* Institut National de la Santé et de la Recherche Médicale, Génétique Moléculaire et Génétique Epidémiologique, Université de Bretagne Occidentale, Etablissement Français du SangBretagne, Brest, France
Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
Centre Hospitalier Universitaire de Melun, Service de Gastroenterologie, Melun, France
|| Centre Hospitalier Universitaire de Morvan, Brest, France
Correspondence: E-mail: claude.ferec{at}univ-brest.fr
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Abstract |
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Key Words: activation peptide chronic pancreatitis comparative genomic analysis human cationic trypsinogen molecular evolution missense mutation
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Introduction |
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Historically, trypsin(ogen) has been among the most extensively studied enzyme models of protein structure and function due to its availability in robust quantities from vertebrate pancreas. Bovine trypsin was purified by crystallization in the early 1930s (Northrop and Kunitz 1931), almost the entire amino acid sequence of bovine trypsinogen was determined by protein sequencing in the 1960s (Walsh et al. 1964), and the three-dimensional structure of bovine trypsin was solved in the 1970s (Huber et al. 1974; Stroud, Kay, and Dickerson 1974). The wealth of structural and functional information also made trypsin(ogen) a good model for studying gene and protein evolution (e.g., Hartley et al. 1965; Neurath 1984).
A number of early studies examined the role of the trypsinogen activation peptide, an N-terminal short sequence that is hydrolyzed as the first step in the activation process of trypsinogen. As early as in the 1950s, bovine trypsinogen was shown to be activated through cleavage of an N-terminal hexapeptide, Val-Asp-Asp-Asp-Asp-Lys-Ile, both by bovine trypsin (autoactivation) (Davie and Neurath 1955) and by its physiological activator, enteropeptidase (enterokinase) (Yamashina 1956). The unusual four aspartyl residues (Asp4) preceding the cleavage bond Lys23-Ile24 (human trypsinogen numbering is used throughout the text) were found to be strictly conserved in trypsinogen activation peptides of pig (Charles et al. 1963), sheep (Schyns, Bricteux-Gregoire, and Florkin 1969), and other mammals (e.g., Bricteux-Gregoire, Schyns, and Flokin 1972), including the human (Guy et al. 1976, 1978). However, this is not always the case in nonmammalian vertebrates (e.g., de Haen, Walsh, and Neurath 1977). More recently, a comparative analysis of trypsinogen activation peptides suggested that there might be a progressive increase in selective pressure for such acidic residues during the course of vertebrate evolution (Roach et al. 1997).
Nevertheless, the initial observation that the Asp4 sequence was conserved in the bovine, porcine, and ovine trypsinogen activation peptides stimulated studies on its role in the mechanism of trypsinogen activation. Qualitative studies using small synthetic peptides indicated that Asp4 slowed significantly the hydrolysis of the Lys23-Ile24 bond by bovine trypsin (Abita, Delagge, and Lazdunski 1969) but enhanced the action of enteropeptidase (Maroux, Baratti, and Desnuelle 1971). These results suggested that although Asp4 might play a protective role against occasional activation of the zymogen within the pancreas (Abita, Delagge, and Lazdunski 1969), it must constitute the signal for specific activation of trypsinogen by enteropeptidase in the duodenum (Maroux, Baratti, and Desnuelle 1971).
Recent advances in two different areas have provided unique opportunities for an in-depth evaluation of this important issue. On the one hand, two pancreatitis-associated mutations K23R (Ferec et al. 1999) and D22G (Teich et al. 2000) were identified in the activation peptide of human cationic trypsinogen (Online Mendelian Inheritance in Man [OMIM] 276000; http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM). On the other hand, a large number of trypsinogen sequences have been isolated from phyla and species that extend over the whole range of evolution from bacteria to mammals (reviewed in Halfon and Craik 1998). Whereas analyzing the effects of disease-associated mutations is helpful in understanding the evolutionary forces that have shaped gene products, comparative genomic analysis traces the evolutionary patterns and processes that affected those genes in the past. The two types of information are complementary, and their integration results in a clearer picture about gene evolution. This work represents such an attempt. We analyzed the functional consequences of a newly identified activation peptide mutation, D19A, as well as the previously found D22G and K23R mutations in recombinant human cationic trypsinogen. The functional data, together with a comprehensive sequence comparison, shed further light on the molecular evolution and biology of trypsinogen activation peptides.
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Materials and Methods |
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Expression and Purification of Trypsinogen
Small-scale expression and in vitro refolding of human cationic trypsinogen and mutants D19A, D22G, and K23R were carried out as reported previously (Sahin-Tóth 2000, 2001; Sahin-Tóth and Tóth 2000). Concentrations of zymogen solutions were measured from their ultraviolet absorbance using a calculated extinction coefficient of 36,160 M1 cm1 at 280 nm. The activation peptide sequence of recombinant zymogen preparations used in this study was Met-Ala-Pro-Phe-(Asp)4-Lys.
