Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum.
Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum.
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发表时间：
1989-12
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通讯作者：
L. Fliegel;K. Burns;D. Maclennan;R. Reithmeier;M. Michalak
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作者：
L. Fliegel;K. Burns;D. Maclennan;R. Reithmeier;M. Michalak
分离并测序了兔骨骼肌肌浆网高亲和力Ca2+结合蛋白（HACBP）的cDNA克隆。cDNA编码了一个418个氨基酸的蛋白，但将推导出的氨基酸序列与纯化蛋白的nh2末端氨基酸序列进行比较发现，合成过程中去除了17个残基的nh2末端信号序列。对编码该蛋白的mRNA的体外翻译研究证实了这一点。结构预测并没有揭示蛋白质中任何潜在的跨膜片段。高亲和力Ca2+结合蛋白Lys-Asp-Glu-Leu的cooh末端序列与被认为是内质网保留信号的序列相同（Munro， S., and Pelham, h.r. B. (1987) Cell 48, 899-907）。所有这些特征提示该蛋白位于肌浆网管腔内。Mr 46,567的成熟蛋白含有109个酸性氨基酸和52个碱性氨基酸。结构预测表明，分子的前半部分形成一个由8条反平行β链组成的球状结构域，在NH2末端具有螺旋-转-螺旋基序。接下来三分之一的序列富含脯氨酸。该片段可细分为带电区，包含17个氨基酸重复序列，其次是富含脯氨酸、丝氨酸和苏氨酸的片段，从Pro-246延伸至Thr-316。37个酸性残基聚集在蛋白质的COOH末端的56个氨基酸中。虽然该蛋白以高亲和力结合1 mol /mol Ca2+，但在该蛋白中未观察到“EF-hand”一致序列。然而，酸性的COOH末端可以解释在蛋白质中观察到的低亲和力，高容量的Ca2+结合。在与其他相关实验室的一致意见下，我们为这种蛋白质选择了钙网蛋白的名称。
A cDNA clone encoding the high affinity Ca2+-binding protein (HACBP) of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 418 amino acids, but a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that a 17-residue NH2-terminal signal sequence was removed during synthesis. This was confirmed by studies of in vitro translation of mRNA encoding the protein. Structural predictions did not reveal any potential transmembrane segments in the protein. The COOH-terminal sequence of the high affinity Ca2+-binding protein, Lys-Asp-Glu-Leu, is the same as that proposed to be an endoplasmic reticulum retention signal (Munro, S., and Pelham, H. R. B. (1987) Cell 48, 899-907). All of these characteristics suggest that the protein is localized in the lumen of the sarcoplasmic reticulum. The mature protein of Mr 46,567 contains 109 acidic and 52 basic amino acids. Structural predictions suggest that the first half of the molecule forms a globular domain of 8 anti-parallel beta-strands with a helix-turn-helix motif at the extreme NH2 terminus. The next one-third of the sequence is proline-rich. This segment can be subdivided into a charged region which contains a 17-amino acid repeat, followed by a proline, serine, and threonine-rich segment extending from Pro-246 to Thr-316. Thirty-seven acidic residues are clustered within 56 amino acids at the COOH terminus of the protein. Although the protein binds 1 mol of Ca2+/mol with high affinity, no "EF-hand" consensus sequence was observed in the protein. The acidic COOH terminus, however, could account for the low affinity, high capacity Ca2+ binding observed in the protein. In agreement with other involved laboratories, we have chosen the name calreticulin for the protein.