High Affinity Small Protein Inhibitors of Human Chymotrypsin C (CTRC) Selected by Phage Display Reveal Unusual Preference for P4′ Acidic Residues

Abstract

Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2 (Leu or Asp), and P4 (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2 (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4 (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K D 20 pM) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K D of 110 pM and a selectivity ranging from 225-to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys 51 , Arg 56 , and Arg 80 ) on the surface of human CTRC that interact with the P4 acidic residue of the inhibitor. The acidic preference of CTRC at P4 is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.Digestion of dietary proteins in the small intestine is catalyzed by proteases secreted from the pancreas. These proteases are produced as inactive proenzymes (zymogens) and their activation is spatially restricted to the duodenum and proceeds in a cascade-like manner. The membrane-localized serine protease enteropeptidase (enterokinase) activates trypsinogens to active trypsins, which, in turn, activate all other proteolytic proenzymes; in the human these include chymotrypsinogens B1, B2, C, and L1, proelastases 2A, 3A, and 3B, and procarboxypeptidases A1, A2, and B1. This classic textbook paradigm of digestive enzyme activation has been recently revised by the discovery of interactions between digestive proteases that can modulate the ultimate levels of active enzymes. In this regard, we demonstrated that human chymotrypsin C (CTRC) 4 regulates activation and degradation of human cationic trypsinogen and trypsin (1, 2). CTRC facilitates trypsin-mediated activation (autoactivation) of cationic trypsinogen by processing the trypsinogen activation peptide, at the Phe 18 -Asp 19 peptide bond, to a shorter form that is more readily cleaved by trypsin (1). Mutation A16V in the trypsinogen activation peptide stimulates CTRC-mediated processing and subjects with this mutation are at increased risk of developing chronic pancreatitis (1, 3). CTRC can also promote degradation of human cationic trypsinogen a...

Mesotrypsin Signature Mutation in a Chymotrypsin C (CTRC) Variant Associated with Chronic Pancreatitis

“…Several studies attempted to clarify the determinants of this specificity. Phage display-selected variants of the locust-derived chymotrypsin inhibitor Schistocerca gregaria proteinase inhibitor-2 (SGPI-2) indicated that negatively charged amino acids on the primed side of the scissile peptide bond are important for CTRC recognition (16). This notion seemed in agreement with the natural preponderance of such residues in the regulatory nick sites.…”

“…Purified CTRC zymogen was activated with 50 l of immobilized trypsin (20230, Thermo Scientific) in 3-ml final volume of 0.1 M Tris-HCl (pH 8.0) for 2 h at 22°C. Concentration of active CTRC was determined using active site titration with ecotin, as described recently (16). The concentration of variants p.R29Q, p.G214R, p.S239C, and mutant p.G214M was estimated by comparing their protein band intensity with that of wild-type CTRC on Coomassie Blue-stained gels.…”

“…The expression plasmids for eglin C and SGPI-2-C4 were kind gifts from Evette Radisky (Mayo Clinic Cancer Center, Jacksonville Florida) and Gábor Pál (Eotvos University, Budapest, Hungary), respectively. SGPI-2-C4 is a phage display-selected CTRC-specific variant of the locust-derived chymotrypsin inhibitor SGPI-2 (16). Purification of these inhibitors followed published protocols (16,24).…”

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis

“…Concentrations of purified trypsinogen preparations were determined from the ultraviolet absorbance at 280 nm using the extinction coefficient 37,525 M Ϫ1 ·cm Ϫ1 SPINK1 binding assays. Binding of wild-type and p.L104P mutant human cationic trypsin to the trypsin inhibitor SPINK1 was characterized by measuring the K D value of the reaction in equilibrium, as we described previously (32,35). Trypsin concentrations were determined by titration against ecotin, and SPINK1 concentration was determined by titration against wild-type human cationic trypsin.…”

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

“…The dialyzed chymotrypsinogen solutions were concentrated using a Vivaspin 20 concentrator (10-kDa molecular mass cutoff). Chymotrypsinogens were activated with trypsin, and active chymotrypsin concentrations were determined by active site titration with ecotin, as described previously (24).…”