Localization of segments essential for polymerization and for calcium binding in the gamma-chain of human fibrinogen.
Localization of segments essential for polymerization and for calcium binding in the gamma-chain of human fibrinogen.
复制标题
人纤维蛋白原伽玛链中聚合和钙结合所必需的片段的定位。
DOI：
10.1021/bi00351a001
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Scheraga,HA
中科院分区：
文献类型：
--
作者：
Váradi,A;Scheraga,HA
贝克化学实验室，康奈尔大学，伊萨卡，纽约14853-1301年收到1985年7月9日摘要：我们分离到一种纤维蛋白原的中间血浆降解产物D2，它不抑制纤维蛋白单体的聚合，但能结合钙离子。在有钙离子存在的情况下，纤维蛋白原被纤溶酶有限地消化，并分离出一个由63-411残基组成的7链残基的“大”片段D(片段D1a)。片段D1a在有钙离子存在的情况下进一步消化，得到片段D1a(其7链含有残基86-411)。在乙二醇双(/3-氨基乙基醚)-7V，N，N‘，N’‘-四乙酸(EGTA)存在下，DJ片段被消化成络合物Ca~(2+)，导致7-链的羧基末端逐渐缩短。片段D2(其7链含有残基86-335/356)是在EGTA存在下从中间消化中分离出来的。在D2的这一制备中，7-链的Lys-338-Cys-339肽键是完整的，即使它在分离肽7303-355中断裂(在Cys-326-Cys-339处具有完整的二硫键)。片段D2不干扰纤维蛋白单体的聚合，而片段DJ是纤维蛋白单体聚合的有效抑制剂。我们的结论是，7链片段356/357-411存在于片段Dj中，但不存在于片段D2中，对于维持位于纤维蛋白原外(D)结节的聚合点是必不可少的。这个片段(356/357-411)比早先报道的两个较短的片段长[Olexa，S.A.，&Budzynski，A.Z.(1981)J.Biol]。化学。256,3544-3549；Horwitz，B.H.，Varadi，A.和Scheraga，HA(1984)Proc.娜塔莉。阿卡德。SCI。美国81,5980-5984]；先前报告的数据在此重新解释。平衡透析结合研究表明，D2片段只有一个钙离子结合部位。由于D3片段(其7链含有残基86-302)不能与钙结合，因此我们认为7303-355/356片段在钙结合中起着关键作用。纤维蛋白原向纤维蛋白的转化始于凝血酶诱导的氨基末端纤维蛋白多肽FPA和FbB分别从Act链和B/3链上移除。由此产生的纤维蛋白单体以半交错重叠的方式结合形成原纤维链。在后来的阶段，原纤维横向聚集。在这种聚合中没有形成共价键。然而，在体内，纤维蛋白网络是通过谷氨酰胺和赖氨酸残基之间的共价交联来加强的。这种转酰胺化反应是由血浆转谷氨酰胺酶因子XIIIA催化的[有关最近的综述，请参阅杜利特尔(1984)]。F这项工作得到了美国国立卫生研究院国家心肺血液研究所的研究资助(HL-30616)的支持。
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301 Received July 9, 1985 abstract: We have isolated an intermediate plasmic degradation product, D2, of fibrinogen that does not inhibit the polymerization of fibrin monomer but does bind Ca2+. Fibrinogen was digested to a limited extent with plasmin inthe presence of Ca2+, and a “large” fragment D (fragment D1A) was isolated with a 7-chain remnant consisting of residues 63-411. Fragment D1A was digested further inthe presence of Ca2+, yielding fragment D;(with its 7-chain containing residues 86-411). The digestion of fragment Dj [in the presence of ethylene glycol bis (/3-aminoethyl ether)-7V, N, N', N ‘'-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the 7-chain. Fragment D2 (with its 7-chain containing residues 86-335/356) was isolated from an intermediate digest in the presence of EGTA. The Lys-338-Cys-339 peptide bond of the 7-chain is intact in this preparation of D2, even though it is split in the isolated peptide 7303-355 (with an intact disulfide bond at Cys-326-Cys-339). Fragment D2 does not interfere with the polymerization of fibrin monomer, whereas fragment Dj is a potent inhibitor of this polymerization. We conclude that the 7-chain segment 356/357-411, present in fragment Dj but absent from fragment D2, is essential for maintenance of a polymerization site located in theouter (D) nodule of fibrinogen. This segment (356/357-411) is longer than two shorter ones reported earlier [Olexa, S. A., & Budzynski, A. Z.(1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B. H., Varadi, A., & Scheraga, HA (1984) Proc. Natl. Acad. Sci. USA 81, 5980-5984]; the data for the earlier reports are reinterpreted here. Finally, fragment D2 possessesa single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its 7-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment 7303-355/356 plays a crucial role in Ca2+ binding. e conversion of fibrinogen to fibrin is started with the thrombin-induced removal of the amino-terminal fibrino-peptides FpA and FpB from the Act-and B/3-chains, respec-tively. The resulting fibrin monomers associate in a half-staggered overlapping manner to form protofibril strands. In a later stage, the protofibrils aggregate laterally. No covalent bonds are formed in this polymerization. However, in vivo, the fibrin network is enforced by covalent cross-links between glutamine and lysine residues. This transamidation is catalyzed by the plasma transglutaminase factor XIIIa [for a recent review, see Doolittle (1984)]. fThis work was supported by a research grant (HL-30616) from the National Heart, Lung, and Blood Institute of the National Institutes of Health.