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Journal of Pediatric Gastroenterology & Nutrition:
Invited Reviews

Role of Tissue Transglutaminase in Celiac Disease

Molberg, Øyvind; McAdam, Stephen N.; Sollid, Ludvig M.

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Institute of Immunology, University of Oslo, Rikshospitalet, Oslo, Norway

Received September 14, 1999;

revised November 29, 1999; accepted December 1, 1999.

Address correspondence and reprint requests to Dr. Øyvind Molberg, Institute of Immunology, Rikshospitalet, N-0027 Oslo, Norway.

Celiac disease (CD), a gluten-sensitive enteropathy, is a multifactorial disease affecting 1:200 to 1:400 persons within the European populations (1). The chronic small intestinal inflammation of CD is the result of an inappropriate T-cell–mediated immune response against ingested gluten proteins from wheat and similar proteins in barley and rye (2,3). Normally, our immune system displays tolerance to edible proteins. To understand how and why CD develops, it is therefore critical to identify the genetic and environmental factors involved in the failure of tolerance to gluten proteins. There is a strong genetic influence on the susceptibility to CD and the predisposing genes involve both HLA and non-HLA genes (4,5). It is notable that, in common with most other HLA-associated diseases, CD is not a result of a gene defect, but rather the combined effect of normally functioning variant gene products (6). The primary HLA-association in CD is to the HLA-DQA1*0501, DQB1*0201 genes encoding the DQ2 molecule and to a lesser extent to the DQA1*0301, DQB*0302 genes encoding the DQ8 molecule (7). Interestingly, it seems that non-HLA genes together contribute more to genetic susceptibility than do the HLA-genes, but the contribution from each single, predisposing non-HLA gene appears to be modest (1).

Disease-specific CD4+ memory T cells that recognize gluten proteins can be isolated from biopsy specimens of affected intestine (8–12). A striking feature of these gluten-specific T cells is that most of them use the disease-associated DQ2 molecule (or the DQ8 molecule in the few DQ8+ patients) as their restriction elements (8–10). Moreover, CD has an autoimmune component characterized by the occurrence of disease-specific autoreactive antibodies (Abs), whose serum titers fluctuate with exposure to gluten in the diet. In 1996, Dieterich et al. (13) demonstrated that these autoreactive serum IgA Abs targeted the enzyme tissue transglutaminase (tTG). The diagnostic implications of this seminal study are currently under intense investigation (14,15). The finding has also renewed the interest in defining the roles of this enzyme in CD.

This review is primarily focused on recent advances in our understanding of how tTG may be involved in disease pathogenesis. First, however, we provide an overview of the reactions catalyzed by tTG and the various functions of the enzyme, along with a brief description of the potential roles of tTG in human diseases other than CD.

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The most established property of tTG and the other transglutaminases is to catalyze a Ca2+-dependent cross-linking of a specific glutamine residue in the substrate protein to a primary amine (Fig. 1) (16,17). The acceptor amine can be either a protein-bound lysine (Lys) or a polyamine. Notably, the reaction between substrate and tTG occurs in two distinct steps (16). The first step is binding of a Gln side-chain amino group from the substrate to the catalytic site of tTG, which is followed by release of NH3 and formation of a substrate–enzyme intermediate. The second step is the reaction between substrate and an available amine, or in the absence of amines, the reaction with H2O. The substrate can become cross-linked to Lys in other proteins, to polyamines, or if the substrate itself contains available Lys residues to itself through intra-or intersubstrate cross-linking. If the substrate reacts with H2O, deamidation of the reactive Gln to yield glutamic acid (Glu) will occur. It should be noted that the reaction with H2O occurs with a slower rate than the reaction with acceptor amines (18), for this reason the deamidation of substrate by tTG has often been disregarded (19). Importantly, the result of the two-step reaction between tTG and substrate is always a posttranslational modification of a specific Gln residue in the substrate.

