Renal epidermal growth factor precursor: proteolytic processing in an in vitro cell-free system.
The enzymatic processing of the membrane-bound renal epidermal growth factor precursor (proEGF) could be an important step in the control of nephrogenic repair consecutive to kidney insult. The enzyme machinery responsible for that processing was examined in a cell-free system consisting of renal membranes isolated from kidney homogenates by differential centrifugation, and incubated in vitro. After a 24-h incubation at 37 degrees C, 6-14% of membrane-bound proEGF was processed and soluble products with EGF immunoreactivity were released. As revealed by HPLC and Western blotting analysis, the products of proEGF proteolysis consisted of 6 kDa EGF (the molecular weight of mature EGF) and two polypeptides with molecular weights around 45 kDa. Interestingly the 45 kDa EGF forms, like the 6 kDa EGF, exhibited mitogenic activity toward growth-arrested NRK-52E renal cell line. The kinetic study of proEGF degradation gave data consistent with the 45 kDa product(s) being processing intermediate(s) between proEGF and 6 kDa EGF. The enzymatic activity responsible for proEGF nicking was inhibited by divalent heavy metal ions (Cu2+ or Zn2+) and several protease inhibitors (aprotinin, PMSF, leupeptin, soybean trypsin inhibitor), suggesting that proEGF is processed by kallikrein-like serine proteases present in the membrane preparations. Along with previous studies, the current observations suggest that renal kallikreins might play a role in renal tubular regeneration by promoting the release of soluble EGF in renal tissue.
Journe
F
F
Department of Biological Chemistry, School of Sciences, University of Mons-Hainaut, Mons, Belgium.
Wattiez
R
R
Piron
A
A
Carion
M
M
Laurent
G
G
Heuson-Stiennon
J A
JA
Falmagne
P
P
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002474:Cell-Free System; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D015151:Immunoblotting; D007610:Kallikreins / Q000737:chemistry; D007668:Kidney / Q000201:enzymology*; D008297:Male; D010446:Peptide Fragments / Q000302:isolation & purification; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar
D010446:Peptide Fragments; D011498:Protein Precursors; C042652:epidermal growth factor precursor; D004815:Epidermal Growth Factor; D007610:Kallikreins