Urotensin-II-converting enzyme activity of furin and trypsin in human cells in vitro.
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 microM carboxyl terminal fragment (CTF)-ProhU-II with recombinant furin (2 U/ml, 3 h, 37 degrees C) at pH 7.0 and pH 8.5, but not at pH 5.0, or when the incubating medium was depleted of Ca2+ 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 degrees 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 CTF-ProhU-II to hU-II, and this was inhibited with 35 microM aprotinin. hU-II was detected in blood samples incubated with CTF-ProhU-II (3 h, 37 degrees 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.
Russell
Fraser D
FD
Department of Medicine, University of Queensland, The Prince Charles Hospital, Chermside, Queensland, Australia. russell@medicine.uq.edu.au
Kearns
Philip
P
Toth
Istvan
I
Molenaar
Peter
P
D002478:Cells, Cultured; D004848:Epithelium / Q000201:enzymology*; D045683:Furin / Q000378:metabolism*; D006801:Humans; D011498:Protein Precursors; D014357:Trypsin / Q000378:metabolism*; D014579:Urotensins / Q000378:metabolism*
D011498:Protein Precursors; D014579:Urotensins; C029677:urotensin II; D014357:Trypsin; D045683:Furin