Human Cationic Trypsinogen

Artigo Acesso aberto Revisado por pares

Human Cationic Trypsinogen

2002; Elsevier BV; Volume: 277; Issue: 8 Linguagem: Inglês

10.1074/jbc.m110959200

ISSN

1083-351X

Autores

Zoltán Kukor, Miklós Tóth, Gábor Pál, Miklós Sahin‐Tóth,

Tópico(s)

Gastrointestinal disorders and treatments

Resumo

Mutation of Arg117, an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg117 was investigated in wild-type cationic Tg and in the activation-resistant Lys15 → Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg117 can be examined selectively. We found that trypsinolytic cleavage of the Arg117-Val118 bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca2+, at pH 8.0, the hydrolysis equilibrium (K hyd = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca2+ reduced the rateof cleavage at Arg117 at least 20-fold, and shiftedK hyd to 0.7. These observations indicate that the Arg117-Val118 bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated K i of 80 μm, while no inhibition was observed with K15Q-Tg carrying the Arg117→ His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg117. Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg117 results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg117-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg117 → His mutation. Mutation of Arg117, an autocatalytic cleavage site, is the most frequent amino acid change found in the cationic trypsinogen (Tg) of patients with hereditary pancreatitis. In the present study, the role of Arg117 was investigated in wild-type cationic Tg and in the activation-resistant Lys15 → Gln mutant (K15Q-Tg), in which Tg-specific properties of Arg117 can be examined selectively. We found that trypsinolytic cleavage of the Arg117-Val118 bond did not proceed to completion, but due to trypsin-catalyzed re-synthesis an equilibrium was established between intact Tg and its cleaved, two-chain form. In the absence of Ca2+, at pH 8.0, the hydrolysis equilibrium (K hyd = [cleaved Tg]/[intact Tg]) was 5.4, whereas 5 mm Ca2+ reduced the rateof cleavage at Arg117 at least 20-fold, and shiftedK hyd to 0.7. These observations indicate that the Arg117-Val118 bond exhibits properties analogous to the reactive site bond of canonical trypsin inhibitors and suggest that this surface loop might serve as a low affinity inhibitor of zymogen activation. Consistent with this notion, autoactivation of cationic Tg was inhibited by the cleaved form of K15Q-Tg, with an estimated K i of 80 μm, while no inhibition was observed with K15Q-Tg carrying the Arg117→ His mutation. Finally, zymogen breakdown due to other trypsinolytic pathways was shown to proceed almost 2000-fold slower than cleavage at Arg117. Taken together, the findings suggest two independent, successively functional trypsin-mediated mechanisms against pathological Tg activation in the pancreas. At low trypsin concentrations, cleavage at Arg117 results in inhibition of trypsin, whereas high trypsin concentrations degrade Tg, thus limiting further zymogen activation. Loss of Arg117-dependent trypsin inhibition can contribute to the development of hereditary pancreatitis associated with the Arg117 → His mutation. Autosomal-dominant hereditary pancreatitis (HP) 1HPhereditary pancreatitisTgtrypsinogenTrtrypsinTg*two-chain trypsinogen with the Arg117-Val118 peptide bond cleavedTr*two-chain trypsin with the Arg117-Val118peptide bond cleavedPSTIpancreatic secretory trypsin inhibitor 1HPhereditary pancreatitisTgtrypsinogenTrtrypsinTg*two-chain trypsinogen with the Arg117-Val118 peptide bond cleavedTr*two-chain trypsin with the Arg117-Val118peptide bond cleavedPSTIpancreatic secretory trypsin inhibitor is associated with several mutations in the PRSS1 gene encoding human cationic trypsinogen (Tg). According to a recent report from the Midwest Multicenter Pancreatic Study Group (1Applebaum-Shapiro S.E. Finch R. Pfützer R.H. Hepp L.A. Gates L. Amann S. Martin S. Ulrich C.D., II Whitcomb D.C. Pancreatology. 