Studies on the assembly and secretion of fibrinogen.
cDNAs of fibrinogen A alpha and gamma chains were individually subcloned into a eukaryotic expression vector by using the polymerase chain reaction. Triple cotransfection into COS cells of the two plasmids together with a B beta chain expression plasmid, constructed as described previously (Danishefsky, K.J., Hartwig, R., Banerjee, D., and Redman, C. (1990) Biochim. Biophys. Acta 1048, 202-208), resulted in the secretion of complete fibrinogen into the media and the formation of four additional intracellular complexes which we also showed to be present in the hepatocyte cell line Hep 3B. The complexes, which have Mr = 232, 150, 135, and 128 (x 10(-3) conform with the Mr expected for A alpha B beta gamma 2, B beta gamma 2 and gamma 3, respectively. A A mechanism of assembly is proposed based on the assumption that all these complexes are precursors of complete fibrinogen. Each of the expressed fibrinogen chains in transfected COS cells interacts noncovalently with binding protein (BiP, GRP 78), but not to the same extent; gamma chain binds less BiP than the A alpha and B beta chains. Assembly of fibrinogen is not absolutely required for its secretion. In addition to complete fibrinogen, the conditioned media of hepatocytes and of transfected COS cells contained free A alpha, free gamma, and two of the above-mentioned complexes, A alpha gamma 2 and A alpha B beta gamma 2.
Hartwig
R
R
Lucy and Ishmael Herman Laboratory of Cardiovascular Molecular Biology, Department of Medicine, New York Medical College, Valhalla 10595.
Danishefsky
K J
KJ
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000235:genetics* / Q000378:metabolism; D005341:Fibrinolysin; D005786:Gene Expression Regulation; D006031:Glycosylation; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D014162:Transfection
D005340:Fibrinogen; D004247:DNA; D005341:Fibrinolysin