Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis

Abstract

The human pancreas expresses two major trypsinogen isoforms, cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2). Mutations in PRSS1 cause hereditary pancreatitis by altering cleavage of regulatory nick sites by chymotrypsin C (CTRC) resulting in reduced trypsinogen degradation and increased autoactivation. Despite 90% identity with PRSS1 and a strong propensity for autoactivation, mutations in PRSS2 are not found in hereditary pancreatitis suggesting that activation of this isoform is more tightly regulated. Here, we demonstrated that CTRC promoted degradation and thereby markedly suppressed autoactivation of human anionic trypsinogen more effectively than previously observed with cationic trypsinogen. Increased sensitivity of anionic trypsinogen to CTRC-mediated degradation was due to an additional cleavage site at Leu-148 in the autolysis loop and the lack of the conserved Cys-139-Cys-206 disulfide bond. Significant stabilization of anionic trypsinogen against degradation was achieved by simultaneous mutations of CTRC cleavage sites Leu-81 and Leu-148, autolytic cleavage site Arg-122, and restoration of the missing disulfide bridge. This stands in stark contrast to cationic trypsinogen where single mutations of either Leu-81 or Arg-122 resulted in almost complete resistance to CTRC-mediated degradation. Finally, processing of the trypsinogen activation peptide at Phe-18 by CTRC inhibited autoactivation of anionic trypsinogen, although cationic trypsinogen was strongly stimulated. Taken together, the observations indicate that human anionic trypsinogen is controlled by CTRC in a manner that individual natural mutations are unlikely to increase stability enough to promote intra-pancreatic activation. This unique biochemical property of anionic trypsinogen explains the lack of association of PRSS2 mutations with hereditary pancreatitis.

Keywords: chymotrypsin; chymotrypsin C; pancreas; serine protease; trypsin; trypsinogen.

FIGURE 1.

Regulation of human cationic (A) and anionic (B) trypsinogen isoforms by CTRC. Hereditary pancreatitis-associated PRSS1 mutations that alter activation and/or degradation of cationic trypsinogen are indicated. CTRC cleavage sites are highlighted in red, trypsin cleavage sites in green, the activation peptide in yellow, and the position of the missing disulfide bridge in anionic trypsinogen in blue. See text for further details.

FIGURE 2.

Autoactivation of human cationic (PRSS1, A and B) and anionic trypsinogen (PRSS2, C and D). Trypsinogen (1 μm) was incubated at 37 °C with 10 nm initial trypsin in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, 0–100 mm NaCl, and 0.05% Tween 20 (final concentrations). A and C, at the indicated times aliquots (10 μl) were removed, and trypsin activity was determined and expressed as described under “Experimental Procedures.” B and D, trypsinogen (150 μl) was precipitated with 10% trichloroacetic acid (final concentration) and analyzed by reducing SDS-PAGE and Coomassie Blue staining. Gels shown correspond to activity curves measured in the absence of NaCl for PRSS1 and in 20 mm NaCl for PRSS2. Autolytic bands resulting from cleavage of the Arg-122–Val-123 and Lys-193–Asp-194 peptide bonds are indicated. N-term, N-terminal fragment; C-term, C-terminal fragment. Cleavage sites were determined by N-terminal sequencing after transferring the bands to PVDF membranes. See “Experimental Procedures” for statement on experimental uncertainty and reproducibility.

FIGURE 3.

Effect of CTRC on the autoactivation of human cationic trypsinogen (PRSS1). Wild-type (A), L81A (B), and R122A (C) mutant cationic trypsinogen (1 μm) was incubated at 37 °C with 0–100 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, and 0.05% Tween 20 (final concentrations). At the indicated times aliquots (10 μl) were removed, and trypsin activity was determined and expressed as described under “Experimental Procedures.” Experimental uncertainty and reproducibility are given under “Experimental Procedures.”

FIGURE 4.

Effect of CTRC on the autoactivation of human anionic trypsinogen (PRSS2). Wild-type (A), L81A (B), and R122A (C) mutant anionic trypsinogen (1 μm) was incubated at 37 °C with 0–100 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, 25 mm NaCl, and 0.05% Tween 20 (final concentrations). At the indicated times, aliquots (10 μl) were removed, and trypsin activity was determined and expressed as described under “Experimental Procedures.” Experimental uncertainty and reproducibility are discussed under “Experimental Procedures.”

