title: Construction, expression, and properties of a recombinant chimeric human protein C with replacement of its growth factor-like domains by those of human coagulation factor IX.
abstract: The cDNA encoding a chimeric human protein C (PC), in which its epidermal growth factor-(EGF) like regions have been replaced with equivalent structures from human factor IX (fIX), was constructed and the gene product was expressed in human 293 cells. A molecular subpopulation of the recombinant chimeric protein (r-[PC/delta EGF-1,2/delta fIXEGF-1,2]) was purified that contained the full complement (9 residues/mol) of gamma-carboxyglutamic acid (Gla). After conversion by thrombin to its activated form (r-[APC/delta EGF-1,2/delta fIXEGF-1,2]), this latter enzyme was found to possess approximately 10% of the activity of wild-type recombinant APC (wtr-APC) in an APTT assay. In assay systems employing purified components, the activity of the mutant enzyme toward prothrombinase cofactor Va (fVa) and tenase cofactor VIII (fVIII) was approximately 30% and < 10%, respectively, of that of wtr-APC. The chimeric protein displayed full reactivity with a Ca(2+)-dependent monoclonal antibody to the Gla domain of PC, yielding a C50 for Ca2+ that was very similar to that obtained with wtr-PC (ca. 3.7 mM). Titrations of the dependency on Ca2+ of the intrinsic fluorescence of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] allowed calculation of a C50 value of 0.34 mM, again very similar to that of wtr-PC. As with wtr-PC, Ca2+ inhibited the thrombin-catalyzed activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2] with aKi of 148 microM, as compared to a Ki of 125 microM for wtr-PC. At a saturating level of Ca2+, activation of r-[PC/delta EGF-1,2/delta fIXEGF-1,2/] by the thrombin/thrombomodulin (thrombin/TM) complex occurred at approximately 70% of the rate of that of wtr-PC.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
authors.forename: S
authors.initials: S
authors.lastname: Yu
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: A
authors.initials: A
authors.lastname: Jhingan
authors.forename: W T
authors.initials: WT
authors.lastname: Christiansen
authors.forename: F J
authors.initials: FJ
authors.lastname: Castellino
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002478:Cells, Cultured; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism; D005164:Factor IX / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005169:Factor VIII / Q000378:metabolism; D015943:Factor Va / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D011486:Protein C / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D004792:Enzyme Precursors; D011486:Protein C; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D015943:Factor Va; D005169:Factor VIII; D005164:Factor IX; D002118:Calcium