Fig. 1
Fig. 1
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All the transglutaminases display a high degree of specificity such that only a limited number of proteins can act as substrate. The rules governing the binding of protein-bound Gln residues to tTG are complex and far from clear (20–22). In contrast, it appears that most protein-bound Lys and polyamines can be accommodated in the catalytic site to cross-link the substrate (23). In addition to the catalytic site and a minimum of two Ca2+-binding sites, tTG has an N-terminal fibronectin association site (24) and a site that binds and hydrolyses guanosine triphosphate (GTP) (25). This latter GTPase activity of tTG is independent of the transglutaminating activity, but binding of GTP inhibits substrate binding to the catalytic site. In tTG-transfected cells, the intracellular GTPase activity of tTG mediates α1-adrenergic receptor–mediated stimulation of phospholipase C (25). Notably, whereas the catalysis of substrate modification by tTG takes place both in intracellular and extracellular compartments, the GTP hydrolysis by tTG is strictly an intracellular function (19).

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The transglutaminases are a large family of related enzymes that are distributed widely in tissues and body fluids of all mammals (19,23). The genes encoding transglutaminases are well conserved between species (for instance 80% homology between guinea pig tTG and human tTG) (19), reinforcing the notion that these enzymes have important biological functions. In humans the transglutaminase family includes tTG, blood coagulation factor XIII, keratinocyte TG, epidermal TG, prostate TG, erythrocyte band 4.2, and a novel transglutaminase from the skin (26). The different human transglutaminases share a common motif in the catalytic site and have similar gene structures, indicating that they stem from the same ancestral gene. They are, however, encoded by separate genes and display unique expression patterns as well as differences in substrate specificity. The human tTG gene encodes a 684-amino-acid nonglycosylated single-polypeptide chain (27). It is not clear whether the gene contains any polymorphisms. The tTG gene is expressed in a broad range of tissues, under complex but tight regulation that is influenced by many factors including retinoids, transforming growth factor (TGF)-β1, interleukin-6, and DNA methylation (27–31). Constitutive expression of tTG is found in endothelial cells in blood vessels, in smooth muscle cells, and in some other organ-restricted cells (32). In CD, it is interesting that tTG has been detected in all layers of the small intestinal wall. The enzyme is most heavily expressed in the submucosa, and only 1% of the small intestinal tTG is located within the epithelium. It is noteworthy that crypt epithelial cells do not express tTG, but the intracellular expression increases as the cells maturate and migrate toward the small intestinal villi (32).

The physiological roles of tTG are not fully characterized. In fact, many basic questions remain unresolved. Tissue transglutaminase does not have a typical signal sequence for routing through the normal secretory pathway of cells, but despite this, tTG appears to be actively transported to the extracellular space through an unknown mechanism and not simply shed from dead cells (19). After its synthesis, tTG is found in the cytosol as an inactive enzyme. The effect of Ca2+ ions and GTP on enzyme activation is well described (33). Otherwise, the requirements for intra-and extracellular activation of tTG are largely unknown. An exciting observation is that binding of tTG to specific membrane lipids is involved in the regulation by decreasing the dependence of Ca2+(34). Taken together, available data suggest that the biosynthesis, secretion, and activation of tTG are strictly but independently regulated processes.

A number of in vitro studies implicate tTG in various, often critical biological processes as diverse as cell growth and differentiation (35), formation of extracellular matrix (36), tissue repair (37), apoptosis (38), and, independently, signal transduction through the α1-adrenergic receptor (25). Because all these processes appear to be critical for homeostasis, it could be envisaged that one or more of them could be involved in CD.

While bearing in mind that CD is an immune-mediated disorder, it should also be noted that the activity of tTG has been associated with receptor-mediated endocytosis (39), phagocytosis (40), antigen presentation (41,42), and differentiation of lymphocytes, monocytes, and B cells (43–45). It is particularly interesting that macrophages display increased tTG activity after inflammatory stimuli and that this increase in enzyme activity leads to enhanced Fc receptor-mediated endocytosis in the macrophages (40,46).

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Tissue TG has been linked to a number of diseases, including neurologic disorders (47–50), cancer (51,52), human immunodeficiency virus (HIV) infection (38), inflammatory bowel disease (53), liver cirrhosis (29), cataract of the eye (30), and various autoimmune disorders (54). The role of tTG in certain neurologic disorders, including Huntington's disease has been extensively studied. The huntingtin proteins associated with the central nervous system involvement of this disorder are good substrates for tTG (49,50). Interestingly, huntingtin proteins from patients with Huntington's disease have a polyglutamine stretch above a critical length, a feature that enhances their activity as substrates for tTG (50). Moreover, brains from patients with Huntington's disease display higher tTG activity than control brains (49). Another example of pathologic protein cross-linking in the central nervous system is found in Alzheimer's disease. In this disorder it is the disease-associated tau proteins that become cross-linked by tTG (47).

