also studied in great detail how this works and discovered some very profound and provocative phenomena that relate, more generally, to the broader issue of diet and health. This began with my work in the Philippines coordinating a nationwide program for feeding malnourished children and observing that those few families and their children consuming protein diets at levels similar to the U.S. got more cancer. A subsequent experimental animal study in India confirmed what I suspected that I was seeing. This then led to a long series of experimental animal studies that was largely confirmed in the studies of others, both in humans and in experimental animals.”




“Cooked food, yeah, there’s some information that when we’re overcooking foods, certain kinds of foods, we can, in fact, get some noxious chemicals on the basis of the information we now have, heterocyclic amines, as we call that class of compounds, and in the older literature there was another class of compounds referred to as the polycyclic aromatic hydrocarbons, that may result from essentially the burning of food, or having food exposed to fire. And so it’s the kind of information that would suggest that obviously cooking food a little bit is probably OK most of the time, but if we overdo it it’s not a good idea, and it’s one of the reasons I suggest we kind of stay toward the raw food side as much as possible.”


How have your colleagues responded to your efforts to reverse chronic diseases through diet: TCC: “As far as the research community is concerned, mostly with silence, although as I write this, I am suddenly seeing an increasing number of medical practitioners beginning to carry the banner forward. These people have, for the most part, seen first hand what they can do for their patients when they adopt this practice. I've lost some research colleagues but I've gained a lot more new colleagues.”


“Incidentally, that kind of eating was much better appreciated and accepted and promoted as long ago as the ancient Greek times. Some of the leading Greek philosophers and others who thought about medicine, diet and disease wrote surprisingly impressive observations on the relationship between eating that kind of food and good health. It all sort of disappeared around 300 or 400 AD, for some strange reason lots of things disappeared about that time, and I think that we're just now beginning to come out of the dark ages and going back to this; rediscovering what was well known to these medical people and philosophers.”



Hiromi Shinya

Japanese Physician



“I have examined the stomachs and colons and taken the dietary history of more than 300.000 patients”


"I have examined more than 300,000 people's stomachs and intestines for 35 years and realize that our health depends largely on our dietary life


“I have discovered a strong relationship between health and certain ways of eating and living”


“Diseases, life and health are the result of what you eat every day


 “…diseases of ‘unknown cause’ can sometimes be traced back to dietary history.”


“Looking at the dietary history of cancer patients, I usually find that they have had a diet consisting mainly of animal protein and dairy such as meat, fish, eggs and milk... for women with breast cancer and men with prostate cancer, the probability of discovering an abnormality in their colon is high.”


"Dairy is the worst food you can put in your body."


“There is no other food that is as difficult to digest as milk.”


Casein, which accounts for approximately 80% of the protein found in milk, immediately clumps together once it enters the stomach, making digestion very difficult.”


“We do not inherit disease from our parents, we inherit their dietary habits and all the health problems that come with it.”


Good habits will overcome bad genes


Novak Djokovic

Serbian professional tennis player and world No. 1



“Every time I took a big step toward my dream I felt as though a rope were around my torso pulling me back,” Novak explains. “Physically I couldn’t compete. Mentally I didn’t feel I belonged on the same court as the best players in the game”


“Since the age of thirteen I’d felt constantly stuff y, especially at night. I would wake up groggy, and it would take me a long time to get going. I was always tired. I felt bloated, even when I was training three times a day. I had allergies, and on days when it was humid or the flowers were in full bloom, they would be worse. Yet what was happening to me didn’t make sense. Asthma strikes as soon as you start to exercise; it doesn’t come on three hours into a match. And my problem couldn’t be conditioning. I worked as hard as anyone on the circuit. Yet in the big matches, against the best players, I would hold my own through the first few sets, then collapse. But I wasn’t a hypochondriac, or an asthmatic, or an athlete who just folded when the matches got tough. I was a man who was eating the wrong way


“There was something about me that was broken, unhealthy, unfit. Some called it allergies, some called it asthma, some just called it being out of shape but no matter what we called it no one knew how to fix it.”


“Imagine you’re hammering a nail into a plank of wood and you accidently hit your thumb. It gets swollen, red and angry. That’s what was happening inside me.”


