Enhancement of Human Protein C Function by Site-directed Mutagenesis of the γ-Carboxyglutamic Acid Domain

Abstract

This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (؎ phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.

Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site

“…Similarly, replacement of Glu 32 in factor X has little impact on protein function (12), and Glu 32 of human prothrombin was described as only moderately important (13), having little impact on either K m or V max of the prothrombinase reaction. However, a double mutant of human protein C, S11G/Q32E, shows an ϳ10-fold enhancement in membrane affinity (11), and a double mutant of factor VII, P10Q/K32E, has a 25-fold enhancement in membrane affinity (14). Under appropriate conditions, these proteins show similar improvement in function (11, 14 -16).…”

“…In the case of human protein C, simultaneous changes at residues 11 and 32 are required to realize enhancement (11). It is possible that Ser 11 of human protein C hinders development of a structure, allowing contact of position 32 with calcium or the membrane.…”

“…For example, the P10H mutant of bovine protein C shows an ϳ10-fold enhancement in membrane affinity, whereas the H10P mutant of human protein C shows an ϳ3-fold decline in membrane affinity (9). Although a major difference among the vitamin K-dependent proteins is a Gla residue versus another amino acid at position 32, the Q32E mutation of protein C has no impact on membrane affinity (10,11). Similarly, replacement of Glu 32 in factor X has little impact on protein function (12), and Glu 32 of human prothrombin was described as only moderately important (13), having little impact on either K m or V max of the prothrombinase reaction.…”

Antithrombotic and anticoagulant effects of wild type and Gla-domain mutated human activated protein C in rats

“…Full length cDNAs for the QGNSEDY-hPC and hPC:B148 introduced in the HindIII and XbaI sites in the pRC/CMV vector were created with recombinant DNA-techniques, as previously described [33][34][35][36]. The mutations in the QGNSEDY variant was H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y, where the first letter preceding the amino-acid number is the one-letter abbreviation for the wild-type amino acid, and the letter after the number refers to the amino acid residue introduced by the mutagenesis [33].…”

“…High-expressing colonies were selected for largescale culture and subsequent purification, as previously described [35]. The recombinant protein C variants were activated by thrombin, and then passed through a chromatography column of SP-Sephadex to remove thrombin essentially, as earlier described [36].…”