Abstract
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.
Highlights
Test whether the carboxyl extension was required for Recombinant fibrinogen hasbeen expressed from cDNA fibrinogen assembly or was the resultof limited clones in mammalian cells (Farrell et al, 1989, 1991; Hartwig proteolysis and maturation of the protein during or and Danishefsky, 1991; Roy et al, 1991)
A mutant fibrinogen, inwhich Arg-611 at An unexpected posttranslational modification was discovthe putative cleavage site in the a-chain was conveererdtewdhen the cDNAsfor the a-chainwere originally isolated to Gly, was readily assembled, and, in this case, and sequenced (Kant et al, 1983; Rixon et al, 1983)
A mutant fibrinogen that com- ditional 15 amino acids at its carboxyl terminus, which were pletely lacked the carboxyl extension of the a-chain not found in the amino acid sequence in the mature protein was assembled and secreted from the transfectebdaby circulating in blood
Summary
Test whether the carboxyl extension was required for Recombinant fibrinogen hasbeen expressed from cDNA fibrinogen assembly or was the resultof limited clones in mammalian cells (Farrell et al, 1989, 1991; Hartwig proteolysis and maturation of the protein during or and Danishefsky, 1991; Roy et al, 1991). A transfected derivative of this cell line, which expressed recombinant human fibrinogen, BHK-Fbg, has been described previously (Farrell et al, 1991).
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