Biosynthesis of human fibrinogen. Subunit interactions and potential intermediates in the assembly.

S Huang,E.R Mulvihill,D.H Farrell,D.W Chung,E.W Davie

doi:10.1016/s0021-9258(18)52960-4

S Huang, E.R Mulvihill + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)52960-4

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Abstract

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.

Highlights

Stable transfected baby hamster kidney (BHK) cells fibrinogen are encoded by three independent genes (Chung et expressing human a, 8, and y fibrinogen chains to- al., 1983a, 1983b, 1990; Rixon et al, 1983; Kant et al, 1983)
The individual chainsare the transfected BHK cell lines as well as in Hep G 2 processed, glycosylated, assembled, and eventually secreted cells. These included: 1)formation of ay dimers linked into the circulating blood as mature fibrinogen molecules. It by a disulfide bond(s), 2 ) formation of By dimers linked by a disulfide bond(s), 3) formation of a8y half-molecules linked by disulfide bonds, and 4) formation of mature fibrinogen, which was secreted into the cell culture medium, Analysis of the chaincomposition confirmed the stoichiometry of the ay, By, and a8y complexes
Disulfide-linkead8 Hartwig and Danishefsky, 1991), different combinations of complexes were not found in transfected BHK cells or fibrinogen chains including y3,ayp, and/or Pyz were proposed in Hep G 2 cells, suggesting that theformation of disul- to be intermediates

Summary

SUBUNITINTERACTIONS AND POTENTIALINTERMEDIATESIN THE ASSEMBLY*

The individual chainsare the transfected BHK cell lines as well as in Hep G 2 processed, glycosylated, assembled, and eventually secreted cells These included: 1)formation of ay dimers linked into the circulating blood as mature fibrinogen molecules. Expressionof fibrinogenand fibrinogen chains by these cell lines was established by Western blot analysis and metabolic labelingI.ntracellular extracts were examined and of free a and y chains, as well as three complexeswith apparent masses greater than 250 kDa. In the reduced gel, the 154-kDa complex migrated as a and y chains present in approximatelyequal amounts. Complex Formation in Cell Lines CoexpressingTwo of the ThreeFibrinogenChins-The intracellular fibrinogenchains being expressedwereanalyzed by immunoprecipitation of radiolabeledcell lysates, using human fibrinogen-specific antisera and Protein A-Sepharose, followedby two-dimensional SDS-PAGE. Characteristics of the stable transfected BHK cell lines expressing human fibrinogen chains. BHK 570cells were transfected with expression plasmids for each of the threechains of fibrinogen, selected with various concentrations of methotrexate (MTX), andcharacterized as described under "Materials and Methods."

PM a

Bip b

BHK a y MN

Monomer Dimer Trimer a

DISCUSSION

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