Expression of four mutant fibrinogen gammaC domains in Pichia pastoris confirms them as causes of hypofibrinogenaemia.
ABSTRACT Mutations in the fibrinogen gene cluster can cause low plasma fibrinogen concentrations, known as hypofibrinogenaemia. It is important to verify whether a detected sequence variant in this cluster is deleterious or benign and this can be accomplished using protein expression systems. In this study, four mutations in the fibrinogen gammaC domain that had previously been described in patients with hypofibrinogenaemia were introduced into a gammaC construct and expressed in a Pichia pastoris yeast system to investigate their effects on protein stability and secretion. These experiments showed that the fibrinogen Middlemore (N230D), Dorfen (A289V), Mannheim II (H307Y), and Muncie (T371I) mutations were not secreted, supporting their causative role in hypofibrinogenaemia. Overexpression of the N230D, A289V and H307Y mutants revealed that the majority of the synthesised protein was retained in the endoplasmic reticulum, with only a minor proportion reaching the trans-Golgi network. Regardless, none of this protein was secreted which confirms that the four mutations investigated are indeed responsible for hypofibrinogenaemia.
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ABSTRACT: Introduction : We examined a 6-month-old girl with inherited fibrinogen abnormality and no history of bleeding or thrombosis. Routine coagulation screening tests showed a markedly low level of plasma fibrinogen determined by functional measurement and also a low level by antigenic measurement (functional/antigenic ratio = 0.295), suggesting hypodysfibrinogenemia. Materials and methods : DNA sequence analysis was performed, and γT305A fibrinogen was synthesized in Chinese hamster ovary cells based on the results. We then functionally analyzed and compared with that of nearby recombinant γN308K fibrinogen. Results : DNA sequence analysis revealed a heterozygous γT305A substitution (mature protein residue number). The γT305A fibrinogen indicated markedly impaired thrombin-catalyzed fibrin polymerization both in the presence or absence of 1 mM calcium ion compared with that of γN308K fibrinogen. Protection of plasmin degradation in the presence of calcium ion or Gly-Pro-Arg-Pro peptide (analogue for so-called knob ‘A’) and factor XIIIa-catalyzed fibrinogen crosslinking demonstrated that the calcium binding sites, hole ‘a’ and D:D interaction sites were all markedly impaired, whereas γN308Kwas impaired at the latter two sites. Molecular modeling demonstrated that γT305 is localized at a shorter distance than γN308 from the high affinity calcium binding site and hole ‘a’. Conclusion : Our findings suggest that γT305 might be important for construction of the overall structure of the γ module of fibrinogen. Substitution of γT305A leads to both dysfibrinogenemic and hypofibrinogenemic characterization, namely hypodysfibrinogenemia. We have already reported that recombinant γT305A fibrinogen was synthesized normally and secreted slightly, but was significantly reduced.Thrombosis Research 01/2014; · 3.13 Impact Factor