Abstract
Intra-pancreatic activation of trypsin is an early event in pancreatitis. Trypsinogen can be activated to trypsin either through autoactivation (trypsin-mediated trypsinogen activation) or by the lysosomal protease cathepsin B (CTSB). Experimental separation of CTSB-mediated activation from autoactivation in mice is possible through knocking in mutations that render trypsinogen sensitive to CTSB but resistant to trypsin. Here we present biochemical studies on novel mouse cationic trypsinogen (isoform T7) mutants engineered for selective CTSB activation. First, we demonstrated that mutation K24G, which alters the activation site Lys in T7 trypsinogen, abolished autoactivation while activation by CTSB was stimulated 4-fold at pH 4.0. Interestingly, CTSB-mediated activation of the K24G mutant became more sensitive to inhibition by increasing pH. Next, Ala-scanning of the five Asp residues preceding the activation site Lys revealed that mutation D22A accelerated CTSB-mediated activation by 2-fold. Finally, combination of mutations D22A and K24G resulted in a trypsinogen mutant that exhibited 14-fold increased activation by CTSB and normal pH sensitivity. We conclude that we successfully engineered a mouse T7 trypsinogen mutant (D22A,K24G), which is robustly activated by CTSB but cannot undergo autoactivation. These studies set the stage for the generation of a preclinical mouse model of CTSB-dependent pancreatitis.
Highlights
Activation of the digestive protease trypsinogen to trypsin inside the pancreatic acinar cells is an early event in experimental pancreatitis[1]
We found that mutations D19A and D20A had no effect on cathepsin B (CTSB)-mediated trypsinogen activation (Fig. 2A), while mutation D22A stimulated the reaction about 2-fold (Fig. 2B, Table 1)
We engineered the mouse cationic trypsinogen for robust and selective activation by CTSB. This was achieved by strategically mutating the trypsinogen activation peptide so that trypsin-mediated activation was abolished while CTSB-mediated activation was accelerated
Summary
Activation of the digestive protease trypsinogen to trypsin inside the pancreatic acinar cells is an early event in experimental pancreatitis[1]. Genetically altered mice (Ctrb1-del) that exhibited increased cerulein-induced trypsin activation in their pancreas developed more severe pancreatitis[8]. The observations from animal experiments suggest that trypsin plays a significant role in pancreatitis development and severity only under conditions when intra-pancreatic trypsin activation is stimulated due to genetic alterations. A knowledge gap remains whether CTSB-mediated trypsinogen activation observed in rodent models is relevant to the human condition To address this problem, we set out to generate mouse models in which the two trypsinogen activation processes (autoactivation versus CTSB-mediated activation) can be experimentally separated and their effect on pancreatitis studied selectively. As a first step towards a CTSB-dependent mouse pancreatitis model, here we performed biochemical studies to identify T7 trypsinogen mutants that cannot autoactivate but are selectively and robustly activated by CTSB
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