Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

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IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.