Cathepsin B cleavage of the trypsinogen activation peptide
BMC Gastroenterology volume 2, Article number: 16 (2002)
Cathepsin B is thought to play a central role in intrapancreatic trypsinogen activation and the onset of pancreatitis. A recent investigation of the cathepsin B mediated activability of wildtype trypsinogen and their mutations N29I, N29T and R122H, which are associated to hereditary pancreatitis, revealed no differences. This action seems to be restricted to the K23-I24 peptide bond, which is the trypsinogen activation bond. Here we investigated the influence of the mutations D22G and K23R of the trypsinogen activation peptide on the cleavability by cathepsin B.
To investigate the functional impact of the TAP mutations on cathepsin B mediated cleavage of the trypsinogen activating K23-I24 bond, the corresponding peptides pWT, APFDDDDKIVGG; pD22G, APFDDDGKIVGG; and pK23R, APFDDDDRIVGG were digested with cathepsin B for 30 min at pH 3.8 and 5.0, and the fragments were analysed by high-performance liquid chromatography.
Without cathepsin B, less than 1 % of the peptides were hydrolysed. After a 30-minute digestion with cathepsin B at pH 5, 96% of pWT, 48% of pK23R, but only 2.4% of pD22G were hydrolysed. At pH 3.8, the cathepsin B cleavage of pWT and pK23R was less than at pH 5, whereas the cleavage of pD22G was completely inhibited
Cathepsin B mediated trypsinogen activation seems not to be a crucial pathogenic step in hereditary pancreatitis patients with the trypsinogen mutations D22G and K23R.
In families with hereditary pancreatitis, a cationic trypsinogen mutation is a predominant risk factor for the manifestation of chronic pancreatitis . Almost all disease-associated mutations are located within the active enzyme (http://www.uni-leipzig.de/pancreasmutation); three other gained interest, as they change the amino acid sequence of the trypsinogen activation peptide (TAP), but not the active trypsin molecule. In detail, the A16V mutation alters the cleavage bond A15-A16 of the signal peptide . The D22G variant destroys the highly conserved tetraaspartic group of TAP , whereas K23R directly affects the trypsinogen activation bond K23-I24  (table 1). As reported earlier, D22G and K23R facilitate the hydrolysis of the K23-I24 bond by active trypsin, what could lead to pancreatitis by enhanced trypsinogen autoactivation .
The lysosomal cysteine protease cathepsin B is thought to play a central role in intrapancreatic trypsinogen activation and the onset of pancreatitis. It activates human trypsinogen in vitro  and was found to be redistributed to a zymogen-granule enriched subcellular compartment during the early course of experimental pancreatitis . Studies with cathepsin B deficient mice presented evidence, that the premature and intracellular activation of trypsinogen largely depends on the presence of cathepsin B . A recent study showed that cathepsin B is abundantly present in the secretory compartment of the healthy human pancreas and is secreted together with trypsinogen and active trypsin into the pancreatic juice of patients with chronic pancreatitis . The authors conclude, that trypsinogen and cathepsin B could potentially interact. This interaction may be modified by pancreatitis associated trypsinogen mutations. The investigation of the wild type trypsinogen molecule and three distinct mutants (i.e., N29I, N29T and R122H) found that cathepsin B mediated trypsinogen activation was not influenced by the respective mutants .
The studies by Kukor et al. imply, that the proteolytic cleavage of trypsinogen by cathepsin B is restricted to the K23-I24 trypsinogen activation peptide bond in the TAP . In contrast to the mutations N29I, N29T and R122H, which are located away from this cleavage site, trypsinogen mutations affecting the K23-I24 bond could potentially affect the cathepsin B action on trypsinogen activation. The aim of this study was to evaluate the cathepsin B mediated cleavage of the K23-I24 bond in the presence of the pancreatitis associated TAP mutations D22G and K23R.
Materials and Methods
Unless otherwise indicated, all reagents were obtained from Sigma-Aldrich (Deisenhofen/Germany). Human liver cathepsin B (Sigma-Aldrich, catalog number C8571, enzymatic activity 3,930 units/mg protein) was used in a final concentration of 0.14 ng/μl. The oligopeptides shown in table 1 were commercially synthesized (MWG Biotech, Ebersberg, Germany) and purified by high-performance liquid chromatography (HPLC), and their molecular masses were verified by mass spectrometry. To investigate the functional impact of the TAP mutations on cathepsin B mediated cleavage of the trypsinogen activating K23-I24 bond, the corresponding peptides pWT, pD22G and pK23R were digested with cathepsin B. These dodecapeptides are homologous to the N-terminal part of wild type human cationic trypsinogen and the pancreatitis associated mutations D22G and K23R and include the activation peptide cleavage site (table 1).
pH 3.8 has been previously used in the activation of human trypsinogen . The concentration of cathepsin B in lysosomes is high (about 1 mM)  and pH 5 mimics the approximate intralysosomal pH, the putative cellular compartment of trypsinogen activation in acute pancreatitis [10–14]. Therefore, all experiments have been carried out at both pH 3.8 and 5.0.