Trypsinogen Autoactivation
Aliquots of wild-type or mutant human cationic trypsinogen (2 µM final concentrations) were incubated at 37°C in 0.1 M Tris-HCl (pH 8.0) or 0.1 M Na-acetate buffer (pH 5.0), in the absence or presence of 1 mM CaCl2 in a final volume of 100 µl. At indicated times, 2.5 µl aliquots were removed for trypsin activity assays. Trypsin activity was determined using the synthetic chromogenic substrate N-CBZ-Gly-Pro-Arg-p-nitroanilide (Sigma, St. Louis, Mo.) at 140 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. To prevent nonspecific binding of trypsinogen to tube walls at pH 5.0, bovine serum albumin (2 mg/ml) was included in the activation mixtures (see figure 4A in Kukor et al. 2002a).
Activation of Recombinant Trypsinogen with Enteropeptidase
Under conditions that are optimal to assay enteropeptidase activation, the trypsinogen mutants studied here exhibited a strong tendency for autoactivation. To minimize this confounding factor, enteropeptidase activation was determined by continuous monitoring of trypsin generation at dilute trypsinogen concentrations, as described previously (Sahin-Tóth 2000). Approximately 100 nM wild-type or mutant cationic trypsinogen (final concentrations) were mixed with 100 ng/ml enteropeptidase (final concentration) in a microplate well containing 200 µl of 0.1 M Tris-HCl (pH 8.0), 1 mM CaCl2, and 180 µM synthetic trypsin substrate N-CBZ-Gly-Pro-Arg-p-nitroanilide (final concentrations). The activation reaction was followed at 22°C by continuous monitoring of p-nitroanilide release at 405 nm as a measure of trypsin activity. Trypsinogen samples without added enteropeptidase exhibited a small increase in their absorbance readings over the 1 min period, and these values were subtracted from the absorbance readings of the enteropeptidase treated samples.
Acquisition of Trypsinogen Activation Peptide Sequences
Trypsinogen activation peptide sequences were mainly collated from major online protein databanks. The sequences were identified by searching with the keywords "trypsin" and "trypsinogen" separately in both GenBank and Swiss-Prot. In addition, several vertebrate activation peptide sequences were gathered through an extensive search of the literature.
To identify the functionally most relevant trypsinogen genes, target sequences were manually evaluated and selected for comparative analysis based upon the following criteria. First, only genes that have been physically identified at the mRNA and/or protein level were included. Second, if more than three trypsinogen genes were physically identified in a species, usually the first reported three were included for sequence comparison. Finally, all of the "fast-evolving" group III trypsinogen genes in cold-adapted fishes (Roach 2002 and references therein) were treated separately.
Uncertainties or mistakes in the designation of certain activation peptides were resolved or corrected by predicting the cleavage site for the removal of the signal peptide using the computer program SignalP version 2.0.b2 (Nielsen et al. 1997) and by multiple sequence alignments using ClustalW.
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Results and Discussion |
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In our routine screening for pancreatitis-associated genetic risk factors, a novel mutation termed D19A (GenBank accession number AY234116) was found in the activation peptide sequence of the human cationic trypsinogen gene from a 27-year-old white French patient with chronic pancreatitis. Details on patient information and mutation identification are available in Supplementary Material online. Figure 1 indicates the location of the D19A mutation and the two previously found D22G and K23R mutations in the trypsinogen activation peptide.
Functional Analysis of the D19A, D22G, and K23R Activation Peptide Mutants in the Context of Recombinant Human Cationic Trypsinogen
Early studies on the bovine trypsinogen activation peptide were performed using small synthetic peptides as models (Abita, Delagge, and Lazdunski 1969; Maroux, Baratti, and Desnuelle 1971). The same approach was used to analyze the two previously identified D22G and K23R mutations (Teich et al. 2000; Teich, Bodeker, and Keim 2002). Recently, methodology has been developed for the recombinant expression, in vitro refolding, and purification of human cationic trypsinogen. This type of recombinant trypsinogen preparation has been used in a growing number of studies that investigated the effects of hereditary pancreatitis-associated mutations (Sahin-Tóth 2000; Sahin-Tóth and Tóth 2000; Szilágyi et al. 2001; Kukor et al. 2002a, 2002b; Simon et al. 2002). It was further employed in the present study to analyze the functional effects of the three activation peptide mutations.
