Chymotrypsin C (caldecrin) stimulates autoactivation of human cationic trypsinogen

Abstract

Trypsin-mediated trypsinogen activation (autoactivation) facilitates digestive zymogen activation in the duodenum but may precipitate pancreatitis if it occurs prematurely in the pancreas. Autoactivation of human cationic trypsinogen is inhibited by a repulsive electrostatic interaction between the unique Asp218 on the surface of cationic trypsin and the conserved tetra-aspartate (Asp19-22) motif in the trypsinogen activation peptide (Nemoda, Z., and Sahin-Tóth, M. (2005) J. Biol. Chem. 280, 29645-29652). Here we describe that this interaction is regulated by chymotrypsin C (caldecrin), which can specifically cleave the Phe18-Asp19 peptide bond in the trypsinogen activation peptide and remove the N-terminal tripeptide. In contrast, chymotrypsin B, elastase 2A, or elastase 3A (proteinase E) are ineffective. Autoactivation of N-terminally truncated cationic trypsinogen is stimulated approximately 3-fold, and this effect is dependent on the presence of Asp218. Because chymotrypsinogen C is activated by trypsin, and chymotrypsin C stimulates trypsinogen activation, these reactions establish a positive feedback mechanism in the digestive enzyme cascade of humans. Furthermore, inappropriate activation of chymotrypsinogen C in the pancreas may contribute to the development of pancreatitis. Consistent with this notion, the pancreatitis-associated mutation A16V in cationic trypsinogen increases the rate of chymotrypsin C-mediated processing of the activation peptide 4-fold and causes accelerated trypsinogen activation in vitro.

Figure 1

Autoactivation of native cationic (PRSS1) and anionic (PRSS2) trypsinogens from human pancreatic juice. Trypsin-mediated trypsinogen activation was measured at 37 °C, using 2 μM trypsinogen and 10 nM trypsin initial concentrations. The buffers used were Na-acetate, (pH 4.0 and 5.0); Na-MES (pH 6.0); Na-HEPES (pH 7.0) and Tris-HCl (pH 8.0). Solid symbols represent native trypsinogens purified from pancreatic juice samples PS7 (diamonds), PS13 (squares) and PS19 (circles for PRSS1 and triangles for PRSS2). For comparison, recombinant trypsinogens expressed in E. coli Rosetta(DE3) are also shown (open circles for PRSS1 and open triangles for PRSS2). A. Time courses of PRSS1 autoactivation measured in 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl₂ and 2 mg/mL bovine serum albumin. Trypsin activity was expressed as percent of potential maximal activity, which was determined by activation with human enteropeptidase. B. pH dependence of PRSS1 autoactivation. Initial rates were calculated from time-courses of autoactivation using progress curve analysis, as described in [15]. C. Time-courses of autoactivation of PRSS2 measured in 0.1 M Tris-HCl (pH 8.0) and 10 mM CaCl₂. D. pH dependence of autoactivation of PRSS2.

Figure 2

N-terminal heterogeneity of trypsinogens purified from pancreatic juice PS19. Approximately 5 μg native (N) or recombinant (R) trypsinogens were heat-denatured at 95 °C for 5 min in Laemmli sample buffer either in the presence or absence of 100 mM dithiotreithol (DTT) and electrophoresed on 13 % SDS-polyacrylamide mini-gels. Gels were stained with Brilliant Blue R. Native trypsinogens exhibiting double bands were transferred to PVDF membrane and subjected to N-terminal sequencing. In addition to the native, intact sequence, a new truncated form was detected, with the N-terminal Ala¹⁶-Pro¹⁷-Phe¹⁸ tripeptide deleted.

Figure 3

MonoQ anion-exchange chromatography of pancreatic juice samples PS19, PS7 and PS13. After injection of the 4 mL sample volume, the column was developed with a linear gradient of 0–0.5 M NaCl in 20 mM Tris-HCl (pH 8.0) at a flow-rate of 1 mL/min. The elution profile of proteins was followed by UV absorbance at 280 nm, indicated by the black dashed line. Chymotrypsin activity was determined using the synthetic chromogenic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide before (solid orange line) and after (solid red line) activation with trypsin, as described in Experimental Procedures. The three peaks with chymotrypsin-like activity were designated I, II and III. There was no measurable chymotrypsin activity in the PS7 and PS13 fractions before activation with trypsin. Trypsin activity was assayed by N-CBZ-Gly-Pro-Arg-p-nitroanilide hydrolysis after activation by enteropeptidase (solid blue line). There was no detectable trypsin activity before enteropeptidase-activation. The enzyme activities are indicated as absorbance change at 405 nm in 1 min (mOD/min).

