Influence of Six Mutations of the Protein C Gene on the Gla Domain Conformation and Calcium Affinity

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Abstract
The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to Hn, a monoclonal antibody (mAb) known to recognize the sequence Phe 4 to Arg 9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by Hn. Mutation of Arg 229 to Gin and Ser 252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to Hn mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl210 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation. Conversely, the patient bearing the mutation Arg-1 to Cys had an abnormal elution profile, showing that calcium binding affinity was impaired probably due to a defective carboxylation (the behaviour of the Glu 7 to Asp mutant lacking one Gla residue validated our experimental approach). These results confirm that mutations in the serine protease domain do not modify the conformation of the N-terminal part of the Gla domain. Arg-1 to His, Arg-1 to Cys and Glu 7 to Asp mutations strongly modified affinity for calcium ions, whereas the Arg-5 to Trp mutation probably modifies the conformation of the Gla domain.

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