Effects of the Three Activation Peptide Mutations on Trypsinogen Autoactivation
We first analyzed the effects of the three activation peptide mutations on trypsinogen autoactivation. As shown in figure 2A, at pH 8.0, all three activation peptide mutations significantly increased autoactivation of human cationic trypsinogen. Increased activation was particularly striking with mutations D22G and K23R. Addition of 1 mM Ca2+ stimulated autoactivation of wild-type trypsinogen and mutant D19A (fig. 2B). In contrast, only a slight stimulation was evident with mutant K23R, whereas autoactivation of mutant D22G was marginally inhibited by the divalent cation. The effect of the mutations on autoactivation was also examined at pH 5.0 (fig. 2C), since hereditary pancreatitis-associated mutations studied previously caused increased autoactivation rates in an acidic milieu (Sahin-Tóth 2000, 2001; Sahin-Tóth and Tóth 2000; Szilágyi et al. 2001). Again, all three mutations increased autoactivation, although the stimulation by mutation D19A was less pronounced at this pH.
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Our finding that the K23R mutant trypsinogen exhibited dramatically increased autoactivation is consistent with the known Arg preference of trypsin. A model peptide carrying the K23R substitution was also cleaved by trypsin at an enhanced rate (Teich et al. 2000). The primary (S1) specificity pocket of trypsin interacts with positively charged side chains, such as Lys or Arg. Trypsin has a twofold to 10-fold preference for Arg over Lys at the P1 position of peptide substrates (Craik et al. 1985). The structural basis for this phenomenon is the stronger interactions of the Arg side chain with Asp194 (Asp189 in chymotrypsin numbering) at the bottom of the specificity pocket. Thus, the guanidinium group of a P1 Arg can form direct interactions with the carboxylate group of Asp194, whereas the contact between the amino group of a P1 Lys and Asp194 is mediated by a water molecule. In addition, the observation that Ca2+ does not cause significant further stimulation of autoactivation in the K23R mutant also suggests that the perfect geometry of the Arg23-Asp194, P1-S1 interaction counteracts the inhibitory effect of the tetra-aspartate residues.
In summary, functional characterization of the three pancreatitis-associated activation peptide mutations confirmed the hypothesis that the evolutionarily conserved, negatively charged tetra-aspartate sequence in mammalian trypsinogens protects against trypsinogen autoactivation within the pancreas (Abita, Delagge, and Lazdunski 1969). The use of Lys instead of Arg at the P1 position of human cationic trypsinogen activation peptide also proved to have such a protective effect.
Effects of the Three Activation Peptide Mutations on Trypsinogen Activation with Enteropeptidase
It is possible that the well-conserved Asp4 sequence in mammalian trypsinogen activation peptides is strongly selected for enteropeptidase recognition. Thus, we also analyzed the effects of the three activation peptide mutations on activation of trypsinogen by the physiological activator enteropeptidase. In these experiments, trypsinogen was incubated with bovine enteropeptidase in the presence of a synthetic trypsin substrate (N-CBZ-Gly-Pro-Arg-p-nitroanilide) and development of trypsin activity was continuously monitored by following release of the yellow p-nitroanilide. In this assay, trypsinogen concentrations were relatively low; thus, interference from autoactivation was less likely to occur. As shown in figure 3, at pH 8.0 in 1 mM Ca2+, the D19A mutation had no significant effect on enteropeptidase activation. In contrast, mutation K23R stimulated and mutation D22G almost completely inhibited enteropeptidase-mediated trypsinogen activation.
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Integration of Functional Characterization of Disease-Associated Mutations with Comparative Sequence Analysis of Trypsinogen Activation Peptides
Table 1 is a summary of currently available trypsinogen activation peptide sequences we collated from major databanks and original publications. Since trypsinogens found in bacteria and fungi do not possess a "typical" activation peptide, only two representative sequences, one from the bacterium Streptomyces griseus and one from the mold Fusarium oxysporum, were included in the table. In addition, although multiple trypsinogen genes are present in several insects, nearly all insect trypsinogen activation peptides terminate in Gly at P2 and Arg at P1; therefore, only one sequence from Drosophila melanogaster was listed.
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Lys in the P1 Position Has Evolved to Minimize Trypsinogen Autoactivation
As shown in table 1, trypsinogen activation peptides in bacteria and fungi contain no Lys or Arg residues at the P1 position. Consequently, they are not capable of autocatalyzing their own activation. Although these trypsins are significantly different from the pancreatic trypsins of mammals, they can hydrolyze synthetic peptide substrates having arginine or lysine at the P1 position and can be inhibited by typical trypsin inhibitors (e.g., Lee, Kang, and Kim 1998).
All other species, with the exception of the sea lamprey, have evolved the autoactivating property within trypsinogen, characterized by the presence of Lys or Arg as the P1 residue in their activation peptides. At the present time, it is not clear why trypsinogens adapted the ability to autoactivate, although one may speculate that this may be a mechanism initially evolved for activating trypsinogen. In support of this view is the observation that in fungi, insects, and crustaceans, trypsinogen activation peptides lack an acidic residue at P2 (table 1) and thus may not be recognized by enteropeptidase.
