Processing of the carboxyl 15-amino acid extension in the alpha-chain of fibrinogen.
The cDNA for the alpha-chain of human fibrinogen codes for 15 amino acids at the carboxyl terminus that are not found in the native protein circulating in plasma. In the present studies, experiments were designed to test whether the carboxyl extension was required for fibrinogen assembly or was simply the result of limited proteolysis and maturation of the protein during or following secretion. Baby hamster kidney cells were transfected with cDNAs coding for the alpha-, beta-, and gamma-chains, and the recombinant fibrinogen secreted into the cell culture medium was identified either with an antibody against the mature molecule or with an antibody directed toward the carboxyl extension of the alpha-chain. The secreted protein contained primarily two species of alpha-chains, including one with the carboxyl extension and one that was the same as that in plasma fibrinogen. A mutant fibrinogen, in which Arg-611 at the putative cleavage site in the alpha-chain was converted to Gly, was also readily assembled, and, in this case, the protein was secreted with the alpha-chain carboxyl extension. Similarly, a mutant fibrinogen that completely lacked the carboxyl extension of the alpha-chain was assembled and secreted from the transfected baby hamster kidney cells. These two mutants with modified alpha-chains were readily clotted by thrombin and cross-linked by factor XIIIa. These results indicate that the alpha-chain carboxyl extension in fibrinogen is not required for assembly or secretion of the molecule. Accordingly, it was concluded that the cleavage of the alpha-chain removing the carboxyl-terminal 15 amino acids is a normal and specific processing event occurring during the maturation of the protein.
Farrell
D H
DH
Department of Biochemistry, University of Washington, Seattle 98195.
Huang
S
S
Davie
E W
EW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001777:Blood Coagulation; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005340:Fibrinogen / Q000096:biosynthesis / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008647:Mesocricetus; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005340:Fibrinogen; D004247:DNA