ENHANCING THE ACTIVITY OF PROTEIN-C BY MUTAGENESIS TO IMPROVE THE MEMBRANE-BINDING SITE - STUDIES RELATED TO PROLINE-10

Bovine and human protein C show high homology in the amino acids of th eir GLA domains (amino-terminal 44 residues), despite the about 10-fol d higher membrane affinity of the human protein. A proposed membrane c ontact site and mechanism suggested that this difference was largely d ue to the presence of proline at position 10 of bovine protein C versu s histidine at position 10 of human protein C [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L . (1997) Biochemistry 36, 5120-5127], This study examined the impact o f replacing proline-10 in bovine protein C with histidine, and the rev erse change in human protein C. In both cases, the protein containing proline-10 showed lower membrane affinity, about 10-fold lower for bov ine protein C and 5-fold lower for human protein C. As expected, activ ated human protein C (hAPC) containing proline at position 10 showed 2 .4-3.5-fold lower activity than wild type hAPC, depending on the assay used. Most interesting was that bovine APC containing histidine-10 di splayed up to 15-fold higher activity than wild type bAPC. This demons trated the ability to improve both membrane contact and activity by mu tation. This general strategy should be applicable to other vitamin K- dependent proteins, providing opportunities to study function as well as to produce proteins that may find use as promoters and inhibitors o f blood coagulation in pathological states.