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

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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