A close correlation between the expression of tTG and the rate of apoptosis have been reported in HIV-infected CD4+ T cells (38), this supports the hypothesis that tTG-mediated cross-linking is a critical event during induction of apoptosis. It has been proposed that the tTG-mediated modification of intracellular proteins during apoptosis can lead to formation of neoantigens that may trigger autoimmunity (54,55). This possibility has hitherto been supported only by circumstantial evidence.

The deamidating activity of tTG implicated in CD (see later description) has so far not been associated with other diseases. However, the lenses of the eye have high TG activity, and it has been shown that specific Gln residues in water-soluble eye lens proteins are deamidated (56). Whether these deamidations, which are associated with cataract development, are catalyzed by tTG is presently unknown (57). In addition, recent studies have described deamidation of specific Glns in human proteins by a bacterial TG. Notably, these deamidations dramatically change the functional properties of the human proteins (58).

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In a pioneering study in 1985, Bruce et al. (59) demonstrated that homogenates of intestinal biopsy specimens contain an enzyme that has the ability to cross-link labeled polyamines to a defined transglutaminase substrate. They also showed that specimens from treated and untreated patients with CD had higher transglutaminase activity than control specimens. Notably, the homogenates also catalyzed cross-linking of the labeled polyamines to gliadin proteins, indicating that gliadin could also act as a substrate for transglutaminase. An Italian study confirmed the finding of transglutaminase activity in biopsy specimens from patients with CD and further reported decreased serum transglutaminase activity in the same patients (60). However, it should be noted that serum transglutaminase activity is mainly mediated by factor XIII and not by tTG.

Later, gliadin proteins were identified as good substrates for tTG. In fact, up to 36% of the Gln residues in gliadin were accessible to modification by tTG, when evaluated either by polyamine incorporation or by liberation of NH3 during deamidation of the gliadin (61). In both cases, large polymers, probably gliadin-gliadin complexes (formed by cross-linking of Gln to one of the few gliadin Lys residues) and/or tTG-gliadin complexes were detected.

The identification of tTG as the target of CD-specific IgA anti-endomysial Abs (EMAs) revived the idea that these large complexes may play a role in the pathogenesis of CD (13). Dieterich et al. (62) demonstrated that tTG-gliadin complexes could be formed in vitro and argued that these complexes may contain neoepitopes that trigger mucosal T cells and degrade tolerance. The production of the anti-tTG IgA Abs must be dependent on cognate T-cell help to facilitate the isotype switching of the autoreactive B cells. Dieterich et al. suggested that the necessary help for the B-cell production of anti-tTG IgA could be provided by autoreactive T cells specific for tTG. We, however, doubt the existence of autoreactive T cells. Because tTG is expressed in the thymic epithelium, it is likely that tTG-reactive T cells are deleted during negative selection in the thymus (32). Moreover, the consequence of having autoreactive T cells specific for such a ubiquitously expressed enzyme as tTG, would most likely be systemic autoimmunity (63). As an alternative, we have suggested a model that is not dependent on the presence of tTG-reactive T cells. Instead the production of anti-tTG antibodies is driven entirely by intestinal gliadin-specific T cells (63). Our hypothesis is based on the premise that the tTG-gliadin complexes form in vivo function analogous to the hapten-carrier complexes described in mouse immunology (64). The principle is simple. The tTG-gliadin complexes must be bound to the tTG-specific B cells to be endocytosed and processed. This processing allows both tTG and gliadin fragments to be presented by DQ2 and the other class II molecules expressed by the tTG-specific B cells. The gliadin-DQ2 complexes presented by the tTG-specific B cells are recognized by the gliadin-specific T cells present in the celiac mucosa. The activation of these T cells results in proliferation and provides in return the necessary help for the tTG-specific B cell to produce anti-tTG Abs (63). Recently, two reports have supported the notion that anti-tTG Abs can be produced by B cells present in the celiac lesions. Both the studies demonstrated that anti-tTG Abs were detectable in supernatants from intestinal CD biopsy specimens cultured ex vivo (65,66). In one of these studies the production of anti-tTG was independent of biopsy challenge (66), whereas in the other the production of anti-tTG was found to be dependent on gliadin challenge (65). The possibility of a functional role of the anti-tTG Abs in disease pathogenesis will be discussed later.

