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 reticulum Ca(2+)-ATPase 3 (SERCA3) 3'-end transcripts, 3b and 3c, has been recently published. Data were lacking, however, for the presence of these SERCA3 variants in different tissue or cell types at the protein level. Here we report the co-expression of three human SERCA3 protein isoforms in platelets and T lymphoid Jurkat cells. Isoform-specific polyclonal anti-peptide antibodies have been generated that recognize specifically the SERCA3a, 3b or 3c splice variants at their C-termini, and this has been confirmed by peptide-competition experiments as well. None of these antibodies cross-reacted with the housekeeping SERCA2b isoform co-expressed endogenously with SERCA3 proteins in non-muscle cells. Although all three SERCA3 isoforms could be detected in platelets, the 3a form was the most abundantly expressed species. Its size matched the apparent size of SERCA3a over-expressed in HEK-293 cells. Immunoprecipitation of the SERCA3 variants from platelet membranes using a PL/IM 430-affinity matrix provided evidence that the putative pan-anti-SERCA3 antibody, PL/IM 430, recognizes all SERCA3 protein isoforms. The epitope 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 isoforms was more than 10 times lower in Jurkat cells than in platelets, whereas expression of the ubiquitous SERCA2b was nearly identical. This work highlights new Ca(2+)-transporting proteins of haematopoietic cells and provides specific antibodies for their detection.
Kovács
T
T
National Institute of Haematology and Immunology, H-1113 Budapest, Daróczi u. 24, Hungary. T.Kovacs@biomembrane.hu
Felföldi
F
F
Papp
B
B
Pászty
K
K
Bredoux
R
R
Enyedi A
Enouf
J
J
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D000939:Epitopes / Q000737:chemistry; D014644:Genetic Variation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013601:T-Lymphocytes / Q000201:enzymology*; D014158:Transcription, Genetic; D014162:Transfection
D000939:Epitopes; D007527:Isoenzymes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506949:ATP2A3 protein, human; D000252:Calcium-Transporting ATPases