FOTGCREN HYPOTHESIS

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

 

Aim of this hypothesis: link the crosslinking enzymatic activity of human transglutaminase binding covalently certain food proteins to endogenous proteins with all possible diseases.

 

SUPPOSED NON-LINKED FACTS:

 

1) INTACT DIETARY PROTEIN ABSORPTION

There is now no reasonable doubt that small quantities of intact proteins do cross the gastrointestinal tract in animals and adult humans (1).

There now is irrefutable evidence that small amounts of intact peptides and proteins do enter the circulation under normal circumstances (1).

GLUTEN:

Hemmings results indicating massive scale absorption of 40-70% of bovine IgG or gliadin as high-molecular-weight fragments in adult rats (macromolecular fragments of protein were absorbed on a large-scale; 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) (1).

CASEIN:

The experiments by Gardner (1975, 1978, 1982) showed absorption, including significant transmucosal passage, of peptide bound N. The amount crossing in intact form depended on the concentration of the protein digest in the intestinal lumen and on the origin of the partial digest being perfused through the lumen. Casein and soy-bean digests appeared to give rise to significant passage of intact peptide while muscle "peptone" did not. ("peptones" (that is, mixtures of polypeptides)). Also, different digests of casein gave rise to different amounts of intact-peptide passage (2).

One study on entry of peptides into superior mesenteric blood of anesthetized guinea pigs during absorption of an enzymic digest of casein suggested that more than 10% of the absorbed amino N could have been absorbed in the form of small peptides (2).

2) HUMAN TRANSGLUTAMINASE: THE UBIQUOUS PROTEIN CROSSLINKING ENZYME

Ubiquitous: existing or being everywhere, especially at the same time; being or found everywhere at the same time: omnipresent, existing or being everywhere at the same time: constantly encountered: Synonyms: omnipresent, widespread, everywhere.

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective crosslinking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis (Reiss 2011).

Transglutaminases (TGs) are enzymes that catalyze the formation of isopeptide bondsa type of covalent linkage between two protein molecules (4).

This reaction is called crosslinking or transamidation.

The crosslinked products are highly resistant to mechanical challenge and proteolytic degradation and their accumulation is found in a number of tissues and processes where such properties are important including skin, hair, blood clotting and wound healing (3).

The cross-links formed in this reaction are extremely resistant to proteolytic cleavage or mechanical disruption (4).

3) GLUTEN & CASEIN: PERFECT TRANSGLUTAMINASE SUBSTRATES OF CROSSLINKING

GLUTEN & CASEIN:

TG2 recognizes only specific glutamine residues, which helps to limit its activity since proteins with appropriate glutamines are fairly uncommon (4).

Gluten and casein are transglutaminase substrates (3).

4) CROSSLINKING OF PROTEINS & DISEASE: A FORESEEABLE CONNECTION

It can be speculated that, under conditions where undigested gliadin peptides have access to the circulation due to a barrier disturbance, these peptides can potentially be cross- linked to proteins in organs where TG2 is available for the reaction. In this way, gliadin peptides may also be an initiating factor for other autoimmune diseases (8).

It has been suggested that the up-regulation of TG2 in CD may generate additional antigenic neo-epitopes by cross-linking or deamidating viral, nutritional or endogenous proteins, and thereby contribute to initiation of autoimmune diseases (8).

Deregulation of transglutaminase activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin (3).

This topic has attracted much interest, and in very recent years has yielded interesting new data with respect to the relevance of Tgases in chronic diseases, in particular in (a) inflammatory diseases, including wound healing, tissue repair and fibrosis, and autoimmune conditions; (b) chronic degenerative diseases (e.g. arthritis, atherosclerosis and neurodegenerative pathologies); and (c) tumour biology. In the majority of these diseases the prevalent role of tTgase appears to be related to its interaction with, and stabilization of, the cell matrix, rather than as a major player in apoptosis (3).

Neurodegenerative disorders are characterized by progressive neuronal loss and the aggregation of disease specific pathogenic proteins in hallmark neuropathologic lesions. Many of these proteins, including amyloid Αβ, tau, α-synuclein and huntingtin, are cross-linked by the enzymatic activity of transglutaminase 2 (TG2) (4).

5) BUG PROTEINS; IN VIVO CROSSLINKING LESSONS

Some pathogens hijack host transglutaminases to enhance their virulence such as Candida albicans, which is cross-linked to host oral epithelial cells via the hyphal protein Hwp1 as a necessary precursor to systemic candidiasis (Staab 2013).

GLUTEN & CASEIN:

Gluten and casein share homology in their aminoacid sequences with Candida Albicans Hwp1 protein.

These homologies are aligned to the aminoacids targeted by the crosslinking activity of human transglutaminases.

6) STRESS: THE BOOSTER OF TRANSGLUTAMINASE

Cellular stress leads to an upregulation of TG2 (4).

                                                                                                                

1) INTACT DIETARY PROTEIN ABSORPTION:

The most relevant meaning for the term "absorption" in gastrointestinal physiology to the present question is whether intact peptides produced in the lumen of the gastrointestinal tract during digestion reach peripheral tissues; overall passage from intestinal lumen to venous blood or lymphatics, that is, transmucosal or transepithelial transport (2).

 

Although it used to be stated dogmatically in most physiology textbooks that proteins are digested wholly to free amino acids within the lumen of the intestine and that free amino acids therefore are the sole form of assimilation of protein, there is now abundant evidence that this is not true (2).

 

Peptides predominate over free amino acids in the small intestinal lumen during assimilation of a protein meal (2).

 

Although there is enough evidence that intact peptides are not a major nutritional form in which the amino N enters the circulation, it is now clear that significant amounts of some peptides can and do cross the intestine in intact form (2).

 

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. 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 (1).

 

The small quantities of intact proteins that do cross the gastrointestinal tract in animals and adult humans are 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 (1).

 

Intact protein absorption must now be regarded as a normal physiological process in humans and animals (1).

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 (1).

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 (1).

 

It is clear that many substances that until recently were regarded as "nonabsorbable" can be absorbed in small but readily measurable amount. Indeed, some substances, such as poly(ethylene) glycols once used as "unabsorbable markers" in experimental gastroenterology, are now being used as probes for measurement of intestinal permeability or "leakiness". Permeability measured by these probes is now known to be increased in a number of common gastrointestinal disorders, and it also appears that significant numbers of apparently asymptomatic individuals may have relatively "leaky" intestines (2).

 

There is now adequate sound evidence that some peptides, including some biologically active ones, can be and are absorbed across the small intestine in intact (that is, unhydrolyzed or incompletely hydrolyzed) form. The amounts are generally, possibly invariably, small. Although they are regarded as nutritionally irrelevant, it does not follow that they are biologically insignificant. Quantitative data on this process are quite inadequate and, especially since indirect evidence suggests strongly that several commonly ocurring factors are likely to augment it, further data are urgently needed (2).

 

The amount of intact peptide crossing the intestine depends on a balance between (1) peptide digestibility, (2) intestinal permeability or "leakiness," and (3) mucosal digestive capacity, all factors that are known to be subject to change (2).

Indirect evidence suggests that the extent of absorption of intact peptides is likely to depend on a balance among (a) digestibility of peptide or protein, (b) intestinal permeability, and (c) mucosal digestive capacity (2).

 

IN VITRO:

Although it is clear that the vast majority of peptides are hydrolyzed by the brush-border and cytoplasmic peptidases during absorption, numerous studies using several different in vitro preparations have shown that small amounts of intact peptides do cross rat and hamster small intestine (2).

 

IN VIVO:

Experiments performed both with single dipeptides and with partial digests of proteins (in vivo on intestine in anesthetized animals) have provided corroboration for the tentative conclusions drawn in the corresponding experiments in vitro, namely, that small but measurable quantities of peptides can cross the small intestine and that the magnitude of this phenomenon is inversely related to the susceptibility of particular peptides to enzymic hydrolysis (2).

 

REGIONS OF GASTROINTESTINAL TRACT

GARDNER 1988 (1):

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 (1).

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 (1).

 

DIFFERENT CELLS IN INTESTINE:

GARDNER 1988 (1):

THE M-CELL ROUTE - THE GUT AS AN IMMUNE ORGAN

The gastrointestinal tract is a major site of immunologically competent tissue -hence the expresion gut-associated lymphoid tissue (1).

Throughout the intestine, the lamina propia contains a substantial population of lymphocytes and macrophages (1).

The small intestine contains many Peyer's Patches, clearly discernible nodules of lymphoid tissue (1).

Peyer's Patches were covered by a special type of cell. 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 (1).

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 (1).

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 (1).

One important question is that of the relative contributions made by M cells and by "ordinary" columnar epithelial absorptive cells to macromolecular absorption (1).

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. 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 (1).

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 (1).

 

ROUTES OF INTACT PROTEIN ABSORPTION:

Research of the last 30 years led to the schematic view of absorption models provided below. Proteins are digested by multiple hydrolases, associated with membranes of columnar epithelial cells and secreted into the gut lumen. Specific carrier molecules transport amino acids, peptides and small proteins. In contrast, proteins are incorporated by M-cells present in the follicle-associated envelope of Peyer’s patches or through endocytosis by columnar epithelial cells. The incorporation of proteins by diffusion through intercellular spaces may be supported by intrinsic proteolytic activity of the administered proteases to solve the tight junctions or by reorganization processes of the epithelial layer (persorption) (Lorkowski 2012).

Schematic view on absorption mechanisms of the gastrointestinal tract for amino acids, peptides and protein molecules (Lorkowski 2012)

Although the maximum size of peptide transported by the specific peptide carriers in the brush-border membrane probably is tri- or possibly tetrapeptide, much larger peptides (and, indeed, proteins) do cross the mucosa. However, in their cases other transport mechanisms -for example, the transcellular route vía pinocytosis (or, less plausible, lipid-soluble route) or the paracellular route through "tight" junctions -are involved (2).

GARDNER 1988 (1):

TRANSCELLULAR OR PARACELLULAR ROUTES:

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 (1).

Both (a) the paracellular pathway through the "tight" junctions -arguably inaptly named because of their permeability and their major role in fluid  and ion transport -and through cell extrusion zones and areas of damaged mucosa, and (b) the transcellular pathway may be involved in intact protein absorption. However, most evidence favors the latter route as dominant, especially in healthy intestine, although the process is a complex one involving metabolic energy expenditure, cytoplasmic tubule formation, and lysosomal processing. Bockman & Winborn observed ferritin passing through hámster intestinal cells by pinocytosis, with none passing between the cells. Likewise, the corroboration found in more recent work by Desjeux and colleagues suggests that only a small fraction of absorbed horseadish peroxidase crossed by the paracellular route in their rabbit ileal experiments in vitro. Hence, the transcellular route seems to be more important than the paracellular route, although increases in it caused by disease or with excessive exfoliation may even make it a predominant route. Their observations on biopsy material from malnourished infants suggested that decreases in intracellular processing were the basis for the increased transepithelial passage of the peroxidase marker (1).

The permeability probes discussed above for use in humans appear to reflect paracellular leakiness , which undoubtedly is increased at least to small molecules in many diseases. This route also can be used by particles and macromolecules: Volkheimer, who considered that motility was a driving force for particulate absorption, coined the term "persorption" for this process (1).

GARDNER 1988 (2):

PARACELLULAR:

The paracellular or intercellular path via  the inaptly named "tight" junctions which are particularly susceptible to reversible "opening" or "loosening" in the presence of hyperosmotic intestinal luminal contents (2).