Although the studies mentioned indicate a role of tTG in disease pathogenesis, their results were not easily reconciled with the prevailing view of CD as a T-cell–mediated disorder. We wanted to examine whether such a link existed, particularly because we knew at that time that intestinal T cells of patients with CD recognize deamidated gliadins and because tTG was capable of deamidating these proteins. Intriguingly, tTG had a potent and specific effect on the intestinal T-cell recognition of gliadin. We demonstrated that tTG in vitro catalyzed an ordered and specific deamidation of certain glutamine (Gln) residues in wheat gliadin peptides. These specific modifications generated an antigen that was optimal for gut-derived, DQ-restricted T cells (67). Fine-specificity analysis of an immunorelevant γ-gliadin epitope (DQ2-γ-I) recognized by a DQ2-restricted intestinal T cell (68) unveiled that the tTG-mediated deamidations occur in positions that were beneficial for the binding of the peptide to DQ2 and critical for recognition by the intestinal T cell (67). Independently, van de Wal et al. demonstrated that tTG-modification of a gliadin epitope recognized by a DQ8-restricted intestinal T-cell clone greatly enhanced the reactivity of the T cell (69).

Recently, two different intestinal T-cell epitopes (DQ2-α-I and DQ2-α-II) located within two recombinant α-gliadins (α-9 and α-2, respectively) were identified (70). The α-gliadin peptides containing the epitopes were distinct but partially overlapping and shared a common seven-amino-acid motif—α-2 (62–75) and α-9 (57–68). In their native form, these peptides were not recognized by intestinal T cells, but after deamidation by tTG both became very efficient T-cell antigens. Intriguingly, tTG was found to catalyze deamidation of the same single Gln residue (Gln65) in epitope DQ2-α-I (α-9[57-68]E65) and DQ2-α-II (α-2[62-75]E65). Moreover, it appeared that the DQ2-α-I and DQ2-α-II epitopes were very frequently recognized by intestinal T cells from adult DQ2+ Norwegian patients with CD. Combined analysis of T-cell recognition and DQ2 binding assays indicated that the Glu65 residue of the DQ2-α-I epitope was accommodated in relative position 6 in the DQ2 peptide-binding groove and the Glu65 residue of DQ2-α-II in relative position 4. The DQ2-binding assays demonstrated that the native α-2 and α-9 gliadin peptides bound poorly to DQ2, whereas the deamidated epitopes bound with reasonable, but by no means exceptional, binding affinity (70).

The data from the mapping of three distinct intestinal T-cell epitopes allows the following conclusions. In their native form, the three peptides bind poorly to DQ2 and are at best only barely recognized by intestinal T cells. After in vitro tTG treatment, they are, however, transformed into epitopes that bind reasonably well to DQ2 and are recognized by DQ2-restricted T cells isolated from biopsy specimens of patients with CD. It appears that the deamidation of the gliadin peptides have a dual effect. The introduction of negatively charged Glu residues at defined positions in the gliadin peptides markedly increases their binding affinity to the disease-associated DQ2 molecule (71). This is in line with the proposed peptide-binding motif for DQ2 that predicts a preference for negatively charged residues in certain positions (72–75). Notably, the deamidated residues in the three defined epitopes are located in different relative positions. This indicates that the immune response against gliadin in CD is not directed against one pathogenic gliadin peptide or a single “pathogenic motif.” The tTG-mediated deamidation of the three different gliadin peptides is also critical for recognition by intestinal T cells. Collectively, these data suggest that tTG, by deamidating certain residues in ingested gliadin proteins, could be a key factor in the breakdown of tolerance in CD.

The data above demonstrated the in vitro effect of tTG-catalyzed deamidations of gliadin. A critical question, however, is whether tTG can also deamidate gliadin proteins in vivo. Immunohistochemical staining of intestinal biopsy specimens from patients with CD demonstrated a spatial relationship between extracellular tTG, DQ-expressing cells, and T cells in the subepithelial region (67). Moreover, we have preliminary data to suggest that T cells from CD lesions recognize gliadin epitopes deamidated in situ by endogenous tTG (Molberg et al., submitted, 2000). Taken together, these observations provide persuasive evidence that the deamidation of gliadin peptides in vivo is mediated by endogenous tTG in a microenvironment containing the necessary factors to initiate an immune response.

