Recombinant prolylcarboxypeptidase activates plasma prekallikrein.
The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment.
Shariat-Madar
Zia
Z
Department of Internal Medicine, Division of Hematology and Oncology, University of Michigan, 5301 MSRB III, 1150 W Medical Center Drive, Ann Arbor, MI 48109-0649, USA.
Mahdi
Fakhri
F
Schmaier
Alvin H
AH
D000818:Animals; D001483:Base Sequence; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D004331:Drosophila melanogaster; D004730:Endothelium, Vascular / Q000201:enzymology; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D009025:Morpholines; D009838:Oligodeoxyribonucleotides; D011288:Prekallikrein / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014471:Umbilical Veins
D017931:DNA Primers; D009025:Morpholines; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D011288:Prekallikrein; D002268:Carboxypeptidases; C022448:lysosomal Pro-X carboxypeptidase