FOOD PROTEINS TRANSGLUTAMINASE CROSSLINKED WITH ENDOGENOUS PROTEINS HYPOTHESIS
CERTAIN FOOD PROTEINS CROSSLINKED (ENZIMATICALLY COVALENTLY FOREVER BONDED) WITH HUMAN ENDOGENOUS PROTEINS BY A HUMAN ENDOGENOUS ENZYME, HUMAN TRANSGLUTAMINASE, AS UNIVERSAL BIOCHEMICAL PATHOGENIC MECHANISM
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).
AIM OF THIS HYPOTHESIS:
This hypothesis is intended to demonstrate that most human diseases are directly or indirectly due to the crosslinking (forever covalently binding) of certain food proteins with certain endogenous proteins by a human endogenous enzyme called transglutaminase. The question is simple, is it really possible that one of our own enzymes bind a food protein to one of our own proteins forever in vivo? What would be the consequences if that happened?
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, exhibits G protein activity, and has been implicated in the modulation of cell adhesion, migration, invasion and cancer metastasis (Lin 2011).
Due to the lack of availability of studies investigating the effect of this mechanism on food proteins, to begin to address the questions this hypothesis propose we focus initially on analyzing bug studies related to similar issues.
Ok, lets look at those bugs!:
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).
CANDIDA ALBICANS INTERACTIONS WITH HOST CELLS:
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):
YEAST TO HYPHAL MORPHOLOGICAL TRANSITION: HYPHAE
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; THE HALF MAMMALIAN HALF FUNGAL PROTEIN:
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 “FUNGAL” DOMAIN: SERINE/THREONINE-RICH DOMAINS I AND II:
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 “MAMMALIAN” DOMAIN: ANTIGENIC, REPETITIVE, PROLINE/GLUTAMINE RICH, OR TRANSGLUTAMINASE SUBSTRATE DOMAIN:
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).
CELLULAR LOCALIZATIONS OF THE Hwp1 SPECIES:
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).
FUNCTION OF THE REPEATS:
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).
Hwp1 HYPHAL LOCATION:
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).
Hwp1 ON HYPHAL SURFACES: STAAB 1996 IMMUNOFLUORESCENCE ASSAY OF CANDIDA ALBICANS GROWN IN LABORATORY CULTURES (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).
Hwp1 ON HYPHAL SURFACES: STAAB 2004 IMMUNOFLUORESCENCE ASSAY OF CANDIDA ALBICANS GROWN IN LABORATORY CULTURES (11):
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):
Hwp1 ON HYPHAL SURFACES: STAAB 1996 IMMUNOFLUORESCENCE ASSAY OF MAMMALIAN HOST TISSUES COLONIZED WITH CANDIDA ALBICANS (3):
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):
Hwp1 ON HYPHAL SURFACES: DANIELS 2003 ANTI-Hwp1 ANTIBODY STAINING OF CANDIDA ALBICANS (5):
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).
OTHER ANTIGENS ON HYPAHE SURFACE:
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 & TRANSGLUTAMINASE
Hwp1 IS A TRANSGLUTAMINASE SUBSTRATE:
Hwp1 is a substrate for mammalian transglutaminase (1).
Potential transglutaminase susbtrates 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)
STAAB 1999 RECOMBINANT Hwp1 PROTEIN ASSAY (1):
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).
WHICH TRANSGLUTAMINASES ARE INVOLVED?:
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).
BUCCAL EPITHELIAL CELLS (BECs) TGASE:
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).
TRANSGLUTAMINASE 1 OR KERATINOCYTE TRANSGLUTAMINASE (TG1):
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).
TRANSGLUTAMINASE 2 OR TISSUE TRANSGLUTAMINASE (TG2):
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 SURFACE LOCALIZATION ON HYPHAE AS TRANSGLUTAMINASE SUBSTRATE:
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).
STAAB 1999 CANDIDA ALBICANS TRANSGLUTAMINASE SUBSTRATE ASSAY (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.
STAAB 2013 CANDIDA ALBICANS GERM TUBE TRANSGLUTAMINASE ASSAYS (13):
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):
Hwp1 HOMOLOGIES RELATED TO TRANSGLUTAMINASE:
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).
- HOMOLOGY Hwp1 & SMALL PROLINE-RICH PROTEINS (SPRs):
WHAT ARE SPRs?:
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 AND TRANSGLUTAMINASE:
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 VERSUS SPRs:
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).
CONTACT TIME NEEDED FOR CROSSLINKING HAPPENS:
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).
