AUTOANTIBODIES TO IA-2 IN IDDM - LOCATION OF MAJOR ANTIGENIC DETERMINANTS

Thirty-three IDDM sera that immunoprecipitated full-length IA-2 were t ested for reactivity with different fragments of the IA-2 molecule, Th e fragments were prepared by PCR amplification of IA-2 cDNA and by exp ression in a rabbit reticulocyte transcription/translation system, Whe reas all 33 sera reacted with the intracellular domain (amino acid 604 to 979), none of the sera reacted with the extracellular domain of IA -2 (amino acid 31 to 577). Analysis of the reactivity of IDDM sera wit h tile different regions of the intracellular domain showed that 94% ( 31 of the 33) reacted with the COOH-terminus (amino acid 771 to 979), 40% reacted with the NH2-terminus (amino acid 604 to 776), and 40% rea cted with the middle portion (amino, acid 692 to 875). Of the 31 sera that reacted with the COOH-terminus, 14 of these reacted only with the COOH-terminus and with no other region, Of the 13 sera that reacted w ith the NH2-terminus, only one reacted exclusively with the NH2-termin us. Treatment of the different domains of IA-2 with trypsin showed tha t only the COOH-terminus was resistant to trypsin, arguing that it is from this region of the IA-2 molecule that the 40-kDa tryptic fragment from insulinoma cells is derived, From these experiments, it is concl uded that the major antigenic determinant of IA-2 is located at the CO OH-terminus and that minor antigenic determinants are located at the N H2-terminus and middle portion of the intracellular domain.