Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range

• Published in: Biochemistry (Easton) Vol. 62; no. 15; pp. 2289 - 2300
• Main Authors: Yoon, Michael C., Phan, Von, Podvin, Sonia, Mosier, Charles, O’Donoghue, Anthony J., Hook, Vivian
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.08.2023
• Subjects: Cathepsin B - metabolism; Cathepsins - metabolism; Cysteine; Endopeptidases - metabolism; Lysosomes - metabolism; Peptides; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.3c00139

The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is, therefore, desirable to develop specific substrates for cathepsin B that measure its activity over broad pH ranges. Current substrates used to monitor cathepsin B activity consist of Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, but they lack specificity since they are cleaved by other cysteine cathepsins. Furthermore, Z-Arg-Arg-AMC monitors cathepsin B activity at neutral pH and displays minimal activity at acidic pH. Therefore, the purpose of this study was to design and validate specific fluorogenic peptide substrates that can monitor cathepsin B activity over a broad pH range from acidic to neutral pH conditions. In-depth cleavage properties of cathepsin B were compared to those of the cysteine cathepsins K, L, S, V, and X via multiplex substrate profiling by mass spectrometry at pH 4.6 and pH 7.2. Analysis of the cleavage preferences predicted the tripeptide Z-Nle-Lys-Arg-AMC as a preferred substrate for cathepsin B. Significantly, Z-Nle-Lys-Arg-AMC displayed the advantageous properties of measuring high cathepsin B specific activity over acidic to neutral pHs and was specifically cleaved by cathepsin B over the other cysteine cathepsins. Z-Nle-Lys-Arg-AMC specifically monitored cathepsin B activity in neuronal and glial cells which were consistent with relative abundances of cathepsin B protein. These findings validate Z-Nle-Lys-Arg-AMC as a novel substrate that specifically monitors cathepsin B activity over a broad pH range.