Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly.
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.
Huang
S
S
Department of Biochemistry, University of Washington, Seattle 98195.
Mulvihill
E R
ER
Farrell
D H
DH
Chung
D W
DW
Davie
E W
EW
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; D005340:Fibrinogen