Cellular basis for protein C deficiency caused by a single amino acid substitution at Arg15 in the gamma-carboxyglutamic acid domain.
Protein C is a zymogen of an anticoagulant vitamin K-dependent serine protease. Inherited protein C deficiency is often associated with a high risk for venous thromboembolism. It is characteristic of protein C deficiency that most single amino acid replacements result in type I (secretion defect) deficiency. To determine the molecular and cellular bases of protein C deficiency, we expressed recombinant human protein C mutants in which Arg15 was mutated to either Gly, Trp, Gln, Leu, or Pro by a single base exchange. Arg15 is one of the conservative residues in the gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent coagulation factors, and is also one of the high frequency multiple mutation sites in protein C deficiency. In transient expression studies using human kidney 293 cells, the relative amounts of Arg15 mutants secreted into the medium and determined by enzyme-linked immunosorbent assay (ELISA) were as follows: Gly, 42%; Trp, 14%; Gln, 54%; Leu, 22%; and Pro, 13%, the amount of wild-type (Wt) protein C being taken as 100%. Thus, the order of the secreted amounts of the recombinant mutants was determined to be Wt > Gln > Gly > Leu > Trp, Pro. Pulse-chase experiments using both transiently-transfected and a pool of stably-transfected 293 cells, and stably-transfected BHK cells showed the same order of secretion efficiency. Since this order correlated well with that of the hydrophobicity scale of amino acid side chains, a conformational alteration of the Gla domain resulting in impaired secretion may be dependent on the hydrophobicity of the replaced amino acid. In transient cells, the relative radioactivities of pulse-labeled bands of all recombinant protein C were almost equal, suggesting that the same translational efficiency for Wt and all Arg15 mutants. All of the Arg15-mutated protein C precursors were shown to be located in the same organelle as protein disulfide isomerase (PDI), an endoplasmic reticulum-resident protein, and were sensitive to endoglycosidase H digestion. These results suggest that mutations of the highly conserved Arg15 in the Gla domain of protein C caused a secretion defect to variable degrees depending on replaced amino acid residue.
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo.
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Tokunaga
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Tsukamoto
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Koide
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011486:Protein C / Q000737:chemistry / Q000235:genetics*; D020151:Protein C Deficiency; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013924:Thrombophlebitis / Q000209:etiology / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D011486:Protein C; D011994:Recombinant Proteins; D001120:Arginine