Biochemical analyses of cystatin-C dimers and cathepsin-B reveals a trypsin-driven feedback mechanism in acute pancreatitis

Abstract

Acute pancreatitis (AP) is characterised by self-digestion of the pancreas by its own proteases. This pathophysiological initiating event in AP occurs inside pancreatic acinar cells where intrapancreatic trypsinogen becomes prematurely activated by cathepsin B (CTSB), and induces the digestive protease cascade, while cathepsin L (CTSL) degrades trypsin and trypsinogen and therefore prevents the development of AP. These proteases are located in the secretory compartment of acinar cells together with cystatin C (CST3), an endogenous inhibitor of CTSB and CTSL. The results are based on detailed biochemical analysis, site-directed mutagenesis and molecular dynamics simulations in combination with an experimental disease model of AP using CST3 deficient mice. This identifies that CST3 is a critical regulator of CTSB and CTSL activity during AP. CST3 deficient mice show a higher intracellular CTSB activity resulting in elevated trypsinogen activation accompanied by an increased disease severity. This reveals that CST3 can be cleaved by trypsin disabling the inhibition of CTSB, but not of CTSL. Furthermore, dimerised CST3 enhances the CTSB activity by binding to an allosteric pocket specific to the CTSB structure. CST3 shifts from an inhibitor to an activator of CTSB and therefore fuels the intrapancreatic protease cascade during the onset of AP.

Fig. 1. Cystatin C expression in the secretory compartment of pancreatic acinar cells.

A Representative immunofluorescent labelling of cystatin C (red) and pancreatic α-amylase (green) in sections of mouse pancreatic tissue, the experiment was repeated at least three times independently with a similar result (the scale bar represents 20 µm and 10 µm). B Western blot analysis of subcellular fractions (ZG zymogen granules, Lys lysosomal fraction, and Cyt cytosolic fraction) from tissue lysates of mouse pancreas, taken from untreated control mice and animals 1 h after induction of pancreatitis by caerulein, the experiment was repeated four times independently with a similar result. Enzyme activity measurements of CTSB (p = 0.0073) (C), CTSL (D), chymotrypsin (p < 0.0001) (E) and trypsin (p < 0.0001) (F) in mouse subcellular fractions (n = 4). G Immunofluorescent labelling of cystatin C (red) and pancreatic α-amylase (green) in sections of human pancreatic tissue the experiment was repeated three times independently with a similar result (the scale bar represents 50 µm and 10 µm). H Western blot analysis of five different human pancreatic fluid samples. C–F show four independent experiments; significance was calculated by two-tailed Student t-test for independent samples. Results are shown as mean ± SD. Significance levels of p < 0.05 are marked by an asterisk. Source data are provided as Source Data file.

Fig. 2. Protease activation in cystatin C deficient mice.

A Western blot analysis of cystatin C in the pancreas of wild-type (WT) and Cst3−/− mice, GAPDH reference as loading control. B Trypsin and chymotrypsin activities were measured after enterokinase activation as well as amylase content in the pancreas of WT and Cst3−/− mice (n = 4 biological replicates). C Immunofluorescent labelling of pancreatic α-amylase (green) and CTSB (violet) in pancreas sections of wild type and Cst3−/− mice (scale bar represents 20 µm). D Protease activities were measured using fluorochrome substrates for CTSB (n = 8 biological replicates, without CCK, p = 0.0011, +CCK p = 0.0285), CTSL (n = 8 biological replicates, +CCK p = 0.0114), trypsin (n = 8 biological replicates, +CCK p = 0.0145), and chymotrypsin (n = 7 biological replicates, +CCK p = 0.0071) in homogenates of isolated acini from wild-type and Cst3−/− mice ± stimulation for 30 min with 0.001 mM CCK. E Protease activities of cathepsin B (n = 5 biological replicates, 20 min p = 0.0034, 40 min p = 0.0399), trypsin (n = 5 biological replicates, 20 min p = 0.0057, 40 min p = 0.0092) and propidium iodide (n = 10 biological replicates) uptake were measured in freshly prepared living acinar cells of wild-type and Cst3−/− mice. F Activities of cathepsin B (1 h ZG, p = 0.0275) and cathepsin L (0 h ZG, p = 0.032, 0 h Lys p = 0.003, 1 h ZG p < 0.001) were measured by fluorochrome substrates in subcellular fractions (ZG zymogen granules, Lys lysosomal fraction, and Cyt cytosolic fraction) of wild-type and Cst3−/− untreated control mice and 1 h after induction of pancreatitis (n = 5 biological replicates). G Activity measurements of trypsin (1 h ZG, one-tailed students t-test p = 0.0492) and chymotrypsin (1 h ZG, p = 0.0336) in the same subcellular fractions (n = 5 biological replicates). Data represent five or more independent experiments; significance was calculated by two-tailed Student t-test for independent samples unless otherwise mentioned. Results are shown as mean ± SD. Significance levels of p < 0.05 are marked by an asterisk. Source data are provided as  Source Data file.