Gliadin-specific T cells isolated from the peripheral blood of patients with CD are neither predominantly DQ restricted nor sensitive to tTG treatment of the gliadin (67,76). This strongly argues that they recognize gliadin epitopes different from the epitopes recognized by intestinal T cells. A possible explanation for this is that the gliadin-specific T cells isolated from the celiac lesions represent a distinct subpopulation of memory T cells that selectively home to the intestinal microenvironment from whence they were initially triggered. Gliadin-specific T cells can be isolated from the mucosa of patients with CD who have consumed a gluten-free diet for 10 to 20 years, indicating that at least some of them are localized in the lamina propria throughout the patients' lifespans. It is tempting to speculate that tTG, by interacting with extracellular proteins and perhaps plasma membrane proteins can be involved in the formation of a reticular network that colocalizes memory T cells and DQ-expressing cells in the celiac lesions.

In the introduction, we stated that an understanding of the pathogenesis of CD was dependent on the identification of the involved genetic and environmental factors. From such a perspective, the demonstration of the connection between intestinal T-cell reactivity and tTG-mediated deamidation of gliadin generates as many new questions as answers.

An attractive but speculative hypothesis is that tolerance to gluten is broken in patients with CD when endogenous tTG starts to form deamidated gluten peptides that bind to DQ2 molecules in the mucosa and thereby trigger local CD4+ T cells (Fig. 2). Based on this hypothesis, the chronic intestinal inflammation can be viewed as the final consequence of complex interactions between the products of disease predisposing genes and environmental factors. That biopsy specimens from both untreated patients with CD and patients consuming a gluten-free diet display higher tTG activity than those of control subjects indicates that some of the genes predisposing to CD may be identified among the factors that affect the transcription regulation or functional activity of the enzyme.

Fig. 2
Fig. 2
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The triggering of pathogenic immune responses may be related to induction of the deamidating activity of tTG. Feasibly, deamidation only happens if tTG activity is high, substrate is in excess, and the level of acceptor amines is low. During episodes of intestinal inflammation, such situations could occur. Inflammation induces tTG activity, disturbs epithelial integrity, and, notably, increases the need for exogenous and endogenous polyamines, in that cross-linking of polyamines by tTG is critical for normal repair functions of the intestinal mucosa (37). The polyamine content in a normal diet is not high, and a rate-limiting enzyme restricts synthesis of polyamines (77). An inflammation may thus cause a local depletion of polyamines concurrently with an increased leakage of gluten proteins across the epithelial barrier. Notably, the possibility that the deamidating activity identified in celiac lesions is not mediated by tTG but by some other as yet unidentified enzyme should not be discarded. No other human enzyme that catalyzes deamidation of specific protein-bound Gln residues has been identified, but it should be remembered that bacterial TG, which could be present in the gut lumen, preferentially deamidates the certain subtrates, instead of cross-linking them (58).

The activity of tTG is implied in critical biological functions that, if dysfunctional, may disrupt homeostasis in the intestinal mucosa. Although there are currently few data to support these ideas, it is worth mentioning a few examples. Increased apoptosis of epithelial cells may destroy the integrity of the epithelium and cause increased leakage of gluten proteins. Moreover, tTG-associated changes in the apoptotic rate of local immune-competent cells may skew the balance from tolerance to aggressive immune responses. The activation of TGF-β, a pleiotropic growth factor, is partly driven by tTG (78), and alterations in the level of active TGF-β have specific effects on T cells, B cells, and antigen-presenting cells (79). Finally, it has been demonstrated that binding of calreticulin to tTG in vitro inhibits both the TG and the GTPase activity of the enzyme (80). Notably, calreticulin has been described as an additional target for Abs in CD (81). The binding of calreticulin to tTG, in the presence of gliadin, enables formation of hapten-carrier complexes comparable to the previously described tTG-gliadin complexes. It may thus be envisaged that gliadin-specific T cells provide cognate help to production of anti-calreticulin Abs by calreticulin-specific B cells in a fashion similar to the help provided to the production of anti-tTG Abs.

