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

Bálint Zoltán Németh, Zoltán Attila Nagy, Bence Kiss, Gabriella Gellén, Gitta Schlosser, Alexandra Demcsák, Andrea Geisz, Eszter Hegyi, Miklós Sahin-Tóth, Gábor Pál

Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] 2023 Sep

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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. Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

Citation

Bálint Zoltán Németh, Zoltán Attila Nagy, Bence Kiss, Gabriella Gellén, Gitta Schlosser, Alexandra Demcsák, Andrea Geisz, Eszter Hegyi, Miklós Sahin-Tóth, Gábor Pál. Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors. Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.]. 2023 Sep;23(6):742-749