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Studies for the tertiary structure of Bβ-and γ-chain that are important for assembly and/or secretion of fibrinogen.

研究对于纤维蛋白原的组装和/或分泌很重要的 Bβ 和 γ 链的三级结构。

基本信息

批准号：
16590451
负责人：
OKUMURA Nobuo
金额：
$ 1.34万
依托单位：
SHINSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590451/
关键词：
fibrinogen γ-chain Bβ-chain tertiary structure assembly secretion フィビリノゲン

项目摘要

1. I examined the role of S-S bond between γ153Cys and γ182Cys for formation of tertiary structure of γC module. The γ-chain substituted γ153Cys by Ala can not form Aαγ-or Bβγ-complex in the CHO cells. These results demonstrate that none of the intact fibrinogen was assembled and subsequently secreted.2. I examined the role of γ319Asn and γ320Asp for formation of tertiary structure of γC module. Co-transfection of vectors expressing the γ-chain deleted γ319Asn and γ320Asp with normal γ-chain revealed that abnormal γ-chain was synthesized and assembled into fibrinogen with normal Aα-and Bβ-chain in the CHO cells, however, secretion of aberrant fibrinogen was significantly reduced in comparison of that of normal fibrinogen.3. To examine the role of γ-chain residue, 387Ile, for assembly and secretion of fibrinogen, γ387Ile was substituted by Arg, Leu, Met, Ala, or Asp. Variant γ-chains with Arg, Leu, Met, and Ala were assembled into fibrinogen inside the CHO cells and subsequently secrete … More d into medium, however, assembly and secretion of variant fibrinogen with Asp was markedly impaired. These observations indicate that the residue at γ387Ile is more critical for fibrinogen assembly and secretion than the length of the γC-tail (γ387-411).4. To examine the role of Bβ-chain residue, 455Arg corresponding to γ387Ile, for assembly and secretion of fibrinogen, I made mutant vectors, Bβ-456terminal, Bβ-455terminal, and substitution by Ile, Asp, Lys, or Ala. Variant fibrinogen with Bβ-456terminal was assembled and secreted into medium, however, variant fibrinogen with Bp-455terminal was not. Unfortunately, I can not establish the CHO cell lines expressing Bβ455Ile-, Asp-, Lys-, or Ala-variant Bβ-chain to be used for studies of assembly and secretion of variant fibrinogen.5.I also found the novel dysfunctional fibrinogen deleted Bβ111Ser residue. This variant fibrinogen has impaired fibrin polymerization, especially lateral aggregation, however, I guess assembly and secretion of this variant fibrinogen might not be aberrant. Less

1. 我研究了γ153Cys和γ182Cys之间的S-S键对于γC模块三级结构形成的作用。 γ链被Ala取代的γ153Cys在CHO细胞中不能形成Aαγ-或Bβγ-复合物。这些结果表明，没有完整的纤维蛋白原被组装并随后被分泌。2．我研究了 γ319Asn 和 γ320Asp 在 γC 模块三级结构形成中的作用。表达缺失γ链的γ319Asn和γ320Asp与正常γ链的载体共转染发现，CHO细胞中合成了异常的γ链，并与正常的Aα链和Bβ链组装成纤维蛋白原，但与正常纤维蛋白原相比，异常纤维蛋白原的分泌显着减少。 3．为了检查 γ 链残基 387Ile 在纤维蛋白原组装和分泌中的作用，γ387Ile 被 Arg、Leu、Met、Ala 或 Asp 取代。带有 Arg、Leu、Met 和 Ala 的变体 γ 链在 CHO 细胞内组装成纤维蛋白原，随后分泌到培养基中，然而，带有 Asp 的变体纤维蛋白原的组装和分泌明显受损。这些观察结果表明，γ387Ile 上的残基对于纤维蛋白原组装和分泌比γC 尾部(γ387-411) 的长度更重要。4。为了检查 Bβ 链残基（对应于 γ387Ile 的 455Arg）对于纤维蛋白原组装和分泌的作用，我制作了突变载体 Bβ-456 末端、Bβ-455 末端，并用 Ile、Asp、Lys 或 Ala 进行替换。具有 Bβ-456 末端的变体纤维蛋白原被组装并分泌到培养基中，然而，具有 Bβ-456 末端的变体纤维蛋白原被组装并分泌到培养基中。 BP-455terminal 不是。不幸的是，我无法建立表达Bβ455Ile-、Asp-、Lys-或Ala-变体Bβ-链的CHO细胞系用于研究变体纤维蛋白原的组装和分泌。5.我还发现了新型功能失调的纤维蛋白原删除了Bβ111Ser残基。这种变异纤维蛋白原损害了纤维蛋白聚合，特别是横向聚集，但是，我猜这种变异纤维蛋白原的组装和分泌可能不会异常。较少的