Cetojevic suggested that Djokovic eliminate gluten from his diet. After commissioning some blood work, he recommended that Djokovic also eliminate dairy products and cut down on tomatoes. (In solidarity, Miljan Amanovic, Djokovic’s trainer, underwent an assessment and had to forsake egg whites and pineapple.) The program was hard to fathom—his parents owned a pizza parlor!—but Djokovic was desperate enough to try it, and, once he did, he experienced it as a complete rebirth. As he recalls in “Serve to Win” (subtitle: “The 14-Day Gluten-Free Plan for Physical and Mental Excellence”),


 “It wasn’t a new racquet, a new workout, a new coach, or even a new serve that helped me lose weight, find mental focus, and enjoy the best health of my life. It was a new diet,” says Djokovic in his new book, “Serve to Win: The 14-Day Gluten-Free Plan for Physical and Mental Excellence.” After gaining a reputation of being unpredictable, prone to sickness and even out of shape — something that commentators often blamed on asthma — Djokovic went gluten-free in 2010. The next year, he won 10 tennis titles, three Grand Slam events and 43 consecutive matches. He’s now ranked No. 1 in the world by the Association of Tennis Professionals. “My life had changed because I had begun to eat the right foods for my body, in the way that my body demanded,” he writes.


“If you think you’re just going to exercise away your troubles, you’d better think again. I was training at least five hours a day, every single day, and I still wasn’t fit enough. Was I carrying an extra nine pounds because I wasn’t exercising enough? No. I was heavy, slow, and tired because I was eating the way most of us eat. I ate like a Serb (and an American)— plenty of Italian food like pizza, pasta, and especially bread, as well as heavy meat dishes at least a couple of times a day. I snacked on candy bars and other sugary foods during matches, thinking they would help to keep my energy up, and figured my training schedule had earned me a handful off every cookie tray that passed by. But what I didn’t realize was that eating this way causes a phenomenon called inflammation. Basically, your body reacts to food it doesn’t like by sending you signals: stuffiness, achy joints, cramping bowels. Doctors have linked inflammation to everything from asthma to arthritis to heart disease and Alzheimer’s”


“My life has changed because I now eat the right foods for my body. I feel fresher, more alert and more energetic than I have in my life. You certainly don’t have to be a tennis pro to make the changes I did to improve your body, your health and outlook on life.”


“Mentally, you’ll be fresh, you’ll be happier, you’ll be calmer," said Djokovic. Physically, you’ll be stronger, faster, more dynamic, your muscles will work better. That’s what I feel."


 “I was lighter, quicker, clearer in mind and spirit. . . . I could tell the moment I woke up each morning that I was different than I had been, maybe since childhood. I sprang out of bed, ready to tear into the day ahead.” One day, as an experiment, he ate a bagel. He writes, “I felt like I’d spent the night drinking whiskey!”


The diet changed my life in a really positive way and affected positively my career and my overall feeling on and off the court," he said. "I particularly wanted to share this kind of food regime and this kind of change that affected my life positively with the people, just present them my own experience”


"If you can mentally overcome this greed and eat only the food that is good for your metabolism, then you will have the best results, not just in tennis but in life as well"


Since going on a gluten-free diet, “my allergies abated, my asthma disappeared; my fears and doubts were replaced by confidence. I have not had a serious cold or flu in nearly three years”.


"Thousands of new strains (of wheat) have made it to the human commercial food supply without a single effort at safety testing."


 “Imagine you’re hammering a nail into a plank of wood, and you accidentally hit your thumb. It hurts, right? Your thumb gets swollen and red and angry. That’s inflammation. Now imagine that occurring inside your body, where you can’t see it. That’s what happens when we eat foods our bodies don’t like. When I fell apart at the Australian Open, my body was telling me that I was beating myself up from the inside out. I had to learn to listen to it. Once I did, everything changed. And I don’t mean just my tennis career. My entire life changed. You could call it magic— it sure felt like magic. But it was nothing more than trying different foods to find the ones that worked for me, and applying that knowledge to my daily diet. Bottom line: I figured out which foods hurt me and which helped. Once you know the correct foods to eat, when to eat them, and how to maximize the benefits, you’ll have a blueprint for remaking your body, and your life”


“You start by eliminating gluten from your diet for two weeks. (This is simpler than you think, as you’ll read a little later on.) After that, you attack the excess sugar and dairy in your diet for two weeks, and see how you feel. (Here’s a hint: You’ll feel great.)”

First version of the page 7 - 11 - 2013

Current version 26 - 1 - 2015


hits counter