The anti-tTG IgA Abs from patient sera preferentially bind Ca2+-activated tTG (14,15). This strongly indicates that they recognize conformation-dependent epitopes in the enzyme. In certain autoimmune diseases, including Mb Addison, autoimmune thyroiditis, and type I diabetes (82–84), similar conformation-dependent serum auto-Abs that target disease-relevant enzymes have been identified. A subset of these conformation-dependent auto-Abs have been shown to interfere with the catalytic activity of the enzyme (82,83). Thus, a critical question that would have a profound impact on our understanding of the pathogenesis of CD is whether anti-tTG Abs modulate the activity of tTG in any way (85).

Given that anti-tTG Abs affect tTG, many different outcomes are possible. The antibodies could inhibit or enhance the cross-linking, deamidating and/or GTPase activity of the enzyme. In any case, a feedback loop between anti-tTG Abs and the activity of tTG could be envisaged. In CD the most obvious feedback system is connected to the tTG-mediated deamidation of gluten peptides. As outlined earlier, this is followed in vivo by binding of the deamidated peptides to DQ2 and specific activation of intestinal T cells that provides help for the tTG-specific B cells to produce the anti-tTG Abs that finally bind to activated tTG. The effect of this feedback system is dependent on how the Abs influence the rate of deamidation of the gliadin peptides. If antibody binding inhibits the catalytic activity of tTG, the effect should be beneficial, because fewer gliadin peptides become deamidated. In contrast, if binding enhances tTG activity, a vicious circle perpetuated by gliadin exposure is formed.

Indications of an inhibiting effect on the cross-linking activity of tTG by serum anti-tTG IgA Abs from patients with CD have been reported in a recent in vitro study (86). It should be noted, however, that this study did not provide formal evidence that the observed effects were due to interactions between anti-tTG and tTG (86). Many relevant arguments against a role of anti-tTG IgA Abs in CD can also be put forth. One is that CD occurs with an increased frequency among patients with primary IgA deficiency (1). In this subgroup of patients, intestinal lesions develop that are indistinguishable from those in other patients, a fact that argues against a critical role of anti-tTG IgA in disease pathogenesis. It should be noted, however, that it is not clear whether the anti-tTG IgG found in the IgA-deficient patients with CD may substitute for the anti-tTG IgA produced by the other patients. Another concern is the possible effects of binding of serum anti-tTG Abs to tTG expressed outside the small intestinal mucosa. It could be speculated that if this binding of serum Abs to enzyme influences tTG activity, it would cause systemic responses. Along the same lines is the suggestion that serum anti-tTG Abs could cross-react with the other transglutaminases. This possibility is particularly interesting with respect to dermatitis herpetiformis. Patients with this CD-associated skin disorder have levels of serum anti-tTG IgA comparable with other patients with CD (87). Therefore, it can not be excluded that the enigmatic (and diagnostic) IgA deposits in the skin of patients with dermatitis herpetiformis are in fact anti-tTG Abs cross-reacting with a transglutaminase expressed in skin (87).

The elucidation of the role of tTG in CD will be facilitated by the functional characterization of the anti-tTG Abs. If they affect enzymatic activity, then CD must be redefined as a T-cell–mediated disorder modulated by autoimmune B cells. Ongoing research should clarify these critical issues.

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We have focused on a discussion of tTG and its potential role in CD. The data obtained so far demonstrate at least two different, although not necessarily independent, roles of this enzyme in disease pathogenesis. First, activated tTG is the main target of disease-specific autoantibodies. Second, the deamidating activity of the enzyme has been shown to create gliadin peptides that bind DQ2 to be recognized by disease-specific intestinal T cells. Future work should establish the connection between these two immune-related functions of tTG and, as discussed, should search for further roles of the enzyme. Notably, the phenomenon of tTG-mediated modification of substrate has been linked to several diseases, but it is only in CD that the role of the deamidating activity of the enzyme has been defined.

On a more general level, it is interesting to note that an increasing number of reports identify strong, specific immune responses against posttranslationally modified proteins (88–91). The modification of proteins could occur spontaneously, or as is the case in CD, could be catalyzed by enzymes. A multitude of enzymes capable of catalyzing specific protein modifications exists, and abnormal activation of any of these in microenvironments with ongoing immune activation could modify self or non–self proteins to create neoepitopes. Because they by definition are novel to the immune system, all such neoepitopes could be involved in the breaking of tolerance and precipitation of disease.

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