Epitope mapping of monoclonal antibodies to human plasma membrane Ca2-ATPase

Overlapping fragments of the fourth isoform of human plasma membrane Ca(2+)-ATPase (hPMCA4) and several fragments of hPMCA1 were expressed in bacterial cells and purified by metal affinity chromatography. Enzyme immunoassays of the fragments helped map epitopes for 4 monoclonal antibodies (2D8, 8B8, 7C8 and 5E6). The epitope for 2D8 was localized within the 222-249 site (i.e., in the putative transduction domain), the epitopes for 8B8 and 7C8 were localized within the 330-353 site, in which phospholipids are presumably bound, and the 5E6 epitope was found within the 791-843 site, where the putative hinge region is situated. 2D8 recognizes hPMCA1 and hPMCA4 isoforms, while 8B8 and 7C8 are specific for hPMCA4. The amino acid sequences of these epitopes and phage-displayed mimotopes were compared.