2001; 1: 439-443Crossref PubMed Scopus (64) Google Scholar), the great majority (76%) of Tg mutations affects the Arg117 residue in the Tg sequence (chymotrypsin numbering), changing it to either His (R117H, 75% (2Whitcomb D.C. Gorry M.C. Preston R.A. Furey W. Sossenheimer M.J. Ulrich C.D. Martin S.P. Gates L.K., Jr. Amann S.T. Toskes P.P. Liddle R. McGrath K. Uomo G. Post J.C. Ehrlich G.D. Nat. Genet. 1996; 14: 141-145Crossref PubMed Scopus (1317) Google Scholar)) or rarely to Cys (R117C, 1% (3Simon, P., Weiss, U. F., Sahin-Tóth, M., Perry, M., Nayler, O., Lenfers, B., Schnekenburger, J., Mayerle, J., Domschke, W., and Lerch, M. M. (2001) J. Biol. Chem., in pressGoogle Scholar, 4Le Marechal C. Chen J.M. Quere I. Raguenes O. Ferec C. Auroux J. BMC Genet. 2001; 2: 19Crossref PubMed Scopus (39) Google Scholar, 5Pfützer R. Myers E. Applebaum-Shapiro S. Finch R. Ellis I. Neoptolemos J. Kant J.A. Whitcomb D.C. Gut. 2002; 50: 271-272Crossref PubMed Scopus (81) Google Scholar)). The second most frequent target of HP mutations is Asn21, where mutations Asn21→ Ile (N21I, 20% (6Gorry M.C. Gabbaizedeh D. Furey W. Gates L.K., Jr Preston R.A. Aston C.E. Zhang Y. Ulrich C. Ehrlich G.D. Whitcomb D.C. Gastroenterology. 1997; 113: 1063-1068Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 7Teich N. Mossner J. Keim V. Hum. Mutat. 1998; 12: 39-43Crossref PubMed Scopus (99) Google Scholar, 8Ferec C. Raguenes O. Salomon R. Roche C. Bernard J.P. Guillot M. Quere I. Faure C. Mercier B. Audrezet M.P. Guillausseau P.J. Dupont C. Munnich A. Bignon J.D. Le Bodic L. J. Med. Genet. 1999; 36: 228-232PubMed Google Scholar)) and Asn21 → Thr (N21T, 1% (5Pfützer R. Myers E. Applebaum-Shapiro S. Finch R. Ellis I. Neoptolemos J. Kant J.A. Whitcomb D.C. Gut. 2002; 50: 271-272Crossref PubMed Scopus (81) Google Scholar)) have been identified so far. In addition, several other rare Tg mutations have been described in patients with HP or other forms of chronic pancreatitis (8Ferec C. Raguenes O. Salomon R. Roche C. Bernard J.P. Guillot M. Quere I. Faure C. Mercier B. Audrezet M.P. Guillausseau P.J. Dupont C. Munnich A. Bignon J.D. Le Bodic L. J. Med. Genet. 1999; 36: 228-232PubMed Google Scholar, 9Witt H. Luck W. Becker M. Gastroenterology. 1999; 117: 7-10Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 10Chen J.M. Raguenes O. Ferec C. Deprez P.H. Verellen-Dumoulin C. Andriulli A. Gastroenterology. 1999; 117: 1508-1509Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11Pfützer R.H. Whitcomb D.C. Gastroenterology. 1999; 117: 1507-1508Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Teich N. Ockenga J. Hoffmeister A. Manns M. Mössner J. Keim V. Gastroenterology. 2000; 119: 461-465Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Chen J.M. Piepoli Bis A., Le Bodic L. Ruszniewski P. Robaszkiewicz M. Deprez P.H. Raguenes O. Quere I. Andriulli A. Ferec C. Clin. Genet. 2001; 59: 189-193Crossref PubMed Scopus (64) Google Scholar, 14Le Maréchal C. Bretagne J.-F. Raguénès O. Quéré I. Chen J.-M. Ferec C. Mol. Genet. Metab. 2001; 74: 342-344Crossref PubMed Scopus (32) Google Scholar). hereditary pancreatitis trypsinogen trypsin two-chain trypsinogen with the Arg117-Val118 peptide bond cleaved two-chain trypsin with the Arg117-Val118peptide bond cleaved pancreatic secretory trypsin inhibitor hereditary pancreatitis trypsinogen trypsin two-chain trypsinogen with the Arg117-Val118 peptide bond cleaved two-chain trypsin with the Arg117-Val118peptide bond cleaved pancreatic secretory trypsin inhibitor The discovery of HP mutations provided strong support that Tg and/or trypsin (Tr) plays a central role in the pathogenesis of human pancreatitis. Although the precise mechanisms leading to pancreatitis have not been unraveled, increased Tr activity in the pancreas due to altered properties of mutant Tg/Tr emerged as a generally accepted model. Consequently, the biochemical defects, gain or loss of function, caused by mutations of Arg117 and Asn21 have been the target of intense research in recent years. Early studies used recombinant rat anionic Tg as a model for human cationic Tg (15Várallyai É. Pál G. Patthy A. Szilágyi L. Gráf L. Biochem. Biophys. Res. Commun. 