- HOMOLOGY Hwp1 & ACIDIC PROLINE RICH PROTEINS (APRPs):
WHAT ARE APRPs?:
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 AND TRANSGLUTAMINASE:
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.
APRPs AS DEFENSE MECHANISM (10):
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 VERSUS APRPs:
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).
PHAN 2007 SALIVA ASSAYS (20):
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).
ANOTHER TRANSGLUTAMINASE SUBSTRATE PROTEIN IN CANDIDA ALBICANS BLASTOSPORE FORM?:
- TGASE-CATALYZED CROSSLINKING OF PUTRESCINE TO CANDIDA ALBICANS SURFACE PROTEINS BRADWAY 1993 ASSAY (10):
[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-CATALYZED CROSSLINKING OF ACIDIC PROLINE RICH PROTEINS (APRPs) TO CANDIDA ALBICANS SURFACE PROTEINS BRADWAY 1993 ASSAY (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 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):
ADHERENCE BRADWAY 1993 ASSAY: IMPLICATION OF TGASE CATALYZED CROSSLINKING IN CANDIDA ALBICANS BLASTOSPORE FORM / BUCCAL EPITHELIAL CELLS ADHESION (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).
ANOTHER TRANSGLUTAMINASE SUBSTRATE PROTEIN IN CANDIDA ALBICANS HYPHAL FORM?:
STAAB 2004 ADHESION ASSAY: USING RECOMBINANT Hwp1 (rHwp1N13) (11):
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).
Hwp1 & ADHESION:
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)
ROLE OF Hwp1 IN ADHESION OF CANDIDA ALBICANS TO BUCCAL EPITHELIAL CELLS:
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).
STAAB 1999 ADHESION ASSAY: MUTANTS LACKING Hwp1 (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).
STAAB 1999 ADHESION ASSAY: USING RECOMBINANT Hwp1 (rHwp1ΔC37) (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):
STAAB 1999 ADHESION ASSAY: STABILIZED ADHESION BEING PREVENTED BY TGase INHIBITORS (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):
STAAB 2004 ADHESION ASSAY: USING RECOMBINANT Hwp1 (rHwp1N13) (11):
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).
Hwp1 NON-TRANSLUTAMINASE ADHESION:
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).
LATEX COATED BEADS:
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 & INVASION:
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).
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).
- ACTIVE PENETRATION:
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 & ENDOCYTOSIS:
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):
Hwp1 – ALS3 SIMILARITIES:
- 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).
Hwp1 – ALS3 DIFFERENCES:
- 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).
Als3 IS REQUIRED FOR CANDIDA ALBICANS TO BE ENDOCYTOSED BY ENDOTHELIAL CELLS AND ORAL EPITHELIAL CELLS:
PHAN 2007 DIFFERENTIAL FLUORESCENCE ASSAY (20):
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):
Als3 IS NECESSARY FOR CANDIDA ALBICANS TO BIND TO N-CADHERIN ON ENDOTHELIAL CELLS AND E-CADHERIN ON ORAL EPITHELIAL CELLS:
Previously, we have reported that N-cadherin is one of the endothelial cell surface proteins that mediate endocytosis of C. Albicans (20).
PHAN 2007 AFFINITY PURIFICATION APPROACH (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).
ORAL EPITHELIAL CELLS:
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).
N-CADHERIN AND E-CADHERIN WITH ENDOCYTOSED CANDIDA ALBICANS HYPHAE REQUIRES
PHAN 2007 INDIRECT IMMUNOFLUORESCENCE ASSAY (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):
ENDOTHELIAL & EPITHELIAL CELLS:
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).
ALS1, ALS3 AND CADHERINS:
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).
ALS - N-CADHERIN INTERACTION:
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).
ALS - E-CADHERIN INTERACTION:
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).
PHAN 2007 CONCLUSION:
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).
OTHER CANDIDA ALBICANS ALS INVASINS:
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).
COMPARING ENDOCYTOSIS CAPACITY:
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:
DAMAGE INDUCED BY INVASION: PHAN 2007 51Cr RELEASE ASSAY (20):
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).
KILLED ORGANISM ENDOCYTOSIS:
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 ENDOCYTOSIS:
Latex beads coated with the recombinant N-terminal portion of Als3 are avidly endocytosed by epithelial cells (14).
PHAN 2007 ALS BEADS COATED ASSAYS (20):
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).
ABSENCE OF TANDEM REPEATS IN COATED BEADS ONLY WITH N-TERMINAL PORTION:
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).