 

TRANSCELLULAR ROUTE: ENDOCYTOTIC MECHANISMS

Transmucosal passage of intact peptides has been remarkably neglected, probably because the bulk of evidence has indicated that entry of peptide to the body accounts for a nutritionally irrelevant fraction of the total amount of amino N absorbed during assimilation of a protein meal. Attention has been focused on absorption of free amino acids. While this may be appropriate from the point of view of contribution to the body's N balance, it neglects the possible consequences of small quantities of biologically active peptides, some of which may be highly potent molecules, entering the circulation (2).

GARDNER 1988 (1):

ENDOCYTOTIC MECHANISMS:

The scheme described by Walker & Isselbacher meets most of the histochemical and electron microscopic observatíons on macromolecule transport (1):

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 (1).

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 (1).

 

INTERSPECIES DIFFERENCES: DIFFERENT TRANSPORT MECHANISMS:

GARDNER 1988 (1):

A sight must not be lost of one particular difference between humans and other mammals. Most species (not humans) 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 (1).

Hence, the mechanisms and routes of intact protein transport in neonatal animals may be fundamentally different from those operating in humans (1).

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 (1).

 

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 (1).

 

ANTIBODIES TO DIETARY PROTEINS IN BLOODSTREAM:

Numerous authors have shown the presence in blood of antibodies to food proteins. There have been suggestions that the occurrence or levels of such antibodies may be increased in disease, but, to date, the concept of "macromolecular absorption in intestinal disease" has not attracted general attention. Certainly, this information indicates that molecular size is no absolute bar to passage across the gastrointestinal tract (2).

GARDNER 1988 (1):

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 (1).

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 (1).

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 (1).

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 (1).

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) (1).

MEASUREMENTS OF MACROMOLECULES IN BLOOD

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 (1).

GARDNER 1988 (2):

The liver and kidney are also major sites of peptide hydrolase activity. While their physiological functions are not known, these too may act as a defense mechanism to minimize the half-life of peptides that escape digestion. Thus, intravenously administered peptides are quite rapidly cleared from the blood (e.g., Adibi and Krzysik, 1977) (2).

 

DISEASE
Although there are good reasons for supposing that the amount of intact peptide absorbed across the intestine may be greatly increased in various pathological circumstances, satisfactory quantitative evidence on this aspect is lacking at present (2).

Works performed in numerous different centers all agree in showing that many common pathological situations do lead to enhanced intestinal permeability or "leakiness," which is often reversible (2).

Apart from showing that intestinal permeability is increased in various pathological states and by hypertonic solutions, these investigations additionally point to the likelihood, indeed the near certainty, that peptides too can be expected to cross the intestine unless the rate of their hydrolysis is so high that permeation definitely becomes the rate-limiting step for every molecule (2).

Relatively common disorders and other situations (for example, hyperosmolar luminal contents) lead to an increase in intestinal permeability or "leakiness." (2).

The high biological potency of many small peptides and the fact that biologically active peptides (for example, "exorphins") can be produced during protein digestion make it imperative that the significance of absorption of intact peptides be very carefully reconsidered (2).

GARDNER 1988 (1):

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 (1).

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 (1).

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 (1).

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 (1).

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 (1).

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 (1).

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 (1).

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 (1).

The possible association between gluten, schizophrenia, and celiac disease merits reexamination (2).

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 (1).

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 (1).

 

CERTAIN FOOD PROTEINS
Are peptides absorbed across the gastrointestinal tract? If the answer is "yes," then we need to know the likely magnitude of the process, whether any specific types of peptide are particularly well absorbed, and in what physiological or pathological circumstances the process may be enhanced (2).

 

Data gained by four independent approaches all lead to the same conclusion: namely, that significant amounts of some (but not all) peptides can cross the intestine in intact form and, if biologically active, may retain this activity. These approaches are (1) biological activity exerted by orally administered peptides in vivo; (2) transintestinal passage of intact peptides across perfused intestine or intestinal sacs etc. in vitro; (3) transintestinal passage of intact peptides across ligated or perfused loops of intestine in vivo; and (4) observations on absorption of other macromolecules including oligosaccharides and intact proteins (2).

 

The peptides that have been shown to cross the intestine in intact form in relatively large amounts have been characteristically indigestible peptides, that is, relatively resistant to enzymic hydrolysis (2).

 

GLUTEN:

GARDNER 1988 (1):

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 (1).

 

Although the name “closure” 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 (1).

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 (1).

 

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 (1).

 

CASEIN:

GARDNER 1988 (2):

ABSORPTION OF INTACT PEPTIDES IN VITRO:

The experiments by Gardner (1975, 1978, 1982) showed absorption, including significant transmucosal passage, of peptide bound N. The amount crossing in intact form depended on the concentration of the protein digest in the intestinal lumen and on the origin of the partial digest being perfused through the lumen (2).

Casein and soy-bean digests appeared to give rise to significant passage of intact peptide while muscle "peptone" did not. ("peptones" (that is, mixtures of polypeptides)). Also, different digests of casein gave rise to different amounts of intact-peptide passage (2).

However, apart from strongly suggesting that some (but not all) proteins give rise to peptides that cross the intestine in intact form and that the fraction of amino N crossing in this form might even approach 30%, these experiments do not permit firm conclusions to be drawn. Also, in spite of other evidence that the organ was being maintained in vitro in a physiologically viable condition (and in spite of the observation that no intact peptide crossed during perfusion with the muscle peptone), the fact remains that one cannot prove beyond question that the experimental conditions in vitro had not made the organ or its intercellular junctions abnormally "leaky" (2).

One study on entry of peptides into superior mesenteric blood of anesthetized guinea pigs during absorption of an enzymic digest of casein suggested that more than 10% of the absorbed amino N could have been absorbed in the form of small peptides, although the difficulties in quantitatively analyzing heterogeneous mixtures of small peptides in blood make it risky to attempt to draw conclusions about the actual fraction absorbed in peptide-bound form (Gardner et al., 1983). The general conclusion that some intact peptides were absorbed in vivo seems inescapable (2).

Less is known about the peptidase activity in plasma. However, it is interesting that Kreil et al. (1983) reported that beta-casomorphins (estimated by radioimmunoassay) were quite rapidly degraded by bovine or rat plasma even though these peptides were resistant to digestion by gastrointestinal proteases and peptidases. The enzyme responsible was suggested to be dipeptidyl-dipeptidase IV (2).

 

2) HUMAN TRANSGLUTAMINASE: THE UBIQUITOUS PROTEIN CROSSLINKING ENZYME

Transglutaminases (TGs) are (enzymes) found in a wide range of species from bacteria to humans (4).

Transglutaminases (Tgases) are a widely distributed group of enzymes which have now been found in microorganisms, plants, invertebrates, amphibians, fish, birds and mammals (3).

Transglutaminases catalytic triad necessary for cross-linking activity and the nearby amino acids are particularly highly conserved, suggesting the importance of these residues (4).

BODY LOCALIZATION:

In mammals, eight distinct Tgase isoenzymes have been identified at the genomic level; however, only six have so far been isolated and characterized at the protein level, after purification either from natural sources or as recombinant proteins. The fully characterized enzymes include (a) the circulating zymogen Factor XIII, which is converted, by a thrombin-dependent proteolysis, into the active Tgase Factor XIIIa, (plasma Tgase) involved in stabilization of fibrin clots and in wound healing; (b) the keratinocyte Tgase (type 1 Tgase) which exists in membrane-bound and soluble forms, is activated severalfold by proteolysis and is involved in the terminal differentiation of keratinocytes; (c) the ubiquitous tissue Tgase (tTgase; type 2 Tgase), whose role is still debated; (d) the epidermal hair follicle Tgase (type 3 Tgase), which also requires proteolysis to become active and, like type 1, is involved in the terminal differentiation of the keratinocyte; (e) the prostatic secretory Tgase (type 4 Tgase), essential for fertility in rodents; and (f) the recently characterized type 5 Tgase (3).

Below: Tgases characterized at the protein level: In addition to the eight dierent enzymes listed below, a further Tgase-like protein has been characterized from red blood cells. This protein, named erythrocyte-bound 4.2, has strong sequence identity with the Tgase family of proteins, but is inactive because of a substitution of alanine for the active-site cysteine: it forms a major component of the erythrocyte membrane skeleton (3):

INTESTINE:

In the small intestine, TG2 (tissue transglutaminase) is mainly localized extracellularly in lamina propria and shows increased levels in patients with CD (celiac disease) (8).

 

BRAIN:

Tissue transglutaminase (TG2) is found in many neural tissues including the brain, spinal cord, and peripheral nerves. Within the brain, it is present in many regions including frontal and temporal cortex, hippocampus, substantia nigra and cerebellum. It is primarily neuronal, but can be found in glia as well (4).

Transglutaminase 2 (TG2) is highly expressed in the human central nervous system (CNS) (4).

Type 2 transglutaminase (tTgase) has been isolated from mammalian brain (3).

 

OTHER:

TG2 is also expressed in many other tissues including fibroblasts, endothelial and smooth muscle cells of blood vessels, and in the kidney, colon, liver, heart, lung, and spleen (4).

 

UBIQUITOUS TRANSGLUTAMINASE:

UBIQUITOUS DEFINITION:

Ubiquitous: existing or being everywhere, especially at the same time; being or found everywhere at the same time: omnipresent, existing or being everywhere at the same time: constantly encountered: Synonyms: omnipresent, widespread, everywhere.

UBIQUITOUS TRANSGLUTAMINASE:

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective crosslinking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis (Reiss 2011).

Of the known TGs, TG2 is the most ubiquitous (4).

Tissue transglutaminase (TGase) is an ubiquitous enzyme presents in all tissues, with both intra- and extracellular localization (Naiyer 2008).

Tissue transglutaminase is an ubiquitous cellular enzyme (Dieterich 2006), found both intracellularly and extracellularly in many tissues and organs, including the small intestine (Bethune 2008).

Ubiquitous tissue transglutaminase (tTG) is one member of the large transglutaminase (TG) family, which catalyze posttranslational modification of proteins by establishing ϵ(γ-glutamyl)lysine cross-linking and/or covalent incorporation of polyamines (Wu 2004).

Transglutaminase 2 is a ubiquitous multifunctional mammalian protein that catalyzes the formation of intermolecular isopeptide bonds between glutamine and lysine residues of a few selected proteins (Sollid 2011)

Transglutaminase is a naturally occurring and ubiquitous enzyme which is responsible for the transfer of acyl groups (and resulting crosslinking) between glutamine and lysine residues (Bernard 1998)
"Tissue transglutaminase" is an ubiquitous enzyme present in all mammalian tissues. A recent systematic immunohistochemical study has shown that this wide spread biochemical occurrence was determined by its presence in ubiquitous cell types such as endothelial and smooth muscle cells. A few specific cell types (mesangial cells, renomedullary interstitial cells, thymic subcapsular epithelium, colonic perycristal fibroblasts) also express the enzyme costitutively. Moreover, it is well known that the enzyme may be induced or activated in other cell types by several stimuli (Columbano 2012)

Tissue transglutaminase (tTG) is a calcium dependent ubiquitous enzyme which catalyses posttranslational modification of proteins and is released from cells during inflammation (Di Sabatino 2012).

Tissue transglutaminase (tTG) is an ubiquitous enzyme that crosslinks lysine and glutamine residues in peptides and proteins (Ritter 2008)

Transglutaminase 2 (TG2) is a unique member of the transglutaminase family owing to its specialized biochemical, structural and functional elements, ubiquitous tissue distribution and subcellular localization, and substrate specificity (Odii 2014)

Transglutaminase 2 (TG2, “tissue” transglutaminase, EC 2.3.2.13) is an ubiquitous cellular protein also present in the extracellular matrix where it catalyzes Ca2+-dependent protein cross-linking via N′(γ-glutamyl)lysine bonds or the deamidation of glutamine residues (Simon-Vecsei 2012)

TG2 is a ubiquitous member of the transglutaminase family of enzymes and is implicated in such diverse processes as inflammation, wound healing, apoptosis, neurodegenerative disorders and cancer (Chhabra 2009).

