A gamma-carboxyglutamic acid (gamma) variant (gamma 6D, gamma 7D) of human activated protein C displays greatly reduced activity as an anticoagulant.
Site-specific mutagenesis has been employed to alter the cDNA of human protein C (PC), such that the gamma-carboxyglutamic acid (gamma) pair at positions 6 and 7 of the recombinant (r) protein would be changed to aspartic acid residues. This variant, [gamma 6D, gamma 7D]r-PC, and its wild-type (wt) counterpart have been expressed in human kidney 293 cells. After purification, forms of wtr-PC that were fully gamma-carboxylated and beta-hydroxylated and of [gamma 6D, gamma 7D]r-PC that lacked only the two altered gamma-residues at amino acid sequence positions 6 and 7 were obtained. Subsequent to its conversion to activated PC (APC), [gamma 6D, gamma 7D]r-APC displayed a greatly reduced activity in the activated partial thromboplastin time of PC-deficient plasma, as compared to wtr-APC and human plasma APC. In addition, the activity of [gamma 6D, gamma 7D]r-APC toward inactivation of purified human factor VIII was reduced to less than 5% of that of wtr-APC and human plasma APC. These results, with the first reported mutations at gamma-residues of PC produced by recombinant DNA technology, indicate that the paired gamma-residues at positions 6 and 7, which are highly conserved in all vitamin K dependent coagulation proteins, are very important to generation of fully functional APC. Additional results demonstrate further that lack of gamma-carboxylation at positions 6 and 7 of PC does not substantially affect this same processing reaction at other relevant glutamic acid residues.
Zhang
L
L
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001777:Blood Coagulation; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005169:Factor VIII / Q000378:metabolism; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010314:Partial Thromboplastin Time; D011486:Protein C / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology; D013329:Structure-Activity Relationship; D014162:Transfection
D011486:Protein C; D011994:Recombinant Proteins; D015055:1-Carboxyglutamic Acid; D005169:Factor VIII; D004247:DNA