Cathepsin B Dipeptidyl Carboxypeptidase and Endopeptidase Activities Demonstrated across a Broad pH Range

• Published in: Biochemistry (Easton) Vol. 61; no. 17; pp. 1904 - 1914
• Main Authors: Yoon, Michael C., Hook, Vivian, O’Donoghue, Anthony J.
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.09.2022
• Subjects: Cathepsin B - chemistry; Cathepsins - metabolism; Endopeptidases; Hydrogen-Ion Concentration; Hydrolysis; Proteolysis; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.2c00358

Cathepsin B is a lysosomal protease that participates in protein degradation. However, cathepsin B is also active under neutral pH conditions of the cytosol, nuclei, and extracellular locations. The dipeptidyl carboxypeptidase (DPCP) activity of cathepsin B, assayed with the Abz-GIVR↓AK­(Dnp)-OH substrate, has been reported to display an acidic pH optimum. In contrast, the endopeptidase activity, monitored with Z-RR-↓AMC, has a neutral pH optimum. These observations raise the question of whether other substrates can demonstrate cathepsin B DPCP activity at neutral pH and endopeptidase activity at acidic pH. To address this question, global cleavage profiling of cathepsin B with a diverse peptide library was conducted under acidic and neutral pH conditions. Results revealed that cathepsin B has (1) major DPCP activity and modest endopeptidase activity under both acidic and neutral pH conditions and (2) distinct pH-dependent amino acid preferences adjacent to cleavage sites for both DPCP and endopeptidase activities. The pH-dependent cleavage preferences were utilized to design a new Abz-GnVR↓AK­(Dnp)-OH DPCP substrate, with norleucine (n) at the P3 position, having improved DPCP activity of cathepsin B at neutral pH compared to the original Abz-GIVR↓AK­(Dnp)-OH substrate. The new Z-VR-AMC and Z-ER-AMC substrates displayed improved endopeptidase activity at acidic pH compared to the original Z-RR-AMC. These findings illustrate the new concept that cathepsin B possesses DPCP and endopeptidase activities at both acidic and neutral pH values. These results advance understanding of the pH-dependent cleavage properties of the dual DPCP and endopeptidase activities of cathepsin B that function under different cellular pH conditions.