Als3 & ENDOCYTOSIS & CADHERINS CONCLUSIONS:
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).
Als3 & ENDOCYTOSIS & CLATHRIN-DEPENDENT ENDOCYTOSIS PATHWAY:
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).
INVASION & ACTIVE PENETRATION:
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).
SAPS & ACTIVE PENETRATION:
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).
E-cadherin & ACTIVE PENETRATION:
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).
INVASION INTO/BETWEEN EPITHELIAL CELLS MECHANISM:
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).
E-CADHERIN & TRANSGLUTAMINASE: THE LINK BETWEEN ENDOCYTOSIS AND ACTIVE PENETRATION?:
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).
E-cadherin & ACTIVE PENETRATION:
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).
E-CADHERIN & TRANSGLUTAMINASE:
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:
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).
E-CADHERIN - TRANSGLUTAMINASE TYPE 1 COLOCALIZATION:
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):
TRANSGLUTAMINASE TYPE 1 CONCENTRATED AT CADHERIN-BASED ADHERENS JUNCTONS:
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 TYPE 1 CROSSLINKING ACTIVITY CONCENTRATED AT CADHERIN-BASED ADHERENS JUNCTIONS:
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).
CROSSLINKING ACTIVITY OF ENDOGENOUS TGASE:
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).
HIIRAGI 1999 CONCLUSIONS & SUGGESTIONS:
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).
Hwp1 & TRANSLOCATION:
Microbial translocation is defined as the migration of viable microorganisms or bacterial endotoxins (i.e., bacterial lipopolysaccharide (LPS), peptidoglycan, and lipopeptides) from the intestinal lumen to the mesenteric lymph nodes (MLN) and other extraintestinal sites.
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).
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).
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):
INTESTINAL COLONIZATION OF C57BL/6 MICE WITH CANDIDA ALBICANS:
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:
NICHE NO SPECIFIC:
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:
TRANSGLUTAMINASE RELATED SEQUENCE COMPARISON 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)
P46593|HWP1_CANAL QEPCDDYPQQQQQQEPCDY 45-63
P02666|CASB_BOVIN NKKIEKFQSEEQQQTEDEL 42-60
P05814|CASB_HUMAN KQKVEKVKHEDQQQGEDEH 33-51
:: :. ::*** :
P46593|HWP1_CANAL EPCDYPQQQPQEPCDYPQQP 71-90
P02666|CASB_BOVIN QDKIHPFAQTQSLVYPFPGP 61-80
P05814|CASB_HUMAN QDKIYPSFQPQPLIYPFVEP 52-71
* * * *
P46593|HWP1_CANAL QQQQQQEPCDYPQQQQQEEPCDYPQQQP 53-80
P02666|CASB_BOVIN QQQTEDELQDKIHPFAQTQSLVYPFPGP 53-80
P05814|CASB_HUMAN QQQGEDEHQDKIYPSFQPQPLIYPFVEP 44-71
*** * * * ** *
Compare Beta casein Human and Bovine with Hwp1 here:
P46593|HWP1_CANAL DYPQQPQEPCDNPPQ 105-119
P02666|CASB_BOVIN NIPPLTQTPVVVPPF 88-102
P05814|CASB_HUMAN NILPLAQPAVVLPVP 80-94
: * * * **
P46593|HWP1_CANAL EPCDYPQQ 59-66; 71-78; 82-89; 92-99; 102-109
P02666|CASB_BOVIN IPNSLPQN 81-88
P05814|CASB_HUMAN PYGFLPQN 73-80
* . **
P46593|HWP1_CANAL PQQPQEPCDNPPQPDVP 107-123
P02666|CASB_BOVIN LPQNIPPLTQTPVVVPP 85-101
P05814|CASB_HUMAN LPQNILPLAQPAVVLPV 77-93
TRANSGLUTAMINASE SUBSTRATE EQUIVALENCE Hwp1 & casein:
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).
HISTOLOGICAL COMPARISON: Hwp1 versus Casein:
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.