Nevertheless, based upon the absence of an Arg-P1 residue in the vast majority of trypsinogen activation peptides (table 1), the biochemical and structural data demonstrating the preference of trypsin for Arg over Lys, and the association of the K23R mutation in the human cationic trypsinogen with pancreatitis, it seems reasonable to conclude that Lys at P1 has evolved to minimize autoactivation within the pancreas. In contrast, the selective use of Lys at P1 is clearly not critical for enteropeptidase recognition, since an activation peptide having Arg at P1 is more readily activated by enteropeptidase. This evolutionary scenario suggests that even a minor increase in trypsinogen autoactivation within the pancreas may be potentially dangerous, whereas a small decrease in affinity for enteropeptidase is inconsequential due to its high catalytic activity and relative abundance in the duodenum.
An intriguing exception in the evolution of trypsinogen activation peptides is observed in the sea lamprey, one of the most primitive vertebrates. Strikingly, all of its five trypsinogen activation peptides end in a histidine (see table 1), a residue after which trypsin or enteropeptidase cannot cleave. As suggested by Roach et al. (1997), this exception may be associated with the sea lamprey's life cycle, during which adults can go months to years without eating. Therefore, there may exist a different, tightly controlled mechanism for trypsinogen activation in the sea lamprey. Alternatively, these trypsinogens may not be activated, indicating a possible divergence from the digestive function of trypsins, analogous to the group III trypsinogens of cold-adapted fishes (discussed later).
The Asp4 Sequence in Mammalian Trypsinogen Activation Peptides Has Evolved to Inhibit Autoactivation and to Enhance Enteropeptidase Cleavage
The comprehensive comparative analysis of trypsinogen activation peptides performed in this study revealed how the Asp4 sequence strictly conserved in mammalian trypsinogen activation peptides has evolved progressively. As indicated in table 1, the insect and crustacean activation peptides do not possess an acidic residue immediately before Lys-P1 or Arg-P1. The two urochordates, tunicate and star ascidian, have an Asp residue at P2. Additional Asp residues have gradually evolved during the course of vertebrate evolution: two in the sea lamprey; three or four in fishes, amphibians, and birds; and strictly four in the mammals. The adaptation and subsequent increase of the number of Asp residues preceding the Lys23-Ile24 cleavage bond is associated with a progressively decreased tendency to autoactivate, as evidenced by available biochemical and structural data on wild-type, D19A, and D22G mutant trypsinogens.
An interesting exception to the rule is the presence of the Asp4 motif in the primitive vertebrate dogfish. We could not exclude the possibility that the common ancestor of the dogfish and Osteicthyes possessed the tetra-aspartyl sequence in its trypsinogen activation peptide. Nevertheless, based upon the general evolutionary pattern of trypsinogen activation peptides from bacteria to mammals, we believe that the presence of the Asp4 sequence in the dogfish is a coincidence. Alternatively, this may be due to convergent evolution resulting from common selective pressures. Additional trypsinogen activation peptide sequences from the most vertebrate-like invertebrates such as hagfish and Amphioxus and other species of Chondrichthyes may help to clarify this issue.
Fixed Substitutions in Key Residues of Trypsinogen Activation Peptides May Suggest the Evolution of New Functions
The pancreatitis-associated activation peptide mutations are rare alleles in the population that are constantly selected against. However, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion.
Vertebrate trypsins have been classified into two groups according to their genomic location and, to a lesser degree, their charge at physiologic pH. Group I trypsins are usually, but not always, anionic at physiologic pH, whereas group II trypsins are usually, but not always, cationic at physiologic pH (Roach 2002 and references therein). However, some trypsin sequences determined from fishes that spend all or part of their lives at temperatures near 0°C cannot be classified into either of these two groups and were designated as group III (table 2). Principal component analysis of amino acid compositions, molecular trees, and multidimensional scaling of molecular sequence distances indicated that group III trypsins have evolved recently from other vertebrate trypsins. The increased rate of evolution in these trypsins, relative to group I and group II trypsins, might be correlated with the development of a new functionpossibly extreme cold adaptation (Roach 2002 and references therein).
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Interestingly, the key substitutions in the group III trypsinogen activation peptides are Asp to Gly at P2 and Lys to Arg at P1 with respect to the other fish trypsinogens and the mammalian trypsinogens. Since Gly-P2 and Arg-P1 are present only in insect and salmon louse trypsinogens (table 1), and the D22G and K23R mutations in the human cationic trypsinogen have been associated with pancreatitis, it appears that group III trypsinogens are dangerous to the organism. We believe that concomitant evolutionary changes within the trypsin sequence might have counteracted the potentially deleterious effects of the new activation peptides. As a result, the group III "trypsins," whose primary structure is notably different from those of the other fish trypsins, might function differently than the classic pancreatic trypsins. Therefore, it is important to make a distinction between rare alleles that encode functional digestive trypsinogens and fixed alleles that may have evolved new functions, possibly unrelated to digestion.