A solution of 2 mg dodecapeptide /mL of buffer (40 mM sodium acetate + 40 mM cysteine) with pH 3.8 and pH 5.0, was used . Cathepsin B was added, and the mixture was incubated for 30 min at 37°C. As control, the peptides were incubated without addition of cathepsin B. The samples were then centrifuged using the Microcon centrifugal filter device YM-10 (catalogue no. 42406, Millipore, Bedford, MA) to remove cathepsin B from peptides or hydrolytic fragments. Next, 100 μl of the eluates was then separated by HPLC. After HPLC separation, the undigested peptides were eluted at a retention time of approximately 14 minutes. The N-terminal octapeptide was eluted after 11.6 minutes, and the residual C-terminal tetrapeptide was detected after 8.6 minutes. The method has been described in detail in reference .
Rates of hydrolysis were determined by integration of the respective area under each peak, and the digestion rates were indicated as percentages of initial dodecapeptide concentration.
Results and Discussion
Without cathepsin B, less than 1 % of the peptides was hydrolysed. After a 30-minute digestion with cathepsin B at pH 5, 96% of pWT, 48% of pK23R, but only 2.4% of pD22G were hydrolysed. At pH 3.8, the cathepsin B cleavage of pWT and pK23R was less than at pH 5, whereas the cleavage of pD22G was completely inhibited (figure 1).
In contrast to pancreatitis associated trypsin mutations, pancreatitis associated TAP mutations potentially prevent the hydrolytic activity of cathepsin B. The respective cleavage of the K23-I24 bond was reduced by 50% by the K23R mutation and largely inhibited by the D22G mutation. This indicates a high control of cathepsin B action on trypsinogen activation by the amino acid structure of TAP and highlights the particular functional importance of the D22 residue within the tetraaspartic group D19-D20-D21-D22.
An experimental setup using mutant dodecapeptides but not trypsinogen molecules might be questioned, as the structural organization of the N-terminal part of native trypsinogen is different from that of the synthetic oligopeptide. However, the recombinant expression of human trypsinogen is difficult and has been carried out by only two groups worldwide, with partially conflicting results due to different analytical conditions [8, 15]. Crystallographic studies of the trypsinogen-to-trypsin conversion revealed that TAP can move freely in space and lacks a distinct secondary and tertiary structure . In the past, the activation process of trypsinogen was analysed in numerous studies using oligopeptides  and the method used here has been proven to be highly reproducible .
The investigated TAP mutations have been discovered, as their carriers have chronic pancreatitis. With respect to that clinical picture it is highly unlikely, that a reduced activation of trypsinogen by cathepsin B provokes pancreatitis, but favors the role of trypsinogen autoactivation by active trypsin. Our data support the generally accepted hypothesis, that pancreatitis is caused by increased intrapancreatic trypsin activity. In contrast to this attractive hypothesis of gain-of-function trypsins in hereditary pancreatitis, recent in vitro studies have challenged these assumptions and suggested that a loss of trypsin function could impair the inactivation of other (more pancreatotoxic?) digestive enzymes [18, 19]. These new insights suggest, that structural alterations, which impair the function of trypsin, could eliminate a protective mechanism rather than triggering an aggressive mechanism in initiating pancreatitis. However, our investigations on the cleavability of the trypsinogen activation bond bond by active trypsin  and cathepsin B provide further evidence, that autoactivation rather than cathepsin B mediated trypsinogen activation is the key pathogenic event in the development of hereditary pancreatitis in D22G and K23R-carriers.
In summary, two effects have been shown for the trypsinogen mutants D22G and K23R: (I) The trypsin mediated TAP-cleavage is facilitated. This could overwhelm the pancreatic protease-antiprotease-equilibrium and may lead to pancreatitis . (II) The cathepsin B mediated TAP-cleavage is reduced. The resulting impaired cathepsin B mediated trypsinogen activation seems not be a pancreatitis promoting pathogenic step.
Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Ulrich CD, Martin SP, Gates LK, Amann ST, Toskes PP, Liddle R, McGrath K, Uomo G, Post JC, Ehrlich GD: Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet. 1996, 14: 141-145.
Witt H, Luck W, Becker M: A signal peptide cleavage site mutation in the cationic trypsinogen gene is strongly associated with chronic pancreatitis. Gastroenterology. 1999, 117: 7-10.
