AUTOANTIGENS IN INSULIN-DEPENDENT DIABETES-MELLITUS - MOLECULAR-CLONING AND CHARACTERIZATION OF HUMAN IA-2-BETA

In this study, we describe the isolation, expression, and characteriza tion of a new member of the transmembrane protein tyrosine phosphatase family from human brain, designated IA-2 beta. The 3853-bp cDNA encod es 986 amino acids with a molecular mass of 108,044 daltons (a predict ed pi value of 5.8). The intracellular domain of human IA-2 beta is 74 % identical to human IA-2. Northern blot analysis showed that IA-2 bet a cDNA recognized two transcripts (approximately 5.0 kb and 4.0 kb) in four of five human insulinomas, one glucagonoma, and in normal human brain, pituitary, and pancreas, but not in a variety of other normal t issues. Rabbit antiserum, raised against the intracellular domain of I A-2 beta, reacted with pancreatic islets. Treatment of in vitro-transl ated full-length IA-2 beta protein with trypsin converted it into a 37 -kD fragment. Using recombinant human IA-2 beta, we developed a radioi mmunoprecipitation assay to measure autoantibodies in the sera of pati ents with insulin-dependent diabetes mellitus (IDDM). Seventy-six new- onset IDDM patients were tested. Thirty-seven percent (28 of 76) of th e IDDM sera-but less than 1% of the control sera (1 of 174)-reacted wi th IA-2 beta. The same IDDM sera tested for autoantibodies to IA-2 and glutamic acid decarboxylase (GAD(65)) showed that 64% (49 of 76) and 57% (43 of 76), respectively, were positive. All but two of the IA-2 b eta autoantibody-positive sera also reacted with IA-2, supporting the close sequence similarity between the two molecules. Combination of an y two markers, such as IA-2 beta and IA-2, or IA-2 beta and GAD(65), o r IA-2 and GAD(65), revealed that 67%, 74%, and 87% of IDDM sera were positive for autoantibodies, respectively. Blocking of IDDM sera with recombinant IA-2, IA-2 beta, or GAD(65) resulted in marked inhibition of reactivity of IDDM sera with pancreatic islet sections as measured by islet cell autoantibody immunofluorescence. This result suggests th at these three autoantigens are the major targets of islet-cell autoan tibody reactivity.