FIGURE 5.

Cleavage of human anionic trypsinogen by CTRC. Wild-type (A) and L81A mutant (B) anionic trypsinogen (1 μm) was incubated at 37 °C with 10 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 25 mm NaCl, and 20 nm human SPINK1 trypsin inhibitor (final concentrations). Trypsin inhibitor was included to prevent autoactivation during the cleavage reaction. At the indicated times, trypsinogen (150 μl) was precipitated with 10% trichloroacetic acid (final concentration) and analyzed by reducing SDS-PAGE and Coomassie Blue staining. The two bands generated by cleavage of the Leu-148–Ser-149 peptide bond are indicated. Note that the N-terminal upper band slightly shifts over time as the activation peptide is processed at Phe-18 by CTRC. See text for details. See “Experimental Procedures” for statement on experimental uncertainty and reproducibility. C, model of human anionic trypsinogen with regulatory cleavage sites indicated in red. The position of the missing disulfide replaced by Ser-139 and Ser-206 is highlighted in orange. Bovine cationic trypsinogen (Protein Data Bank file 1TGN (34)) was used as template to generate a model for anionic trypsinogen using the SWISS-MODEL structure homology-modeling server (35). The image was rendered by PyMOL 3.1.

FIGURE 6.

Effect of mutation L148A on the autoactivation of human anionic trypsinogen in the presence of CTRC. Mutant L148A (A), double mutant L81A,L148A (B), and triple mutant L81A,R122A,L148A (C) anionic trypsinogen (1 μm) were incubated at 37 °C with 0–100 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, 25 mm NaCl, and 0.05% Tween 20 (final concentrations). At the indicated times, aliquots (10 μl) were removed, and trypsin activity was determined and expressed as described under “Experimental Procedures.” Experimental uncertainty and reproducibility are given under “Experimental Procedures.”

FIGURE 7.

Effect of restoration of the Cys-139–Cys-206 disulfide bond on the autoactivation of human anionic trypsinogen in the presence of CTRC. Double mutant S139C,S206C (A) and quintuple mutant L81A,R122A, S139C,L148A,S206C (B) anionic trypsinogen (1 μm) were incubated at 37 °C with 0–100 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, 25 mm NaCl, and 0.05% Tween 20 (final concentrations). At the indicated times aliquots (10 μl) were removed, and trypsin activity was determined and expressed as described under “Experimental Procedures.” C, cleavage of the quintuple anionic trypsinogen mutant L81A,R122A,S139C,L148A,S206C by CTRC. Trypsinogen (1 μm) was incubated at 37 °C with 10 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 25 mm NaCl, and 20 nm human SPINK1 trypsin inhibitor (final concentrations). At the indicated times, trypsinogen (150 μl) was precipitated with 10% trichloroacetic acid (final concentration) and analyzed by reducing SDS-PAGE and Coomassie Blue staining. See “Experimental Procedures” for statement on experimental uncertainty and reproducibility.

FIGURE 8.

Cleavage of the trypsinogen activation peptide by CTRC and its effect on autoactivation of human cationic (PRSS1) and anionic (PRSS2) trypsinogen. A, wild-type human cationic and anionic trypsinogen (1 μm) were incubated at 37 °C with 25 nm CTRC in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, and 20 nm human SPINK1 trypsin inhibitor (final concentrations). To remain consistent with the autoactivation experiments, reactions with anionic trypsinogen also contained 25 mm NaCl. At the indicated times trypsinogen (150 μl) was precipitated with 10% trichloroacetic acid (final concentration) and analyzed by SDS-PAGE under non-reducing conditions followed by Coomassie Blue staining. APF-del indicates the band cleaved at Phe-18 by CTRC. The graph shows densitometric quantitation of the trypsinogen band from three experiments (mean ± S.E.). The calculated rates of cleavage were 30.9 nm/min for PRSS1 and 152.8 nm/min for PRSS2. B, wild-type and N-terminally truncated (APF-del) mutant trypsinogen (1 μm) were incubated at 37 °C in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl₂, and 25 mm NaCl (PRSS2) or no salt added (PRSS1). At the indicated times aliquots (10 μl) were removed, and trypsin activity was determined and expressed as described under “Experimental Procedures.” Experimental uncertainty and reproducibility are discussed under “Experimental Procedures.”