Functions of individual gamma-carboxyglutamic acid (Gla) residues of human protein c. Determination of functionally nonessential Gla residues and correlations with their mode of binding to calcium.
Functions of individual gamma-carboxyglutamic acid (Gla) residues of human protein c. Determination of functionally nonessential Gla residues and correlations with their mode of binding to calcium.
复制标题
人类蛋白质 c 的各个 γ-羧基谷氨酸 (Gla) 残基的功能。
DOI：
10.1021/bi00254a007
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Castellino,FJ
中科院分区：
文献类型：
--
作者：
Christiansen,WT;Tulinsky,A;Castellino,FJ
摘要：本实验室以前的研究致力于阐明单个γ-羧基谷氨酸（Gla）残基在人蛋白C（PC）和活化蛋白C（APC）的Gla结构域相关Ca ~（2+）导向特性中的作用。结果的基础上，使用含有高度保守的（Asp）突变的个别Gla残基的PC的重组变体，它以前提出，Gla 6，Gla 14，和Gla 19可能不是必不可少的性质与Ca 2+依赖的构象这些蛋白质的Gla结构域。在这项研究中，我们已经证明，Gla残基14和19的瓦尔的根本突变分别导致野生型重组APC的94%和82%的Gla结构域相关的、Ca 2 +-和磷脂-（PL-）依赖的抗凝（APTT）活性，而[Gla 6-Val] r-APC显示完全丧失了相同的活性。更保守的突变体[Gla 6-Gln] r-APC具有wtr-APC的4%的APTT活性，而[Gla 6-Asp] r-APC几乎完全具有活性。与wtr-PC一样，[Gla 6-瓦尔] r-PC和[Gla 6-Gln] r-PC均显示出Ca 2+依赖的内源荧光猝灭，表明它们采用了Ca 2+诱导的构象。然而，Ca 2+滴定数据表明，这些构象是不相同的，所经历的wtr-PC。此外，[Gla 6-瓦尔] r-PC和[Gla 6-Gln] r-PC与酸性PL囊泡的Ca ~（2+）介导的结合参数也存在缺陷。这些数据进行了解释，在分子水平上使用模型的Gla域的PC的基础上的X-射线晶体结构的Ca 2 +/牛凝血酶原片段1复合物。我们得出结论，Gla 14和Gla 19，和唯一的Ca 2+（Ca-7），这是协调这两个残基，是不是必不可少的Gla域相关的Ca 2+和PL依赖的抗凝性能的PC和APC。Gla 6存在更复杂的情况。该模型表明，该残基的仅单个羧酸盐用于Ca 2+配位，并且该基团与两种不同的Ca 2+离子相互作用，即，Ca-4和Ca-5 Asp的单独羧酸酯可能能够有效地取代Gla 6中的该相同基团，从而为[Gla 6-Asp] r-APC提供功能活性，并且在小得多的程度上，Gln也可能能够起到配位功能。这表明Gla 6是功能上必需的Ca ~（2+）结合位点之一。PC是抗凝丝氨酸蛋白酶APC的酶原形式，是凝血血浆蛋白类的成员，其需要维生素K进行翻译后加工事件，导致其前9个E残基的γ-羧化。这些后面的氨基酸包含在被鉴定为Gla结构域（氨基酸残基1-37）的基因组外显子内。APC的主要活性涉及fV/Va的有限特异性蛋白水解（Kisiel等人，1977）和fVIII/fVIIIa（Vehar & Davie，1980）在由Ca 2+、PL和蛋白S（步行者，1981）刺激的反应中。APC对这些蛋白质的作用导致它们在凝血酶原酶和tenase复合物中的辅因子活性丧失。
Revised Manuscript Received September 30, 1994s abstract: Previous studies from this laboratory havebeen directedtoward elucidation of the roles of individual y-carboxyglutamic acid (Gla) residues in Gla domain-related Ca2+-directed properties of human protein C (PC) and activated protein C (APC). On thebasis of results using recombinant variants of PC containing highly conservative (Asp) mutations of individual Gla residues, it was previously proposed that Gla6, Gla14, and Gla19 may not be essential for properties associated with the Ca2+-dependent conformation of the Gla domain of these proteins. In this study, we have demonstrated thatradical mutations to Val of Gla residues 14 and 19 resulted in 94% and 82%, respectively, of the Gla domain-related, Ca2+-and phospholipid-(PL-) dependent anticoagulant (APTT) activityof wild-typerecombinant (wtr) APC, while [Gla6—-ValJr-APC showed a complete loss of this same activity. The more conservative mutant [Gla6—Gln] r-APC possessed 4% of the APTT activity of wtr-APC, whereas [Gla6—Asp] r-APC was nearly fully active. As with wtr-PC, both [Gla6—Val] r-PC and [Gla6—Gln] r-PC displayed Ca2+-dependent intrinsic fluorescence quenching, suggesting that they adopted a Ca2+-induced conformation. However, Ca2+ titration data suggested that these conformations were not identical to that undergone by wtr-PC. In addition, the Ca2+-mediated binding parameters of [Gla6—Val] r-PC and [Gla6—Gln] r-PC to acidic PL vesicles were found to be defective. These data were interpreted at the molecular level using a model for the Gla domain of PC based on the X-ray crystal structure of the Ca2+/bovine prothrombin fragment 1 complex. We conclude that Gla14 and Gla19, and the sole Ca2+(Ca-7) that is coordinated to these two residues, are not essential for the Gla domain-related Ca2+-and PL-dependent anticoagulant properties of PC and APC. A more complex situation exists with Gla6. The model suggests that only a single carboxylate of this residue is employed in Ca2+ coordination, and this group interacts with two different Ca2+ ions, viz., Ca-4 and Ca-5. The lone carboxylate of Asp may be able to substitute effectively for this same group in Gla6, thus providing functional activity to [Gla6—Asp] r-APC, and to a much lesser degree Gin may also be able to serve a coordination function. This suggests that Gla6 is one of the functionally essential Ca2+ binding sites.PC, 1 the zymogen form of the anticoagulant serine protease APC, is a member of the class of blood coagulation plasma proteins that requires vitaminK for the posttranslational processing events that result in y-carboxylation of its first nine E residues. These latter amino acids are contained within a genomic exon identified as the Gla domain (amino acid residues 1—37). The principal activities of APC involve limited specific proteolysis of fV/Va (Kisiel et al., 1977) and fVIII/fVIIIa (Vehar & Davie, 1980) in reactions that are stimulated by Ca2+, PL, and protein S (Walker, 1981). The action of APC on these proteins results in loss of their cofactor activities in the prothrombinase and tenase com-