Teich N, Ockenga J, Hoffmeister A, Manns M, Mössner J, Keim V: Chronic pancreatitis associated with an activation peptide mutation that facilitates trypsin activation. Gastroenterology. 2000, 119: 461-465.
Ferec C, Raguenes O, Salomon R, Roche C, Bernard JP, Guillot M, Quere I, Faure C, Mercier B, Audrezet MP, Guillausseau PJ, Dupont C, Munnich A, Bignon JD, Le Bodic L: Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis. J Med Genet. 1999, 36: 228-232.
Figarella C, Miszczuk-Jamska B, Barrett AJ: Possible lysosomal activation of pancreatic zymogens. Activation of both human trypsinogens by cathepsin B and spontaneous acid. Activation of human trypsinogen 1. Biol Chem Hoppe Seyler. 1988, 369: 293-298.
Saluja A, Hashimoto S, Saluja M, Powers RE, Meldolesi J, Steer ML: Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis. Am J Physiol. 1987, 253: 508-516.
Halangk W, Lerch MM, Brandt-Nedelev B, Roth W, Ruthenbuerger M, Reinheckel T, Domschke W, Lippert H, Peters C, Deussing J: Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis. J Clin Invest. 2000, 106: 773-781.
Kukor Z, Mayerle J, Kruger B, Toth M, Steed PM, Halangk W, Lerch MM, Sahin-Toth M: Presence of cathepsin B in the human pancreatic secretory pathway and its role in trypsinogen activation during hereditary pancreatitis. J Biol Chem. 2002,
Dean RT: The roles of cathepsins B1 and D in the digestion of cytoplasmic protiens in vitro by lysosomal extracts. Biochem Biophys Res Commun. 1976, 68: 518-523.
Ohkuma S, Poole B: Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc Natl Acad Sci U S A. 1978, 75: 3327-3331.
Schneider DL: ATP-dependent acidification of intact and disrupted lysosomes. Evidence for an ATP-driven proton pump. J Biol Chem. 1981, 256: 3858-3864.
Myers BM, Prendergast FG, Holman R, Kuntz SM, LaRusso NF: Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload. J Clin Invest. 1991, 88: 1207-1215.
Myers BM, Prendergast FG, Holman R, Kuntz SM, Larusso NF: Alterations in hepatocyte lysosomes in experimental hepatic copper overload in rats. Gastroenterology. 1993, 105: 1814-1823.
Moriyama Y, Maeda M, Futai M: Involvement of a non-proton pump factor (possibly Donnan-type equilibrium) in maintenance of an acidic pH in lysosomes. FEBS Lett. 1992, 302: 18-20. 10.1016/0014-5793(92)80274-K.
Szilagyi L, Kenesi E, Katona G, Kaslik G, Juhasz G, Graf L: Comparative in vitro studies on native and recombinant human cationic trypsins. Cathepsin B is a possible pathological activator of trypsinogen in pancreatitis. J Biol Chem. 2001, 276: 24574-24580. 10.1074/jbc.M011374200.
Bode W, Fehlhammer H, Huber R: Crystal structure of bovine trypsinogen at 1–8 A resolution. I. Data collection, application of patterson search techniques and preliminary structural interpretation. J Mol Biol. 1976, 106: 325-335.
Abita JP, Delaage M, Lazdunski M: The mechanism of activation of trypsinogen. The role of the four N-terminal aspartyl residues. Eur J Biochem. 1969, 8: 314-324.
Simon P, Weiss FU, Sahin-Toth M, Parry M, Nayler O, Lenfers B, Schnekenburger J, Mayerle J, Domschke W, Lerch MM: Hereditary pancreatitis caused by a novel PRSS1 mutation (Arg-122 --> Cys) that alters autoactivation and autodegradation of cationic trypsinogen. J Biol Chem. 2002, 277: 5404-5410. 10.1074/jbc.M108073200.
Halangk W, Kruger B, Ruthenburger M, Sturzebecher J, Albrecht E, Lippert H, Lerch MM: Trypsin activity is not involved in premature, intrapancreatic trypsinogen activation. Am J Physiol Gastrointest Liver Physiol. 2002, 282: G367-374.
The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/2/16/prepub
NT carried out the peptide studies and drafted the manuscript. HB carried out the HPLCs. VK conceived of the study, and participated in its design and coordination. All authors wrote the manuscript.
All authors read and approved the final manuscript.
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Teich, N., Bödeker, H. & Keim, V. Cathepsin B cleavage of the trypsinogen activation peptide. BMC Gastroenterol 2, 16 (2002). https://doi.org/10.1186/1471-230X-2-16