Identification of renal cathepsin B as a human prorenin-processing enzyme

• Published in: The Journal of biological chemistry 1991-07, Vol.266 (19), p.12633-12638
• Main Authors: POU HSIUNG WANG, DO, Y. S, MACAULEY, L, SHINAGAWA, T, ANDERSON, P. W, BAXTER, J. D, HSUEH, W. A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Cathepsin B - immunology; Cathepsin B - metabolism; Cross Reactions; Cysteine Endopeptidases - metabolism; Cytoplasmic Granules - enzymology; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrolases; Immunohistochemistry; Kidney Cortex - metabolism; Liver - enzymology; Molecular Sequence Data
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)98946-5

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.