Conclusion
In summary, we reported a new pancreatitis-associated activation peptide mutation, D19A, in the human cationic trypsinogen. This new mutation, as well as the previously found D22RG and K23R mutations, was, for the first time, functionally evaluated in the context of recombinant human cationic trypsinogen. The functional data extended the previous observations on the role of the well-conserved Asp4 sequence in mammalian trypsinogen activation peptides and, more importantly, clarified the driving force behind this strong natural selection. Incorporation of this information into a comprehensive sequence comparison provided further insights into the molecular evolution and biology of trypsinogen activation peptides. Our most important findings indicate that the Asp4 sequence in the mammalian trypsinogen activation peptides has evolved for both efficient inhibition of trypsinogen autoactivation within the pancreas and optimal enteropeptidase recognition in the duodenum. The use of Lys at the P1 position has also an advantageous effect against trypsinogen autoactivation. Furthermore, we proposed that fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions. Finally, more information on the structure, function, and evolution of enteropeptidase is essential for a full picture of the molecular evolution of trypsinogen activation peptides, the evolution of the P1 and P2 positions in particular.
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Acknowledgements |
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Footnotes |
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Literature Cited |
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Abita, J. P., M. Delaage, and M. Lazdunski. 1969. The mechanism of activation of trypsinogen: the role of the four N-terminal aspartyl residues. Eur. J. Biochem. 8:314-324.[ISI][Medline]
Berger, A., and I. Schechter. 1970. Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos. Trans. R. Soc. Lond. B Biol. Sci. 257:249-264.[ISI][Medline]
Bodley, M. D., R. J. Naude, W. Oelofsen, and A. Patthy. 1995. Ostrich trypsinogen: purification, kinetic properties and characterization of the pancreatic enzyme. Int. J. Biochem. Cell Biol. 27:719-728.[CrossRef][ISI][Medline]
Bradshaw, R. A., H. Neurath, R. W. Tye, K. A. Walsh, and W. P. Winter. 1970. Comparison of the partial amino-acid sequence of dogfish trypsinogen with bovine trypsinogen. Nature 226:237-239.[ISI][Medline]
Bricteux-Gregoire, S. 1970. Demonstration of 2 trypsinogens in the roe-deer. Arch. Int. Physiol. Biochim. 8:399-413 [in French].
Bricteux-Gregoire, S., R. Schyns, and M. Florkin. 1971a. Purification, properties and N-terminal sequence of goat trypsinogen. Biochim. Biophys. Acta 229:123-135 [in French].[ISI][Medline]
Bricteux-Gregoire, S., R. Schyns, and M. Florkin. 1971b. Phylogeny of activation peptides of trypsinogen. Pp. 130149 in E. Schoffeniels, ed. Biochemical evolution and the origin of life. North Holland Publishing Co., Amsterdam.
Bricteux-Gregoire, S., R. Schyns, and M. Florkin. 1971c. N-terminal amino acid sequence of dromedary trypsinogen. Biochim. Biophys. Acta 251:79-82.[ISI][Medline]
Bricteux-Gregoire, S., R. Schyns, and M. Florkin. 1972. Phylogeny of trypsinogen activation peptides. Comp. Biochem. Physiol. B 42:23-39.[CrossRef][Medline]
Bricteux-Gregoire, S., R. Schyns, and M. Florkin. 1974. N-terminal amino acid sequence of trypsinogen from the elephant seal Mirounga leonina L. (Carnivora). Biochim. Biophys. Acta 351:87-91.[ISI][Medline]
Bricteux-Gregoire, S., R. Schyns, M. Florkin, M. Emmens, G. W. Welling, and J. J. Beintema. 1975. N-terminal amino acid sequence of trypsinogen from the lesser rorqual, Balaenoptera acutorostrata (Cetacea). Simultaneous isolation of trypsinogen, chymotrypsinogen and ribonuclease from pancreas. Biochim. Biophys. Acta 386:244-255.[ISI][Medline]
Charles, M., M. Rovery, A. Guidoni, and P. Desnuelle. 1963. On trypsinogen and trypsin of pig. Biochim. Biophys. Acta 69:115-129 [in French].[CrossRef][ISI]
Chen, J. M., and C. Ferec. 2000. Wanted: a consensus nomenclature for cationic trypsinogen mutations. Gastroenterology 119:277-278.[ISI][Medline]
Chen, J. M., T. Montier, and C. Ferec. 2001. Molecular pathology and evolutionary and physiological implications of pancreatitis-associated cationic trypsinogen mutations. Hum. Genet. 109:245-252.[CrossRef][ISI][Medline]
Chen, L., A. L. DeVries, and C. H. Cheng. 1997. Evolution of antifreeze glycoprotein gene from a trypsinogen gene in Antarctic notothenioid fish. Proc. Natl. Acad. Sci. USA 94:3811-3816.
Cheng, C. H., and L. Chen. 1999. Evolution of an antifreeze glycoprotein. Nature 40:443-444.