TG2 is the most ubiquitous of the TG family members and expressed in many tissues such as bone, cartilage, kidney, colon, liver, heart, lung, spleen, blood and nervous tissue. TG2 is expressed by many cell types such as osteoblasts, chondrocytes, mesenchymal stem cells (MSCs), neuronal and glial cells, phagocytes, monocytes, neutrophils and T-cells and pancreatic β-cells (Myneni 2015)

Tissue transglutaminase (TG2) is a Ca2+-dependent enzyme and probably the most ubiquitously expressed member of the mammalian transglutaminase family. The most ubiquitous mammalian transglutaminase is tissue transglutaminase (TG2) and has been the subject of much research due to its association with a variety of disease states such as metastatic cancer, celiac disease, fibrosis and neurodegenerative disorders (Badarau 2011)

TGM2 is a unique transglutaminase family member. Within the TGM family, TGM2 is the most ubiquitous enzyme and is found in numerous tissues, including the human peripheral and central nervous system (Tovar-Vidales 2008)

Transglutaminase 2 (TG2) is a ubiquitous enzyme involved in diverse biological processes (Kim 2014)

Transglutaminase 2 (TG2) is a ubiquitous member of the transglutaminase family found in many tissues and cell types (Iismaa 2002)

CELLULAR LOCALIZATION:

TG2 can be found both intra-and extra-cellularly. Early work showed that when NIH 3T3 cells were stably transfected with inducible TG2, it could be found in both the intracellular and extracellular compartments after induction. Various studies have confirmed that TG2 can be found extracellularly by identifying γ-glutamyl-ε-lysine bonds, the hallmark linkage formed by TGs, in the extracellular matrix (ECM) as well as on the cell surface by direct visualization of TG2 via immunocytochemistry and electron microscopy. The behavior of TG2 in these different compartments varies. In duodenal biopsy specimens, for example, TG2 localized on the cell surface does not demonstrate enzymatic activity, whereas TG2 in the ECM does (Maiuri, et al., 2005). Undoubtedly, the extracellular environment contributes to the activity of TG2 in each of these compartments and this must be kept in mind in studying the native biological functions of TG2 (4).

It has been reported that TGM2 expression is mostly detected in the cytoplasm of cells, but it is also found in the plasma membrane and the nucleus. In addition, TGM2 can be secreted into the ECM (Tovar-Vidales 2008).

CYTOSOL:

Tissue transglutaminases (TG) (e.g., liver TG, keratinocyte TG, epidermal TG, prostate TG and erythrocyte TG) are located inside the cells, and therefore, act as intracellular enzymes (Matsuka 2010).

Within the cell, TG2 is essentially a cytosolic protein with about 80% found in this compartment. However, it has been suggested that TG2 in the cytosol is enzymatically inactive due to low concentration of calcium (~100nM), which is necessary for its enzymatic activity, and high concentration of GTP (~100uM), which as discussed above is a negative regulator. This fact has led to the hypothesis that cytosolic TG2 is only activated upon cellular stress, which is frequently associated with energy depletion leading to decreased GTP and a resultant loss of calcium homeostasis. These regulatory factors are also associated with neurodegenerative diseases and, therefore, provide a link between TG2 and these disorders (4).

MEMBRANES:

TG2 can also associate with membranes within the cell. Approximately 10-15% of cellular TG2 is in the plasma membrane and about 5% is in nuclear membranes. The microenvironment at the plasma membrane may be permissive for the enzymatic activity of TG2, since the membrane lipid sphingosylphosphocholine decreases the calcium requirement of its activity. In accordance with this finding, retinoic acid can induce TG2 to associate with the membranes of HeLa cells, and this treatment also increases GTP-binding, GTP-hydrolyzing, and transamidating activities of TG2 relative to untreated cells (4).

NUCLEUS:

TG2 enters the nucleus as well. Its presence in this compartment has been demonstrated in subcellular fractionation studies, by its interaction with the nuclear transport protein importin-α3, and by immunoelectron microscopy. Additionally, TG2 functions as a G-protein in the nucleus, and cross-links and phosphorylates nuclear proteins (4).

This nuclear localization and activity appear particularly relevant in light of recent studies indicating that TG2 may play an important role in transcriptional regulation, and that it may contribute to Huntington's disease (HD) through this function (4).

TRANSGLUTAMINASE MAIN REACTION:

Transglutaminases are best known for their ability to catalyze protein cross-linking reactions that impart chemical and physical resilience to cellular structures (Fernandes 2015).

Transglutaminases (TGs) are enzymes that catalyze the formation of isopeptide bondsa type of covalent linkage between two protein molecules (4).

Transglutaminase catalyzes the formation of ε-(γ-glutamyl)lysyl crosslinks in a reaction termed crosslinking or transamidation.

Crosslinking or transamidation of proteins occurs through an acyl-transfer reaction between the γ-carboxamide group of peptide-bound glutamine (acyl donor) and the ε-amino group of peptide-bound lysine (acyl acceptor), resulting in a ε-(γ-glutamyl)lysine isopeptide bond (3).

γ-glutamyl-ε-lysine bonds are the hallmark linkage formed by TGs (4).

 

 

 

 

The crosslinked products are highly resistant to mechanical challenge and proteolytic degradation and their accumulation is found in a number of tissues and processes where such properties are important including skin, hair, blood clotting and wound healing (3).

Theoretically, this transglutaminase bond is reversible because there is very little free energy change during its formation. But under physiologic conditions, it tends to be irreversible due to the loss of ammonia and because the changes in protein conformation make reversal of the bond thermodynamically unfavorable. As a result, the cross-links formed in this reaction are extremely resistant to proteolytic cleavage or mechanical disruption (4).

Tgases display strict specificity in recognition of glutamine protein substrates (however, the rules which govern selection of only a few peptidyl glutamine residues are still unclear), and poor specificity for the acyl-acceptor amine group (3).

The acyl-acceptor amine group which can either be the ε-amino group of peptidyl lysine or also can be a low-molecular mass primary amine (frequently a polyamine). In the former instance, the reaction products are often cross-linked high molecular-mass protein aggregates, while in the latter, protein-polyamine conjugates are generated, which can also be further polymerized (3).

Biochemical and cell-biological studies indicate that both reactions involving protein cross-linking and polyamidation are relevant in vivo, and competition between these amine substrates may take place within cells in a number of important physiological functions where they act as a `biological glue', including that of cell death, cell-matrix interactions in the stabilization of the epidermis and of hair and in the general maintenance of tissue integrity (3).

TRANSGLUTAMINASE FUNCTION:

As might be expected from the many functions and ubiquitous nature of TG2, diverse physiological roles have been attributed to this protein. Frequently, the stable cross-links formed by TG2 serve a structural purpose, but this transglutaminase activity can also alter the function of substrate molecules (4).

 

TG2 can also play a role in wound healing (Upchurch et al., 1991; Haroon et al., 1999) (4).

Extracellularly localized TG2 may be involved in stabilization of the extracellular matrix (Chen & Mehta, 1999; Lesort, et al., 2000; Fesus & Piacentini, 2002) (4).

 

DAMAGE tTgase: the ubiquitous tissue Tgase (tTgase; type 2 Tgase)

tTgase becomes involved in cell survival/cell death and maintenance of tissue integrity following cell stress or damage (3).

Cells under stress/insult release the tTgase into the matrix. The end result is maintenance of tissue integrity via protein cross-linking and matrix deposition (3).

Once externalized from the cell, tTgase has been shown to bind and cross-link a number of extracellular proteins, in particular fibronectin, for which it has a high binding affinity. Other extracellular proteins found both at the cell surface and in the surrounding matrix have been reported to be substrates for tTgase. The physiological implications related to matrix protein crosslinking indicate that its function is to stabilize these proteins, i.e. increasing their proteolytic, chemical and mechanical resistance (But not only, because it is also to facilitate cell adhesion and cell motility (ability of cells to spread and adhere) althogh in this case transamidating activity did not need to be intact for the enzyme to undertake these cell-adhesion roles. t has also been demonstrated that cell migration on fibronectin is dependent on the presence of the enzyme at the cell surface but not its transamidating activity (3).

OTHER PHYSIOLOGICAL FUNCTIONS tTgase: the ubiquitous tissue Tgase (tTgase; type 2 Tgase)

The search for a physiological function of type 2 tTgase is certainly not yet over. Most studies dedicated to this issue have tried to extend and attribute general meanings to experiments carried out on relatively narrow and specialized fields. Early investigations suggested that tTgase may have a role in cell proliferation. Others generated the impression that the enzyme was involved in receptor-mediated endocytosis. A further role was postulated for the enzyme in the Ca2+-mediated exocytotic events involved in the stimulus-secretion coupling involved in insulin release. Interestingly the tTgase knockout mice (-/-) do show symptoms of mild onset diabetes as they age, which is thought to be related to perturbations in insulin release from their pancreatic β-cells (3).

ENDOCYTOSIS:

TG2 can also play a role in endocytosis (Chen & Mehta, 1999; Akimov & Belkin, 2001; Siegel & Khosla, 2007) (4).

INFLAMATION:

TG2 has been implicated in the control of inflammatory processes (Siegel & Khosla, 2007; Iismaa, et al., 2009; Park, et al., 2011). For example, transfection of microglia with TG2 leads to increased activity of the pro-inflammatory factor NF-κB (Lee, et al., 2004) (4).

Synaptic activity:

A role of TG2 in synaptic activity might be expected since neuronal depolarization is accompanied by a large increase in intracellular calcium in microdomains within the depolarizing cells, raising the concentration to a level at which TG2 can be active as a cross-linking enzyme. Experimentally, isopeptide bonds are found in hippocampal slices following induction of long-term potentiation (LTP), which has led to the suggestion that TG2 is in part responsible for stabilizing the long-term changes that occur in synapses in LTP. In addition, synapsin I, a protein important for anchoring synaptic vesicles in the synapse, is a substrate for TGs. Further, inhibiting TGs with monodansylcadaverine rescues synaptosomes from tetanus toxin-induced impairment of neurotransmitter release. The proposed mechanism is that tetanus toxin normally activates TGs, and the cross-linking function interferes with neurotransmitter release, a process that is prevented when TG is inhibited. Accordingly, TGs may be important in the regulation of neurotransmitter release (4).

 

TRANSGLUTAMINASE REGULATION:

As might be expected of a protein such as TG2 that is widely expressed and has multiple functions, it is highly regulated at many levels (4).

pH:

The transamidation reaction has been demonstrated to increase with increasing pH (8).

INFLAMMATION:

A number of regulators increase TG2 expression, many of which vary based on cell type, although some generalizations can be made. One important aspect of TG2 regulation is the frequent involvement of inflammatory mediators (4).

This becomes highly relevant in light of the important contribution of inflammation in neurodegenerative diseases through which TG2 can potentially be upregulated in these disorders (4).

Treatment with lipopolysaccharide (LPS), which induces the release of pro-inflammatory signals, also causes an increase in TG2 mRNA and protein levels in rat astrocytes. This effect could be suppressed by treatment with the antioxidant ethyl pyruvate, suggesting a role for oxidative stress in TG2 regulation as well (4).

CALCIUM:

The cross-linking activity of TG2 is highly regulated and a primary means of regulation is calcium. Decreased calcium concentration results in decreased transamidating activity. Conversely, conditions such as neurodegenerative disorders, in which calcium homeostasis is lost leading to increased free intracellular calcium, are associated with dysregulation of TG2 activity. Binding to GDP or GTP is another means of regulating the cross-linking activity of TG2. When TG2 binds to these nucleotides, a conformational change occurs that leads to blocking its active site by distorting a putative calcium binding site resulting in reduced affinity for calcium (4).

