DEFINITION OF MULTIPLE ICA512 PHOGRIN AUTOANTIBODY EPITOPES AND DETECTION OF INTRAMOLECULAR EPITOPE SPREADING IN RELATIVES OF PATIENTS WITHTYPE-1 DIABETES/

The related tyrosine phosphatase-like proteins, islet cell antigen 512 (ICA512) and phosphatase homologue in granules of insulinoma (phogrin ), are major targets of autoantibodies in patients with type 1 diabete s. In the current study, we have examined the overlapping specificitie s and antigenic epitopes of autoantibodies to ICA512 and phogrin and d etermined whether intramolecular epitope spreading occurs during the d evelopment of diabetic autoimmunity. ICA512 autoantibodies and phogrin autoantibodies were detected in 65-70% (n = 110) of patients with new -onset type 1 diabetes and 60-65% (n = 42) of prediabetic relatives of patients with type 1 diabetes. Of the sera, 10% reacted with ICA512 b ut not phogrin, whereas only 1% of sera reacted with phogrin but not I CA512. The binding of phogrin autoantibodies in 88 dual (ICA512 and ph ogrin) autoantibody-positive sera could be completely blocked by exces s recombinant ICA512, whereas the blocking of ICA512 autoantibodies wi th recombinant phogrin was only partial (mean inhibition of 58.9 +/- 3 .7%, mean +/- SE). Binding and competition analysis using multiple chi meric ICA512/phogrin constructs demonstrated that a major unique epito pe for ICA512 autoantibodies is localized to amino acids 762-887. A co nformational epitope associated with the carboxy-terminal 31 amino aci ds of ICA512 was recognized by one-third of sera, and a minor epitope is located on amino acids 601-762 of ICA512. The major epitopes for ph ogrin-selective autoantibodies were localized to amino acids 640-922 o f phogrin, Sequential serum samples were analyzed in 22 relatives who expressed lCA512/phogrin autoantibodies. Intramolecular epitope spread ing was found for 5 of 13 relatives who have progressed to type 1 diab etes. Among nine relatives who have remained nondiabetic, three demons trated a decrease in the number of epitopes recognized. These studies highlight the complexity of autoantibody recognition of ICA512/phogrin and are consistent with the hypothesis that ICA512/phogrin may be rec ognized as a consequence of beta-cell destruction.