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.


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. Cathepsin B and cathepsin K cleavage profiles at pH 4.6 and pH 7.2 assessed for preferred residues at P4 to P4' positions adjacent to P1-P1' cleavage sites.
(a) Cathepsin B cleavage profiles at pH 4.6 and pH 7.2. The preference of cathepsin B cleavages for amino acid residues at each of the P4 to P4' positions adjacent to the P1-P1' cleavage site was deduced from MSP-MS data cleavage profiling data. Z-scores provided quantitation of preferred (green shades) and non-preferred (yellow shades) residues illustrated in a heat map.
The z-scores were utilized for iceLogo illustration of the primary preferred residues at the P4 to P4' positions shown above the mid-line, with non-preferred residues shown below the mid-line.
The purple letters indicate significance of p < 0.05, and the black letters indicate p < 0.3.
(b) Cathepsin K cleavage profiles at pH 4.6 and pH 7.2. The preference of cathepsin K cleavages for residues at P4 to P4' positions was defined from MSP-MS data, indicated by z-scores shown in a heat map for preferred residues (green shades) and non-preferred residues (yellow shades).
The primary preferred residues, based on z-scores, were plotted in iceLogo illustrations. The purple letters indicate significance of p < 0.05, and the black letters indicate p < 0.3. Figure S3. Cathepsins L, S, and V cleavage profiles at pH 4.6 and pH 7.2 assessed for preferred residues at P4 to P4' positions.

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The preferences of each cathepsin protease for residues at P4 to P4' positions were defined by MSP-MS data, and indicated z-scores for preferred residues (green shades) and non-preferred residues (yellow shades). IceLogo illustrated the main preferred residues (above the line) and preferred residues with significance of p < 0.05 are shown in purple letters.

Cathepsin X activity is illustrated with its standard substrate Mca-RPPGFSAFK(Dnp)-OH,
showing activity at pH 4.6 and pH 5.5 (but not at pH 7.2). Cathepsin X was assessed for proteolytic cleavage of the Z-Nle-Lys-Arg-AMC, Z-Phe-Arg-AMC, or Z-Arg-Arg-AMC substrates (40 µM substrate concentrations) at the three pHs tested of pH 4.6, 5.5, and 7.2. Cathepsin X did not display cleavage of these fluorogenic substrates.

Figure S5. Specific cathepsin B activity monitored with novel Z-Nle-Lys-Arg-AMC substrate in human neuroblastoma and mouse microglia cells. Cathepsin B activity in cell homogenates from human neuroblastoma SHSY-5Y cells (panel a), human neuroblastoma SK-N-MC cells (panel b), and mouse microglia BV2 cells (panel c) was assessed with the substrates
Z-Nle-Lys-Arg-AMC, Z-Arg-Arg-AMC, and Z-Phe-Arg-AMC (60 µM) at pH 4.6, 5.5, and 7.2. Data show cathepsin B activity as pmol AMC/µg enzyme protein (30 min incubation) as the mean + SD (n=3). Significance is indicated by *p < 0.05 (student's t-test) comparing proteolytic activity with the of substrates Z-Arg-Arg-AMC and Z-Phe-Arg-AMC compared to Z-Nle-Lys-Arg-AMC.

Arg-Lys-AMC, Z-Arg-Arg-AMC, and Z-Phe-Arg-AMC.
(a) pH-dependence of mouse cathepsin B activity with Z-Nle-Lys-Arg-AMC compared to Z-Arg-Arg-AMC and Z-Phe-Arg-AMC substrates. Cathepsin B specific activity with the three different substrates (40 µM) were assessed at pH 4.6, 5.5, and 7.2. Data points are shown as the mean ± SD (n = 3).
(b) Kinetic kcat/Km values for cathepsin B activity with the substrates Z-Nle-Lys-Arg, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC. kcat/Km values for each of the substrate(s at pH 4.6 and pH 7.2 were calculated for mouse cathepsin B as described in the methods. (The asterisk indicates that the kcat /Km was determined from the linear slope of the curve since the curve did not plateau out.) Table S1. Kinetic values of human cathepsin B assessed with substrates Z-Nle-Lys-Arg-AMC, Z-Arg-Arg-AMC, and Z-Phe-Arg AMC at pH 4.6 and 7.2.

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Kinetic values were determined from three independent determinations assessed for Michaelis-Menten kinetics with standard deviation (conducted using GraphPad Prism). n.d. = not determined.