Specificity of tissue transglutaminase explains cereal toxicity in celiac disease.

Celiac disease is caused by a selective lack of T cell tolerance for gluten. It is known that the enzyme tissue transglutaminase (tTG) is involved in the generation of T cell stimulatory gluten peptides through deamidation of glutamine, the most abundant amino acid in gluten. Only particular glutamine residues, however, are modified by tTG. Here we provide evidence that the spacing between glutamine and proline, the second most abundant amino acid in gluten, plays an essential role in the specificity of deamidation. On the basis of this, algorithms were designed and used to successfully predict novel T cell stimulatory peptides in gluten. Strikingly, these algorithms identified many similar peptides in the gluten-like hordeins from barley and secalins from rye but not in the avenins from oats. The avenins contain significantly lower percentages of proline residues, which offers a likely explanation for the lack of toxicity of oats. Thus, the unique amino acid composition of gluten and related proteins in barley and rye favors the generation of toxic T cell stimulatory gluten peptides by tTG. This provides a rationale for the observation that celiac disease patients are intolerant to these cereal proteins but not to other common food proteins.

New chelation strategy allows for quick and clean 99mTc-labeling of synthetic peptides.

Analogues of bombesin have been synthesized in which a N2S2 (bis-mercaptoacetyl functionalized diaminopropionic acid) or a N3S (mercaptoacetyl-Gly-Gly-Gly) radiometal-chelating center has been incorporated that allows radiolabeling of these peptides with 99mTc without the need for conjugation or harsh reaction conditions. A mild radiolabeling is possible by using an acetyl-moiety as sulfur protecting group, which can be removed by mild hydroxylamine-treatment at room temperature before radiolabeling. Retained receptor binding is demonstrated in competitive binding experiments with 99mTc-radiolabeled peptides and PC-3 cells with bombesin receptors.

T-cell recognition of HLA-DQ2-bound gluten peptides can be influenced by an N-terminal proline at p-1.

Recent research has implicated a large number of gluten-derived peptides in the pathogenesis of celiac disease, a preponderantly HLA-DQ2-associated disorder. Current evidence indicates that the core of some of those peptides is ten amino acids long, while HLA class II normally accommodates nine amino acids in the binding groove. We have now investigated this in detail, using gluten-specific T-cell clones, HLA-DQ2-specific peptide-binding assays and molecular modelling. T-cell recognition of both a gamma-gliadin peptide and a low-molecular-weight glutenin peptide was found to be strictly dependent on a ten-amino acids-long peptide. Subsequent peptide-binding studies indicated that the glutenin peptide bound in a conventional p1/p9 register, with an additional proline at p-1. Testing of substitution analogues demonstrated that the nature of the amino acid at p-1 strongly influenced T-cell recognition of the peptide. Moreover, molecular modelling confirmed that the glutenin peptide binds in a p1/p9 register, and that the proline at p-1 points upward towards the T-cell receptor. Database searches indicate that a large number of potential T-cell stimulatory gluten peptides with an additional proline at relative position p-1 exist, suggesting that the recognition of other gluten peptides may depend on this proline as well. This knowledge may be of importance for the identification of additional T-cell stimulatory gluten peptides and the design of a peptide-based, tolerance-inducing therapy.

The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses.

In patients with celiac disease, inflammatory T cell responses to HLA-DQ2-bound gluten peptides are thought to cause disease. Two types of HLA-DQ2 molecules exist, termed HLA-DQ2.5 and HLA-DQ2.2. Whereas HLA-DQ2.5 predisposes to celiac disease, HLA-DQ2.2 does not. We now provide evidence that the disease-associated HLA-DQ2.5 molecule presents a large repertoire of gluten peptides, whereas the non-disease-associated HLA-DQ2.2 molecule can present only a subset of these. Moreover, gluten presentation by HLA-DQ2 homozygous antigen-presenting cells was superior to presentation by HLA-DQ2/non-DQ2 heterozygous antigen-presenting cells in terms of T cell proliferation and cytokine secretion. Gluten presentation by HLA-DQ2.5/2.2 heterozygous antigen-presenting cells induced intermediate T cell stimulation. These results correlated with peptide binding to the antigen-presenting cells. Finally, we demonstrate that HLA-DQ trans dimers formed in HLA-DQ2.5/2.2 heterozygous individuals have properties identical with HLA-DQ2.5 dimers. Our findings explain the strongly increased risk of disease development for HLA-DQ2.5 homozygous and HLA-DQ2.2/2.5 heterozygous individuals, and they are indicative of a quantitative model for disease development, where HLA-DQ expression and the available number of T cell-stimulatory gluten peptides are critical limiting factors. This model may have important implications for disease prevention.

Characterization of cereal toxicity for celiac disease patients based on protein homology in grains.

BACKGROUND AND AIMS: Celiac disease is caused by T-cell responses to wheat gluten-derived peptides. The presence of such peptides in other widely consumed grains, however, has hardly been studied. METHODS: We have performed homology searches to identify regions with sequence similarity to T-cell stimulatory gluten peptides in the available gluten sequences: the hordeins of barley, secalins of rye, and avenins of oats. The identified peptides were tested for T-cell stimulatory properties. RESULTS: With 1 exception, no identical matches with T-cell stimulatory gluten peptides were found in the other grains. However, less stringent searches identified 11 homologous sequences in hordeins, secalins, and avenins located in regions similar to those in the original gluten proteins. Seven of these 11 peptides were recognized by gluten-specific T-cell lines and/or clones from patients with celiac disease. Comparison of T-cell stimulatory sequences with homologous but non-T-cell stimulatory sequences indicated key amino acids that on substitution either completely or partially abrogated the T-cell stimulatory activity of the gluten peptides. Finally, we show that single nucleotide substitutions in gluten genes will suffice to induce these effects. CONCLUSIONS: These results show that the disease-inducing properties of barley and rye can in part be explained by T-cell cross-reactivity against gluten-, secalin-, and hordein-derived peptides. Moreover, the results provide a first step toward a rational strategy for gluten detoxification via targeted mutagenesis at the genetic level.

The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides.

BACKGROUND & AIMS: Gluten (GLU)-specific T-cell responses in HLA-DQ2 positive adult celiac disease (CD) patients are directed to an immunodominant alpha-gliadin (GLIA) peptide that requires deamidation for T-cell recognition. The aim of the current study was to determine which GLU peptide(s) are involved early in disease. METHODS: We have characterized the GLU-specific T-cell response in HLA-DQ2 positive children with recent onset CD. RESULTS: We found that 50% of these patients do not respond to the alpha-GLIA peptide but to a diverse set of GLIA and glutenin (GLT) peptides, including 6 novel epitopes. Moreover, individual patients respond to distinct (combinations of) GLU peptides. T-cell cross-reactivity toward homologous GLIA and GLT peptides was observed, which might play a role in the initial spreading of the GLU-specific T-cell response. Although all pediatric patients displayed deamidation-dependent responses, deamidation-independent responses were found in the majority of patients as well. Finally, T-cell responses to 3 of these novel GLU peptides were found in adult CD patients. CONCLUSIONS: The diversity of the GLU-specific T-cell response is far greater than was previously appreciated. Both adult and young CD patients can respond to a diverse repertoire of GLU peptides. The observation that T-cell responses to 3 of the novel peptides are independent of deamidation indicates that T-cell responses can be initiated toward native GLU peptides. The possibility that deamidation drives the GLU response toward immunodominant T-cell stimulatory peptides after disease initiation is discussed.