TRANSGLUTAMINASE RELATED SEQUENCE COMPARISON Hwp1 & Gluten:
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)
P46593|HWP1_CANAL VPQVDGQGETE 22-32
P04727|GDA7_WHEAT VPQLQPKNPSQ 22-32
P04725|GDA5_WHEAT VPQLQPQNPSQ 25-35
P02863|GDA0_WHEAT VPQLQPQNPSQ 25-35
P18573|GDA9_WHEAT VPQLQPQNPSQ 25-35
***:: : ::
P46593|HWP1_CANAL QEPCDDYPQQQQQQEP 45-60
P04727|GDA7_WHEAT QQPQEQVPLVQQQQFP 33-48
P04725|GDA5_WHEAT QQPQEQVPLVQQQQFP 36-51
P02863|GDA0_WHEAT QQPQEQVPLVQQQQFL 36-51
P18573|GDA9_WHEAT QQPQEQVPLVQQQQFP 36-51
*:* :: * ****
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 YPQQQQQEEPCDYPQQQPQEPCDYPQQPQEPCDYPQQPQEPCDYPQQP 63-110
P04727|GDA7_WHEAT FPPQQPYPQPQPFPSQQPYLQLQPFPQPQPFLPQLPYPQPQSFPPQQP 54-101
P04725|GDA5_WHEAT FPPQQPYPQPQPFPSQQPYLQLQPFPQPQPFPPQLPYPQPQSFPPQQP 57-104
P02863|GDA0_WHEAT FPPQQPYPQPQPFPSQLPYLQLQPFPQPQLPYSQPQPFRPQQPYPQPQ 57-104
P18573|GDA9_WHEAT FPPQQPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQLPYPQPQLPYPQ 57-104
* ** * * *** *** ** *
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 PPQPDIPCDNPPQPDIPC 137-154; 147-161
P04727|GDA7_WHEAT QQQQQILQQILQQQLIPC 134-151
P04725|GDA5_WHEAT QQQQQILQQILQQQLIPC 135-152
P02863|GDA0_WHEAT QQQQQILQQILQQQLIPC 129-146
P18573|GDA9_WHEAT QQQQQILQQILQQQLIPC 146-163
* :* : * ***
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).
NIEUWENHUIZEN 2003 (2):
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:
NIEUWENHUIZEN 2003 (2):
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:
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).
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.
ROCKY MOUNTAIN ANALYTICAL 2014:
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.
HISTOLOGICAL COMPARISON: Hwp1 versus Gluten:
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:.
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).
FUSION, DISRUPTION OR PORE FORMATION AT PLASMA MEMBRANE; DIRECT MEMBRANE ENTRY:
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).
VIRUSES INTO ENDOCYTIC COMPARTMENTS; ENDOCYTOSIS:
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 oﬀers. 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 traﬃcking events within intracellular vesicles (endocytic compartments).
Endocytic compartments are pleiomorphic structures that fuse with one another to promote ligand traﬃcking. Subsequent to internalization, endosomes often undergo complex
traﬃcking and sorting events. Two principal post-internalization endocytic traﬃcking routes exist in the cell, which can be termed recycling or lysosome-targeted. Regulation of sorting and traﬃcking 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 diﬀer 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 suﬃcient 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 traﬃcking 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).
VARIABILITY OF gp160:
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).
VARIABILITY OF gp120:
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
THE SECOND CONSERVED REGION (C2) REGION OF THE HIV/SIV gp120 ENVELOPE PROTEIN:
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 PVVSTQLLLN PEPTIDE OF THE C2 REGION OF THE HIV gp120 ENVELOPE PROTEIN:
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 KTIIVQLN PEPTIDE OF THE C2 REGION OF THE HIV gp120 ENVELOPE PROTEIN:
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).
THE VASOACTIVE INTESTINAL PEPTIDE (VIP) VERSUS THE C-TERMINUS OF THE C2 REGION OF THE HIV-1 gp120:
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).
DISEASE PROGRESSION VERSUS THE C-TERMINUS OF THE C2 REGION OF THE HIV-1 gp120:
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
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 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).
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).
TWO WAYS OF HIV INVASION:
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):
- HIV WAY ONE: FUSION WITH PLASMA MEMBRANE
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)
DIRECT FUSION FACTS:
- 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).
- HIV WAY TWO: RECEPTOR MEDIATED ENDOCYTOSIS:
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):
gp120 & gp41 & TRANSGLUTAMINASE
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 NOT A TRANSGLUTAMINASE SUBSTRATE:
gp160 is completely ineffective as transglutaminase substrate (23).
gp120 & gp41 ARE TRANSGLUTAMINASE SUBSTRATES:
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).
NON-SUBSTRATE RESIDUES (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).
SUBSTRATE RESIDUES (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).
THE PVVSTQLLLN PEPTIDE: MARINIELLO 1993 (gp120) (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)
THE KTIIVQLN PEPTIDE: FOTGCREN 2014:
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).