1998; 243: 56-60Crossref PubMed Scopus (95) Google Scholar, 16Sahin-Tóth M. J. Biol. Chem. 1999; 274: 29699-29704Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 17Sahin-Tóth M. Gráf L. Tóth M. Biochem. Biophys. Res. Commun. 1999; 264: 505-508Crossref PubMed Scopus (49) Google Scholar, 18Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 275: 668-671Crossref PubMed Scopus (12) Google Scholar), but, lately, techniques have been developed that allowed recombinant expression and in vitro refolding of the human enzyme (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 278: 286-289Crossref PubMed Scopus (168) Google Scholar, 21Sahin-Tóth M. Pancreatology. 2001; 1: 461-465Crossref PubMed Scopus (21) Google Scholar, 22Szilágyi L. Kénesi E. Katona G. Kaslik G. Juhász G. Gráf L. J. Biol. Chem. 2001; 276: 24574-24580Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Experiments using these recombinant cationic Tg preparations have addressed four major aspects of Tg/Tr biochemistry, with the following developments: (i) Autocatalytic degradation (autolysis) of Tr was inhibited by mutations R117H (20Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 278: 286-289Crossref PubMed Scopus (168) Google Scholar, 21Sahin-Tóth M. Pancreatology. 2001; 1: 461-465Crossref PubMed Scopus (21) Google Scholar), R117C (3Simon, P., Weiss, U. F., Sahin-Tóth, M., Perry, M., Nayler, O., Lenfers, B., Schnekenburger, J., Mayerle, J., Domschke, W., and Lerch, M. M. (2001) J. Biol. Chem., in pressGoogle Scholar), and N21T (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), whereas mutation N21I (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 278: 286-289Crossref PubMed Scopus (168) Google Scholar, 21Sahin-Tóth M. Pancreatology. 2001; 1: 461-465Crossref PubMed Scopus (21) Google Scholar, 22Szilágyi L. Kénesi E. Katona G. Kaslik G. Juhász G. Gráf L. J. Biol. Chem. 2001; 276: 24574-24580Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) had no such effect. (ii) Autocatalytic activation (autoactivation) of Tg was enhanced by all four mutations (N21T, N21I, R117H, R117C) studied so far (3Simon, P., Weiss, U. F., Sahin-Tóth, M., Perry, M., Nayler, O., Lenfers, B., Schnekenburger, J., Mayerle, J., Domschke, W., and Lerch, M. M. (2001) J. Biol. Chem., in pressGoogle Scholar, 19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 278: 286-289Crossref PubMed Scopus (168) Google Scholar, 21Sahin-Tóth M. Pancreatology. 2001; 1: 461-465Crossref PubMed Scopus (21) Google Scholar, 22Szilágyi L. Kénesi E. Katona G. Kaslik G. Juhász G. Gráf L. J. Biol. Chem. 2001; 276: 24574-24580Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Interestingly, increased autoactivation of N21I was prominent only at pH 5.0. A study using model peptides corresponding to the N terminus of Tg suggested that the rare K15R and D14G mutations might also increase Tg autoactivation (12Teich N. Ockenga J. Hoffmeister A. Manns M. Mössner J. Keim V. Gastroenterology. 2000; 119: 461-465Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), however, supporting data on recombinant enzymes are still lacking. (iii) Cathepsin B, a lysosomal cysteine protease, is believed to be responsible for initiating zymogen activation in animal models of experimental pancreatitis (see Ref. 23Halangk M.W. Lerch M.M. Brandt-Nedelev B. Roth W. Ruthenbuerger M. Reinheckel T. Domschke W. Lippert H. Peters C. Deussing J. J. Clin. Invest. 