Formation of the human fibrinogen subclass Fib420:: Disulfide bonds and glycosylation in its unique (αE chain) domains

COS cell transfection has been used to monitor the assembly and secretion of fibrinogen molecules, both those of the subclass containing the novel alpha(E) chain and those of the more abundant subclass whose alpha chains lack alpha(E)'s globular C-terminus. That region, referred to as the alpha(E)C domain, is closely related to the ends of beta and gamma chains of fibrinogen (beta C and gamma C), Transfection of COS cells with alpha(E), beta, and gamma cDNAs alone results in secretion of the symmetrical molecule (alpha(E)beta gamma)(2), also known as Fib(420) Cotransfection with cDNA for the shorter cr chain yielded secretion of both (alpha beta gamma)(2) and (alpha(E)beta gamma)(2) but no mixed molecules of the structure alpha alpha(E)(beta gamma)(2). Exploiting the COS cells' fidelity with regard to Fib(420) production, identification was made of the highly conserved Asn667 as the sole site of N-linked glycosylation in the cue chain. No evidence from Cys --> Ser replacements was found for interchain disulfide bridges involving the four cysteines of the alpha(E)C domain, However, for fibrinogen secretion, the alpha(E), \beta, and gamma subunits do exhibit different requirements for integrity of the two intradomain disulfide bridges located at homologous positions in their respective C-termini, indicating dis similar structural roles in the process of fibrinogen assembly. (C) 1998 by The American Society of Hematology.