Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis

Abstract

Mutations in the PRSS1 gene encoding human cationic trypsinogen are associated with hereditary and sporadic chronic pancreatitis. High-penetrance PRSS1 mutations found in hereditary pancreatitis alter activation and/or degradation of cationic trypsinogen, thereby promoting intrapancreatic trypsinogen activation. In contrast, a number of rare PRSS1 variants identified in subjects with sporadic chronic pancreatitis cause misfolding and endoplasmic reticulum (ER) stress. Mutation p.L104P is unique among natural PRSS1 variants, since it affects the substrate binding site of trypsin. The aim of the present study was to establish the clinical significance of variant p.L104P through functional analysis. We found that p.L104P trypsin exhibited decreased activity on peptide and protein substrates; however, autoactivation was slightly accelerated. Remarkably, binding of the physiological trypsin inhibitor serine protease inhibitor Kazal type 1 (SPINK1) was decreased by 70-fold. In the presence of the trypsinogen-degrading enzyme chymotrypsin C, mutant p.L104P autoactivated to higher trypsin levels than wild-type trypsinogen. This apparent resistance to degradation was due to slower cleavage at Arg(122) rather than Leu(81) Finally, secretion of mutant p.L104P from transfected cells was markedly reduced due to intracellular retention and aggregation with concomitant elevation of ER stress markers. We conclude that PRSS1 variant p.L104P exhibits a variety of phenotypic changes that can increase risk for chronic pancreatitis. Mutation-induced misfolding and associated ER stress are the dominant effects that support a direct pathogenic role in chronic pancreatitis.

Keywords: autoactivation; chronic pancreatitis; endoplasmic reticulum stress; hereditary pancreatitis; intracellular aggregation; misfolding; trypsinogen activation.

Fig. 1.

Role of Leu¹⁰⁴ in the S2 subsite of the trypsin substrate binding site. The Michaelis complex between a catalytically inactive bovine trypsin mutant and the Pittsburgh variant of α₁-antitrypsin is shown (Protein Data Bank file 1OPH). The side chains contributing to the S2 subsite of trypsin are shown in red, and the P1 Arg and P2 Pro residues of the inhibitor are indicated in green. See text for details. The broken line indicates an H bond between the main chain atoms of positions 104 and 100 that stabilizes the 97–107 loop. The image was rendered by UCFS Chimera 1.10.2 (www.cgl.ucsf.edu/chimera).

Fig. 2.

Effect of mutation p.L104P on the activation of pancreatic proenzymes by trypsin. A and B: wild-type and mutant human cationic trypsin (25 nM) were incubated with 2 μM human chymotrypsinogen B1 (CTRB1) or chymotrypsinogen C (CTRC) in 0.1 M Tris·HCl (pH 8.0), 1 mM CaCl₂, and 0.05% Tween 20 (final concentrations) at 37°C. At the indicated times, 2-μl aliquots were removed, and chymotrypsin activity was measured using 150 μM Suc-Ala-Ala-Pro-Phe-p-nitroanilide substrate. C: wild-type and mutant human cationic trypsin (25 nM) were incubated with 2 μM human procarboxypeptidase A1 (proCPA1) in 0.1 M Tris·HCl (pH 8.0) and 1 mM CaCl₂ (final concentrations) at 37°C. At the indicated times, 100-μl aliquots were precipitated with 10% trichloroacetic acid, electrophoresed on 15% SDS-PAGE gels, and stained with Coomassie blue. CPA1, activated carboxypeptidase A1. Representative graphs and gel picture from two or three experiments are shown.

Fig. 3.

Effect of mutation p.L104P on the digestion of bovine β-casein with trypsin. Wild-type and mutant human cationic trypsin (2 nM) were incubated with β-casein (0.2 mg/ml) in 0.1 M Tris·HCl (pH 8.0) and 1 mM CaCl₂ (final concentrations) at 37°C. At the indicated times, 100-μl aliquots were precipitated with 10% trichloroacetic acid, electrophoresed on 15% SDS-PAGE gels, and stained with Coomassie blue. A representative gel from two experiments is shown.

Fig. 4.