2000; 106: 773-781Crossref PubMed Scopus (456) Google Scholar, and references therein). It was reported (22Szilágyi L. Kénesi E. Katona G. Kaslik G. Juhász G. Gráf L. J. Biol. Chem. 2001; 276: 24574-24580Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) that the N21I cationic Tg mutant was activated almost 3-fold faster by cathepsin B than wild-type Tg, even in the presence of pancreatic secretory Tr inhibitor (PSTI). However, these observations could not be independently confirmed, because in more recent experiments mutants N21I, R117H, R117C and wild-type Tg exhibited no appreciable differences in activation by cathepsin B (3Simon, P., Weiss, U. F., Sahin-Tóth, M., Perry, M., Nayler, O., Lenfers, B., Schnekenburger, J., Mayerle, J., Domschke, W., and Lerch, M. M. (2001) J. Biol. Chem., in pressGoogle Scholar). 2Z. Kukor, M. Tóth, and M. Sahin-Tóth, unpublished observations. 2Z. Kukor, M. Tóth, and M. Sahin-Tóth, unpublished observations. (iv) The ability of PSTI to inhibit the N21I Tr mutant was also investigated, but no difference was found when compared with wild-type Tr (22Szilágyi L. Kénesi E. Katona G. Kaslik G. Juhász G. Gráf L. J. Biol. Chem. 2001; 276: 24574-24580Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Taken together, the bulk of evidence suggests that increased autoactivation is a common mechanism by which HP-associated mutations can lead to pathological Tr accumulation in the pancreas. In addition, Tr stabilization appears to be an accessory pathway, associated with mutations R117H, R117C, and N21T. Although it is now generally accepted that HP mutations lead to a gain of function, a recent report (3Simon, P., Weiss, U. F., Sahin-Tóth, M., Perry, M., Nayler, O., Lenfers, B., Schnekenburger, J., Mayerle, J., Domschke, W., and Lerch, M. M. (2001) J. Biol. Chem., in pressGoogle Scholar) challenged this notion by demonstrating that almost 70% of mutant R117C was misfolded and inactive after recombinant expression andin vitro refolding. It remains unclear, however, whether or not the same folding defect is manifested in vivo. In any event, the correctly folded fraction of the R117C mutant exhibited gain-of-function properties that were similar to those of the R117H mutant. Despite considerable progress in the field, the mechanism(s) by which the mutations cause the described functional changes are still unclear. In the present study, we set out to investigate the role Arg117 might play in Tg activation and how mutations in Arg117 can enhance zymogen activation. Unexpectedly, we found that Tr not only cleaved the Arg117-Val118 bond but also re-synthesized it until an equilibrium was reached between the cleaved and intact Tg forms. These properties of Arg117 resemble the reactive site of reversible canonical protease inhibitors (24Laskowski M., Jr. Qasim M.A. Biochim. Biophys. Acta. 2000; 1477: 324-337Crossref PubMed Scopus (305) Google Scholar) and suggest that Arg117 plays an inhibitory role in controlling zymogen activation in the pancreas. Evidence to support this notion is presented here, and a model describing the dual role of Tr in protecting against pathological Tg activation is discussed. In this article the classic chymotrypsin numbering was used to denote amino acid residues in the human cationic Tg sequence. In more recent genetic and clinical literature the actual Tg sequence numbering has been used by convention (25Chen J.M. Ferec C. Pfützer R.H. Whitcomb D.C. Gastroenterology. 2000; 119: 277-279Abstract Full Text Full Text PDF PubMed Google Scholar). In the chymotrypsin numbering system Asp14, Lys15, Asn21, Arg117, Val118, Lys188, and Asp189 correspond to Tg residues Asp22, Lys23, Asn29, Arg122, Val123, Lys193, and Asp194, respectively. Following the designation of cleaved canonical protease inhibitors (24Laskowski M., Jr. Qasim M.A. Biochim. Biophys. Acta. 2000; 1477: 324-337Crossref PubMed Scopus (305) Google Scholar), the two-chain autolyzed form of Tg with the Arg117-Val118 bond cleaved was denoted here as Tg*. In previous studies bovine Tg and Tr, autolyzed at the Arg117-Val118 bond, were also named Val-neotrypsinogen (26Higaki J.N. Light A. Anal. Biochem. 1985; 148: 111-120Crossref PubMed Scopus (12) Google Scholar) or δ-trypsin (27Kumazaki T. Ishii S.-I. J. Biochem. 