Fig. 3. Regulation of protease activity by cystatin C during pancreatitis.

Measurement of CTSB activity at different pH conditions (pH 3–9) in zymogen granule fractions of wild-type mice (pH 7, p = 0.0169) (A) and Cst3−/− mice (B) before and after induction of pancreatitis (n = 4 biological replicates). Measurement of CTSL activity under different pH conditions in ZG-fraction of wild-type mice (C) and Cst3−/− mice (D) before and after induction of pancreatitis (n = 4 biological replicates). E Coomassie staining illustrates the time-dependent cleavage of recombinant cystatin C by trypsin over a time period of 60 min, the experiment was repeated three times independently with a similar result. F Western blot analysis of the time-dependent cleavage of cystatin C by trypsin, the experiment was repeated three times independently with the similar result. G Measurement of CTSB activity at pH 5.5 in the presence of cystatin C, trypsin and preincubated cystatin C with trypsin, (n = 6 biological replicates, CTSB vs. CTSB + CST3 p = 0.0025, CTSB vs. CTSB  + Try/CST3 p < 0.0001, CTSB + Try + Try/CST3 p < 0.0001) (H) measurement of CTSL activity at pH 4.0 in the presence of cystatin C, trypsin and preincubated cystatin C with trypsin (n = 4 biological replicates, CTSL vs. CTSL + CST3 p = 0.0225, CTSL vs. CTSL + Try/CST3 p = 0.0282). Data represent four independent experiments; significance was calculated by two-tailed Student t-test for independent samples or one-way Anova followed Dunn–Šidák correction for multiple testing. Results are shown as mean ± SD. Significance levels of p < 0.05 are marked by an asterisk. Source data are provided as Source Data file.

Fig. 4. Trypsin-mediated cleavage of cystatin C and its oligomerization state.

A Amino acid sequence of murine cystatin C expressed in E.coli SHuffle T7 Express with highlighted His-tag for purification (green) and potential trypsin-cleavage sites (yellow). B AlphaFold2 structure of murine cystatin C (AlphaFoldDB: AF-P21460 [https://www.uniprot.org/uniprotkb/P21460/entry]) with potential trypsin cleavage sites. C SDS-PAGE analysis of cystatin C wild-type and mutants where several possible cleavage sites were substituted with alanine residues. Shown are the purified proteins not treated with trypsin (−) and treated with trypsin (+), the experiment was repeated three times independently with a similar result. D AlphaFold2 structure of murine cystatin C dimer with coloured cystatin C chains in orange and blue and annotated potential cleavage sites. E Chromatogram of the separation of cystatin C monomer and dimer fractions by size exclusion chromatography. Activity tests of CTSB at pH 5.5 (con vs. monomer p < 0.0001, con vs. monomer + try p < 0.0001, con vs. dimer p < 0.0001, con vs dimer + try p < 0.0001, monomer vs. monomer + try p = 0.0173, dimer vs. dimer + try p = 0.0003, n = 3 biological replicates) (F) and CTSL at pH 4.0 (con vs monomer p < 0.0001, con vs monomer + try p < 0.0001, con vs. dimer p < 0.0001, con vs. dimer + try p < 0.0001, n = 3 biological replicates) (G) with cystatin C monomer and dimer and trypsin-mediated cleaved cystatin C monomer and dimer. Comparison of pH-related dimerisation of full-length cystatin C (H) and trypsin-cleaved cystatin C (I). Shown are the dimer/monomer ratios obtained in sodium acetate buffer pH 4.0–5.5 (NaOAc, dark blue and red, respectively) and the dimer/monomer ratios obtained in sodium phosphate buffer pH 5.5–8.0 (NaPi, light blue and orange, respectively). Data represent three independent experiments; significance was calculated by one-way Anova followed Dunn–Šidák correction for multiple testing. Results are shown as mean ± SD. Significance levels of p < 0.05 are marked by an asterisk. Source data are provided as Source Data file.

