Identification of renal cathepsin B as a human prorenin-processing enzyme.
Prorenin, the inactive biosynthetic precursor of renin, is proteolytically cleaved in the renal juxtaglomerular cells to renin. The activity of renin is rate-limiting for generation of angiotensin II in the circulation. We identified a renal thiol protease which activates and accurately cleaves the 43-amino acid prosegment of human recombinant prorenin. In the current studies, 6.5 mg of this protease was purified from human renal cortex using a three-step procedure dependent upon Leu-Leu-arginyl affinity chromatography. This represented an overall 766-fold purification and resulted in three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of molecular weights 30,000, 25,000, and 24,000. All three bands cross-reacted with an anti-human liver cathepsin B antibody upon immunoblot analysis; electrolution of each band and amino-terminal sequence analysis confirmed that the Mr 30,000 protein was mature cathepsin B and the Mr 25,000 and 24,000 bands were cathepsin B subunits. The pH optimum for the hydrolysis of pure human recombinant prorenin by pure renal cathepsin B was 6, and the Michaelis-Menten constant, Km, of the reaction was 1.4 x 10(-9) M. Immunostaining of human kidney using a sheep anti-human cathepsin B antibody demonstrated the presence of cathepsin B in the juxtaglomerular areas of the kidney, as well as in the renal proximal tubules. Electron microscopic immunohistochemistry using the same antibody demonstrated cathepsin B in dense secretory granules of the juxtaglomerular cells. Renin was also shown to be present in these granules. This study provides both biochemical and morphological evidence that renal cathepsin B is a human prorenin-processing enzyme.
Wang
P H
PH
Department of Medicine, Los Angeles County, Medical Center 90033.
Do
Y S
YS
Macaulay
L
L
Shinagawa
T
T
Anderson
P W
PW
Baxter
J D
JD
Hsueh
W A
WA
D000595:Amino Acid Sequence; D002401:Cathepsin B / Q000276:immunology / Q000378:metabolism*; D003429:Cross Reactions; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry; D007672:Kidney Cortex / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data
D003546:Cysteine Endopeptidases; C063281:prorenin processing enzyme; D002401:Cathepsin B