Engineering mouse chymotrypsin B1 for improved trypsinogen degradation

Area of Science:

• Biochemistry
• Enzymology
• Protease Engineering

Background:

• Digestive proteases like chymotrypsin (CTR) are crucial for pancreatic function and protection.
• Human CTRB2 exhibits superior trypsinogen degradation compared to CTRB1, attributed to Arg236.
• Previous studies showed Arg236 introduction into CTRB1 enhanced human anionic trypsinogen degradation.

Purpose of the Study:

• To investigate if mouse CTRB1 activity can be improved by introducing Arg236 (G236R mutant) and/or widening the substrate binding pocket (A244G mutant).
• To assess the substrate specificity and efficiency of engineered mouse CTRB1 variants.
• To explore potential applications in preclinical models for pancreatitis.

Main Methods:

• Site-directed mutagenesis was used to create G236R and A244G mutants of mouse CTRB1.
• Enzyme kinetics and degradation assays were performed using mouse anionic (T8) and cationic (T7) trypsinogens, and bovine beta-casein as substrates.
• The proteolytic activity and substrate specificity of wild-type and mutant CTRB1 enzymes were compared.

Main Results:

• The G236R mutant showed a 32-fold increase in degrading mouse anionic trypsinogen (T8) but no change for cationic trypsinogen (T7) or casein.
• The A244G mutation reduced the activity of mouse CTRB1 against both trypsinogen isoforms and casein.
• The double mutant G236R-A244G exhibited improved anionic trypsinogen degradation but was less efficient than the G236R single mutant and showed reduced casein degradation.

Conclusions:

• In mouse CTRB1, Arg236 enhances protease activity in a substrate-specific manner, particularly for anionic trypsinogen.
• The Gly244 residue negatively impacts overall CTRB1 activity.
• These findings can guide the development of preclinical models with improved trypsinogen degradation for pancreatitis resilience.