Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: Studies related to proline-10

L. Shen, A. M. Shah, B. Dahlback, Gary L Nelsestuen

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Abstract

Bovine and human protein C show high homology in the amino acids of their GLA domains (amino-terminal 44 residues), despite the about 10-fold higher membrane affinity of the human protein. A proposed membrane contact site and mechanism suggested that this difference was largely due to the presence of proline at position 10 of bovine protein C versus 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 of replacing proline-10 in bovine protein C with histidine, and the reverse change in human protein C. In both cases, the protein containing proline-10 showed lower membrane affinity, about 10-fold lower for bovine protein C and 5-fold lower for human protein C. As expected, activated 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 displayed up to 15-fold higher activity than wild type bAPC. This demonstrated the ability to improve both membrane contact and activity by mutation. 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 of blood coagulation in pathological states.

Original languageEnglish (US)
Pages (from-to)16025-16031
Number of pages7
JournalBiochemistry
Volume36
Issue number51
DOIs
StatePublished - Dec 1 1997

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