Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly.
Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly.
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DOI：
10.1016/s0021-9258(18)52960-4
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发表时间：
1993-04
期刊：
影响因子：
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通讯作者：
Shaoming Huang;E. Mulvihill;D. Farrell;D. Chung;E. Davie
中科院分区：
文献类型：
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作者：
Shaoming Huang;E. Mulvihill;D. Farrell;D. Chung;E. Davie
建立了稳定表达人α、β和伽马纤维蛋白原链的稳定转基因仓鼠肾(BHK)细胞，以任意两种不同的组合或单独表达。在BHK细胞株和Hep G2细胞中观察到几种类型的亚基相互作用。这些过程包括：1)通过二硫键连接的α-伽马二聚体的形成(S)，2)通过二硫键连接的β-伽马二聚体的形成(S)，3)通过二硫键连接的α-β-伽马半分子的形成，以及4)成熟的纤维蛋白原的形成，它也被分泌到细胞培养液中。链组成的分析证实了α-伽马、β-伽马和α-β-伽马络合物的化学计量比。这些数据与α-伽马和β-伽马二聚体以及α-β-伽马半分子是纤维蛋白原组装和生物合成的中间产物的说法一致。相反，在转基因的BHK细胞或Hep G2细胞中没有发现二硫键连接的α-β复合体，这表明这两条链之间二硫键的形成很可能发生在α-β-伽马和/或β-伽马复合体形成半分子时，以及当α-β-伽马半分子二聚生成成熟分子时。当每条链在没有其他两条链的情况下表达时，也可以鉴定出由二硫键连接的每条链的二聚体、三聚体和较大的低聚物。α-伽马和β-伽马复合体的优先形成，而不是单个链的低聚物，表明低聚物不太可能是纤维蛋白原组装的中间产物。基于这些结果，提出了纤维蛋白原组装的模型。
Stable transfected baby hamster kidney (BHK) cells expressing human alpha, beta, and gamma fibrinogen chains together, in various combinations of any two, or individually, were established. Several types of subunit interactions were observed in the intracellular extracts of the transfected BHK cell lines as well as in Hep G2 cells. These included: 1) formation of alpha gamma dimers linked by a disulfide bond(s), 2) formation of beta gamma dimers linked by a disulfide bond(s), 3) formation of alpha beta gamma half-molecules linked by disulfide bonds, and 4) formation of mature fibrinogen, which was also secreted into the cell culture medium. Analysis of the chain composition confirmed the stoichiometry of the alpha gamma, beta gamma, and alpha beta gamma complexes. These data are consistent with the proposal that the alpha gamma and beta gamma dimers as well as the alpha beta gamma half-molecules are intermediates in the assembly and biosynthesis of fibrinogen. In contrast, disulfide-linked alpha beta complexes were not found in transfected BHK cells or in Hep G2 cells, suggesting that the formation of disulfide bonds between these two chains most likely occurs when alpha beta gamma half-molecules are formed from alpha gamma and/or beta gamma complexes and when alpha beta gamma half-molecules dimerize to generate the mature molecule. Dimers, trimers, and larger oligomers of each individual chain linked by disulfide bonds were also identified when each chain was expressed in the absence of the other two chains. Preferential formation of alpha gamma and beta gamma complexes, rather than oligomers of individual chains, suggested that the oligomers were less likely to be intermediates in the assembly of fibrinogen. A model for fibrinogen assembly is presented based on these results.