Enouf J, Bredoux R, Papp B, Djaffar I, Lompré A M, Kieffer N, Gayet O, Clemetson K, Wuytack F, Rosa J P
U348 INSERM, Hôpital Lariboisière, Paris, France.
Biochem J. 1992 Aug 15;286 ( Pt 1)(Pt 1):135-40. doi: 10.1042/bj2860135.
Previous biochemical studies suggested that the human platelet Ca2+ATPase system may be cell-specific. To test this hypothesis, we first undertook the molecular cloning of Ca2+ATPase from human erythroleukaemia (HEL) cells, because this human cell line exhibits megakaryocytic features and expresses a Ca2+ATPase that cross-reacts with platelet Ca(2+)-ATPase. For this cloning, an HEL-cell cDNA library was screened with a rat cardiac Ca2+ATPase cDNA probe. The insert of the longest clone isolated was 3.9 kb and its sequence displayed a 100% identity with that of the non-muscle human Ca2+ATPase 2-b isoform, termed SERCA2-b (sarco-endoplasmic-reticulum Ca2+ATPase). The 3.9 kb cDNA covered a subtotal coding region and part of the 3' non-coding end of the SERCA2-b mRNA. It cross-hybridized with the 4 kb transcript species of cardiac SERCA2-a and with non-muscle SERCA2-b mRNAs, but not with fast-skeletal-muscle SERCA1 mRNA. We next confirmed that SERCA2-b was a component of the platelet Ca2+ATPase system because (1) the platelet clones isolated from a platelet cDNA library exhibited a 100% homology with HEL-cell cDNA; (2) SERCA2-b mRNA was amplified by PCR on total platelet RNA and (3) platelet Ca2+ATPase cross-reacted with a polyclonal SERCA2-b-specific antiserum. Platelets therefore contain a Ca2+ATPase definitely identified as the SERCA2-b isoform of Ca2+ATPase, thus eliminating the possibility that they only contain a single specific Ca2+ATPase.
以往的生化研究表明，人类血小板Ca2+ATP酶系统可能具有细胞特异性。为了验证这一假设，我们首先对来自人类红白血病（HEL）细胞的Ca2+ATP酶进行了分子克隆，因为这种人类细胞系具有巨核细胞特征，并表达一种与血小板Ca(2+)-ATP酶发生交叉反应的Ca2+ATP酶。为了进行这种克隆，用大鼠心脏Ca2+ATP酶cDNA探针筛选了一个HEL细胞cDNA文库。分离出的最长克隆的插入片段为3.9 kb，其序列与非肌肉型人类Ca2+ATP酶2-b同工型（称为SERCA2-b，即肌浆网Ca2+ATP酶）的序列具有100%的同一性。3.9 kb的cDNA覆盖了SERCA2-b mRNA的部分编码区和3'非编码端。它与心脏SERCA2-a的4 kb转录本以及非肌肉SERCA2-b mRNA发生交叉杂交，但不与快骨骼肌SERCA1 mRNA杂交。接下来我们证实SERCA2-b是血小板Ca2+ATP酶系统的一个组成部分，因为（1）从血小板cDNA文库中分离出的血小板克隆与HEL细胞cDNA具有100%的同源性；（2）通过PCR在总血小板RNA上扩增出了SERCA2-b mRNA；（3）血小板Ca2+ATP酶与多克隆SERCA2-b特异性抗血清发生交叉反应。因此，血小板含有一种明确鉴定为Ca2+ATP酶的SERCA2-b同工型的Ca2+ATP酶，从而排除了它们只含有单一特异性Ca2+ATP酶的可能性。
