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 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.
Shen
L
L
Department of Clinical Chemistry, Malmö General Hospital and University of Lund, Malmö, Sweden.
Shah
A M
AM
Dahlbäck
B
B
Nelsestuen
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D015943:Factor Va / Q000037:antagonists & inhibitors; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D011392:Proline / Q000737:chemistry; D011486:Protein C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D008081:Liposomes; D010743:Phospholipids; D011486:Protein C; D011994:Recombinant Proteins; D006639:Histidine; D015943:Factor Va; D011392:Proline