In common with many other important cellular functions found in mammalian cells, Tgases require the binding of Ca2+ for their activity, but at concentrations normally in the supraphysiological, not the physiological, range associated with most intracellular processes. Moreover their Ca2+ activation is also modulated by further regulatory processes, which in essence means that they are virtually inactive under normal conditions and only activated following major disruptions in physiological homoeostatic mechanisms (3).

The calcium requirement for the cross-linking activity of TG2 is greater than that usually found in resting cells, and normal cellular GTP and ATP levels are high enough to be inhibitory. As a result, it has been suggested that under normal conditions intracellular TG2 is inactive and, therefore, simply overexpressing it may not necessarily lead to an increase in cross-links. However, certain physiologic processes may transiently increase intracellular calcium levels sufficiently to activate TG2 (4).

OTHER FACTORS:

A number of other factors are implicated to regulate the crosslinking activity of TG2 (4).

GTP binding downregulates cross-linking activity presumably by affecting the tertiary structure of TG2 (4).

GDP: upon binding to GDP, TG2 assumes a compact form that is enzymatically inactive because its C-terminal β-barrels shift to block access of substrates to its catalytic core (4).

Sphingosylphosphocholine, which is a membrane lipid, lowers the calcium requirement for TG2 activation, reduces the inhibitory effect of GTP binding, and inhibits the proteolysis of TG2. This suggests that while TG2 may not be active in the cytosol, its interaction with membranes may allow for enzymatic activity (4).

As another potential means of regulation, there is some evidence that proteins become better substrates for TG2 when oxidized, while S-nitrosylation of TG2 itself inhibits its enzymatic activity. Multiple cysteine residues in TG2 can be nitrosylated, and this modification makes TG2 more susceptible to inhibition by GTP. Zinc may also inhibit TG2 by preventing it from binding to calcium. Finally, TG2 recognizes only specific glutamine residues, which helps to limit its activity since proteins with appropriate glutamines are fairly uncommon (4).

Retinoids are highly important regulators of TG2 expression in a broad range of cell types and species. Treating rats with retinoic acid leads to an increase in TG2 expression and activity in hepatocytes, whereas feeding rats a diet deficient in vitamin A causes a decrease in TG2 levels, which parallels a decrease in retinoic acid receptor mRNA (4).

TRANSGLUTAMINASE SUBSTRATES:

GRIFFIN 2002 (3):

Below: an overview of endogenous substrate proteins for mammalian type 2 tTgase is given in Table below. They are classified according to their cellular distribution and function; it is evident that a huge number of tTgase substrate proteins are those involved in cell motility, in the interaction of cells with extracellular matrix structures, and in key steps of energetic intermediate metabolism. Despite their great functional relevance, attempts to relate tTgase-catalysed protein modification to changes in physiological functions have so far been deceiving and are limited depending on the experimental system. It is also pertinent to mention that tTgase can modify a number of exogenous proteins, including alimentary proteins, like wheat and soya-bean proteins, milk casein and whey proteins and proteins from pathogenic micro-organisms (e.g. Candida albicans surface proteins, envelope proteins and aspartyl-pro-teinase from HIV and the hepatitis-C-virus core protein) (3):

 

 

3) GLUTEN & CASEIN: PERFECT TRANSGLUTAMINASE SUBSTRATES OF CROSSLINKING

RESISTANCE TO DIGESTION:

GLUTEN:

Many gliadin peptide fragments from gluten remain undigested (Shan et al., 2002).

A common feature among gluten epitopes is the presence of multiple proline and glutamine residues, which make them exceptionally impregnable by gastric, pancreatic and intestinal digestive proteases (Ciccocioppo 2005).

Gluten proteins are poorly digested in the intestine in humans with or without CD because these proteins are relatively resistant to human proteolytic enzymes in the small intestine. Incomplete digestion generates high molecular weight oligopeptides (Ross 2014).

Gliadin and other prolamines are partially resistant to degradation by the intestinal peptidases because of their high proline and glutamine content. Incomplete gigestion of these proteins occurs because both the gastrointestinal peptidases, and the brush border dipeptidyl peptidase IV (DPPIV) and dipeptidyl carboxypeptidase I, have poor affinity for the peptide bonds adjacent to proline and glutamine, thus resulting in accumulation of the immunogenic 33-mer and the 26-mer long oligopeptide fragments (Ross 2014).

Because of the high proline content gliadin peptides are highly resistant to digestive processing by pancreatic and brush border proteases (Wyllie 2015).

Because of its high content in proline and glutamine, gliadins show an unusual resistance to gastrointestinal enzymes. It has been demonstrated that the lack of endo-prolylpeptidase activity in gastric and pancreatic enzymes and in the human brush border, prevents efficient enzymatic attack of proline-rich domains in gluten proteins (Wyllie 2015).

For example certain gliadin peptides (e.g., the 33-mer A-gliadin peptide) and other gliadin peptides (e.g., A-gliadin peptide P31-43) (Nanayakkara 2013).

Certain gliadin peptides, both the 33-mer peptide containing P57-68 and the 25-mer peptide containing P31-43 (P31-55) are resistant to hydrolysis by gastric, pancreatic and intestinal proteases, these peptides remain active in vivo in the intestine after gluten ingestion (Nanayakkara 2013).

TRANSGLUTAMINASE SUBSTRATES FOR CROSSLINKING:

GLUTEN:

The repetitive presence of multiple proline and glutamine residues makes gluten peptides a preferred substrate of tTG, whose main function is to catalyse the covalent and irreversible crosslinking of a glutamine residue in glutamine-donor proteins with a lysine residue in glutamine-acceptor proteins which results in the formation of an  ε-(γ-glutamyl)-lysine (isopeptidyl) bond  (Ciccocioppo 2005).

MAIURI 2005 (6):

p31-43 and pα-9 are gluten-derived gliadin peptides.

p31-43 and pα-9 are both substrates of TG2 in patients with celiac disease and in controls (6).

p31–43 (LGQQQPFPPQQPY)

Detection of TG enzymatic activity (green) in (A) control and (D) untreated celiac disease biopsy specimens by using (A , D) biotinylated p31-43 (A and D) without

pretreatment as TG substrates instead of biotinylated MDC (6).

In (A) control and (D) celiac duodenum, TG enzymatic activity is detected within the extracellular matrix, mainly of the subepithelial compartment, with detectable label within the epithelium when biotinylated p31-43 is used as TG substrate (6).

 

Detection of TG enzymatic activity (green) in (F-H) untreated celiac disease biopsy specimens by using (F–H ) biotinylated p31-43 with pretreatment with (F) R283, (G) CUB 7402 mAb, or (H) 6B9 mAb as TG substrate instead of biotinylated MDC (6).

R283: a TG2 inhibitor;  CUB 7402: an anti-TG mAb;  6B9: an anti-surface TG2 mAb (6).

(F) No label is detected when tissue sections are pretreated with R283 inhibitor (6).

(G) Pretreatment of sections with CUB 7402 mAb is only partially effective in preventing p31-43 tissue binding (6).

(H) No inhibitory effect is provided by 6B9 mAb pretreatment, and the pattern is similar to that observed after incubation with p31-43 without pretreatment (6).

 

 

pα-9(57–68) QLQPFPQPQLPY

Detection of TG enzymatic activity (green) in (C) control biopsy specimens and (E) untreated celiac disease biopsy specimens by using (C and E) biotinylated p-α9 as TG substrate instead of biotinylated MDC (6).

(C and E) A similar pattern (as when biotinylated p31-43 is used) is observed in (C) control and (E) celiac disease biopsy specimens when biotinylated pα-9 is used as substrate (6).

 

deamidated pα-9(57–68) QLQPFPQPELPY

Detection of TG enzymatic activity (green) in (B) control biopsy specimens by using (B) the biotinylated deamidated form of pα-9 (100 μg/mL) as TG substrate instead of biotinylated MDC (6).

(B) In control biopsy specimens, no label is detected when the deamidated form of pα-9 is used; the same pattern is observed in celiac disease biopsy specimens (6).

A similar pattern of inhibition by R283, CUB 7402, or 6B9 mAb is found for pα-9 or pα-2. pα-2 shows the same pattern as pα-9. TCD biopsy specimens show the same behavior as control tissues. (Indirect immunouorescence; original magnication 200x) (6).

REFERENCES TO MAIURI STUDY:

L682777 is a TG2 active-site inhibitor (van den Akker 2011).

SAKLY 2006

Mauiri and coworkers showed that p31–43 binds to the cell membrane as measured by immunouorescence. Peptide binding was inhibited by mAbs to TG2 and Fas [35].

35 Maiuri L, Ciacci C, Ricciardelli I e t al. Unexpected role of surface transglutaminase type II in celiac disease. Gastroenterol 2005; 129:1400–13.

SIEGEL 2007

In another study by Maiuri and coworkers (Maiuri et al., 2005), the authors showed that the 2-[(2-oxopropyl)thio]imidazolium inhibitor L682777 (28 in Table 3, a.k.a. R283) was able to prevent the in situ crosslinking of gluten peptides to endogenous proteins in thin tissue sections taken from both celiac sprue patients and controls.

Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, Rispo A, et al. Unexpected Role of Surface Transglutaminase Type II in Celiac Disease. Gastroenterology 2005;129(5):1400–1413.

BAKSHI 2012

In another study, Maiuri and coauthors showed that the 2-[(2-oxopropyl)thio]imidazolium inhibitor L682777 was able to prevent the in situ crosslinking of gluten peptides to endogenous proteins in tissue sections taken from both CD patients and controls.38 

38. Maiuri L, Ciacci C, Ricciardelli I, et al. Unexpected role of surface  transglutaminase type II in celiac disease. Gastroenterology . 2005;129:1400-1413.

MAKHARIA 2014

In another study, Maiuri et al. (84) showed that the 2-[(2-oxopropyl)thio] imidazolium inhibitor, L682777, was able to prevent the in situ crosslinking of gluten peptides to endogenous proteins in thin tissue sections taken from both celiac disease patients and controls.

84. Maiuri L, Ciacci C, Ricciardelli I, Vacca L, Raia V, Rispo A, et al. Unexpected role of surface transglutaminase type II in celiac disease. Gastroenterology (2005) 129:1400–13.

SKOVBJERG 2004 (8):

Using synthetic peptides containing the immunodominant gliadin epitope together with monoclonal antibodies specific for the deamidated form of the epitope or for both forms, we characterised both the deamidation and cross-linking to intestinal proteins (8).

p59-72 is a gluten-derived gliadin peptide.

α2(59–72)=QPFPQPQLPYPQPQ=PepQ

SKOVBJERG 2004 EXPERIMENT DETAILS:

Synthetic epitope peptides: synthetic alpha-gliadin peptides:

- Synthetic peptide Gliadin 33-mer fragment PepQ =α2(59–72)=QPFPQPQLPYPQPQ

- Synthetic peptide Gliadin 33-mer fragment PepQ in a deamidated (QPFPQPELPYPQPQ- amid) (PepE).

Transglutaminase: Guinea pig liver transglutaminase (GPLT) (8).

Small intestinal mucosa: pigs were anaesthetised, the middle part of duodenum was removed, and the mucosal layer stripped from the underlying layers (8).

SKOVBJERG 2004 RESULTS:

Our aim was to study how the two main catalytic activities of transglutaminase (deamidation and transamidation (cross-linking) of an immunodominant gliadin epitope) are influenced by the presence of acceptor amines in the intestinal mucosa (8).

We analyse deamidation and cross-linking of these peptides to proteins. Our results show that QPFPQPQLPYPQPQ-amide was deamidated when incubated with purified TG2, with fresh mucosal sheets and with mucosal homogenates. A fraction of the non-deamidated epitope was cross-linked to proteins, including TG2. The results suggest that intestinal TG2 is responsible for generation of the active deamidated epitope. As the epitope often occurs in a repeat structure, the result may be cross-linking of a deamidated, i.e., activated cell epitope (8).

