PMID: 8950780Nov 1, 1996Paper

Functional consequences of mutations in amino acid residues that stabilize calcium binding to the first epidermal growth factor homology domain of human protein C

Thrombosis and Haemostasis
J P GengFrancis J Castellino

Abstract

Charge-to-alanine mutations of three amino acid residues, viz, D46, D48, and D/Hya71, which are known to be important in stabilizing Ca2+ binding to epidermal growth factor (EGF) domains of vitamin K-dependent blood coagulation proteins, have been engineered into recombinant human protein C (r-PC). The resulting variants were then employed to assess the importance of this Ca2+ binding site in the activation properties of r-PC and in the activity of activated protein C (APC). Another mutation, of D48 to E, was constructed in order that a more conservative mutation at the Ca2+ binding site could be similarly examined. The mutant proteins were fully processed with regard to proper signal peptide cleavage, gamma-carboxylation, and beta-hydroxylation, except, of course, for the D71A mutant in this latter case. The D48E variant possessed an additional residue of gamma-carboxyglutamic acid (Gla), showing that E48 was gamma-carboxylated. All of the mutants were reactive against a monoclonal antibody (MAb) specific for a Ca(2+)-dependent epitope within the amino-terminus of the Gla domain of r-PC, demonstrating that a proper Ca(2+)-dependent conformation was adopted in this region of the protein. None of the mutants, except for [D48 gam...Continue Reading

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