1979; 85: 581-590Crossref PubMed Scopus (3) Google Scholar), respectively. Mutant K15Q was constructed in the previously described pTrap-T7 expression plasmid (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). A synthetic oligonucleotide linker encoding the mutation was ligated into the NcoI andEcoRI sites. Mutant K15Q/R117H was created by subcloning theEcoRI-SacI fragment from the pTrap-T7/R117H plasmid (20Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 278: 286-289Crossref PubMed Scopus (168) Google Scholar) into the pTrap-T7/K15Q plasmid. Small scale expression of Tg was carried out essentially as previously reported (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 278: 286-289Crossref PubMed Scopus (168) Google Scholar, 21Sahin-Tóth M. Pancreatology. 2001; 1: 461-465Crossref PubMed Scopus (21) Google Scholar). For larger scale expression, 800-ml cultures of Rosetta(DE3) (Novagen) cells harboring pTrap-T7 with given Tg constructs were grown in Luria-Bertani medium with 50 μg/ml carbenicillin and 34 μg/ml chloramphenicol to an A 600 nm of 0.5, induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside, and grown for an additional 5 h. Rosetta(DE3) host strains are BL21(DE3) derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in Escherichia coli. Cells were harvested by centrifugation, resuspended in 60 ml of 0.1 m Tris-HCl (pH 8.0), 5 mm K-EDTA, and disrupted by one passage through a French press cell at 25,000 p.s.i. Inclusion bodies were pelleted by centrifugation and washed twice with 25 ml of 0.1 mTris-HCl (pH 8.0), 5 mm EDTA. Solubilization and in vitro refolding of Tg was performed as described previously (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar,22Szilágyi L. Kénesi E. Katona G. Kaslik G. Juhász G. Gráf L. J. Biol. Chem. 2001; 276: 24574-24580Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) in 0.9 m guanidine-HCl, 0.1 m Tris-HCl (pH 8.0) containing 1 mml-cystine and 1 mml-cysteine. The properly folded Tg was finally purified on an ecotin affinity column, as described (18Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 275: 668-671Crossref PubMed Scopus (12) Google Scholar, 19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 20Sahin-Tóth M. Tóth M. Biochem. Biophys. Res. Commun. 2000; 278: 286-289Crossref PubMed Scopus (168) Google Scholar). Concentrations of zymogen solutions were measured from their ultraviolet absorbance using a calculated extinction coefficient of 36,160 m−1 cm−1 at 280 nm. Tg yields were lower than previously reported (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), typically 2–4 mg of purified zymogen per liter culture. Wild-type Tg was allowed to autoactivate in 1 mm EDTA, 0.1 m Tris-HCl (pH 8.0), at 37 °C for 2 h. K15Q-Tg was digested with cationic Tr at a ratio of ∼1:250 Tr to K15Q-Tg in 1 mm EDTA, 0.1 mTris-HCl (pH 8.0), at 37 °C for 20 min. Samples were immediately loaded onto a MonoQ HR 5/5 column (Amersham Biosciences, Inc.) equilibrated with 20 mm Tris-HCl (pH 8.8). Tg and Tg* were eluted and separated from each other with 25 ml of a 0–0.5m linear gradient of NaCl. Under these conditions Tr did not bind to the column and was recovered in the flow-through. Fractions (0.5 ml) containing Tg* or K15Q-Tg* were pooled and dialyzed against 1 mm HCl overnight. Human cationic Tg (2 μmfinal concentration in 100-μl final volume) was incubated at 37 °C in 0.1 m Tris-HCl (pH 8.0) or 0.1 m sodium acetate buffer (pH 5.0), in the presence of 5 mmCaCl2 where indicated. At given times 2.5-μl aliquots were removed for Tr activity assays. Tr activity was determined using the synthetic chromogenic substrateN-carbobenzyloxy-Gly-Pro-Arg-p-nitroanilide (200 μm final concentration). Kinetics of the chromophore release was followed at 405 nm in 0.1 m Tris-HCl (pH 8.0), 1 mm CaCl2, at 22 °C. Where indicated, samples were precipitated with trichloroacetic acid (10% final concentration), incubated on ice for 10 min, and centrifuged for 10 min at 14,000 rpm in an Eppendorf bench top centrifuge at 4 °C. Laemmli sample buffer (20 μl), NaOH (1 μl, 2 n), and dithiothreitol (2 μl, 1 m) were