We demonstrated that the epitope was deamidated in an environment similar to the intestinal wall, albeit at a rather low rate. The lower rate was explained by concomitant binding to a multitude of proteins (8).

 

Cross-linking of PepQ to transglutaminase:

The cross-linking (between gliadin epitope and transglutaminase) occurs, as demonstrated in this paper and recently by others (Fleckenstein et al 2004), also outside the active site of TG2 (8).

B. Fleckenstein, S.W. Qiao, M.R. Larsen, G. Jung, P. Roepstorff, L.M. Sollid, Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides, J. Biol. Chem. 279 (2004) 17607–17616.

TG2: It has earlier been reported that gliadin peptides bind to TG2 (8).

B. Fleckenstein, S.W. Qiao, M.R. Larsen, G. Jung, P. Roepstorff, L.M. Sollid, Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides, J. Biol. Chem. 279 (2004) 17607–17616.

J.L. Piper, G.M. Gray, C. Khosla, High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: implications for celiac sprue, Biochemistry 41 (2002) 386–393.

R. Ciccocioppo, A. Di Sabatino, C. Ara, F. Biagi, M. Perilli, G. Amicosante, M.G. Cifone, G.R. Corazza, Gliadin and tissue transglutaminase complexes in normal and coeliac duodenal mucosa, Clin. Exp. Immunol. 134 (2003) 516 –524.

Such a binding (cross-linking of a gliadin T cell epitope to transglutaminase) has been demonstrated using a labelled epitope peptide added to recombinant human TG2, and has been suggested to be due to temporary binding to the active site of the enzyme or to lysines in the protein (8).

B. Fleckenstein, S.W. Qiao, M.R. Larsen, G. Jung, P. Roepstorff, L.M. Sollid, Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides, J. Biol. Chem. 279 (2004) 17607–17616.

J.L. Piper, G.M. Gray, C. Khosla, High selectivity of human tissue transglutaminase for immunoactive gliadin peptides: implications for celiac sprue, Biochemistry 41 (2002) 386–393.

Evidence for cross-linking was also provided by Ciccocioppo (8).

R. Ciccocioppo, A. Di Sabatino, C. Ara, F. Biagi, M. Perilli, G. Amicosante, M.G. Cifone, G.R. Corazza, Gliadin and tissue trans- glutaminase complexes in normal and coeliac duodenal mucosa, Clin. Exp. Immunol. 134 (2003) 516 –524.

Corresponding experiments with TG2 show that the epitope peptide is cross-linked to GPLT independent of a native active site conformation, and is in accordance with our demonstration of binding also to other proteins and with recent findings on TG2 of Fleckenstein (8).

Cross-linking of PepQ to purified TG2. PepQ was incubated with GPLT with or without addition of heat inactivated GPLT: The epitope peptide was bound to GPLT (inactive and active) as observed for other proteins. In control experiments without addition of high amounts of inactive GPLT, the antibody reactivity was low, demonstrating that most of the registered epitope peptide was cross-linked to the inactivated GPLT (8).

 

Cross-linking of PepQ to other proteins:

Cross-linking of PepQ to chicken egg albumin and BSA: PepQ was incubated with chicken egg albumin or bovine serum albumin (BSA) in the presence of Guinea pig liver transglutaminase (GPLT). The results for chicken egg albumin and BSA clearly show that, in the presence of GPLT, PepQ was cross-linked to these proteins. The high molecular weight peaks seen both in the presence of chicken egg albumin and BSA were only present after incubation with GPLT, and represent the polymerised protein which has also incorporated the epitope peptide (8).

Incubation of bovine serum albumin (BSA) and chicken egg albumin with epitope peptide (alpha-gliadin peptides) and active transglutaminase clearly demonstrated that the epitope peptide (alpha-gliadin peptides) bound to these proteins, as the binding profile in both cases, follows the protein profile (8).

Cross-linking is therefore expected to occur to all proteins having epsilon-amino groups that are reactive with the gliadin epitope (8).

That this really is the case is demonstrated in this paper by its binding to BSA and chicken egg albumin (8).

 

The fact that cross-linking is expected to occur to all proteins having epsilon-amino groups that are reactive with the gliadin epitope and that this really is the case is demonstrated in this paper by its binding to BSA and chicken egg albumin (8).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

 

Cross-linking of PepQ to mucosal proteins:

PepQ  was incubated with mucosal homogenate and GPLT: The result strongly supports the suggestion that the presence of primary amines in the mucosal homogenate inhibits deamidation, We suggest that the inhibition of deamidation by mucosal proteins is due to cross-linking by TG2 (8).

When the elution fractions were analysed the result fits the assumption that the critical glutamine residue was involved in cross-linking (8).

 

Relation between deamidation and cross-linking:

Proteolytically digested gluten was incubated with or without various concentrations of lysine. The reaction rate in this experiment was measured by the rate of liberation of ammonia. The results demonstrate that the maximal transamidation rate for TG2 was about four times higher than the deamidation rate (in the absence of lysine) (8).

Interestingly, the studied immunodominant epitope occurs in an oligomerised non-digestible form having more than one deamidation/transamidation site, thus potentially allowing simultaneous cross-linking and deamidation by transglutaminase (8).

 

In this paper, it is demonstrated that TG2, when present in the intestinal mucosa, can deamidate an immunodominant peptide epitope. In the presence of primary amines, this reaction is inhibited, although not completely, and cross-linking to intestinal proteins seems to dominate (8).

 

CASEIN:

It has been shown that bovine casein is a good substrate for various transglutaminases in the presence or the absence of added amines (7).

IKURA 1980 (7):

In this study, using transglutaminase purified from fresh guinea pig livers (tissue transglutaminase or TG2), the reactivity of purified bovine casein components (αs1-, β-, and κ-caseins) in the transglutaminase reaction catalyzed by guinea pig liver enzyme was compared (7).

Both αs1- and β-caseins had the same high reactivity, but a much lower reactivity was found for k-casein. Since amine was not added to the transglutaminase reaction system, the amount of ammonia released during the reaction corresponds to the number of inter­ or intramolecular ε-(γ-glutamyl)lysyl crosslinks formed by the action of the enzyme on casein (7).

After 120 min under the experimental conditions mentioned, 3.5 ε-(γ-glutamyl)lysyl crosslinks per molecule were formed with αs1-casein, 3.8 with β-casein, and 1.0 with κ-casein. With each casein component, the amount of ammonia released during the reaction corresponded to the decrease of free amino groups (7).

Transglutaminase polymerizes each of the casein components by the formation of intermolecular cross-links (formation by transglutaminase of intermolecular crosslinks for each of the casein components) (7).

Sodiumdodecyl sulfate­polyacrylamide gel electrophoresis analysis indicated that each casein component was polymerized through formation of intermolecular crosslinks by transglutaminase (7).

 

 

X) CROSSLINKING HYPOTHESIS AND CELIAC & OTHER AUTOIMMUNE REACTIONS:

SKOVBJERG 2004 (8):

Cross-linking of a gliadin T cell epitope to transglutaminase may help the production of anti-TG2 by B cells (8).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

If gliadin peptides also bind to other proteins, a similar mechanism may be involved in generation of antibodies against other endogenous proteins (8).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

A short segment, PQPQLPY, was previously identified as a core sequence of the immunodominant alpha-gliadin epitope, which is responsible for most of the stimulatory activity on intestinal and peripheral CD4+ T lymphocytes (8).

Deamidation of the underlined glutamine by TG2 was shown to be important for recognition by the T cells (8).

Patients with CD have circulating antibodies against TG2, which is considered to be the dominating autoantigen, including the reactivity earlier ascribed to endomysium and reticulin. However, not all anti-TG2 activities of patient sera are absorbed by guinea pig or monoclonal TG2, pointing to the existence of other submucosal autoantigens. In addition, some patients have autoantibodies against the cytoskeletal protein actin and possibly other proteins (8).

The binding of the epitope peptide to TG2 supports the hypothesis of Sollid et al. that T cell immune response to gliadin would drive antibody responses towards TG2 that is cross-linked to gliadin T cell epitopes (8).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

The fact that cross-linking is expected to occur to all proteins having epsilon-amino groups that are reactive with the gliadin epitope and that this really is the case is demonstrated in this paper by its binding to BSA and chicken egg albumin strengthens the hypothesis (Sollid 1997) that gliadin (in celiac disease) also drives antibody responses to proteins other than TG2 (8).

L.M. Sollid, O. Molberg, S. McAdam, K.E. Lundin, Autoantibodies in coeliac disease: tissue transglutaminase—guilt by association? Gut 41 (1997) 851–852.

The importance of posttranslational protein modifications in antigen recognition and autoimmunity has recently been reviewed by Doyle et al (2001).

H.A. Doyle, M.J. Mamula, Post-translational protein modifications in antigen recognition and autoimmunity, Trends Immunol. 22 (2001) 443–449.

Sjfstrfm et al. suggested that the intestinal immune system is tolerant to non-deamidated peptides and that a process leading to deamidation of gliadins could contribute to break of tolerance. However, the use of the particular glutamine residue in the gliadin epitope for cross-linking instead of deamidation may argue against this suggestion (8).

H. Sjfstrfm, K.E. Lundin, a. Molberg, R. Kfrner, S.N. McAdam, D. Anthonsen, H. Quarsten, O. Nore´n, P. Roepstorff, E. Thorsby, L.M. Sollid, Identification of a gliadin T-cell epitope in coeliac disease: general importance of gliadin deamidation for intestinal T-cell recognition, Scand. J. Immunol. 48 (1998) 111 –115.

Our research provides a basis for the suggestion that binding of a peptide to a protein in connection to its modification to a T cell epitope might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens (8).

 

4) CROSSLINKING OF PROTEINS & DISEASE: A FORESEEABLE CONNECTION

Tissue transglutaminase (TG2) is implicated in a number of diseases including many neurodegenerative disorders (4).

The interest in transglutaminases is further stimulated by their involvement in a number of human disease states (e.g. certain neurodegenerative diseases, autoimmune conditions such as coeliac disease, cancer and tissue fibrosis) and this represents a growing area of Tgase research (3).

It is the cross-linking (transamidating) activity of TG2 that has been most strongly implicated in pathologic conditions (involved in disease pathogenesis) (4).

Deregulation of transglutaminase activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin (3).

In very recent years this topic has yielded interesting new data with respect to the relevance of Tgases in chronic diseases, in particular in (a) inflammatory diseases, including wound healing, tissue repair and fibrosis, and autoimmune conditions; (b) chronic degenerative diseases (e.g. arthritis, atherosclerosis and neurodegenerative pathologies); and (c) tumour biology. In the majority of these diseases the prevalent role of tTgase appears to be related to its interaction with, and stabilization of, the cell matrix, rather than as a major player in apoptosis (3).

The local administration of purified enzymes (usually placental Factor XIII, but more recently tTgase) have been used as an exogenous biological `glue' to aid in the repair of surgical wounds, fractures and cartilage lesions. This practice, employing recombinant rather than extracted enzymes, is still being explored in surgical practice and in the treatment of certain intestinal diseases (3).

DAMAGE:

Wound healing requires the involvement of several distinct Tgases, which co-operate with each other to finally reconstitute tissue integrity damaged by traumatic or other pathological injuries. Factor XIIIa is clearly involved in the control of blood loss after the traumatic injury of blood vessels, through the stabilization of fibrin during blood clotting, in the activation of platelets, and in the deposition of granulation tissues, which represents the first stable repair to a local lesion. Tgases 1 and 3 are particularly involved in repair of the epidermal teguments, in conjunction with Tgase 2, which is probably involved in the angiogenic phase of wound repair as well as in its interaction with and stabilization of the extracellular matrix (3).