Fig. 5. Autoinhibition of isolated CTSB and inhibition complexes of CTSB and CTSL with mCST3 as obtained through molecular dynamic simulations.

A Cartoon representation of murine cathepsin B structure (red) with the occluding loop (yellow) in a closed position. Active-site residues (C108, H278, N298) are shown as spheres and coloured by element (C: grey; N: blue; O: red; S: yellow; H: white). B Representative state of CTSB with an open conformation of the occluding loop. C (left) Centre-of-mass distance between the active site and occluding loop atoms across all structures generated during two independent TIGER2h_PE simulations of isolated CTSB. (right) Histogram of distance data with the selected threshold for open states (red). Dashed lines denote the integral and allow access to the open/closed fractions at different distance thresholds. D Inhibition complex of CTSB (red) with monomeric cystatin C (orange) as obtained through TIGER2h_PE simulations. Residues K24 and R28, which are cleaved off by trypsin, are highlighted. E Inhibition complex of cathepsin L with cystatin C as obtained through TIGER2h_PE simulations. F Cartoon representation of the cystatin C dimer structure (orange and yellow) illustrating the swap of the helical domain. The primary binding loop is now extended to connect the two monomers, therefore inhibition of CTSB is no longer possible. Source data are provided as Source Data file.

Fig. 6. Allosteric modulation of CTSB through dCST3 binding into the discovered pocket.

A Snapshot of major structural clusters as obtained by TIGER2h_PE simulations of cathepsin B with different models of the trypsin-processed cystatin C. Binding to an allosteric pocket on CTSB’s surface results in a pronounced opening of the occluding loop. B Average centre-of-mass distance (±SD) between the active site and occluding loop atoms for major cluster states generated throughout different TIGER2h_PE simulations. Binding configurations into the allosteric site (four simulations) show significant differences to isolated CST3 (two simulations). Significance was calculated by one-way Anova followed by Dunn–Šidák correction for multiple testing (p < 0.0001 for all binding configurations into the allosteric site (sim5 n = 5935, sim6 n = 8563, sim7 n = 980, sim8 n = 7026) against isolated CST3 (sim1 n = 7378, sim2 n = 5516)). C Resulting fraction of open states using a distance threshold of 16.5 Å. Allosteric modulation by CST3 results in a 3.15-fold increase in active states. D Allosteric sites predicted by AlloSitePro (green, magenta) in superposition with the binding site predicted by TIGER2h_PE simulations (cyan) and intersection surface area (purple). Mutation target selected based on simulation data are shown as ball-and-stick. E Activity tests with CTSB wild-type and mutated allosteric site (S169A, Y215F, E221V, D222P, H224L, D333P) under the influence of cystatin C monomer- and dimer-fractions. The wild-type shows a three-fold increase in activity when measured in complex with dimeric cystatin C, similar to the increase in open states. This activating effect is diminished with the allosteric site mutated (WT CTSB vs WT CTSB + CST3 monomer p = 0.0048, mut. CTSB vs mut. CTSB + CST3 monomer p = 0.0009, WT CTSB vs WT CTSB + CST3 dimer p < 0.0001, mut. CTSB vs WT CTSB + CST3 dimer p < 0.0001, WT CTSB + CST3 dimer vs mut. CTSB + CST3 dimer p < 0.0001). F Schematic illustration summarises the effect of CST3 processing for the balance of CTSB and CTSL activity and intracellular trypsinogen activation. Figure 6E shows three independent experiments; significance was calculated by one-way Anova followed by Dunn–Šidák correction for multiple testing. Results are shown as mean ± SD. Significance levels of p < 0.05 are marked by an asterisk. Source data are provided as Source Data file.

