Endocrine Abstracts

Background: Post-translational modification (PTM) of antigens has been shown to play a role in the pathogenesis of autoimmune disorders. In coeliac disease (CD), tissue transglutaminase (tTG) deamidates gliadin peptides to activate the immune response against gut endomysium. CD is six times more prevalent in Type 1 diabetes (T1D) patients than in the general population.

Hypothesis: tTG also modifies auto-antigens implicated in the pathogenesis of T1D, leading to an autoimmune response to pancreatic β-cells.

Methods: 20 randomly selected samples from the Bart’s-Oxford cohort were used to study the effects of tTG PTM. tTG was incubated with the following auto-antigens, which had previously been shown to have high correlation with the development of T1D: glutamic acid decarboxylase isoform 65 (GAD65), full length islet antigen (IA-2), intracellular portion of IA-2 (IA-2ic), and both isoforms of zinc transporter 8 (ZnT8W and ZnT8R). Antigens were radiolabelled and incubated with tTG for 20 h at 27 °C in 100 mM Denver buffer, 3.33 nM CaCl₂, at pH 7.3. Antibody binding with incubated antigen was measured using a standard radiobinding assay. Site-directed mutagenesis was carried out in ZnT8W, changing glutamine (Q) to glutamate (E) at residue 375, to mimic the expected action of tTG incubation with wild type ZnT8W.

Results: tTG treatment of ZnT8W, ZnT8R and IA-2ic showed no significant change in antibody: antigen binding. Modest increases in binding were observed with tTG-treated GAD65 and full length IA-2 (6.5 and 14.5% respectively). Decreases in binding of antibody to tTG-treated ZnT8W compared with mutant ZnT8W Q375E in 3 samples (% reduction of 69.9, 42.1 and 19.5), where antibody binding should have been the same, questions what tTG modification events occurred, or whether these sera contained antibodies against the Q-X-P site.

Conclusion: In the case of GAD65, full length IA-2, and certain samples with IA-2ic, the strength of antibody: antigen binding increased after incubation with tTG. However, the exact PTM events occurring with tTG incubation for each antigen, as well as the exact sites of antibody: antigen binding in each sample, require further study.