FIBROSIS AND SCARRING:

It is also likely that Tgases, particularly the type 2 tTgase are also involved in tissue fibrosis and scarring. Examples include these vere chronic inflammatory states found in liver diseases (cirrhosis and fibrosis, alcoholic hepatopathy and type C hepatitis), and in renal and lung fibrosis, the latter ultimately leading to renal and pulmonary failure via deposition of excessive scar tissue. In addition, involvement of tTgase in the pathogenesis of the chronic inflammatory diseases of the joints, including rheumatoid arthritis and osteoarthritis, has been reported. A major role of the enzyme in many of these conditions is apparently linked to its involvement in the activation of pro-inflammatory cytokines such as TGFβ1. In the latter case, tTgase is thought to be important in both the matrix storage and activation of TGFβ1 via a mechanism that involves the cross-linking of the LTBP-1 complex to the matrix, which is a pre-requisite for the release and activation of this fibrogenic cytokine. Activated cytokines such as TGFβ1 can stimulate pyrophosphate release in diseased joints, leading to mineralization and progression of diseases such as arthritis. Activation of growth factors such as TGFβ1 and cytokines such as interleukin-6 and tumour necrosis factor-α in their turn can lead to further induction and expression of tTgase, leading to an effective, but vicious, autocrine loop. It is also noteworthy that Factor XIII is frequently present and active in the synovial fluids of inflamed joints, catalysing stabilization of fibrin, further complicating the biological clinical picture (3).

CELIAC DISEASE

Interest in type 2 tTgase immunoreactivity has grown explosively during the last few years in relation to the pathogenesis and diagnosis of coeliac disease. In the intestinal mucosa of gliadin-sensitive individuals, tTgase is apparently involved in deamidation of glutamine residues in gliadin and in formation of aggregates of Tgase itself and of gliadin, which are highly immunogenic through local activation of T-lymphocytes (3).

AUTOIMMUNE DISEASES

The importance of the introduction of gluten in relation to breast feeding has also been under investigation. Interestingly, food supplementation with gluten-containing foods before the age of 3 months was shown to be associated with significantly increased islet autoantibody risk (8).

A.G. Ziegler, S. Schmied, D. Huber, M. Hummel, E. Bonifacio, Early infant feeding and risk of developing type 1 diabetes—associated autoantibodies, JAMA 290 (2003) 1721– 1728.

In addition, autoantibodies specific to diabetes mellitus type 1 have in one case been reported to disappear following change to a gluten-free diet (8).

P. Banin, R. Perretta, E. Ravaioli, V. De Sanctis, Regression of autoimmunity and abnormal glucose homeostasis in an adolescent boy with silent coeliac disease, Acta Paediatr. 91 (2002) 1141–1143.

Furthermore, patients with gluten ataxia have been shown to have antibodies against Purkinje cells (8).

M. Hadjivassiliou, S. Boscolo, G.A. Davies-Jones, R.A. Grunewald, T. Not, D.S. Sanders, J.E. Simpson, E. Tongiorgi, C.A. Williamson, N.M. Woodroofe, The humoral response in the pathogenesis of gluten ataxia, Neurology 58 (2002) 1221 –1226.

Cross-linking with TG2 has also been suggested to be involved in autoantigen modifications during apoptosis or cellular injury (8).

P.J. Utz, P. Anderson, Posttranslational protein modifications, apoptosis, and the bypass of tolerance to autoantigens, Arthritis Rheum. 41 (1998) 1152 –1160.

However, in none of these cases the mechanism for generation of neo-epitopes was specified. It can be speculated that, under conditions where undigested gliadin peptides have access to the circulation due to a barrier disturbance, these peptides can potentially be cross- linked to proteins in organs where TG2 is available for the reaction. In this way, gliadin peptides may also be an initiating factor for other autoimmune diseases (8).

It has been suggested that the up-regulation of TG2 in CD may generate additional antigenic neo-epitopes by cross-linking or deamidating viral, nutritional or endogenous proteins, and thereby contribute to initiation of autoimmune diseases (8).

D. Schuppan, R. Ciccocioppo, Coeliac disease and secondary auto- immunity, Dig. Liver Dis. 34 (2002) 13 –15.

NEURO

Tissue transglutaminase (TG2) is implicated in a number of neurodegenerative disorders including Huntington's (HD), Alzheimer's (AD), and Parkinson's diseases (PD) based on several lines of evidence (4):

First, TG2 is found throughout most brain regions, particularly in neurons, and accounts for the majority of transglutaminase activity in the mouse brain (4).

Second, the pathophysiology of all these diseases includes the formation of insoluble aggregates, and the covalent cross-linking of pathogenic proteins by TG2 is effectively irreversible and leads to the formation of protein polymers (4).

Third, conditions that promote the cross-linking activity of TG2 intracellularly, such as low GTP and high calcium levels, are frequently met in damaged cells in neurodegenerative diseases where there is energy depletion and loss of calcium homeostasis. Increased calcium concentration can also induce translocation of TG2 to the nucleus where it can repress transcription of potentially protective genes (4).

Fourth, oxidative damage, which is a well-known occurrence in these diseases, can make proteins better substrates for TG2 (4).

And fifth, the role of TG2 in autophagy may be another link between TG2 and neurodegenerative diseases considering the dysregulated autophagy in the pathogenesis of these disorders (4).

This line of reasoning has led to the notion that in neurodegenerative diseases, neuronal damage either due to environmental insults or genetic predisposition leads to conditions conducive to TG2 activation. TG2 then begins to cross-link susceptible substrates into conformations that may be toxic or may prevent important proteins from performing their biological functions. This, in turn, exacerbates the damage to neurons, preventing them from recovering from the initial insult and leading to cell death. To examine this hypothesis, the role of TG2 has been studied in neurodegenerative diseases, and considerable evidence has built up suggesting that it does play a role in these disorders (4).

Abnormal protein aggregates and the accumulating evidence that certain forms of these aggregates can be neurotoxic (4).

Factors that promote the formation of these aggregates have been the subject of intense investigations with the objective of identifying interventions that can slow the progressive disease process. One of these factors is the crosslinking of pathogenic proteins into toxic higher order oligomers by the enzymatic activity of TG2 (4).

Some proteins involved in neurodegenerative diseases can also propagate across neurons (4).

GROSSO 2012 (4):

Neurodegenerative disorders encompass a number of chronic progressive diseases that are characterized by the loss of select neuronal populations in the CNS, which underlie their respective clinical phenotypes, and the aggregation of disease-specific pathogenic proteins in hallmark lesions that highlight the neuropathology of each disorder (4).

Other commonalities among these disorders include loss of calcium homeostasis, energy depletion, and increased generation of reactive oxygen species in affected cells, all of which may contribute to the activation of TG2 (4).

Neurodegenerative disorders are characterized by progressive neuronal loss and the aggregation of disease specific pathogenic proteins in hallmark neuropathologic lesions. Many of these proteins, including amyloid Αβ, tau, α-synuclein and huntingtin, are cross-linked by the enzymatic activity of transglutaminase 2 (TG2). Additionally, the expression and activity of TG2 is increased in affected brain regions in these disorders. These observations along with experimental evidence in cellular and mouse models suggest that TG2 can contribute to the abnormal aggregation of disease causing proteins and consequently to neuronal damage (4).

GRIFFIN 2002 (3):

The mechanisms whereby Tgases are involved in the pathogenesis of several chronic neurodegenerative diseases, which are characterized by the accumulation of highly cross-linked insoluble protein materials. These include senile dementia of the Alzheimer type (Alzheimer disease, AD) and the polyglutamine (polyQ) tail diseases, such as Huntington's disease, rubropallidal atrophy and spinocerebellar palsy. In AD, the expression of tTgase is increased. In the diseased brain, the elevated tTgase activity is manifested by polymerization of a number of proteins, including Aβ peptide, β-amyloid precursor protein and the microtubule-associated tau protein, with formation of neurofibrillary tangles, as well as deposition of amyloid-like materials in the extracellular compartments. These abnormal protein polymers might be relevant to the pathogenesis of AD brains, and their formation has been ascribed to increased tTgase activity (3).

In contrast, the polyQ diseases are primarily characterized by transcriptional defects in the substrates, rather than in the enzyme, with the synthesis of proteins with abnormal tail extensions that represent the sites of Tgase-mediated protein cross-linking. This issue is still controversial, since the presence of multiple glutamine repeats directly promotes stickiness in the altered proteins, which tend to rapidly polymerize. This phenomenon would, however, be further favoured by covalent cross-linking by tTgase. PolyQ extensions could be present in a number of proteins in the diseased brains, including several enzymes associated with energy metabolism. Recent studies have demonstrated that administration of the Tgase inhibitor cystamine to transgenic mice (expressing exon 1 of huntingtin containing an expanded polyglutamine repeat) was found to alter the course of the disease in a favourable way, thus providing further evidence for the involvement of tTgase in this disease (3).

TUMOURS

An additional field of active research on the importance of tTgases in human pathology is that of neoplastic diseases. Numerous reports have dealt with these issues, and the general feeling is that tumour cells, when observed in vitro, generally have a lower tTgase content than their normal counterparts, contain forms of Tgase which are identical with those found in normal cells, together with modified forms, which are some times inactive,and may differ in their subcellular localization. Tumours usually display a definitively larger proportion of Tgase activity in the cell particulate fraction when compared with normal cells, although the absolute amount of enzyme present in this fraction is normally not altered. The decline of Tgase activity in tumours is potentially a bad prognostic biomarker and is possibly related to tumour metastatic potential, dictating the ability of the cells to cross basal membranes and to invade the bloodstream . Given the proposed functions of tTgase, reduced enzyme expression and activity in tumours would indeed lead to reduced cell adhesion, increased migration and a less stable extracellular matrix, thus facilitating the initial invasive stage of the tumour. However, reports of increased tTgase expression in highly invasive tumours have also been reported, e.g. in the breast, and increased tTgase expression has been found in secondary metastatic tumours. Other intriguing issues arise from the reported decreased rates of apoptosis in tumours and the still-debated relationships between Tgases and apoptosis. It is also noteworthy that successful induction of tTgase by powerful inducers such as retinoids (e.g. 9-cis-retinoic acid or alltrans-retinoic) provide an effective switch to cell differentiation and apoptotic death, as observed with squamous-cell carcinoma in vitro and in promyelocytic leukaemia in vivo. The observation that other synthetic retinoids can be even more active than retinoic acid in inducing tTgase activity and apoptosis in cell lines which are insensitive to the therapeutic effects of retinoic acid has stimulated further research on the application of modified retinoids. It is also now clear that other chemotherapeutic agents of different structure might be as effective as antineoplastic drugs, but their relationships to Tgase related pathways are still controversial. Conversely, host tissues frequently display higher tTgase expression and activity in peritumoral regions, possibly as a local wound-healing mechanism, related to the rearrangement of the extracellular matrix, which may even promote angiogenesis and further spreading of the cancerous cells (3).

A recent alternative and useful approach is to modulate endogenous tTgase expression, rather than to administer purified enzyme, by means of specific inducers such as the retinoids. This approach is now a recognized strategy in the therapy of selected malignancies in vivo. Although studies are still at the experimental stage, additional encouraging results have been obtained in some animal tumours, e.g. melanomas, in which metastatic spread is greatly limited by inducing tTgase activity in either the invading tumour or the host. Earlier studies showing that cell transfection leading to overexpression of tTgase in fibrosarcoma cells results in the reduction in tumour growth may have a future application as a tool for gene therapy. The great advantage of such selective therapy, as compared with classic chemotherapy, is its reduced toxicity to normal cells (3).

