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Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

ABSTRACT: Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.

SUBMITTER: Baricos WH

PROVIDER: S-EPMC1149139 | biostudies-other | 1988 May

REPOSITORIES: biostudies-other

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Project description:BackgroundCD151 is a cell-surface molecule of the tetraspanin family. Its lateral interaction with laminin-binding integrin ɑ3β1 is important for podocyte adhesion to the glomerular basement membrane (GBM). Deletion of Cd151 in mice induces glomerular dysfunction, with proteinuria and associated focal glomerulosclerosis, disorganisation of GBM and tubular cystic dilation. Despite this, CD151 is not routinely screened for in patients with nephrotic-range proteinuria. We aimed to better understand the relevance of CD151 in human kidney disease.MethodsNext-generation sequencing (NGS) was used to detect the variant in CD151. Electron and light microscopy were used to visualise the filtration barrier in the patient kidney biopsy, and immunoreactivity of patient red blood cells to anti-CD151/MER2 antibodies was performed. Further validation of the CD151 variant as disease-causing was performed in zebrafish using CRISPR-Cas9.ResultsWe report a young child with nail dystrophy and persistent urinary tract infections who was incidentally found to have nephrotic-range proteinuria. Through targeted NGS, a novel, homozygous truncating variant was identified in CD151, a gene rarely reported in patients with nephrotic syndrome. Electron microscopy imaging of patient kidney tissue showed thickening of GBM and podocyte effacement. Immunofluorescence of patient kidney tissue demonstrated that CD151 was significantly reduced, and we did not detect immunoreactivity to CD151/MER2 on patient red blood cells. CRISPR-Cas9 depletion of cd151 in zebrafish caused proteinuria, which was rescued by injection of wild-type CD151 mRNA, but not CD151 mRNA containing the variant sequence.ConclusionsOur results indicate that a novel variant in CD151 is associated with nephrotic-range proteinuria and microscopic haematuria and provides further evidence for a role of CD151 in glomerular disease. Our work highlights a functional testing pipeline for future analysis of patient genetic variants. A higher resolution version of the Graphical abstract is available as Supplementary information.

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Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

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