All three splice variants of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene are translated to proteins: a study of their co-expression in platelets and lymphoid cells

The molecular cloning of two previously unknown human sarco/endoplasmic ret iculum Ca2+-ATPase 3 (SERCA3) 3'-end transcripts, 3b and 3c, has been recen tly published. Data were lacking, however, for the presence of these SERCA3 variants in different tissue or cell types at the protein level. Here we r eport the co-expression of three human SERCA3 protein isoforms in platelets and T lymphoid Jurkat cells. Isoform-specific polyclonal anti-peptide anti bodies have been generated that recognize specifically the SERCA3a, 3b or 3 c splice variants at their C-termini, and this has been confirmed by peptid e-competition experiments as well. None of these antibodies cross-reacted w ith the housekeeping SERCA2b isoform co-expressed endogenously with SERCA3 proteins in non-muscle cells. Although all three SERCA3 isoforms could be d etected in platelets, the 3a form was the most abundantly expressed species . Its size matched the apparent size of SERCA3a over-expressed in HEK-293 c ells. Immunoprecipitation of the SERCA3 variants from platelet membranes us ing a PL/IM 430-affinity matrix provided evidence that the putative pan-ant i-SERCA3 antibody, PL/IM 430, recognizes all SERCA3 protein isoforms. The e pitope for the PL/IM 430 antibody could be localized in a 40 kDa N-terminal tryptic fragment common to all three SERCA3 variants. Comparative Western- blot analysis showed that the expression level of the SERCA3a, 3b and 3c is oforms was more than 10 times lower in Jurkat cells than in platelets, wher eas expression of the ubiquitous SERCA2b was nearly identical. This work hi ghlights new Ca2+-transporting proteins of haematopoietic cells and provide s specific antibodies for their detection.