Enzymatically active TG2 can be transferred from cancer-derived cells in microvesicles into surrounding normal cells (4).

CELL DEATH(APOPTOSIS) VERSUS CELL SURVIVAL: TRANSGLUTAMINASE VERSUS CANCER

Apoptosis is the process of programmed cell death that may occur in multicellular organisms (Wikipedia 2015).

Apoptosis is a multi-step, multi-pathway cell-death programme that is inherent in every cell of the body (Wikipedia 2015).

Excessive apoptosis causes atrophy, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer (Wikipedia 2015).

Linkage of abnormalities in cell death to human disease, in particular cancer (Wikipedia 2015).

Inhibition of apoptosis can result in a number of cancers, autoimmune diseases, inflammatory diseases, and viral infections. It was originally believed that the associated accumulation of cells was due to an increase in cellular proliferation, but it is now known that it is also due to a decrease in cell death. The most common of these diseases is cancer (Wikipedia 2015).

In cancer, the apoptosis cell-division ratio is altered. Cancer treatment by chemotherapy and irradiation kills target cells primarily by inducing apoptosis (Wikipedia 2015).

GROSSO 2012 (4):

CELL DEATH & CELL SURVIVAL:

A number of mechanisms have been proposed regarding how TG2 contributes to apoptosis. One theory is that TG2 cross-links apoptosis inhibitors causing their inactivation (4).

Alternatively, TG2 can play an important role in later stages of cell death when calcium levels rise resulting from loss of calcium homeostasis, and GTP levels fall due to energy depletion. At this point, TG2 begins to transamidate proteins leading to extensive irreversible cross-linking. In doing so, it stabilizes apoptotic bodies and prevents leakage of intracellular contents (4).

It is possible that all these processes collectively contribute to the role of TG2 in apoptosis (4).

The involvement of TG2 in apoptosis is particularly relevant in neurodegenerative diseases (4).

Conversely, evidence for a role of TG2 in cell survival has also emerged. As mentioned above, cellular stress upregulates TG2. However, this response may represent an attempt at cellular preservation rather than a contribution to apoptosis. In support of this notion, TG2 activity is increased in the rat sciatic nerve and goldfish optic nerve post-injury during regeneration, and in rat superior cervical ganglia an hour after axotomy. More recent work has demonstrated that treatment with retinoic acid leading to an increase in TG2 expression is protective in some models of apoptosis, and that the GTPase activity of TG2 is necessary for this effect. Additionally, TG2 inhibits the up-regulation of pro-apoptotic factors following hypoxia (4).

Few explanations are offered as to how TG2 may exert its cell protective effects. TG2 binding to Rb may not inactivate the latter but rather prevent its degradation by caspases in a transamidation-dependent manner. Because Rb plays a role in preventing apoptosis, blocking its degradation may inhibit cell death. There is also evidence suggesting that the above-mentioned interaction between TG2 and the proapoptotic factor Bax may actually be cytoprotective by downregulating the latter protein, and that TG2 may also cross-link and inactivate the apoptosis initiator cytochrome c. Additionally, under certain conditions, including the absence of Bax, TG2 cross-links caspase 3, preventing apoptosis induced by thapsigargin (4).

In an attempt to reconcile these seemingly conflicting findings it has been suggested that whether TG2 acts as an apoptotic or cell-survival factor is determined by multiple factors including the cell/tissue type where it is expressed and its cellular localization. Accordingly, the identification of specific TG2 substrates in different cell types may provide clues to help explain disparate outcomes in different experimental conditions. TG2 acting in the nucleus or extracellular space appears to have a cell survival role, whereas in the cytoplasm it functions as an apoptotic protein. Additionally, the transamidating activity of TG2 can have either a proapoptotic or anti-apoptotic function depending on these various factors (4).

CELL PROLIFERATION:

TG2 is implicated in a myriad of other physiologic activities. One of these is as a cell cycle regulator through which it helps to regulate cell proliferation and differentiation (4).

AGIOGENESIS:

TG2 can also play a role in angiogenesis (Haroon, et al., 1999) (4).

Extracellularly localized TG2 may be involved in cell migration (Martin, et al., 2006) (4).

GENES

Transcription regulation:

TG2 plays a role in transcription regulation as well. This property is well studied in HD models, where inhibiting TG2 activity increases transcription of putative neuroprotective genes through modification of histones. Indeed, histones are known substrates of TG2 leading to their cross-linking and subsequent silencing of genes. The first transcription regulator shown experimentally to be modulated by TG2 was nuclear factor κB (NF-κB) through cross-linking of its inhibitor I-κB. Additional factors now recognized to be regulated by TG2 include PPARγ and Sp1 (4).

 

5) BUG PROTEINS; IN VIVO CROSSLINKING LESSONS

Some pathogens hijack host transglutaminases to enhance their virulence such as Candida albicans, which is cross-linked to host oral epithelial cells via the hyphal protein Hwp1 as a necessary precursor to systemic candidiasis (Staab 2013).

Hwp1 Hyphal wall protein 1:

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

Dimorphic: Candida albicans fungus has two main 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 (Staab 1996).

 

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)

 

Hwp1 is a transglutaminase substrate (N-terminal third of Hwp1 to be precise) (5).

Anatomy of Hwp1: Left: three-dimensional structure (Sundstrom 2002) and Right: aminoacid sequence (UniProtKB/Swiss-Prot).

Hwp1 IS A TRANSGLUTAMINASE SUBSTRATE:

Hwp1 is a substrate for mammalian transglutaminase (5).

 

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

 

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

 

STAAB 1999 RECOMBINANT Hwp1 PROTEIN ASSAY (5):

Employing recombinant Hwp1 (rHwp1) it has been showed that the mature N-terminal third of Hwp1 serves as a substrate for mammalian TGases (Staab 2004).

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 (5).

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 (5).

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 (5).

 

Hwp1 SURFACE LOCALIZATION ON HYPHAE AS TRANSGLUTAMINASE SUBSTRATE:

Hwp1 is a transglutaminase substrate located on hyphal surfaces of Candida Albicans (5).

The NH2-terminal domain of native Hwp1 appears to be a surface-exposed TGase substrate available for interactions with exogenous mammalian Tgases (5).

STAAB 1999 CANDIDA ALBICANS TRANSGLUTAMINASE SUBSTRATE ASSAY (5):

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 (5).

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 (5):

 

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.

 

Candida Albicans adhesion (filamentous, hyphal forms)

 

Candida Albicans adhesion (filamentous, hyphal forms)

 

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) (5).

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 (5).

STAAB 1999 ADHESION ASSAY: MUTANTS LACKING Hwp1 (5):

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 (5).

STAAB 1999 ADHESION ASSAY: USING RECOMBINANT Hwp1 (rHwp1ΔC37) (5):

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 (5).

Below: Radiolabeled rHwp1ΔC37 were incubated with BECs under adhesion assay conditions with or without iodoacetamide. Then associated rHwp1ΔC37 with BECs envelopes were determined (5):

STAAB 1999 ADHESION ASSAY: STABILIZED ADHESION BEING PREVENTED BY TGase INHIBITORS (5):

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 (5).

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 (5).

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 (5).

Below: Candida Albicans strains with or without Hwp1 were used for comparison in TGase assays for stabilized adhesion. Transglutaminase inhibitors also were used (5):

 

Hwp1 & Casein:

TRANSGLUTAMINASE RELATED SEQUENCE COMPARISON Hwp1 & Casein:

FOTGCREN 2014:

BETA-CASEINS:

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 (5):

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 (5).

Below: Incorporation of [14C]putrescine by rHwp1∆C37, casein, and BSA mediated by TGase (5):

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 (5).

Hwp1 & Gluten:

TRANSGLUTAMINASE RELATED SEQUENCE COMPARISON Hwp1 & Gluten:

FOTGCREN 2014:

ALPHA/BETA-GLIADINS:

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
                  ***:: :  ::

QQQPQE

Hwp1 strain P46593 versus different alfa-gliadins around QQQPQE motif; QQQPQE in Hwp1 (77-82) versus QQQPQE in alfa-gliadin-P04727 (32-37):

P46593|HWP1_CANAL QEEPCDYPQQQPQEPCDYPQQPQEP 69-93

P04727|GDA7_WHEAT  QLQPKNPSQQQPQEQVPLVQQQQFP 24-48

P04725|GDA5_WHEAT  QLQPQNPSQQQPQEQVPLVQQQQFP 27-51

P02863|GDA0_WHEAT  QLQPQNPSQQQPQEQVPLVQQQQFL   27-51

P18573|GDA9_WHEAT  QLQPQNPSQQQPQEQVPLVQQQQFP 27-51

 

P46593 QEEPCDYPQQQPQEPCDYPQQPQEPCDYPQQPQEPCDYPQQPQEPCDNPPQPDVPCDNPPQPDVPCDNPPQPDIPCDNPPQPDIPCDNPPQPDQP 69-163

P04727 QLQPKNPSQQQPQEQVPLVQQQQFPGQQQQFPPQQPYPQPQPFPSQQPYLQLQPFPQPQPFLPQLPYPQPQSFPPQQPYPQQRPKYLQPQQPISQ 24-118

P04725 QLQPQNPSQQQPQEQVPLVQQQQFPGQQQQFPPQQPYPQPQPFPSQQPYLQLQPFPQPQPFPPQLPYPQPQSFPPQQPYPQQQPQYLQPQQPISQ 27-121

P02863 QLQPQNPSQQQPQEQVPLVQQQQFLGQQQPFPPQQPYPQPQPFPSQLPYLQLQPFPQPQLPYSQPQPFRPQQPYPQPQPQYSQPQQPISQQQQQQ 27-121

P18573 QLQPQNPSQQQPQEQVPLVQQQQFPGQQQPFPPQQPYPQPQPFPSQQPYLQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFRPQQPYPQSQPQY 27-121

 

Other alignment of QQQPQE in alfa-gliadin-P04727 (32-37) with another part of Hwp1 strain P46593:

P46593|HWP1_CANAL YQEPCDDYPQQQQQQEP 44-60
P04727|GDA7_WHEAT QQQPQEQVPLVQQQQFP 32-48
P04725|GDA5_WHEAT QQQPQEQVPLVQQQQFP 35-51
P02863|GDA0_WHEAT QQQPQEQVPLVQQQQFL 35-51
P18573|GDA9_WHEAT QQQPQEQVPLVQQQQFP 35-51
 
P46593 YQEPCDDYPQQQQQQEPCDYPQQQQQEEPCDYPQQQPQEPCDYPQQPQEPCDYPQQPQEPCDYPQQPQEPCDNPPQPDVPCDNPPQPDVPCDNPPQ 44-139
P04727 QQQPQEQVPLVQQQQFPGQQQQFPPQQPYPQPQPFPSQQPYLQLQPFPQPQPFLPQLPYPQPQSFPPQQPYPQQRPKYLQPQQPISQQQAQQQQQQ 32-127

 

QQQPQE substrate of transglutaminase Dorum 2010:

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
                    * :*  :   *  ***

PQXP

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).

Hwp1   PQEPCDYPQQPQEPCDYPQQPQEPCDYPQQPQEPCD
33mer  LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

 

6) STRESS: FOOD versus MIND

Cellular stress also leads to an upregulation of TG2 (4).

Treatment with lipopolysaccharide (LPS), which induces the release of pro-inflammatory signals, also causes an increase in TG2 mRNA and protein levels in rat astrocytes. This effect could be suppressed by treatment with the antioxidant ethyl pyruvate, suggesting a role for oxidative stress in TG2 regulation as well (4).

Expressing TG2 constitutively can cause cell death and increase the susceptibility of cells to toxins (4).

