Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors

• Published in: Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] 2023-09, Vol.23 (6), p.742-749
• Main Authors: Németh, Bálint Zoltán, Nagy, Zoltán Attila, Kiss, Bence, Gellén, Gabriella, Schlosser, Gitta, Demcsák, Alexandra, Geisz, Andrea, Hegyi, Eszter, Sahin-Tóth, Miklós, Pál, Gábor
• Format: Article
• Language: English
• Subjects: Animals; Cattle; Chymases - antagonists & inhibitors; Chymases - chemistry; Chymotrypsin - chemistry; Directed protein evolution; Human chymotrypsin-like enzyme; Humans; Pancreatitis - prevention & control; Peptide Library; Phage display; Protease Inhibitors - chemistry; Protease Inhibitors - isolation & purification; Protease Inhibitors - pharmacology; Serine proteinase; Serine proteinase inhibitor; Substrate Specificity; Trypsinogen
• ISSN: 1424-3903; 1424-3911; 1424-3911
• DOI: 10.1016/j.pan.2023.08.004

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.