Urotensin-II-Converting Enzyme Activity of Furin and Trypsin in Human Cells in Vitro

Abstract

Human urotensin-II (hU-II) is processed from its prohormone (ProhU-II) at putative cleavage sites for furin and serine proteases such as trypsin. Although proteolysis is required for biological activity, the endogenous "urotensin-converting enzyme" (UCE) has not been investigated. The aim of this study was to investigate UCE activity in cultured human cells and in blood, comparing activity with that of furin and trypsin. In a cell-free system, hU-II was detected by high-performance liquid chromatography-mass spectrometry after coincubating 10 M carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37°C) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca 2ϩ ions and supplemented with 2 mM EDTA at pH 7.0. hU-II was readily detected in the superperfusate of permeabilized epicardial mesothelial cells incubated with CTF-ProhU-II (3 h, 37°C), but it was only weakly detected in the superperfusate of intact cells. Conversion of CTF-ProhU-II to hU-II was attenuated in permeabilized cells using conditions found to inhibit furin activity. In a cell-free system, trypsin (0.05 mg/ml) cleaved CTFProhU-II to hU-II, and this was inhibited with 35 M aprotinin. hU-II was detected in blood samples incubated with CTFProhU-II (3 h, 37°C), and this was also inhibited with aprotinin. The findings revealed an intracellular UCE in human epicardial mesothelial cells with furin-like activity. Aprotinin-sensitive UCE activity was detected in blood, suggesting that an endogenous serine protease such as trypsin may also contribute to proteolysis of hU-II prohormone, if the prohormone is secreted into the circulation.

The G Protein-Coupled Receptor UT of the Neuropeptide Urotensin II Displays Structural and Functional Chemokine Features

“…The mature peptide UII results from the proteolysis of preproprotein UII at the tribasic site KKR by a specific urotensin converting enzyme (UCE), which is not still identified (4, 5). Study on the conversion of a 25 amino acid C-terminal fragment of preproprotein to mature peptide revealed that the endoprotease Furin and the serine protease trypsin, may act, respectively, as intracellular and extracellular UCE (12). This enzymatic cleavage appears necessary to confer biological activity (13).…”

Human Urotensin II as a Link between Hypertension and Coronary Artery Disease

“…3), with a molecular weight of ~1,388 (32). This peptide, isolated originally from fish urophysis (a neurosecretory organ of the caudal spinal cord), is derived from pre-pro U-II, which consists of 124 and 139 amino acid residues, by urotensinconverting enzyme (33). U-II has been identified as an endogenous ligand for the G-protein coupled receptor 14 (GPR14)/ sensory epithelia neuropeptide-like receptor (SENR) orphan receptor, with seven transmembrane domains, which was cloned recently and renamed the UT receptor (34).…”

Relationship Between Hypertension and Atherosclerosis: From a Viewpoint of the Most Potent Vasoconstrictor Human Urotensin II

“…2), with a molecular weight of ~1388 [22]. This peptide is derived from pre-pro U-II, consisting of 124 and 139 amino acid residues, by urotensin-converting enzyme [23]. U-II has been identified as an endogenous ligand for an orphan receptor G-protein coupled receptor 14 (GPR14), with seven transmembrane domains, which was cloned recently and renamed UT receptor [24].…”