 

Below: Examples of altered intestinal epithelial barrier (A) A normal intestinal epithelium from a control rat. (B) Altered intestinal barrier in rats exposed to chronic stress. Note the abnormal morphology with multiple vacuoles suggesting increased endocytotic activity (left panel). Numerous bacteria attaching to the epithelial surface, and also internalizing (arrow heads), with actin accumulation at the contact sites (right panel) (Keita & Soderholm 2010):

 

Autophagy regulation

TG2 may be involved in the regulation of autophagy. In the absence of TG2, cell stressors cause a greater induction of autophagy than in cells expressing TG2, suggesting that TG2 inhibits autophagy. One mechanism for this is the ability of TG2 to cross-link beclin 1, which is a key player in autophagosome formation, and sequester it in aggresomes; thus, blocking TG2 activity increases autophagy. However, mesenchymal embryonic fibroblasts (MEF) from TG2 knock-out mice have impaired maturation of pre-autophagic vesicles to autophagolysosomes, suggesting that TG2 is needed for the maturation of autophagic vesicles. While further studies are required to determine the precise role of TG2 in autophagy, one possible explanation is that TG2 inhibits the early-stage induction of autophagy, but is necessary for the completion of this process. Regardless of the exact role of TG2 in autophagy, it does appear to be important. For example, the R6/1 mousemodel of HD demonstrates ultrastructural changes in affected regions in cortex and striatum that are suggestive of altered autophagy including a large number of lysosomes and dilation of the endoplamic reticulum in the cytoplasm of condensed neurons. Deleting TG2 in these mice is associated with fewer of these changes. It therefore follows that the abnormalities in autophagy that are associated with cell death in models of neurodegenerative diseases including HD and PD may be contributed by TG2 (4).

 

7) WHY NOT YET?

The products which accumulate in vivo or in situ in cells and tissues following activation of transglutaminases are predominatly highly cross-linked insoluble polymers, formed by either direct or polyamine dependent linkage. Their structure is complicated, so that the identification of the proteins involved in the polymerization process has been very problematic (3).

 

 

 

REFERENCES:

              1.      GARDNER 1988: Gardner MLG (1988) Gastrointestinal absorption of intact proteins. Annual Review of Nutrition Vol. 8: 329-350. http://www.ncbi.nlm.nih.gov/pubmed/3060169

              2.      GARDNER 1988: Gardner MLG (1988) Intestinal absorption of peptides. Nutritional modulation of neural function / edited by John E. Morley, M. Barry Sterman, John H. Walsh; Academic Press; UCLA Forum in Medical Sciences Number 28; pages 29-38. https://books.google.es/books?id=vImTIsfi0PUC&printsec=frontcover&hl=es#v=onepage&q&f=false

              3.      GRIFFIN 2002: Griffin et al. (2002) Transglutaminases: Nature’s biological glues. Biochem. J. 368, 377-396. http://www.ncbi.nlm.nih.gov/pubmed/12366374

              4.      GROSSO 2012: Grosso et al. (2012) Transglutaminase 2: Biology, Relevance to Neurodegenerative Diseases and Therapeutic Implications. Pharmacology & Therapeutics 133, 392–410. http://www.ncbi.nlm.nih.gov/pubmed/22212614

              5.      STAAB 1999: Staab et al. (1999) Adhesive and mammalian transglutaminase substrate properties of Candida albicans Hwp1. Science 283:1535-1538. http://www.ncbi.nlm.nih.gov/pubmed/10066176

              6.      MAIURI 2005: Maiuri et al. (2005) Unexpected Role of Surface Transglutaminase Type II in Celiac Disease. Gastroenterology 129:1400-1413. http://www.ncbi.nlm.nih.gov/pubmed/16285941

              7.      IKURA 1980: Ikura et al. (1980) Crosslinking of casein components by transglutaminase. Agricultural and Biological Chemistry v.44, p.1567-1573. https://www.jstage.jst.go.jp/article/bbb1961/44/7/44_7_1567/_article

              8.      SKOVBJERG 2004: Skovbjerg et al. (2004) Deamidation and cross-linking of gliadin peptides by transglutaminases and the relation to celiac disease. Biochimica et Biophysica Acta 1690, 220 –230. http://www.ncbi.nlm.nih.gov/pubmed/15511629

              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”

 

Jean Seignalet

French physician (1936-2003)

 

"Food is an integral part of medicine and is more than less salt for hypertensives and less sugar for diabetics "

 

"I have sought to understand scientifically how a inadequate food could lead to a pathology."

 

“I have no doubt about this: food is both preventive and curative”

 

“In addition to the genetic predisposition of each one environmental factors are dominant in 90% of diseases

 

Two out of three cancers depend on food

 

“The acquired cancers (non-hereditary) (about 95%), even if found predisposed genes, are essentially caused by environmental factors: food, tobacco, asbestos or virus such as in the case of uterine cervical cancer”

 

“Radiation, chemicals, viruses and non-intestinal bacterias can only explain 40% of acquired cancers. Therefore, for the remaining 60% it seems logical to consider bacterial and food waste resulting intestinal origin of modern food”

 

"Modern food acts on a key body, the small intestine, providing molecules which can not degrade our enzymes. Large molecules, from food and bacterial origin cross the intestinal barrier and enter the blood. Are deposited in various tissues and clog the body. "

 

“Intracellular poisoning is the main reason for cell cancerization. Some foreign macromolecules progressively bother blocking the operation of various mechanisms and accumulation of waste breaks certain physiological balance. I am persuaded that this prolonged poisoning by lead cell ends alterations of nuclear DNA and cause genetic abnormalities that lead to cancer

 

“I have healed from a serious nervous depression by means of a dietary regime that excluded cereals and dairy products, which was rich in raw products”

 

Cow's milk is a very nutritious food ... for calves in the growing season. Humans digest milk only from our species, and at the the nursing period. The main milk protein, casein, is difficult to digest completely

 

You get interesting results yet their medical colleagues do not always believe in the benefits of feeding. JS: "Do not believe in this theory and it could be indifferent to them, I can understand. What surprises me most is that they do not want to experience. I have done my duty by exposing my theory. "

 

 

T. Colin Campbell

American biochemist

1934

 

I consider nutrition to be THE premiere science in medicine – end of story.”

 

“I am most interested in, namely, the comprehensiveness of the nutrition effect on health and disease

 

“I focused on the role of nutrition on health maintenance and disease occurrence.”

 

“What we choose to eat also is one of the most emotionally intense topics of human discourse, ranking up there with sex, religion and politics. Yet, properly practiced nutrition, as a dietary lifestyle, can do more to create health and save health care costs than all the contemporary medical interventions put together.”

"We’ve distorted our diet seriously through the ages, and we have all the problems we have because of that distortion."

 

“The shorthand of the whole thing is we’re eating the wrong foods, basically, animal-­based foods, plus all this processed food, we’re eating the wrong food, and then turning around and relying on this silly notion that we can take a single chemical after we get a disease and hopefully make ourselves well. Ah, we get can get some benefits from that from time to time, but that’s not the long-­term solution to maintain health, it just doesn’t work that way.”

 

“I want people to talk about and to think about how should we be eating in a very empirical scientific sense, and not with an ideological bent to it.”

 

“All humans share virtually the same biochemistry and physiology, regardless of ethnicity, race and gender. They differ, both as individuals and as groups of people, in the DEGREE to which they respond to dietary insult. But the direction of the effect is essentially the same.”

 

BACKGROUND:

 

"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.”

 

CANCER:

 

“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.”

 

GENES:

 

“Every disease begins with genes but this is not a death sentence. For the most part, genes, good and bad, are controlled by nutrition [i.e., the food we consume]. With the right nutrition resources, our marvelous bodies, while always striving for health, manage which genes to express and which ones to keep quiescent. There is also considerable evidence that the initiation and expression of many autoimmune diseases may be influenced by nutrition (and therefore would be repressed by eating whole foods), sometimes initiated early in life, from what mothers eat while pregnant and nursing. We need more awareness of this phenomenon, and then we need to do more research on mother-child interactions.”

 

“We should not be relying on the idea that genes are determinants of our health. We should not be relying on the idea that nutrient supplementation is the way to get nutrition, because it’s not. I’m talking about whole, plant-based foods. The effect it produces is broad for treatment and prevention of a wide variety of ailments, from cancer to heart disease to diabetes.”

 

“There are hundreds of studies now showing that people who move from one country to another where the disease risk is very different, take on the risk of the disease of the country to which they move while they keep their genes the same. In other words, diseases don't occur because of a genetic predisposition, it may for individuals be somewhat different, but regardless of our genetic predisposition, we can control whether or not that we get the disease simply through dietary and lifestyle changes.”

“It is true that we have discovered a tremendous amount of information but this does not mean discovering what it all means. Indeed, our focus on details has created an enormous pile of contradictory observations--permitting too many people to construct ideas that please their palates and wallets more than educate their brains.”

 

RESEARCH:

 

“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:

 

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.”

 

COOKING EFFECT:

 

“Cooked food, yeah, there’s some information that when we’re overcooking foods, certain kinds of foods, we can, in fact, get some noxious chemicals on the basis of the information we now have, heterocyclic amines, as we call that class of compounds, and in the older literature there was another class of compounds referred to as the polycyclic aromatic hydrocarbons, that may result from essentially the burning of food, or having food exposed to fire. And so it’s the kind of information that would suggest that obviously cooking food a little bit is probably OK most of the time, but if we overdo it it’s not a good idea, and it’s one of the reasons I suggest we kind of stay toward the raw food side as much as possible.”

 

How have your colleagues responded to your efforts to reverse chronic diseases through diet: TCC: “As far as the research community is concerned, mostly with silence, although as I write this, I am suddenly seeing an increasing number of medical practitioners beginning to carry the banner forward. These people have, for the most part, seen first hand what they can do for their patients when they adopt this practice. I've lost some research colleagues but I've gained a lot more new colleagues.”

 

“Incidentally, that kind of eating was much better appreciated and accepted and promoted as long ago as the ancient Greek times. Some of the leading Greek philosophers and others who thought about medicine, diet and disease wrote surprisingly impressive observations on the relationship between eating that kind of food and good health. It all sort of disappeared around 300 or 400 AD, for some strange reason lots of things disappeared about that time, and I think that we're just now beginning to come out of the dark ages and going back to this; rediscovering what was well known to these medical people and philosophers.”

 

 

Hiromi Shinya

Japanese Physician

1935

 

“I have examined the stomachs and colons and taken the dietary history of more than 300.000 patients”

 

"I have examined more than 300,000 people's stomachs and intestines for 35 years and realize that our health depends largely on our dietary life

 

“I have discovered a strong relationship between health and certain ways of eating and living”

 

“Diseases, life and health are the result of what you eat every day

 

 “…diseases of ‘unknown cause’ can sometimes be traced back to dietary history.”

 

“Looking at the dietary history of cancer patients, I usually find that they have had a diet consisting mainly of animal protein and dairy such as meat, fish, eggs and milk... for women with breast cancer and men with prostate cancer, the probability of discovering an abnormality in their colon is high.”

 

"Dairy is the worst food you can put in your body."

 

“There is no other food that is as difficult to digest as milk.”

 

Casein, which accounts for approximately 80% of the protein found in milk, immediately clumps together once it enters the stomach, making digestion very difficult.”

 

“We do not inherit disease from our parents, we inherit their dietary habits and all the health problems that come with it.”

 

Good habits will overcome bad genes

 

 

Novak Djokovic

Serbian professional tennis player and world No. 1

1987

 

“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 stuffy, 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.)”

 

“Insanity: doing the same thing over and over again and expecting different results.” Albert Einstein

“If you always do what you always did, you will always get what you always got.” Albert Einstein

 

FOTGCREN@ono.com

First version of the page 27 - 8 - 2015

Current version 21 - 8 - 2016

 

hits counter