Engineering mouse chymotrypsin B1 for improved trypsinogen degradation

TL;DR

Researchers engineered mouse chymotrypsin B1 to improve its ability to degrade trypsinogen, which could help prevent pancreatitis.

Contribution

The study introduces specific mutations in mouse CTRB1 that enhance trypsinogen degradation in a substrate-specific manner.

Findings

The G236R mutation improved mouse anionic trypsinogen cleavage by 32-fold.

The A244G mutation reduced activity against trypsinogen and casein.

The double mutant G236R-A244G showed improved but not maximal degradation of anionic trypsinogen.

Abstract

The digestive protease chymotrypsin (CTR) protects the pancreas against harmful trypsin activity by promoting degradation of trypsinogen. Recently, we demonstrated that Arg236 is responsible for the higher proteolytic activity and better trypsinogen degrading capability of human CTRB2 compared to CTRB1. Introduction of Arg236 into CTRB1, which normally carries Asp236, dramatically increased degradation of human anionic trypsinogen. Here, we explored whether we could improve the activity of mouse CTRB1 by changing Gly236 to Arg (G236R mutant) and/or by widening the substrate binding pocket (A244G mutant). We found that mutant G236R cleaved mouse anionic (T8) trypsinogen at Phe150 with 32-fold improved efficiency. In contrast, mutant G236R digested mouse cationic (T7) trypsinogen and bovine beta-casein at the same rate as wild-type mouse CTRB1. Mutation A244G reduced the activity of mouse CTRB1 against the two trypsinogen isoforms and casein. Double-mutant G236R-A244G cleaved mouse anionic (T8) trypsinogen 9.8-fold better than wild-type CTRB1 but 3.3-fold slower than single mutant G236R. Mutant G236R-A244G digested mouse cationic (T7) trypsinogen at the same rate as single-mutant A244G but degraded casein 2.3-fold slower. Taken together, the observations indicate that in the context of mouse CTRB1 the Arg236 residue increases protease activity in a substrate-specific manner, while Gly244 has an overall negative impact. The results will inform the design of preclinical mouse models with higher trypsinogen degradation ability and enhanced resilience against pancreatitis.

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