Craik, C. S., C. Largman, T. Fletcher, S. Roczniak, P. J. Barr, R. Fletterick, and W. J. Rutter. 1985. Redesigning trypsin: alteration of substrate specificity. Science 228:291-297.[ISI][Medline]
Davie, E., and H. Neurath. 1955. Identification of a peptide released during autocatalytic activation of trypsinogen. J. Biol. Chem. 212:515-529.
de Haen, C., K. A. Walsh, and H. Neurath. 1977. Isolation and amino-terminal sequence analysis of a new pancreatic trypsinogen of the African lungfish Protopterus aethiopicus. Biochemistry 16:4421-4425.[ISI][Medline]
Delaage, M., P. Desnuelle, M. Lazdunski, E. Bricas, and J. Savrda. 1967. On the activation of trypsinogen: a study of peptide models related to the N-terminal sequence of the zymogen. Biochem. Biophys. Res. Commun. 29:235-240.[ISI][Medline]
Douglas, S. E., and J. W. Gallant. 1998. Isolation of cDNAs for trypsinogen from the winter flounder, Pleuronectes americanus. J. Mar. Biotechnol. 6:214-219.[ISI][Medline]
Emi, M., Y. Nakamura, M. Ogawa, T. Yamamoto, T. Nishide, T. Mori, and K. Matsubara. 1986. Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. Gene 41:305-310.[CrossRef][ISI][Medline]
Ferec, C., O. Raguenes, and R. Salomon, et al. (15 co-authors). 1999. Mutations in the cationic trypsinogen gene and evidence for genetic heterogeneity in hereditary pancreatitis. J. Med. Genet. 36:228-232.
Fletcher, T. S., M. Alhadeff, C. S. Craik, and C. Largman. 1987. Isolation and characterization of a cDNA encoding rat cationic trypsinogen. Biochemistry 26:3081-3086.[ISI][Medline]
Genicot, S., F. Rentier-Delrue, D. Edwards, J. VanBeeumen, and C. Gerday. 1996. Trypsin and trypsinogen from an Antarctic fish: molecular basis of cold adaptation. Biochim. Biophys. Acta. 1298:45-57.[ISI][Medline]
Gudmundsdottir, A., E. Gudmundsdottir, S. Oskarsson, J. B. Bjarnason, A. K. Eakin, and C. S. Craik. 1993. Isolation and characterization of cDNAs from Atlantic cod encoding two different forms of trypsinogen. Eur. J. Biochem. 217:1091-1097.[Abstract]
Guy, O., D. C. Bartelt, J. Amic, E. Colomb, and C. Figarella. 1976. Activation peptide of human trypsinogen 2. FEBS Lett. 62:150-153.[CrossRef][ISI][Medline]
Guy, O., D. Lombardo, D. C. Bartelt, J. Amic, and C. Figarella. 1978. Two human trypsinogens: purification, molecular properties, and N-terminal sequences. Biochemistry 17:1669-1675.[ISI][Medline]
Halfon, S., and C. S. Craik. 1998. Trypsin. Pp. 1221 in A. J. Barrett, N. D. Rawlings, and J. F. Woessner, eds. Handbook of proteolytic enzymes. Academic Press, London.
Harris, C. I., and T. Hofmann. 1969. Studies on equine trypsinogen and trypsin. Biochem. J. 114:82P.
Hartley, B. S. 1970. Homologies in serine proteinases. Philos. Trans. R. Soc. Lond. B Biol. Sci. 257:77-87.[ISI][Medline]
Hartley, B. S. 1979. Evolution of enzyme structure. Philos. Trans. R. Soc. Lond. B Biol. Sci. 205:443-452.
Hartley, B. S., J. R. Brown, D. L. Kauffman, and L. B. Smillie. 1965. Evolutionary similarities between pancreatic proteolytic enzymes. Nature 207:1157-1159.[ISI][Medline]
Hermodson, M. A., R. W. Tye, G. R. Reeck, H. Neurath, and K. A. Walsh. 1971. Comparison of the amino terminal sequences of bovine, dogfish, and lungfish trypsinogens. FEBS Lett. 14:222-224.[CrossRef][ISI][Medline]
Hernandez-Cortes, P., L. Cerenius, F. Garcia-Carreno, and K. Soderhall. 1999. Trypsin from Pacifastacus leniusculus hepatopancreas: purification and cDNA cloning of the synthesized zymogen. Biol. Chem. 380:499-501.[ISI][Medline]
Huber, R., D. Kukla, W. Bode, P. Schwager, K. Bartels, J. Deisenhofer, and W. Steigemann. 1974. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. crystallographic refinement at 1.9 A resolution. J. Mol. Biol. 89:73-101.[ISI][Medline]
Johnson, S. C., K. V. Ewart, J. A. Osborne, D. Delage, N. W. Ross, and H. M. Murray. 2002. Molecular cloning of trypsin cDNAs and trypsin gene expression in the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae). Parasitol. Res. 88:789-796.[CrossRef][ISI][Medline]
Kim, J. C., S. H. Cha, S. T. Jeong, S. K. Oh, and S. M. Byun. 1991. Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene. Biochem. Biophys. Res. Commun. 181:707-713.[ISI][Medline]
Klein, B., G. Le Moullac, D. Sellos, and A. Van Wormhoudt. 1996. Molecular cloning and sequencing of trypsin cDNAs from Penaeus vannamei (Crustacea, Decapoda): use in assessing gene expression during the moult cycle. Int. J. Biochem. Cell Biol. 28:551-563.[CrossRef][ISI][Medline]
Kukor, Z., J. Mayerle, B. Kruger, M. Tóth, P. M. Steed, W. Halangk, M. M. Lerch, and M. Sahin-Tóth. 2002a. Presence of cathepsin B in the human pancreatic secretory pathway and its role in trypsinogen activation during hereditary pancreatitis. J. Biol. Chem. 277:21389-21396.
