Two amino acids in glutamic acid decarboxylase act in concert for maintainance of conformational determinants recognised by Type I diabetic autoantibodies

Aims/hypothesis. Glutamic acid decarboxylase 65 is a major autoantigen in T ype I (insulin-dependent) diabetes mellitus, autoimmune polyendocrine syndr ome and stiff-man syndrome. These disorders are characterised by the presen ce of multiple autoantibodies to the autoantigen which can be distinguished in a variety of different ways. We have investigated the role of single am ino-acid mutations in glutamic acid decarboxylase 65 in distinguishing the binding of serum antibodies and a variety of patient-derived human IgG mono clonal antibodies directed to different determinants of the autoantigen. Methods. We identified a mutant of glutamic acid decarboxylase 65 that cont ained four single amino-acid mutations from the wild-type molecule. The rol e of these mutations was investigated by site-directed mutagenesis. We inve stigated the binding of patient-derived serum antibodies to glutamic acid d ecarboxylase 65 to a number of single and double amino-acid mutants using i mmunoprecipitation with labelled, recombinant antigen. To overcome the hete rogeneity of different anti-glutamic acid decarboxylase 65 antibodies prese nt in a patient's serum, the binding of a panel of eleven patient-derived h uman monoclonal antibodies recognising different determinants on the autoan tigen was also studied. Results. Two replacements in glutamic acid decarboxylase 65 at Asn247Ser an d Leu574Pro were identified that preferentially influence the anti-glutamic acid decarboxylase 65 serum antibodies of Type I diabetic patients, withou t statistically significantly effecting those recognised in other disorders . Single or double amino-acid replacements Asn247Ser and Leu574Pro in the a utoantigen showed differential affects on expression of epitopes recognised by the human monoclonals. The double replacement of Asn247Ser and Leu574Pr o in glutamic acid decarboxylase 65 resulted in the loss of binding of all eleven human monoclonal antibodies, irrespective of their epitope recogniti on. In contrast, single replacement of Leu574Pro statistically significantl y reduced the binding of some carboxyl terminal-directed antibodies such as MICA 1, MICA 3 and DP-A without influencing the binding of other monoclona ls. Replacement of Asn247Ser did not, however, influence the binding of any patients serum or human monoclonal antibodies. Conclusion/interpretation. Two distantly spaced amino acids, Asn247Ser and Leu574 in glutamic acid decarboxylase 65 were identified that act in concer t to greatly influence the conformational structure of the autoantigen and statistically significantly influence the binding of antibodies present in Type I diabetic sera. The single or double amino-acid mutants can be used t o distinguish some anti-glutamic acid decarboxylase-65 autoantibodies and c ould prove useful in distinguishing Type I diabetic from autoimmune polyend ocrine syndrome and stiff-man syndrome patients' sera as well as to study c hanges in antibody patterns during disease progression.