Properties of recombinant chimeric human protein C and activated protein C containing the gamma-carboxyglutamic acid and trailing helical stack domains of protein C replaced by those of human coagulation factor IX.
The properties of a recombinant (r) chimeric human protein C (PC) containing replacement of its gamma-carboxyglutamic acid (Gla) and helical stack (HS) domains by those of human coagulation factor IX (fIX) have been examined. Titration with Ca2+ of the divalent cation-induced intrinsic fluorescence quenching of this chimera (r-GDIX/PC) allowed determination of the [Ca2+], of 1.8 mM, required to produce this alteration in 50% of the protein molecules. These values were 0.41 and 0.61 mM for wtr-PC and fIX, respectively. The chimera did not react with a Ca(2+)-dependent, Gla domain-directed conformational monoclonal antibody (MAb) to r-PC but did interact with a similar MAb (H5B7) to fIX. The [Ca2+] required to induced H5B7 binding to 50% of the r-GDIX/PC molecules was 6.6 mM, while this same value for fIX was a nearly identical 7.2 mM. The [Ca2+] needed for binding of 50% of r-GDIX/PC to acidic phospholipid (PL) vesicles was 0.58 mM, while that for wtr-PC and fIX were 1.2 and 0.55 mM, respectively. The [protein] required for 50% binding of r-GDIX/PC to PL at 20 mM Ca2+ was 0.29 microM. These same values for r-PC and fIX were 0.38 and 1.8 microM, respectively. The Ca(2+)-mediated inhibition of the thrombin-catalyzed activation of r-GDIX/PC was characterized by a Ki of 118 microM, a value similar to that of 125 microM obtained for this same inhibition of wtr-PC activation. The thrombin-catalyzed activation of both r-GDIX/PC and wtr-PC was stimulated by soluble r-thrombomodulin. Similar to the case of wtr-PC, Ca2+ initially enhanced and, at higher concentrations, inhibited the activation of r-GDIX/PC. The Km and kcat values for this latter activation at optimal [Ca2+] (100 microM) were 4.1 microM and 2.5 s-1, respectively. These same kinetic constants for activation of wtr-PC were 4.3 microM and 2.9 s-1, respectively. These results show that many of the features needed for functional integrity of the Ca2+-bound Gla/HS domains of PC are also present in those same modules of fIX, a finding that points to a generalized functional role for the Ca2+-induced conformation of the structural unit consisting of the Gla and HS domains. The data also suggest that the Ca2+-bound form of the Gla/HS region is an independently folded unit in PC and perhaps in fIX.(ABSTRACT TRUNCATED AT 400 WORDS)
Christiansen
W T
WT
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000032:analysis*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002478:Cells, Cultured; D004789:Enzyme Activation; D005164:Factor IX / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011486:Protein C / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011486:Protein C; D011993:Recombinant Fusion Proteins; D015055:1-Carboxyglutamic Acid; D005164:Factor IX; D002118:Calcium