Fig. 7. The disease severity of AP is increased in Cst3−/− mice.

A H&E histology of pancreatic tissue illustrates the local damage in wild-type and Cst3−/− mice (scale bar represents 50 µm), histology score including oedema, necrosis and leucocyte infiltrating summarises the histological evaluation (0 h WT n = 6, Cst3−/− n = 7, 1 h WT n = 6, Cst3−/− n = 6, 8 h WT n = 7, Cst3−/− n = 7, 24 h WT n = 6, Cst3−/− n = 6) (24 h WT vs Cst3−/− p = 0.0002). B Measurement of amylase activity in serum of mice (0 h WT n = 8, Cst3−/− n = 8, 1 h WT n = 7, Cst3−/− n = 7, 8 h WT n = 10, Cst3−/− n = 10, 24 h WT n = 7, Cst3−/− n = 7) (1 h WT vs. Cst3−/− p = 0.0005, 24 h WT vs. Cst3−/− p = 0.0099). C, D Measurement of trypsin (1 h WT vs Cst3−/− p = 0.0014, 8 h WT vs Cst3−/− p = 0.007, 24 h WT vs. Cst3−/− p = 0.0193) and chymotrypsin (1 h WT vs Cst3−/− p = 0.0307, 8 h WT vs. Cst3−/− p < 0.0001) activity in pancreas tissue homogenate normalised to protein content (n = 5). E Quantification of apoptotic nuclei was performed from paraffin slides by counting TUNEL-positive nuclei in pancreatic tissue (0 h WT n = 7, Cst3−/− n = 7, 8 h WT n = 7, Cst3−/− n = 7, 24 h WT n = 6, Cst3−/− n = 6) (8 h WT vs. Cst3−/− p = 0.0206, 24 h WT vs. Cst3−/− p = 0.0007). F Serum cytokines were measured 8 h after induction of pancreatitis by fluorescent beads. Dot blots show the mean fluorescent units (MFI) of the cytokines IL-1β (p = 0.0108), IL-6 (p = 0.0433), TNFα and IL-10 (p = 0.0087) (n = 9). G, H Quantification of CD68+ macrophages was performed from cryo-embedded pancreatic tissue using immunofluorescent labelling (red, scale bar represents 50 µm) and illustrates the local immune response (0 h WT n = 3, Cst3−/− n = 3, 8 h WT n = 7, Cst3−/− n = 6, 24 h WT n = 6, Cst3−/− n = 7) (8 h WT vs. Cst3−/− p = 0.0017, 24 h WT vs. Cst3−/− p = 0.0408). I MPO activity in lung tissue homogenate (0 h WT n = 6, Cst3−/− n = 5, 1 h WT n = 6, Cst3−/− n = 6, 8 h WT n = 11, Cst3−/− n = 11, 24 h WT n = 6, Cst3−/− n = 6) (1 h WT vs Cst3−/− p = 0.0088) and (J) H&E staining illustrate increased lung inflammation in Cst3−/− mice (scale bar represents 50 µm an 1000 µm). A–F all data points represent biological replicates. Data represent five or more independent experiments; significance was calculated by two-tailed Student t-test for independent samples. Results are shown as mean ± SD. Significance levels of p < 0.05 are marked by an asterisk. Source data are provided as Source Data file.