ALIGNMENT OF THE C-TERMINUS OF THE C2 REGION OF THE HIV / SIV gp120:
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
WITH Q 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
Q9IDV2|ENV_HV1YB IVIRHHGGNLLVQWNETVSINC HIV-1 group N YBF106
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)
: : *
WITHOUT Q KTIIVHLN
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
P12449|ENV_HV2SB WHGRDNRTIISLNKYYNLTILC HIV-2 subtype A SBLISY
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)
: : *
PEPTIDE NTM REGION AVAILABLE INFORMATION:
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
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.
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.
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)
HUMAN VASOACTIVE INTESTINAL PEPTIDE (VIP) IS A TRANSGLUTAMINASE SUBSTRATE AND SHARE HOMOLOGY WITH KTIIVQLN HIV gp120 PEPTIDE (26):
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: Ruoppolo 2003 (26) Identification of Q and K residues in VIP sensitive to tTG activity:
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):
NON-SUBSTRATE RESIDUES (23):
gp41 fragments 27-42, 104-116, 128-144 and 143-154 are completely ineffective as TGase amino-acceptor substrates (23).
SUBSTRATE RESIDUES (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)
THE CS3 REGION OF gp41:
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).
CD4 & TRANSGLUTAMINASE
CD4 COULD BE A TRANSGLUTAMINASE SUBSTRATE:
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).
TRANSGLUTAMINASE CROSSLINKING BETWEEN HIV AND HUMAN PROTEINS:
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).
TRANSGLUTAMINASE & CLATHRIN MEDIATED ENTRY INTO HOST CELLS:
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).
TRANSGLUTAMINASE & APOPTOSIS:
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).
BATMANGHELIDJ 1989 (29):
It has been proposed that transglutaminase TGE is involved in the biochemical pathway of programmed cell death (29).
TRANSGLUTAMINASE & SYNCYTIA:
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.
BATMANGHELIDJ 1989 (29):
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).
LIFE-STYLES & HIV & AIDS & TRANSGLUTAMINASE
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.
BATMANGHELIDJ 1989 (29)
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).
LIMITED NUMBER OF T-CELLS AFFECTED
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).
ANTIBODIES TO HIV AND DISEASE PROGRESSION
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):
TRANSGLUTAMINASE OF SEMEN (TGE)
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).
COMMON BETWEEN HOMOSEXUALS, DRUG ADDICTS AND HEMOPHILIACS:
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:
TRANSGLUTAMINASE SUBSTRATE ABILITY COMPARISON gp120 & gp41 & casein:
MARINIELLO 1993 (gp41) ASSAY OF TGASE ACTIVITY (23):
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).
MARINIELLO 1993 (gp120) ASSAY OF TGASE ACTIVITY (22):
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:
TRANSGLUTAMINASE RELATED SEQUENCE COMPARISON gp120 & 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))
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))
RELATIONSHIPS HIV VERSUS GLIADIN:
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).
CD4+ T CELLS:
HIV recruitment of CD4+ T cells to infect them and increase their number by replication:
HIV preferentially infects HIV-speciﬁc CD4+ T cells in vivo. Simply by virtue of their speciﬁcity, HIV-speciﬁc 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-speciﬁc T cells into infected lymphoid sites, enhanced by inﬂammatory 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-speciﬁc 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.
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).
GASTROINTESTINAL ABSORPTION OF INTACT DIETARY PROTEINS:
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).
THE GUT AS A "BARRIER"
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 GUT AS AN IMMUNE AND NEURAL ORGAN
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).
EVIDENCE FOR INTACT PROTEIN ABSORPTION
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).
MEASUREMENTS OF MACROMOLECULES IN BLOOD
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 GUT AS AN IMMUNE ORGAN - THE M-CELL ROUTE
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).
REGIONS OF GASTROINTESTINAL TRACT
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).
PARACELLULAR OR TRANSCELLULAR ROUTES
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 (13a) 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 (50,52) 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 diseñase 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 (102), 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).
FOOD ALLERGY AND INTOLERANCE
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 (1).
Gluten is a protein (a complex mixture of storage proteins) found in various food grains. 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).
Gluten is a substrate for transglutaminases ().
ABSORPTION OF GLIADIN
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).
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.
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: Batmanghelidj (1989) Aids: is transglutaminase the primary permissive factor for HIV establishment and spread? Science In Medicine Simplified, Vol. 1. pp. 3-9.
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”
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
“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.”
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
"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.”
CONSPIRACY / MONEY:
“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.”
WE WILL WIN:
“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
“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.”
“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”
Serbian professional tennis player and former 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 16 - 8 - 2014