added, and samples were denatured at 90 °C for 5 min. Electrophoretic separation was performed on 12% SDS-PAGE gels. Gels were stained with Coomassie Brilliant Blue R for 30 min and destained with 30% methanol, 10% acetic acid overnight. Gels were dried between two layers of cellophane according to instructions of the Gel-Dry (Invitrogen) gel drying kit. Dried gels were scanned at 600 d.p.i. resolution in gray-scale mode, and images were saved as TIFF files. Quantitation of gel bands was carried out with the ImageQuaNT 5.2 (Molecular Dynamics) software. Rectangles were drawn around each band of interest, and an identical rectangle was used in each lane for background subtraction. The gel image in Fig.1 demonstrates the time course of activation of Tg with Tr, in 5 mm Ca2+ at pH 8.0, 37 °C. Conversion of Tg to Tr upon cleavage after Lys15 is indicated by a mobility shift on 12% SDS-PAGE gels (band Tg → band Tr), because Tr migrates faster than Tg under these electrophoretic conditions. In addition to activation, Tr cleaves Tg also at the Arg117-Val118 autolytic peptide bond, resulting in the appearance of band A on reducing gels. As we demonstrated previously (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), band A corresponds to a mixture of two co-migrating polypeptides with nearly identical sizes, namely the N- and C-terminal fragments of autolyzed Tg. This two-chain form of Tg with the Arg117-Val118 bond cleaved will be referred to as Tg* in this report. Tg* is also activated by Tr, which is evidenced by the appearance of band B on reducing gels.Band B is generated from band A by removal of the activation peptide from the N-terminal Tg* fragment. Thus, band B corresponds to the N-terminal fragment of two-chain, Arg117-cleaved Tr (Tr*). Note that the two chains in Tg* and Tr* are held together by a disulfide bond and they only separate under the reducing conditions used for SDS-PAGE gel analysis. We observed that Tr-catalyzed activation of Tg* seemed to proceed slower than Tr-catalyzed activation of intact Tg. Thus, band B always emerged later in the time course and was noticeably lighter in intensity than the Tr band (Fig. 1). This observation led to the hypothesis that cleavage of Tg at Arg117 hinders Tg activation, as we first believed due to a conformational change in the Tg structure. To test this assumption, we isolated Tg* using anion-exchange chromatography, and compared its activation properties to intact wild-type Tg. Tg* was generated from wild-type Tg by limited autoactivation of Tg in the absence of Ca2+. As we demonstrated previously (19Sahin-Tóth M. J. Biol. Chem. 2000; 275: 22750-22755Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar), under these conditions Tg is cleaved predominantly after Arg117, whereas cleavage after Lys15 is suppressed. Thus, the major product is Tg* and only small amounts of Tr are generated. Tg* was then separated on a MonoQ column at alkaline pH, where it eluted after the intact Tg peak (Fig. 2). The gel inset indicates the purity of these preparations, which was greater than >95%, with small amounts of intact Tg present. Although data are not shown, when the activation process was followed by measuring Tr activity, we did not observe any appreciable differences between the activation characteristics of intact Tg and Tg*. Enterokinase or Tr activated both Tg forms at comparable rates, and Ca2+dependence of autoactivation was indistinguishable. These observations did not support our original hypothesis and indicated that cleavage of Tg at Arg117 had no direct effect on Tg activation. Furthermore, enzyme kinetic parameters of Tr and Tr* were essentially identical, in agreement with previous reports that cleavage of the Arg117-Val118 bond in bovine (28Maroux S. Desnuelle P. Biochim. Biophys. Acta. 1969; 181: 59-72Crossref PubMed Scopus (49) Google Scholar) or porcine (29Ru B.G., Du, J.Z. Zeng Y.H. Chen L.S., Ni, Y.S. Tan G.H. Zhang L.X. Sci. Sin. 1980; 23: 1453-1460PubMed Google Scholar) Tr