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 deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portion of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434-1465, and 1661-1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434-1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.
Bao
L
L
Department of Structural and Cellular Biology, University of South Alabama, Mobile 36688, USA.
Varden
C E
CE
Zimmer
W E
WE
Balczon
R
R
D001323:Autoantibodies; D001324:Autoantigens / Q000235:genetics / Q000276:immunology*; D018797:Cell Cycle Proteins; D018385:Centrosome / Q000276:immunology*; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D009842:Oligopeptides / Q000138:chemical synthesis; D011993:Recombinant Fusion Proteins; D012595:Scleroderma, Systemic / Q000276:immunology*; D017384:Sequence Deletion
D001323:Autoantibodies; D001324:Autoantigens; D018797:Cell Cycle Proteins; D000939:Epitopes; D007106:Immune Sera; D009842:Oligopeptides; C086074:PCM1 protein, human; D011993:Recombinant Fusion Proteins