Effect of mutation p.L104P on autoactivation of trypsinogen. Wild-type and mutant human cationic trypsinogen (2 μM) were incubated with 10 nM initial trypsin in 0.1 M Tris·HCl (pH 8.0), 1 mM CaCl₂, and 0.05% Tween 20 (final concentrations) at 37°C. A: at the indicated times 2-μl aliquots were removed, and trypsin activity was measured with 150 μM N-CBZ-Gly-Pro-Arg-p-nitroanilide substrate as described in materials and methods. Trypsin activity was converted to active trypsin concentration using the maximal plateau activity as the 2 μM reference value. B: autoactivation was also followed by SDS-PAGE. At the indicated times, 100-μl aliquots were precipitated with 10% trichloroacetic acid, electrophoresed on 15% SDS-PAGE gels, and stained with Coomassie blue. *Two chains of trypsin cleaved at the Arg¹²² autolytic site. Representative graph and gel from three experiments are shown.

Fig. 5.

Effect of mutation p.L104P on autoactivation of trypsinogen in the presence of chymotrypsin C (CTRC). Wild-type (A) and mutant (B) human cationic trypsinogen (1 μM) were incubated in the absence or presence of the indicated concentrations of human CTRC and 10 nM cationic trypsin in 0.1 M Tris·HCl (pH 8.0), 1 mM CaCl₂, and 0.05% Tween 20 (final concentrations) at 37°C. At the indicated times, 2-μl aliquots were removed, and trypsin activity was measured as described in materials and methods. Trypsin activity was expressed as percentage of the maximal activity measured in the absence of CTRC. C: autoactivation in the presence of 10 nM CTRC was also followed by SDS-PAGE. At the indicated times, 150-μl aliquots were precipitated with 10% trichloroacetic acid, electrophoresed on 15% SDS-PAGE gels, and stained with Coomassie blue. The faint proteolytic fragment observed in the wild-type sample at 15 kDa corresponds to the COOH-terminal chain of trypsin cleaved at the Arg¹²² autolytic site. Representative graph and gel from two experiments are shown.

Fig. 6.

Effect of mutation p.L104P on the cleavage of the Leu⁸¹-Glu⁸² peptide bond in trypsinogen (Tg) by CTRC. Wild-type and mutant human cationic trypsinogen (2 μM) were incubated with 25 nM human CTRC in 0.1 M Tris·HCl (pH 8.0) (final concentrations) at 37°C. A: at the indicated times, 100-μl reactions were precipitated with 10% trichloroacetic acid (final concentration) and analyzed by SDS-PAGE and Coomassie blue staining. B: densitometric evaluation of the intensity of the intact trypsinogen bands. Representative gel from three experiments is shown. Error bars were omitted for clarity; the error was within 10% of the mean.

Fig. 7.

Effect of mutation p.L104P on the binding of serine protease inhibitor Kazal type 1 (SPINK1) to trypsin. Wild-type (50 pM, A) or mutant (500 pM, B) trypsin were incubated for 16 h with the indicated concentrations of SPINK1 inhibitor. Residual trypsin activity was measured, and K_D values were calculated as described in materials and methods. Representative binding experiments are show. K_D values were calculated from three experiments. See text for errors of the mean.

Fig. 8.

Effect of mutation p.L104P on cellular secretion of trypsinogen. HEK 293T cells were transiently transfected with expression plasmids for wild-type and mutant human cationic trypsinogen, and conditioned media were collected after 48 h. Trypsinogen protein levels were determined by Coomassie blue-stained SDS-polyacrylamide gels (A), by Western blots (B), and by measuring trypsin activity in the medium after activation with enteropeptidase (C). Trypsinogen protein levels secreted in the media were calculated from the enzyme activity (mean ± SD, n = 8, ****P < 0.0001). Representative gel and blot from three experiments are shown.

Fig. 9.

Intracellular retention and aggregation of trypsinogen mutant p.L104P. A: lysates (20 μg total protein) of HEK 293T cells expressing wild-type or mutant human cationic trypsinogen were analyzed by Western blotting. B: cell lysates (20 μg) were centrifuged at 50,000 g for 15 min at 4°C. The distribution of trypsinogen between the supernatant and pellet was then analyzed by Western blotting. Representative blots from three experiments are shown.

Fig. 10.

Endoplasmic reticulum stress markers in HEK 293T cells expressing wild-type or p.L104P mutant trypsinogen. A: levels of immunoglobulin-binding protein (BiP) mRNA were measured using quantitative reverse transcription-PCR. B and C: splicing of X-box-binding protein 1 (XBP1) mRNA was followed by reverse transcription-PCR using primers that amplify both the spliced and unspliced species. Levels of spliced mRNA (XBP1s) were determined by densitometric analysis. Mean values ± SD (n = 5, each performed in duplicate) are indicated. Significance was tested with 1-way analysis of variance (P < 0.0001) followed by Tukey-Kramer post hoc analysis (***P < 0.001). Representative agarose gel from five experiments is shown.