Strategy for recombinant multichain protein synthesis: fibrinogen B beta-chain variants as thrombin substrates.
Thrombotic disease has been found in patients with congenital dysfibrinogens that have abnormalities in the amino terminal domain of the fibrinogen B beta-chain. Surprisingly, these fibrinogens are poor substrates for thrombin. In order to examine the molecular basis for this impaired thrombin-fibrinogen interaction, we synthesized three fibrinogens with single amino acid substitutions in this domain: B beta A68T, B beta P70S, and B beta L72S. B beta-chain expression vectors were altered by PCR-directed mutagenesis of the B beta cDNA. The altered vectors were transfected into a Chinese hamster ovary (CHO) cell line that was constructed as a first step in recombinant fibrinogen synthesis, this CHO line synthesizes fibrinogen A alpha- and gamma-chains. More than 86% of the stably selected clones expressed significant levels of fibrinogen, confirming that a two-step strategy permitted efficient synthesis of variant fibrinogens. In large-scale cultures variant fibrinogen accumulation in serum-free medium fluctuated between 1 and 15 micrograms/mL. Normal and variant recombinant fibrinogens were compared to plasma fibrinogens by following the time course of thrombin-catalyzed release of fibrinopeptides. Only the variant B beta A68T, a change identified in a congenital dysfibrinogen, showed significantly impaired kinetics. The rate of fibrinopeptide A release was decreased 27-fold, and the rate of fibrinopeptide B release was decreased 45-fold relative to normal fibrinogen. Fibrinopeptide release was not significantly altered by the substitutions B beta P70S or B beta L72S. These results suggest that B beta residue Ala68 has a novel and critical role in the interaction between thrombin and fibrinogen.
Lord
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Department of Pathology, University of North Carolina at Chapel Hill 27599-7525, USA.
Strickland
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Jayjock
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D005338:Fibrin Fibrinogen Degradation Products / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism*
D017931:DNA Primers; D005338:Fibrin Fibrinogen Degradation Products; D011994:Recombinant Proteins; D013917:Thrombin