has no obvious functional consequences. However, when the time course of autoactivation was followed on SDS-PAGE gels an unexpected observation was made. As shown in Fig.3 A, at pH 8.0, in 5 mm Ca2+, the majority of Tg* was converted back to intact Tg and activated to intact Tr. Attempts to extend these observations revealed that experiments with wild-type Tg and Tr were difficult to interpret quantitatively. Due to the continuous conversion of Tg to Tr, concentrations of substrate (Tg) and enzyme (Tr) were constantly changing during the reaction. In addition, Tr can cleave the Arg117-Val118 bond not only in Tg but also in Tr. This latter process is also reversible due to the re-synthesis of the Arg117-Val118 bond in Tr* (not shown). In the present study we focused selectively on the interaction of Tr with Tg, and the role of Arg117 in Tg. Similar experiments on the properties of Arg117 in Tr will be presented elsewhere. To gain deeper insight into the extraordinary phenomenon of Tr-catalyzed re-synthesis of Tg*, an activation-resistant Tg mutant was constructed, where Lys15 in the activation peptide was mutated to Gln (K15Q-Tg). In this zymogen, Arg117 is the only readily accessible trypsinolytic site, and thus its properties can be selectively studied. A two-chain form of K15Q-Tg cleaved between Arg117 and Val118 (K15Q-Tg*) was also isolated, as described above. When this Tg preparation was treated with cationic Tr, time-dependent re-synthesis of the intact single-chain form was evident, without any significant degradation (Fig.3 B). No re-synthesis was observed in the absence of Tr or with the catalytically incompetent S195A Tr mutant. As shown in Fig.3 C, the re-synthesis did not proceed to completion, but an apparent equilibrium was reached between intact Tg and Tg* with a hydrolysis equilibrium of 0.73 (K hyd = [Tg*]/[Tg]), corresponding to a ratio of ∼40% Tg* to 60% Tg. When intact K15Q-Tg was digested with catalytic amounts (∼1:160 ratio) of cationic Tr in the absence of Ca2+ at pH 8.0, rapid cleavage at Arg117 was evident (Fig. 4 A). Surprisingly, digestion remained incomplete over the entire time course, as a stable equilibrium was reached between the predominant cleaved form (∼85%) and the remaining intact K15Q-Tg (∼15%), yielding a K hyd of 5.4. Increasing concentrations of Ca2+ reduced the rate of cleavage at Arg117 significantly. Estimation of the relative rates by comparing the initial segments of digestion kinetics obtained in the absence of Ca2+ (Fig. 4 A) or in 5 mm Ca2+ (Fig. 4 D) indicated an at least 20-fold decrease (Fig. 4 E). In addition, Ca2+ also lowered the K hyd values from 5.4 to 0.96 and 0.7 in 0.1 mm Ca2+ (Fig.4 B) and 0.5 mm Ca2+ (Fig.4 C), respectively. It is also apparent from Fig.4 E that in 5 mm Ca2+ an equilibrium was not reached during the time course studied. When digestions in 5 mm Ca2+ were carried out with higher Tr concentrations (Fig. 5), an equilibrium could be demonstrated with a K hyd of 0.7, a value that was essentially identical with that observed in the re-synthesis experiments in Fig. 3. It is noteworthy that theK hyd value remained constant when concentrations of Ca2+ were increased from 0.5 to 5 mm, although the rate of cleavage at Arg117 was further depressed, and the same equilibrium state was reached much slower. It must also be emphasized that in these experiments Tr behaved only as a catalyst facilitating the development of the hydrolysis equilibrium between the intact and Arg117-cleaved forms of K15Q-Tg. Consequently, given sufficient time, similar equilibrium states were reached regardless of the Tr concentration present in the incubation mixtures (data not shown).Figure 5Digestion of the Arg117-Val118 bond in K15Q-Tg in 5 mm Ca2+. The experiment was carried out as described in Fig. 4, except that 0.5 μm cationic Tr (final concentration) was used.View Large Image Figure ViewerDownload Hi-res image

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Human Cationic Trypsinogen