Kukor, Z., M. Tóth, G. Pál, and M. Sahin-Tóth. 2002b. Human cationic trypsinogen. Arg(117) is the reactive site of an inhibitory surface loop that controls spontaneous zymogen activation. J. Biol. Chem. 277:6111-6117.
Lee K. J., S. G. Kang, and I. S. Kim. 1998. Trypsin (Streptomyces exfoliatus and S. albidoflavus). Pp. 2122 in A. J. Barret, N. D. Rawlings, and J. F. Woessner, eds. Handbook of proteolytic enzymes. Academic Press, London.
Le Huerou, I., C. Wicker, P. Guilloteau, R. Toullec, and A. Puigserver. 1990. Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen: structural identity within the trypsin family. Eur. J. Biochem. 193:767-773.[Abstract]
Louvard, M. N., and A. Puigserver. 1974. On bovine and porcine anionic trypsinogens. Biochim. Biophys. Acta 371:177-185.[ISI][Medline]
Lu, D., K. Futterer, S. Korolev, X. Zheng, K. Tan, G. Waksman, and J. E. Sadler. 1999. Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide. J. Mol. Biol. 292:361-373.[CrossRef][ISI][Medline]
MacDonald, R. J., S. J. Stary, and G. H. Swift. 1982. Two similar but nonallelic rat pancreatic trypsinogens: nucleotide sequences of the cloned cDNAs. J. Biol. Chem. 257:9724-9732.
Male, R., J. B. Lorens, A. O. Smalas, and K. R. Torrissen. 1995. Molecular cloning and characterization of anionic and cationic variants of trypsin from Atlantic salmon. Eur. J. Biochem. 232:677-685.[Abstract]
Maroux, S., J. Baratti, and P. Desnuelle. 1971. Purification and specificity of porcine enterokinase. J. Biol. Chem. 246:5031-5039.
Neurath, H. 1984. Evolution of proteolytic enzymes. Science 224:350-357.[ISI][Medline]
Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:1-6.[CrossRef][ISI]
Northrop, J. H., and M. Kunitz. 1931. Isolation of protein crystals possessing tryptic activity. Science 73:262-263.
Nyaruhucha, C. N., M. Kito, and S. I. Fukuoka. 1997. Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. J. Biol. Chem. 272:10573-10578.
Pancer, Z., J. Leuck, B. Rinkevich, R. Steffen, I. Muller, and W. E. Muller. 1996. Molecular cloning and sequence analysis of two cDNAs coding for putative anionic trypsinogens from the colonial Urochordate Botryllus schlosseri (Ascidiacea). Mol. Mar. Biol. Biotechnol. 5:326-33.[ISI][Medline]
Pinsky, S. D., K. S. LaForge, and G. Scheele. 1985. Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas. Mol. Cell. Biol. 5:2669-2676.[ISI][Medline]
Radhakrishnan, T. M., K. A. Walsh, and H. Neurath. 1969. The promotion of activation of bovine trypsinogen by specific modification of aspartyl residues. Biochemistry 8:4020-4027.[ISI][Medline]
Roach, J. C. 2002. A clade of trypsins found in cold-adapted fish. Proteins 47:31-44.[CrossRef][ISI][Medline]
Roach, J. C., K. Wang, L. Gan, and L. Hood. 1997. The molecular evolution of the vertebrate trypsinogens. J. Mol. Evol. 45:640-652.[ISI][Medline]
Rypniewski, W. R., S. Hastrup, C. Betzel, M. Dauter, Z. Dauter, G. Papendorf, S. Branner, and K. S. Wilson. 1993. The sequence and X-ray structure of the trypsin from Fusarium oxysporum. Protein Eng. 6:341-348.[ISI][Medline]
Rypniewski, W. R., A. Perrakis, C. E. Vorgias, and K. S. Wilson. 1994. Evolutionary divergence and conservation of trypsin. Protein Eng. 7:57-64.[ISI][Medline]
Sahin-Tóth, M. 2000. Human cationic trypsinogen: role of Asn-21 in zymogen activation and implications in hereditary pancreatitis. J. Biol. Chem. 275:22750-22755.
