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

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.