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

1. 考察了γ - 153cys和γ - 182cys之间的S-S键对γ - c模组三级结构形成的作用。γ-链被α取代的γ- 153cys在CHO细胞中不能形成a αγ-或b βγ-复合物。这些结果表明没有一个完整的纤维蛋白原被组装并随后被分泌。研究了γ - 319asn和γ - 320asp在γ - c模组三级结构形成中的作用。将缺失γ-链的γ- 319asn和γ- 320asp与正常γ-链的表达载体共转染CHO细胞，发现异常γ-链被合成并组装成正常a α和b β-链的纤维蛋白原，但与正常纤维蛋白原相比，异常纤维蛋白原的分泌明显减少。为了研究γ链残基387Ile在纤维蛋白原组装和分泌中的作用，我们用Arg、Leu、Met、Ala或Asp取代了γ- 387Ile。含有Arg、Leu、Met和Ala的变异γ-链在CHO细胞内组装成纤维蛋白原并分泌出氨基酸，而在培养基中，含有Asp的变异γ-链的组装和分泌明显受损。这些观察结果表明，γ - 387ile的残基对纤维蛋白原的组装和分泌比γ - c尾部的长度（γ - 387-411）更为关键。为了研究γ - 387ile对应的b β链残基455Arg在纤维蛋白原组装和分泌中的作用，我制作了突变载体，b β-456末端，b β-455末端，并被Ile， Asp， Lys或Ala取代。具有b β-456末端的变异体纤维蛋白原被组装并分泌到培养基中，而具有bp -455末端的变异体纤维蛋白原则未被组装并分泌到培养基中。不幸的是，我无法建立表达Bβ455Ile-， Asp-， Lys-或ala -变体b β链的CHO细胞系，用于研究变异纤维蛋白原的组装和分泌。我还发现新的功能失调纤维蛋白原删除了Bβ111Ser残基。这种纤维蛋白原的变异使纤维蛋白聚合受损，尤其是横向聚集，然而，我猜想这种纤维蛋白原的变异的聚集和分泌可能不是异常的。少

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