AUTOANTIBODIES TO IA-2 AND IA-2-BETA IN INSULIN-DEPENDENT DIABETES-MELLITUS RECOGNIZE CONFORMATIONAL EPITOPES - LOCATION OF THE 37-KDA AND 40-KDA FRAGMENTS DETERMINED

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabete s mellitus (IDDM) and the precursors, respectively, of a 40- and 37-kD a tryptic fragment that reacts with IDDM sera, In the present study, b y amino acid sequencing of recombinant IA-2 and IA-2 beta, we determin ed the tryptic cleavage sites involved in the generation of these frag ments, Both cleavage sites are immediately after an arginine residue a t 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 the ir 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, RPTP mu, NU-3, SHP, and 3CH134) are completely susceptibl e to digestion by trypsin, Sequence analysis revealed five conserved c ysteine residues in IA-2 and IA-2 beta that are not present in other P TPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant protei ns resulted in loss of bath resistance to digestion by trypsin and rea ctivity with autoantibodies in IDDM sera, It is concluded that disulfi de bond formation plays a critical role in the maintenance of antigeni c structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.