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

• DOI: 10.1016/j.pan.2023.08.004
• Publication Date: 2023-08-17T07:23:42Z
• Authors: Bálint Zoltán Németh; Zoltán Attila Nagy; Bence Kiss; Gabriella Gellén; Gitta Schlosser; Alexandra Demcsák; Andrea Geisz; Eszter Hegyi; Miklós Sahin-Tóth; Gábor Pál
• Keywords: Human chymotrypsin-like enzyme; Serine proteinase inhibitor; Substrate Specificity; Chymases; Pancreatitis; Peptide Library; Trypsinogen; Animals; Humans; Chymotrypsin; Cattle; Protease Inhibitors; Phage display; Directed protein evolution; Serine proteinase

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.