LOCALIZATION OF AUTOEPITOPES ON THE PCM-1 AUTOANTIGEN USING SCLERODERMA SERA WITH AUTOANTIBODIES AGAINST THE CENTROSOME

Characterization of epitope domains of autoantigens is important for d educing the cellular functions of autoantigens and may be important fo r understanding the autoimmune response. In the reported studies, epit ope analysis of the centrosome autoantigen PCM-1 was performed. For th ese investigations, portions of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots o f the extracts were probed by immunoblot analysis using two human auto immune anticentrosome autoantisera. Immunoblotting identified three in dividual autoepitopes of 26-40 amino acid residues, amino acids 506-54 5, 1434-1465, and 1661-1686, within the PCM-1 protein. ELISA assays us ing non-denatured proteins did not identity any additional autoepitope s in the remainder of the PCM-1 molecule. To analyze the identified au toepitopes further, synthetic peptides were generated that covered eac h of the three autoepitopes and the synthetic peptides then were probe d using the scleroderma sera. Peptides that covered the antigenic regi ons from amino acids 506-545 and 1434-1465 failed to react with the an ticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptide s derived from the sequence of the third autoepitope were recognized b y autoantibodies present in the anticentrosome autoantisera allowing t he identification of the tripeptide KDC as the autoepitope in this reg ion of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients tha t are producing anticentrosome autoantibodies and should allow an inve stigation of the cellular role of the PCM-1 protein.