Humoral autoreactivity to an alternatively spliced variant of ICA512/IA-2 in type I diabetes

Aims/hypothesis. The receptor tyrosine phosphatase like-protein ICA512/IA-2 occurs as a proteolytically-processed 65,000 M(r) type 1 transmembrane glycoprotein in beta cells and is a major autoantigen of Type I (insulin-dependent) diabetes mellitus. We investigated whether alternative splicing could affect humoral autoreactivity to the molecule. Methods. Genomic and cDNA sequence analysis showed the presence of a ICA512 variant in islets and lymphoid tissues with an in-frame deletion of exon 13 which produces a secreted form lacking aa 557-629 including the transmembrane domain (aa 577 to 600). The alternatively spliced protein is detectable by western blotting in normal islets and translated into a protein that is processed to a series of soluble forms of 25,000-35,000 M(r) Radioimmunoprecipitation assays for anti-ICA512 autoantibodies were developed with the widely used ICA512.bdc construct (which has exon 13 deleted) and a series of full-length and modified ICA512/IA-2 molecules. Results. The assays showed that ICA512.bdc and ICA512_604-979 gave the best discrimination between diabetic and control sera. With ICA512_604-979 a somewhat greater proportion of patients expressing antibodies were detected than with ICA512.bdc in the groups studied (70.5% vs 63.2% of prediabetic/new-onset and 25.0 vs 13.9% in patients with diabetes > 20 years). Conversely, a small proportion (3% recent-onset and 6% > 20 years) had antibodies to ICA512.bdc but not ICA512_604-979. Conclusion/interpretation. Important epitopes lie within the exon 13 region and others can be generated by the alternative splicing. As the Δexon 13 variant is probably secreted by the beta cell, it could be recognized by the cellular and humoral arm of the immune system in the absence of cellular damage.