Stout J G, Bassé F, Luhm R A, Weiss H J, Wiedmer T, Sims P J
Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee 53201, USA.
J Clin Invest. 1997 May 1;99(9):2232-8. doi: 10.1172/JCI119397.
Phospholipid (PL) scramblase is a plasma membrane protein that mediates accelerated transbilayer migration of PLs upon binding Ca2+, facilitating rapid mobilization of phosphatidylserine to the cell surface upon elevation of internal Ca2+. In patients with Scott syndrome, a congenital bleeding disorder related to defective expression of membrane coagulant activity, circulating blood cells show decreased cell surface exposure of phosphatidylserine at elevated cytosolic [Ca2+], implying an underlying defect or deficiency of PL scramblase. To gain insight into the molecular basis of this disorder, we compared PL scramblase in Scott erythrocyte membranes to those of normal controls. Whereas membranes of Scott cells were unresponsive to Ca2+-induced activation of PL scramblase at neutral pH, apparently normal PL scramblase activity was induced at pH < 6.0. After extraction with octylglucoside, a membrane protein was isolated from the Scott cells which exhibited normal PL scramblase activity when reconstituted in vesicles with exogenous PLs. Like PL scramblase from normal erythrocytes, PL scramblase from Scott erythrocytes was maximally activated either by addition of Ca2+ (at pH 7.4) or by acidification to pH < 6.0, and similar apparent affinities for Ca2+ and rates of transbilayer transfer of PLs were observed. This suggests that the defect in Scott syndrome is related to an altered interaction of Ca2+ with PL scramblase on the endofacial surface of the cell membrane, due either to an intrinsic constraint upon the protein preventing interaction with Ca2+ in situ, or due to an unidentified inhibitor or cofactor in the Scott cell that is dissociated by detergent.
磷脂(PL)翻转酶是一种质膜蛋白,在结合Ca2+后介导PL跨双层迁移加速,从而在细胞内Ca2+升高时促进磷脂酰丝氨酸快速转移到细胞表面。在患有斯科特综合征(一种与膜凝血活性缺陷相关的先天性出血性疾病)的患者中,循环血细胞在胞质[Ca2+]升高时,细胞表面磷脂酰丝氨酸的暴露减少,这意味着存在PL翻转酶潜在的缺陷或缺乏。为深入了解这种疾病的分子基础,我们将斯科特红细胞膜中的PL翻转酶与正常对照的进行了比较。在中性pH条件下,斯科特细胞的膜对Ca2+诱导的PL翻转酶激活无反应,而在pH < 6.0时可诱导出明显正常的PL翻转酶活性。用辛基葡糖苷提取后,从斯科特细胞中分离出一种膜蛋白,当与外源性PLs在囊泡中重组时,该蛋白表现出正常的PL翻转酶活性。与正常红细胞的PL翻转酶一样,斯科特红细胞的PL翻转酶通过添加Ca2+(在pH 7.4时)或酸化至pH < 6.0可达到最大激活,并且观察到对Ca2+的表观亲和力以及PL跨双层转移速率相似。这表明斯科特综合征中的缺陷与Ca2+与细胞膜内表面的PL翻转酶之间相互作用的改变有关,这要么是由于蛋白质本身存在限制,阻止其与原位Ca2+相互作用,要么是由于斯科特细胞中一种未鉴定的抑制剂或辅因子被去污剂解离所致。