Increased activation of hereditary pancreatitis-associated human cationic trypsinogen mutants in presence of chymotrypsin C

Abstract

Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.

FIGURE 1.

Effect of CTRC on autoactivation of human wild-type cationic trypsinogen (A) and mutants L81A (B) and R122A (C). Wild-type and mutant trypsinogens were incubated at 1 μm with 10 nm initial trypsin in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, and 0.05% Tween 20 (final concentrations) in the absence or presence of the indicated CTRC concentrations at 37 °C. At given times, 2-μl aliquots were removed, and trypsin activity was measured as described under “Experimental Procedures.” Trypsin activity is expressed as a percentage of the maximal activity in the absence of CTRC. Representative experiments from two replicates are shown.

FIGURE 2.

Autoactivation of pancreatitis-associated cationic trypsinogen mutants N29I, N29T, R122C, and R122H in presence of CTRC. Wild-type and mutant trypsinogens were incubated at 1 μm with 10 nm initial trypsin in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, and 0.05% Tween 20 at 37 °C in the absence (A) or presence of CTRC at 5 nm (B) or 25 nm (C) (final concentrations). At the indicated times, 2-μl aliquots were removed, and trypsin activity was measured as described under “Experimental Procedures.” Trypsin activity is expressed as a percentage of the maximal activity in the absence of CTRC. Representative experiments from two or three replicates are shown.

FIGURE 3.

Autoactivation of pancreatitis-associated cationic trypsinogen mutants A16V and V39A in presence of CTRC. For comparison, mutant R122H was also included. Wild-type and mutant trypsinogens were incubated at 1 μm with 10 nm initial trypsin in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, and 0.05% Tween 20 at 37 °C in the absence (A) or presence of CTRC at 5 nm (B) or 25 nm (C) (final concentrations). At the indicated times, 2-μl aliquots were removed, and trypsin activity was measured as described under “Experimental Procedures.” Trypsin activity is expressed as a percentage of the maximal activity in the absence of CTRC. Representative experiments from two replicates are shown.

FIGURE 4.

N-terminal processing of human cationic trypsinogen and pancreatitis-associated mutants by CTRC. Wild-type and mutant trypsinogens were incubated at 2 μm with 25 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, and 20 nm SPINK1 trypsin inhibitor (final concentrations) at 37 °C. The trypsin inhibitor was included to prevent autoactivation. A, at the indicated times, reactions were terminated by precipitation with 10% trichloroacetic acid (final concentration), and samples were analyzed by 15% nonreducing SDS-PAGE and Coomassie Blue staining. Relevant segments of representative gels from two replicates demonstrate the small mobility shift of the trypsinogen band caused by N-terminal processing. B, densitometric analysis of stained gels showing the changes in the intensity of the unprocessed intact trypsinogen band as a percentage of the total intensity of the processed and unprocessed bands. Rates of processing were calculated from linear fits to semilogarithmic plots. Error bars were omitted for clarity. The S.E. was within 10% of the mean.

FIGURE 5.

Cleavage of Leu-81–Glu-82 peptide bond in human cationic trypsinogen and pancreatitis-associated mutants by CTRC. Wild-type and mutant trypsinogens were incubated at 2 μm with 25 nm CTRC in 0.1 m Tris-HCl (pH 8.0) and 20 nm SPINK1 trypsin inhibitor (final concentrations) at 37 °C. The trypsin inhibitor was included to prevent autoactivation. A, at the indicated times, reactions were terminated by precipitation with 10% trichloroacetic acid (final concentration), and samples were analyzed by 15% reducing SDS-PAGE and Coomassie Blue staining. The asterisk indicates the faint band migrating at ∼5 kDa, which is the product of a secondary cleavage at Leu-41 by CTRC. Representative gels of two or more experiments are shown. B, densitometric analysis of stained gels showing the changes in the intensity of the unprocessed intact trypsinogen band. Rates of degradation were calculated from linear fits to semilogarithmic plots. Error bars were omitted for clarity. The S.E. was within 12% of the mean.

FIGURE 6.

Cleavage of Arg-122–Val-123 peptide bond in human cationic trypsinogen and pancreatitis-associated mutants by cationic trypsin. Wild-type and mutant trypsinogens were incubated at 2 μm with 10 nm human cationic trypsin in 0.1 m Tris-HCl (pH 8.0) and 5 μm CaCl₂ (final concentrations) at 37 °C. All trypsinogens contained the K23Q mutation to prevent autoactivation. A, at the indicated times, reactions were terminated by precipitation with 10% trichloroacetic acid (final concentration), and samples were analyzed by 15% reducing SDS-PAGE and Coomassie Blue staining. Note that the two cleavage fragments co-migrated and appeared as a single band at ∼15 kDa. Representative gels of two experiments are shown. B, densitometric analysis of stained gels showing the changes in the intensity of the uncleaved intact trypsinogen band as a percentage of the total intensity of the cleaved (Tg*) and uncleaved (Tg) bands. Error bars were omitted for clarity. The S.E. was within 10% of the mean.

FIGURE 7.

Inactivation of human cationic trypsin and pancreatitis-associated mutants by CTRC. Wild-type and mutant trypsinogens at 1 μm were activated to trypsin with 7 ng/ml (64 pm) human enteropeptidase in 0.1 m Tris-HCl (pH 8.0), 5 μm CaCl₂, and 0.05% Tween 20 for 30 min at 37 °C (final concentrations). Initial trypsin activity was determined, and incubations were continued at 37 °C in the absence or presence of 25 nm CTRC. Loss of trypsin activity was followed by withdrawing 2-μl aliquots and measuring trypsin activity as described under “Experimental Procedures.” Trypsin activity is expressed as a percentage of the initial activity. Rates of trypsin inactivation were calculated from linear fits to semilogarithmic plots. Data points represent the average of two or three experiments. Error bars were omitted for clarity. The S.E. was within 10% of the mean.

FIGURE 8.

Potential interactions between Asn-84, Arg-122, and Asn-33 in trypsinogen. A, ribbon diagram of bovine trypsinogen (Protein Data Bank code 1TGN). Positions 29 and 81 were changed to Asn and Leu, respectively, as found in human cationic trypsinogen. The activation peptide is disordered, and the first N-terminal amino acid residue visible is Val-25. The N-terminal peptide and Asn-33 are colored in blue; the calcium-binding loop and Asn-84 are in green with Leu-81 in black; and the Arg-122 turn is shown in orange with Arg-122 in red. Also shown are the positions of pancreatitis-associated mutations N29I, N29T (with Asn-29 in blue), and V39A (with Val-39 in magenta). The image was rendered using PyMOL 1.3 (Schrödinger, LLC). B, schematic representation of the interactions highlighted in the structural model in A.