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Engineering mouse chymotrypsin B1 for improved trypsinogen degradation.

Nataly C Morales Granda1,2,3, András Szabó2, Zsombor Köller4

  • 1Department of Surgery, University of California Los Angeles, Los Angeles, CA, 90095, USA.

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|March 26, 2025
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Summary

Modifying mouse chymotrypsin B1 (CTRB1) with Arg236 significantly enhanced its ability to degrade anionic trypsinogen, offering potential for pancreatitis prevention. Further mutations impacted activity and substrate specificity.

Keywords:
Chronic pancreatitisChymotrypsinSerine proteaseTrypsinogen

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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.