Over-Expression of Prolylcarboxypeptidase Enhances Plasma Prekallikrein Activation on Chinese Hamster Ovary Cell Membranes.

Abstract

Recent investigations indicate that there is a physiologic plasma prekallikrein (PK) activator on the membrane of endothelial cells (HUVEC). PK complexes with high molecular weight kininogen (HK) and HK is PK's receptor on HUVEC. When assembled on HUVEC, PK is activated to plasma kallikrein independent of FXIIa by the serine protease prolylcarboxypeptidase (PRCP) (Km=9 nM). PRCP has been shown to be a PK activator when isolated from HUVEC (JBC 277:17962, 2002) and produced as a recombinant protein (Blood 103:4554, 2004). To additionally confirm that human PRCP is a physiologic PK activator, PRCP was over-expressed in Chinese hamster ovary cells (CHO). CHO were transfected with full-length PRCP under the control of a CMV promoter and CHO rPRCP was expressed as a fusion protein with C-terminal enhanced green fluorescent protein (EGFP). The presence of rPRCP in transfected CHO was detected by real time RT-PCR, immunoblot and immunoprecipitation. In CHO cells, PRCP mRNA and PK activation are 2–3-fold higher than controls. The increase in PRCP-induced PK activation on the transfected CHO cells parallels the increase in PRCP antigen expression on the transfected cells, as determined by anti-PRCP and anti-GFP antibodies. PK activation of the transfected cells is blocked by siRNA to the translation initiation site of PRCP, anti-PRCP antibody, and Z-Pro-Pro-aldehyde dimethyl acetate, with IC50 of the last 2 inhibitors of 0.01 and 7.0 mM respectively. Cellular localization of PRCP in intact cells using confocal microscopy and flow cytometry indicate that its over-expression results in its placement on the CHO cell plasma membrane. These investigations independently confirm that prolylcarboxypeptidase is expressed on cell membranes and its expression increases PK activation. The endothelial cell membrane expression of PRCP resulting in PK activation contributes to the constitutive level of bradykinin in the intravascular compartment to regulate blood pressure homeostasis.