Recombinant variant fibrinogens substituted at residues γ326Cys and γ339Cys demonstrated markedly impaired secretion of assembled fibrinogen

BackgroundTo study the functions of residues γ326Cys and γ339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring γ326Tyr and γ326Ser variants, along with γ326Ala and γ339Ala variants.MethodsA fibrinogen γ-chain expression vector was altered and transfected into Chinese hamster ovary (CHO) cells. Cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblotting analysis. In addition, pulse-chase analysis was performed.ResultsThe CHO cells synthesized mutant γ-chains and assembled these into fibrinogen in the cells, although these variant fibrinogens were barely secreted into the culture media. Pulse-chase analysis indicated that the rates of both assembly and secretion of the variant fibrinogens were lower than that of normal fibrinogen.ConclusionsThe present study indicated that the 326-339 intrachain disulfide bond has a crucial role in maintaining the tertiary structure of the C-terminal domain of the γ-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of γ326Tyr and γ326Ser suggest that heterozygous fibrinogen molecules containing variant γ-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia.