Zhou Q, Sims P J, Wiedmer T
Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201, USA.
Biochemistry. 1998 Feb 24;37(8):2356-60. doi: 10.1021/bi972625o.
Accelerated transbilayer movement of plasma membrane phospholipids (PL) upon elevation of Ca2+ in the cytosol plays a central role in the initiation of plasma clotting and in phagocytic clearance of injured or apoptotic cells. We recently identified a human erythrocyte membrane protein that induces rapid transbilayer movement of PL at elevated Ca2+. We also presented evidence that this PL scramblase is expressed in a variety of other cells and tissues where transbilayer movement of plasma membrane PL is promoted by intracellular Ca2+ [Zhou, Q., et al. (1997) J. Biol. Chem. 272, 18240-18244]. We have now cloned murine PL scramblase for comparison with the human polypeptide. Both human and murine PL scramblase are acidic proteins (pI = 4.9) with a predicted inside-outside (type 2) transmembrane segment at the carboxyl-terminus. Whereas human PL scramblase (318 AA) terminates in a short exoplasmic tail, murine PL scramblase (307 AA) terminates in the predicted membrane-inserted segment. The aligned polypeptide sequences reveal 65% overall identity, including near identity through 12 residues of an apparent Ca2+ binding motif (D[A/S]DNFGIQFPLD) spanning codons 273-284 (human) and 271-282 (murine), respectively. This conserved sequence in the cytoplasmic domain of PL scramblase shows similarity to Ca2+-binding loop motifs previously identified in known EF hand structures. Recombinant murine and human PL scramblase were each expressed in Escherichia coli and incorporated into proteoliposomes. Measurement of transbilayer movement of NBD-labeled PL confirmed that both proteins catalyzed Ca2+-dependent PL flip-flop similar to that observed for the action of Ca2+ at the cytoplasmic face of plasma membranes. Mutation of residues within the putative EF hand loop of human PL scramblase resulted in loss of its PL mobilizing function, suggesting that these residues directly participate in the Ca2+-induced active conformation of the polypeptide.
胞质溶胶中Ca2+浓度升高时，质膜磷脂（PL）跨双层的加速运动在血浆凝血的启动以及受损或凋亡细胞的吞噬清除过程中起着核心作用。我们最近鉴定出一种人类红细胞膜蛋白，它在Ca2+浓度升高时能诱导PL快速跨双层运动。我们还提供了证据表明，这种PL翻转酶在多种其他细胞和组织中表达，在这些细胞和组织中，质膜PL的跨双层运动受细胞内Ca2+促进[周，Q.等人（1997年）《生物化学杂志》272，18240 - 18244]。我们现在克隆了小鼠PL翻转酶，以便与人类多肽进行比较。人类和小鼠PL翻转酶都是酸性蛋白（pI = 4.9），在羧基末端有一个预测的由内向外（2型）跨膜片段。人类PL翻转酶（318个氨基酸）以一个短的细胞外尾端结束，而小鼠PL翻转酶（307个氨基酸）在预测的膜插入片段处结束。比对后的多肽序列显示总体一致性为65%，包括分别跨越密码子273 - 284（人类）和271 - （此处原文缺失一个数字，推测为282）（小鼠）的一个明显的Ca2+结合基序（D[A/S]DNFGIQFPLD）的12个残基几乎完全相同。PL翻转酶胞质结构域中的这个保守序列与先前在已知EF手结构中鉴定出的Ca2+结合环基序相似。重组小鼠和人类PL翻转酶分别在大肠杆菌中表达并整合到蛋白脂质体中。对NBD标记的PL跨双层运动的测量证实，这两种蛋白都催化Ca2+依赖的PL翻转，类似于在质膜胞质面观察到Ca2+作用时的情况。人类PL翻转酶假定的EF手环内残基的突变导致其PL动员功能丧失, 表明这些残基直接参与多肽的Ca2+诱导的活性构象。
