Abstract
Fibrinogen (340 kDa) is a plasma protein that plays an important role in the final stages of blood clotting. Human fibrinogen is a dimer with each half-molecule composed of three different polypeptides (A alpha, 67 kDa; B beta, 57 kDa; gamma, 47 kDa). To understand the mechanism of fibrinogen chain assembly and secretion and to obtain a system capable of producing substantial amounts of fibrinogen for structure-function studies, we developed a recombinant system capable of secreting fibrinogen. An expression vector (pYES2) was constructed with individual fibrinogen chain cDNAs under the control of a Gal-1 promoter fused with mating factor F alpha 1 prepro secretion signal (SS) cascade. In addition, other constructs were prepared with combinations of cDNAs encoding two chains or all three chains in tandem. Each chain was under the control of the Gal-1 promoter. These constructs were used to transform Saccharomyces cerevisiae (INVSC1; Mat alpha his3-delta 1 leu2 trp1-289 ura3-52) in selective media. Single colonies from transformed yeast cells were grown in synthetic media with 4% raffinose to a density of 1 x 10(8) cells/ml and induced with 2% galactose for 16 h. Yeast cells expressing all three chains contained fibrinogen precursors and nascent fibrinogen and secreted about 30 micrograms/ml of fibrinogen into the culture medium. The B beta and gamma chains, but not A alpha, were glycosylated. Glycosylation of B beta and gamma chains was inhibited by treatment of transformed yeast cells with tunicamycin. Intracellular B beta and gamma chains, but not the A alpha chains in secreted fibrinogen, were cleaved by endoglycosidase H. Carbohydrate analysis indicated that secreted recombinant fibrinogen contained N-linked asialo-galactosylated biantennary oligosaccharide. Recombinant fibrinogen yielded the characteristic plasmin digestion products, fragments D and E, that were immunologically indistinct from the same fragments obtained from plasma fibrinogen. The recombinant fibrinogen was shown to be biologically active in that it could form a thrombin-induced clot, which, in the presence of factor XIIIa, could undergo gamma chain dimerization and A alpha chain polymer formation.
Highlights
Molecule with each half-molecule composed of three different polypeptides
Periodic acid-Schiff staining of the separated chains, treatment of transformed yeast cells with tunicamycin, and endoglycosidase H digestion of intracellular and secreted fibrinogen showed that only B and ␥ chains are glycosylated
Tunicamycin and endoglycosidase H treatment suggest that initial N-linked glycosylation of recombinant fibrinogen occurs in a manner similar to that in hepatocytes
Summary
Materials—The expression vector pYES2 and the yeast strain INVSC1 (Mat␣ his3-⌬1 leu trp289 ura3–52) were obtained from Invitrogen, Inc. (San Diego, CA). Construction of Expression Vectors—Expression vectors containing fibrinogen cDNAs for single chains, for combinations of two chains, and for all three chains were inserted into multiple cloning sites at the. To prepare pYES2A␣, pYES2B, and pYES2␥, full-length cDNAs were released by appropriate restriction enzymes from previously described constructs [8] and ligated to pYES2 plasmid at the 3Ј-end of the Gal-1-SS promoter. The cells were harvested by centrifugation for 5 min at 500 ϫ g, resuspended in SC-ura medium containing 2% galactose, and grown for an additional 16 h for induction of fibrinogen chain synthesis. The cells were harvested, washed with phosphate-buffered saline, lysed with 0.5 ml of IP buffer
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