Sahin-Tóth, M. 2001. The pathobiochemistry of hereditary pancreatitis: studies on recombinant human cationic trypsinogen. Pancreatology 1:461-465.[CrossRef][ISI][Medline]
Sahin-Tóth, M., and M. Tóth. 2000. Gain-of-function mutations associated with hereditary pancreatitis enhance autoactivation of human cationic trypsinogen. Biochem. Biophys. Res. Commun. 278:286-289.[CrossRef][ISI][Medline]
Schneider, A., and D. C. Whitcomb. 2002. Hereditary pancreatitis: a model for inflammatory diseases of the pancreas. Best Pract. Res. Clin. Gastroenterol. 16:347-363.[CrossRef][ISI][Medline]
Schyns, R., S. Bricteux-Gregoire, and M. Florkin. 1969. Purification, properties and N-terminal sequence of sheep trypsinogen. Biochim. Biophys. Acta 175:97-112 [in French].[ISI][Medline]
Shi, Y. B., and D. D. Brown. 1990. Developmental and thyroid hormone-dependent regulation of pancreatic genes in Xenopus laevis. Genes Dev. 4:1107-1113.[Abstract]
Simon, P., F. U. Weiss, M. Sahin-Tóth, M. Parry, O. Nayler, B. Lenfers, J. Schnekenburger, J. Mayerle, W. Domschke, and M. M. Lerch. 2002. Hereditary pancreatitis caused by a novel PRSS1 mutation (Arg-122Cys) that alters autoactivation and autodegradation of cationic trypsinogen. J. Biol. Chem. 277:5404-5410.
Spilliaert, R., and A. Gudmundsdottir. 1999. Atlantic cod trypsin Ymember of a novel trypsin group. Mar. Biotechnol. 1:598-607.[ISI][Medline]
Steiner, J. M., T. L. Medinger, and D. A. Williams. 1997. Purification and partial characterization of feline trypsin. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 116:87-93.[CrossRef][ISI][Medline]
Stevenson, B. J., O. Hagenbuchle, and P. K. Wellauer. 1986. Sequence organisation and transcriptional regulation of the mouse elastase II and trypsin genes. Nucleic Acids Res. 14:8307-8330.[Abstract]
Stroud, R. M., L. M. Kay, and R. E. Dickerson. 1974. The structure of bovine trypsin: electron density maps of the inhibited enzyme at 5 Angstrom and at 27 Angstron resolution. J. Mol. Biol. 83:185-208.[ISI][Medline]
Suzuki, T., A. S. Srivastava, and T. Kurokawa. 2002. cDNA cloning and phylogenetic analysis of pancreatic serine proteases from Japanese flounder, Paralichthys olivaceus. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 131:63-70.[CrossRef][ISI][Medline]
Szilágyi, L., E. Kenesi, G. Katona, G. Kaslik, G. Juhász, and L. Gráf. 2001. 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. 276:24574-24580.
Teich, N., H. Bodeker, and V. Keim. 2002. Cathepsin B cleavage of the trypsinogen activation peptide. BMC Gastroenterol. 2:16.[CrossRef][Medline]
Teich, N., J. Ockenga, A. Hoffmeister, M. Manns, J. Mossner, and V. Keim. 2000. Chronic pancreatitis associated with an activation peptide mutation that facilitates trypsin activation. Gastroenterology 119:461-465.[ISI][Medline]
Titani, K., L. H. Ericsson, H. Neurath, and K. A. Walsh. 1975. Amino acid sequence of dogfish trypsin. Biochemistry 14:1358-1366.[ISI][Medline]
Walsh, K. A., D. L. Kauffman, K. S. V. Sampath Kumar, and H. Neurath. 1964. On the structure and function of bovine trypsinogen and trypsin. Proc. Natl. Acad. Sci. USA 51:301-308.[ISI][Medline]
Wang, K., L. Gan, I. Lee, and L. Hood. 1995. Isolation and characterization of the chicken trypsinogen gene family. Biochem. J. 307:471-479.[ISI][Medline]
Wang, S., C. Magoulas, and D. Hickey. 1999. Concerted evolution within a trypsin gene cluster in Drosophila. Mol. Biol. Evol. 16:1117-1124.[Abstract]
Whitcomb, D. C., M. C. Gorry, and R. A. Preston, et al. (15 co-authors). 1996. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat. Genet. 14:141-145.[ISI][Medline]
Yamashina, I. 1956. The action of enterokinase on trypsinogen. Acta Chem. Scand. 10:739-743.[ISI]