Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, La Jolla, San Diego, California 92093, United States.
Biomedical Sciences Graduate Program, University of California, La Jolla, San Diego, California 92093, United States.
Biochemistry. 2023 Aug 1;62(15):2289-2300. doi: 10.1021/acs.biochem.3c00139. Epub 2023 Jul 17.
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.
组织蛋白酶 B 的生物学和病理学功能发生在酸性溶酶体中和细胞质、核和细胞外位置的中性 pH 下。重要的是，组织蛋白酶 B 在酸性 pH 下显示出与中性 pH 条件下不同的底物裂解特性。因此，开发用于测量其在广泛 pH 范围内活性的特异性组织蛋白酶 B 底物是可取的。目前用于监测组织蛋白酶 B 活性的底物包括 Z-Phe-Arg-AMC 和 Z-Arg-Arg-AMC，但它们缺乏特异性，因为它们被其他半胱氨酸组织蛋白酶切割。此外，Z-Arg-Arg-AMC 监测中性 pH 下的组织蛋白酶 B 活性，在酸性 pH 下显示出最小的活性。因此，本研究的目的是设计和验证特异性荧光肽底物，可在从酸性到中性 pH 的广泛 pH 范围内监测组织蛋白酶 B 的活性。通过在 pH 4.6 和 pH 7.2 下进行质谱多底物分析，比较了组织蛋白酶 B 的深入裂解特性与半胱氨酸组织蛋白酶 K、L、S、V 和 X 的特性。分析裂解偏好性预测三肽 Z-Nle-Lys-Arg-AMC 是组织蛋白酶 B 的首选底物。重要的是，Z-Nle-Lys-Arg-AMC 在酸性至中性 pH 范围内具有测量高组织蛋白酶 B 特异性活性的优势特性，并且仅被组织蛋白酶 B 特异性切割，而不被其他半胱氨酸组织蛋白酶切割。Z-Nle-Lys-Arg-AMC 特异性监测神经元和神经胶质细胞中的组织蛋白酶 B 活性，与组织蛋白酶 B 蛋白的相对丰度一致。这些发现验证了 Z-Nle-Lys-Arg-AMC 是一种新型底物，可在广泛的 pH 范围内特异性监测组织蛋白酶 B 的活性。
