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.
Highlights
Tion [1], requires vitamin K for the formation of nine ␥-carboxyglutamic acids (Gla)1 in its amino-terminal 45 residues
This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties
This study showed success in generation of human protein C with enhanced membrane binding
Summary
Recombinant Proteins—Site-directed mutagenesis was performed using a PCR method as described previously [4]. The following oligonucleotides were synthesized: A, 5Ј-AAA TTA ATA CGA CTC ACT ATA GGG AGA CCC AAG CTT-3Ј (corresponding to sense nucleotides 860 – 895 in the vector pRc/CMV, including the HindIII cloning site); B, 5Ј-GCA TTT AGG TGA CAC TAT AGA ATA GGG CCC TCT AGA-3Ј (antisense to nucleotides 984 –1019 in the vector pRc/CMV including the XbaI cloning site). The mutagenesis primers were as follows: C,
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