Assembly and secretion of recombinant human fibrinogen.
Expression vectors containing full-length cDNAs for each of the human fibrinogen chains were constructed. COS-1 cells were transfected with single vectors, mixtures of two, or with all three vectors and stable cell lines selected. Cells transfected with single vectors, or with mixtures of any two vectors, expressed the appropriate fibrinogen chains but did not secrete them. COS cells transfected with three vectors expressed all of the chains and secreted fibrinogen. COS cells transfected with three vectors contained, intracellularly, a mixture of fibrinogen-related proteins. The four main intracellular products were nascent fibrinogen, an A alpha.gamma complex, free A alpha chains, and free gamma chains. This is a similar pattern to that noted in Hep G2 cells. The intracellular forms of fibrinogen were sensitive to endoglycosidase H, indicating that they reside in a pre-Golgi compartment. Secreted fibrinogen was endoglycosidase H-insensitive, suggesting that the secreted glycoprotein moieties were processed in the normal manner. When mixed with plasma fibrinogen, radiolabeled recombinant fibrinogen was incorporated into a thrombin-induced clot. These studies demonstrate that COS cells transfected with all three fibrinogen chain cDNAs are capable of assembling and secreting a functional fibrinogen molecule.
Roy
S N
SN
Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021.
Procyk
R
R
Kudryk
B J
BJ
Redman
C M
CM
D002460:Cell Line; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D010957:Plasmids; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D005340:Fibrinogen; D004247:DNA