Fine mapping of diabetes-associated IA-2 specific autoantibodies

The related tyrosine phosphatase-like proteins (PTP) IA-2 and IA-2beta are autoantigens of type 1 diabetes. Autoantibodies are predominantly against IA-2. We utilized the close homology between IA-2 and IA-2beta PTP domains to design chimeras and mutants in order to identify humoral IA-2-specific epitopes. Fifteen sera with antibodies to IA-2 specific PTP domain epitopes were tested against IA-2beta(741-848)/IA-2(795-889)/IA-2beta(943-1033), IA-2beta(741-848)/IA-2(795-845)/IA-2beta(900-1033), and IA-2beta(741-898)/IA-2(845-875)/IA-2beta(930-1033)chimeras. Two sera bound IA-2beta(741-848)/IA-2(795-889)/IA-2beta(943-1033)and IA-2beta(741-848)/IA-2(795-845)/IA-2beta(900-1033)only indicating that the IA-2 specific residues 859, 862, and/or 867 were critical for antibody binding. Mutation of glutamine 862 abolished binding in one of these sera. Seven sera bound only the IA-2beta(741-848)/IA-2(795-889)/IA-2beta(943-1033)chimera, indicating that binding required IA-2 specific amino acids within both 795-845 and 846-875, or that IA-2 residues 876-888 were important for binding. Mutation of glutamine 862 abolished binding in two of these sera, and mutation of residues 876, 877, 878, and 880 markedly reduced binding in two others. Six sera bound all three chimeras indicating that they contained multiple IA-2 specific PTP domain antibodies. In three of these sera, mutation of residues at positions 876, 877, 878, 880, and/or residues 862 and 822 reduced antibody binding by more than 50%. These findings indicate that glutamine at position 862, and residues 876-880 of the WPD loop of IA-2 are important for several of the IA-2 specific PTP domain epitopes.