Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation
Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation
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DOI：
10.1073/pnas.95.16.9512
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发表时间：
1998-08-04
影响因子：
11.1
通讯作者：
Medof, ME
中科院分区：
文献类型：
--
作者：
Chen, R;Walter, EI;Medof, ME
糖磷脂酰肌醇（GPI）锚定蛋白在人红细胞和有核细胞上表达的差异在于肌醇羟基的酰化，这是一种由pi特异性磷脂酶C （PI-PLC）调节其可切割性的结构特征。为了确定这种GPI锚定修饰是如何被调节的，研究了两种K562细胞转染物(ATCC和。48)分析了PI-PLC敏感和抗性表面蛋白的交替表现，并确定了GPI蛋白转移与PI-PLC敏感性获得之间的时间关系。在48种转染物中，GPI锚定在衰变加速因子（DAF）和胎盘碱性磷酸酶（PLAP）中的酰化率为60% ~ 95%，在ATCC转染物中，它们分别为60%和33%未被取代。相比之下，薄层色谱分析显示，两种细胞系中假定的GPI供体完全相同，且酰化程度大于或等于95%。在HeLa细胞中携带> - 90%未酰化锚点的蛋白质的新生DAF生物合成研究表明，在37℃（或18℃，不允许内质网的存在）的5分钟内，新生44-kDa proDAF蛋白中> - 50%的锚点表现出PI-PLC敏感性。对截断的FLAP报告蛋白miniPLAP的微粒体加工的体外分析表明，锚定供体最初转移到prominiPLAP是酰基化的，然后逐渐去酰基化。这些发现表明：(i)最初转移到新生蛋白的锚定部分是酰基化的，（ii）成熟表面蛋白的肌醇酰化是通过转移后去酰基化调节的，这通常是细胞特异性的，但也可能是蛋白质依赖性的。（iii） GPI转移后内质网立即发生去乙酰化。
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 ins 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 mere identical and were greater than or equal to 95% acylated, Studies of de novo DAF biosynthesis in HeLa cells bearing proteins with > 90% unacylated anchors showed that within 5 min at 37 degrees C (or at 18 degrees C, 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 FLAP 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.