Overexpression of prolylcarboxypeptidase enhances plasma prekallikrein activation on Chinese hamster ovary cells.
Plasma prekallikrein (PK) complexes with its receptor, high-molecular-weight kininogen (HK), on human umbilical vein endothelial cells (HUVEC). When assembled on endothelial cells, PK is activated to plasma kallikrein independent of factor XIIa by the serine protease prolylcarboxypeptidase (PRCP, Km= 9 nM). PRCP was shown to be a PK activator when isolated from HUVEC (J Biol Chem 277: 17962-17969, 2002) and produced as a recombinant protein (Blood 103: 4554-4561, 2004). To additionally confirm that human PRCP is a physiological PK activator, PRCP was overexpressed in Chinese hamster ovary (CHO) cells. CHO cells were transfected with full-length PRCP under the control of a cytomegalovirus promoter, and CHO recombinant PRCP was expressed as a fusion protein with COOH-terminal enhanced green fluorescence protein (EGFP). The presence of recombinant PRCP in transfected CHO cells was detected by real-time RT-PCR, immunoblot, and immunoprecipitation. PRCP mRNA and PK activation were two- to threefold higher in transfected than in control CHO cells. The increase in PRCP-induced PK activation in the transfected CHO cells paralleled the increase in PRCP antigen expression, as determined by anti-PRCP and anti-green fluorescence protein antibodies. PK activation of the transfected cells was blocked by small interfering RNA to PRCP. Anti-PRCP antibody and Z-Pro-Pro-aldehyde dimethyl acetate also blocked PK activation (IC50= 0.01 and 7.0 mM, respectively). Localization of PRCP in intact cells observed via confocal microscopy and flow cytometry also confirmed overexpression of PRCP on the external membrane. These investigations independently confirm that PRCP is expressed on cell membranes and that PRCP expression increases PK activation.
Shariat-Madar
Z
Z
Hematology/Oncology Division, Department of Internal Medicine, Univ. of Michigan, 5301 MSRB III, 1150 West Medical Center Dr., Ann Arbor, MI 48109-0640, USA. madar@umich.edu
Rahimy
E
E
Mahdi
F
F
Schmaier
A H
AH
D000818:Animals; D016466:CHO Cells; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004789:Enzyme Activation; D005786:Gene Expression Regulation / Q000502:physiology; D011288:Prekallikrein / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D011288:Prekallikrein; D002268:Carboxypeptidases; C022448:lysosomal Pro-X carboxypeptidase