Chen R, Walter E I, Parker G, Lapurga J P, Millan J L, Ikehara Y, Udenfriend S, Medof M E
Institute of Pathology, Case Western Reserve University, 2085 Adelbert Road, Cleveland, OH, 44106, USA.
Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9512-7. doi: 10.1073/pnas.95.16.9512.
The glycophosphatidylinositol (GPI) anchors of proteins expressed on human erythrocytes and nucleated cells differ with respect to acylation of an inositol hydroxyl group, a structural feature that modulates their cleavability by PI-specific phospholipase C (PI-PLC). To determine how this GPI anchor modification is regulated, the precursor and protein-associated GPIs in two K562 cell transfectants (ATCC and .48) exhibiting alternatively PI-PLC-sensitive and resistant surface proteins were analyzed and the temporal relationship between GPI protein transfer and acquisition of PI-PLC sensitivity was determined. Nondenaturing PAGE analyses demonstrated that, whereas in .48 transfectants the GPI anchors in decay accelerating factor (DAF) and placental alkaline phosphatase (PLAP) were >95% acylated, in ATCC transfectants, they were 60 and 33% unsubstituted, respectively. In contrast, TLC analyses revealed that putative GPI donors in the two lines were identical and were >/=95% acylated. Studies of de novo DAF biosynthesis in HeLa cells bearing proteins with >90% unacylated anchors showed that within 5 min at 37 degreesC (or at 18 degreesC, which does not permit endoplasmic reticilum exit), >50% of the anchor in nascent 44-kDa proDAF protein exhibited PI-PLC sensitivity. In vitro analyses of the microsomal processing of miniPLAP, a truncated PLAP reporter protein, demonstrated that the anchor donor initially transferred to prominiPLAP was acylated and then progressively was deacylated. These findings indicate that (i) the anchor moiety that initially transfers to nascent proteins is acylated, (ii) inositol acylation in mature surface proteins is regulated via posttransfer deacylation, which in general is cell-specific but also can be protein-dependent, and (iii) deacylation occurs in the endoplasmic reticulum immediately after GPI transfer.
人红细胞和有核细胞上表达的蛋白质的糖基磷脂酰肌醇（GPI）锚在肌醇羟基的酰化方面存在差异，这一结构特征可调节它们被PI特异性磷脂酶C（PI-PLC）切割的能力。为了确定这种GPI锚修饰是如何被调控的，我们分析了两个K562细胞转染体（ATCC和.48）中前体和与蛋白质相关的GPI，这两个转染体分别表现出对PI-PLC敏感和抗性的表面蛋白，并确定了GPI蛋白转移与获得PI-PLC敏感性之间的时间关系。非变性PAGE分析表明，在.48转染体中，衰变加速因子（DAF）和胎盘碱性磷酸酶（PLAP）中的GPI锚酰化程度>95%，而在ATCC转染体中，它们的未取代率分别为60%和33%。相比之下，TLC分析显示，这两个细胞系中假定的GPI供体是相同的，且酰化程度>/=95%。对带有>90%未酰化锚的蛋白质的HeLa细胞中新生DAF生物合成的研究表明，在37℃下5分钟内（或在18℃下，此时不允许内质网输出），新生的44 kDa proDAF蛋白中>50%的锚表现出对PI-PLC的敏感性。对截短的PLAP报告蛋白miniPLAP的微粒体加工的体外分析表明，最初转移到prominiPLAP的锚供体是酰化的，然后逐渐去酰化。这些发现表明：（i）最初转移到新生蛋白质上的锚部分是酰化的；（ii）成熟表面蛋白中的肌醇酰化是通过转移后去酰化来调控的，一般来说这是细胞特异性的，但也可能是蛋白质依赖性的；（iii）去酰化在GPI转移后立即在内质网中发生。
