Expression of two isoforms of the third sarco/endoplasmic reticulum Ca2+ ATPase (SERCA3) in platelets. Possible recognition of the SERCA3b isoform by the PL/IM430 monoclonal antibody.
Human platelets express several sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3'-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N-3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N-1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/ IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.
Bobe
R
R
U. 348 INSERM, IFR Circulation Lariboisière, Hôpital Lariboisière, Paris, France.
Lacabaratz-Porret
C
C
Bredoux
R
R
Martin
V
V
Ozog
A
A
Launay
S
S
Corvazier
E
E
Kovács
T
T
Papp
B
B
Enouf
J
J
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001792:Blood Platelets / Q000201:enzymology*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D012333:RNA, Messenger; D000252:Calcium-Transporting ATPases