Figure 4

Effect of native and recombinant pancreatic proteases with chymotrypsin or elastase activity on the autoactivation of cationic trypsinogen. Ecotin affinity chromatography was carried out to purify the protease zymogens from the three MonoQ-peaks with chymotrypsin activity (see Fig 3). Peak I was from the flow-through fractions 2–6, peak II contained fractions 13–16, and peak III was pooled from fractions 18–20. In the experiments presented here zymogens purified from PS13 were used. Identical results were obtained with zymogens purified from juice sample PS7 (not shown). Recombinant cationic trypsinogen, chymotrypsinogen C, pro-elastase 2A and pro-elastase 3A were expressed in E. coli Rosetta(DE3) and purified with ecotin-affinity chromatography. A. SDS-PAGE analysis of the ecotin-affinity purified peaks I, II and III from pancreatic juice. Results of N-terminal sequencing are indicated. The N-terminal sequences correspond to chymotrypsinogen C (upper band), pro-elastase 3A (middle band), and chymotrypsinogen B (lower band). The Cys residues in the sequences were inferred from their expected positions. B. Trypsin-mediated trypsinogen activation was carried out as described in Figure 1A in the absence or presence of approximately 20 nM purified peak-fractions (final concentrations). Protein concentrations were estimated based on the UV absorbance at 280 nm, using the theoretical extinction coefficient of the predominant zymogen, as described in Experimental Procedures. Because activation reactions contained 10 nM initial trypsin concentration, peak fractions were added directly as zymogens. Identical results were obtained when fractions were first pre-activated with 10 nM trypsin (not shown). The rates of autoactivation calculated from progress curve analysis were as follows. Control (open circles), 1.7 nM/min; peak I (solid diamonds), 2 nM/min; peak II (solid triangles), 1.7 nM/min; peak III (inverted solid triangles), 3.4 nM/min. C. The effect of purified recombinant pancreatic proteases on autoactivation of cationic trypsinogen. Initial concentrations of reactants were 2 μM cationic trypsinogen, 10 nM cationic trypsin and 40 nM chymotrypsin C (CTRC), elastase 3A (ELA3A) or elastase 2A (ELA2A), added as purified zymogens. Autoactivation was measured as described in Fig 1A; the calculated initial rates were the following. Control (open circles), 1.4 nM/min; CTRC (solid circles), 4.5 nM/min; ELA2A (solid triangles), 1.5 nM/min; ELA3A (solid inverted triangles), 1.4 nM/min.

Figure 5

N-terminal processing of cationic trypsinogen. Proteolytic removal of the N-terminal tripeptide from cationic trypsinogen preparations (2 μM final concentration) was measured in the presence of 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl₂, 1 mM benzamidine and the indicated active proteases. Benzamidine was included in the reaction to prevent autoactivation of trypsinogens. Non-reducing SDS-PAGE was used to separate the two trypsinogen forms on 13% polyacrylamide gels; which adequately resolved the intact trypsinogen, the N-terminally processed trypsinogen and the trypsin bands, as indicated. A. N-terminal processing by chymotrypsin C from peak III. Trypsinogens purified from pancreatic juice (native) or expressed recombinantly in human embryonic kidney 293T cells (HEK) or in E. coli LG-3 (LG) were incubated with chymotrypsin C from peak III at ~40 nM final concentration. B. Lack of N-terminal processing of cationic trypsinogen (from E. coli LG-3) by chymotrypsin B from peak II (40 nM final concentration). C. N-terminal processing of cationic trypsinogen by recombinant pancreatic proteases. Recombinant trypsinogen expressed in E. coli LG-3 (LG) was used at 2 μM concentration. CTRC, chymotrypsin C (30 nM concentration); ELA3A, elastase 3A (100 nM concentration); ELA2A, elastase 2A (100 nM concentration). See Experimental Procedures for details. Chymotrypsinogen B and C from peaks II and III, respectively, were purified from juice sample PS13 for the experiments presented here. Identical results were obtained when zymogens purified from juice sample PS7 were used (not shown).

Figure 6

Effect of Asp²¹⁸ on the chymotrypsin C-mediated stimulation of trypsinogen autoactivation. Trypsin-mediated trypsinogen activation was carried out as described in Fig 1A in the absence (open symbols) or presence (solid symbols) of recombinant chymotrypsin C (+ CTRC) at 40 nM concentration. Recombinant trypsinogens were expressed in E. coli Rosetta(DE3). These preparations exhibit enhanced rates of N-terminal processing by chymotrypsin C, which ensured complete processing even for the rapidly autoactivating D218Y mutant. A. Recombinant wild-type cationic trypsinogen. Autoactivation rates were 1.6 nM/min (control) and 4.3 nM/min (+ CTRC). B. D218Y cationic trypsinogen mutant. Autoactivation rates were 17.4 nM/min (control) and 18.1 nM/min (+ CTRC). Although data are not shown, cationic trypsinogen mutant D218S was also tested and no stimulation of autoactivation by chymotrypsin C was observed. Autoactivation rates of the D218S mutant were 12.1 nM/min and 13.3 nM/min, in the absence and presence of CTRC, respectively. C. Recombinant wild-type anionic trypsinogen. Autoactivation rates were 4.2 nM/min (control) and 3.6 nM/min (+ CTRC). D. Y218D anionic trypsinogen mutant. Autoactivation rates were 0.9 nM/min (control) and 2.3 nM/min (+ CTRC).

Figure 7

Effect of the pancreatitis-associated A16V mutation on the N-terminal processing of cationic trypsinogen by chymotrypsin C. Recombinant wild-type (open symbols) and A16V mutant (solid symbols) cationic trypsinogen (2 μM) was incubated with 30 nM recombinant chymotrypsin C (final concentrations) and at the indicated time points samples were precipitated with trichloroacetic acid and analyzed by SDS-PAGE under non-reducing conditions. The graph illustrates the densitometric analysis of the gels shown. Experimental details are given in the legend to Fig 5. Recombinant trypsinogen preparations from two independent sources were tested; from E. coli LG-3 strain (circles) and from human embryonic kidney (HEK) 293T cells (squares).