Human cationic trypsinogen. Purification, characterization, and characteristics of autoactivation

Human pancreatic cationic trypsinogen has been purified to homogenity from an acetone powder of pancreatic tissue. After an initial ion exchange chromatography step on sulfopropyl (SP)-Sephadex at pH 2.6, cationic trypsinogen was separated from the majority of trypsin activity by passage through an affinity column of lima bean trypsin inhibitor-agarose at high ionic strength. The zymogen was then further purified by affinity chromatography on the same material at low ionic strength. Highly purified trypsinogen was resolved from containing chymotrypsinogen by ion exchange chromatography on SP-Sephadex at pH 6.0. The purified zymogen was shown to be homogeneous by polyacrylamide gel electrophoresis at pH 2.1 and at pH 4.3 as well as by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The autoactivation of human trypsinogen was investigated at pH 5.6 and at pH 8.0. The rate of autoactivation of the human zymogen is rapid at pH 5.6 and is maximal in approximately 1 mM Ca2+. These results are in marked contrast to those previously reported for autoactivation of bovine trypsinogen, which is extremely slow at pH 5.6 and which shows a dependence on at least 50 mM Ca2+ for maximum rate of activation (MacDonald, M. R., AND Kunitz, M. (1941) J. Gen. Physiol. 25, 53-73).     

Human cationic trypsinogen is sulfated on Tyr154

The crystal structure of human pancreatic cationic trypsin showed the chemical modification of Tyr154, which was originally described as phosphorylation [Gaboriaud C, Serre L, Guy-Crotte O, Forest E & Fontecilla-Camps JC (1996) J Mol Biol259, 995-1010]. Here we report that Tyr154 is sulfated, not phosphorylated. Cationic and anionic trypsinogens were purified from human pancreatic juice and subjected to alkaline hydrolysis. Modified tyrosine amino acids were separated on a Dowex cation-exchange column and analyzed by thin layer chromatography. Both human cationic and anionic trypsinogens contained tyrosine sulfate, but no tyrosine phosphate, whereas bovine trypsinogen contained neither. Furthermore, incorporation of [(35)S]SO(4) into human cationic trypsinogen transiently expressed by human embryonic kidney 239T cells was demonstrated. Mutation of Tyr154 to Phe abolished radioactive sulfate incorporation, confirming that Tyr154 is the site of sulfation in cationic trypsinogen. Sulfated pancreatic cationic trypsinogen exhibited faster autoactivation than a nonsulfated recombinant form, suggesting that tyrosine sulfation of trypsinogens might enhance intestinal digestive zymogen activation in humans. Finally, sequence alignment revealed that the sulfation motif is only conserved in primate trypsinogens, suggesting that typsinogen sulfation is absent in other vertebrates.     

Human anionic trypsinogen: properties of autocatalytic activation and degradation and implications in pancreatic diseases.

Human pancreatic secretions contain two major trypsinogen isoforms, cationic and anionic trypsinogen, normally at a ratio of 2 : 1. Pancreatitis, pancreatic cancer and chronic alcoholism lead to a characteristic reversal of the isoform ratio, and anionic trypsinogen becomes the predominant zymogen secreted. To understand the biochemical consequences of these alterations, we recombinantly expressed and purified both human trypsinogens and documented characteristics of autoactivation, autocatalytic degradation and Ca2+-dependence. Even though the two trypsinogens are approximately 90% identical in their primary structure, we found that human anionic trypsinogen and trypsin exhibited a significantly increased (10-20-fold) propensity for autocatalytic degradation, relative to cationic trypsinogen and trypsin. Furthermore, in contrast to the characteristic stimulation of the cationic proenzyme, acidic pH inhibited autoactivation of anionic trypsinogen. In mixtures of cationic and anionic trypsinogen, an increase in the proportion of the anionic proenzyme had no significant effect on the levels of trypsin generated by autoactivation or by enterokinase at pH 8.0 in 1 mm Ca2+- conditions that were characteristic of the pancreatic juice. In contrast, rates of trypsinogen activation were markedly reduced with increasing ratios of anionic trypsinogen under conditions that were typical of potential sites of pathological intra-acinar trypsinogen activation. Thus, at low Ca2+ concentrations at pH 8.0, selective degradation of anionic trypsinogen and trypsin caused diminished trypsin production; while at pH 5.0, inhibition of anionic trypsinogen activation resulted in lower trypsin yields. Taken together, the observations indicate that up-regulation of anionic trypsinogen in pancreatic diseases does not affect physiological trypsinogen activation, but significantly limits trypsin generation under potential pathological conditions.     

Isolation and characterization of a cDNA encoding rat cationic trypsinogen

A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.     

Increased activation of hereditary pancreatitis-associated human cationic trypsinogen mutants in presence of chymotrypsin C

Mutations in human cationic trypsinogen (PRSS1) cause autosomal dominant hereditary pancreatitis. Increased intrapancreatic autoactivation of trypsinogen mutants has been hypothesized to initiate the disease. Autoactivation of cationic trypsinogen is proteolytically regulated by chymotrypsin C (CTRC), which mitigates the development of trypsin activity by promoting degradation of both trypsinogen and trypsin. Paradoxically, CTRC also increases the rate of autoactivation by processing the trypsinogen activation peptide to a shorter form. The aim of this study was to investigate the effect of CTRC on the autoactivation of clinically relevant trypsinogen mutants. We found that in the presence of CTRC, trypsinogen mutants associated with classic hereditary pancreatitis (N29I, N29T, V39A, R122C, and R122H) autoactivated at increased rates and reached markedly higher active trypsin levels compared with wild-type cationic trypsinogen. The A16V mutant, known for its variable disease penetrance, exhibited a smaller increase in autoactivation. The mechanistic basis of increased activation was mutation-specific and involved resistance to degradation (N29I, N29T, V39A, R122C, and R122H) and/or increased N-terminal processing by CTRC (A16V and N29I). These observations indicate that hereditary pancreatitis is caused by CTRC-dependent dysregulation of cationic trypsinogen autoactivation, which results in elevated trypsin levels in the pancreas.     

Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas.

In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.     

Chymotrypsin C (caldecrin) stimulates autoactivation of human cationic trypsinogen

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.     

Expression of mutated cationic trypsinogen reduces cellular viability in AR4-2J cells

Mutations in the human cationic trypsinogen are associated with hereditary pancreatitis. The cDNA coding for human cationic trypsinogen was subcloned into the expression vector pcDNA3. The mutations R122H, N29I, A16V, D22G, and K23R were introduced by site directed mutagenesis. We constructed an expression vector coding for active trypsin by subcloning the cDNA of trypsin lacking the coding region for the trypsin activating peptide behind an appropriate signal peptide. Expression of protein was verified by Western blot and measurement of enzymatic activity. AR4-2J cells were transiently transfected with the different expression vectors and cell viability and intracellular caspase-3 activity were quantified. In contrast to wild-type trypsinogen, expression of active trypsin and mutated trypsinogens reduced cell viability of AR4-2J cells. Expression of trypsin and R122H trypsinogen induced caspase-3 activity. Acinar cells might react to intracellular trypsin activity by triggering apoptosis.     

Comparative studies on the mechanism of activation of the two human trypsinogens

The activation of human trypsinogens 1 and 2 by porcine enterokinase at pH 5.6 shows that the two human zymogens are equivalent substrates for this enzyme and that both proteins are activated faster than the cationic bovine trypsinogen. At pH 8.0 and in the presence of 20 mM calcium the two human trypsinogens are activated by either human trypsin at the same rate but the affinity of both trypsins is higher for trypsinogen 1 than for trypsinogen 2. Two Ca2+ binding sites are identified in the two human zymogens and their pK(Ca2+) values determined. For trypsinogen 1 the values are respectively of 2.8 and 3.3 for the primary and secondary Ca2+ binding sites, and for trypsinogen 2 of 3.4 and 2.7. These values are markedly different from those obtained for bovine cationic trypsinogen, especially in the case of trypsinogen 1. These results point out a different degree of saturation of the calcium binding sites of the 2 human zymogens that must exist in physiological conditions, suggesting different biological activities of the two trypsinogens.     

Limited proteolyses in pancreatic chymotrypsinogens and trypsinogens

In the 1950's, the specific cleavages of the peptide bonds occurring in bovine cationic chymotrypsinogen and trypsinogen were among the first examples of limited proteolyses. According to the split bond(s), the precursor is transformed into enzyme or different forms of zymogen or again into inert protein. The conversion of trypsinogen into trypsin triggers the activations of all the other enzyme precursors of pancreatic juice. In the pancreas, several factors oppose trypsinogen autoactivation, whereas, in the duodenum, all the conditions are favorable for trypsinogen activation by enteropeptidase.     

Presence of cathepsin B in the human pancreatic secretory pathway and its role in trypsinogen activation during hereditary pancreatitis

The lysosomal cysteine protease cathepsin B is thought to play a central role in intrapancreatic trypsinogen activation and the onset of experimental pancreatitis. Recent in vitro studies have suggested that this mechanism might be of pathophysiological relevance in hereditary pancreatitis, a human inborn disorder associated with mutations in the cationic trypsinogen gene. In the present study evidence is presented that cathepsin B is abundantly present in the secretory compartment of the human exocrine pancreas, as judged by immunogold electron microscopy. Moreover, pro-cathepsin B and mature cathepsin B are both secreted together with trypsinogen and active trypsin into the pancreatic juice of patients with sporadic pancreatitis or hereditary pancreatitis. Finally, cathepsin B- catalyzed activation of recombinant human cationic trypsinogen with hereditary pancreatitis-associated mutations N29I, N29T, or R122H were characterized. In contrast to a previous report, cathepsin B-mediated activation of wild type and all three mutant trypsinogen forms was essentially identical under a wide range of experimental conditions. These observations confirm the presence of active cathepsin B in the human pancreatic secretory pathway and are consistent with the notion that cathepsin B-mediated trypsinogen activation might play a pathogenic role in human pancreatitis. On the other hand, the results clearly demonstrate that hereditary pancreatitis-associated mutations do not lead to increased or decreased trypsinogen activation by cathepsin B. Therefore, mutation-dependent alterations in cathepsin B-induced trypsinogen activation are not the cause of hereditary pancreatitis.     

SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas

ZG29p, a novel pancreas-specific zymogen granule protein, has been proposed to act as a 'helper protein' in granule formation. To address its function in more detail, we searched for putative binding partners of ZG29p. In zymogen complexes isolated by nondenaturing isoelectric focusing, ZG29p was associated with a protein complex consisting of amylase and cationic trysinogen. Amylase also coeluted with ZG29p after immunoaffinity chromatography using an antibody to recombinant ZG29p. Cross-linking experiments with granule content proteins revealed a direct interaction between recombinant ZG29p and amylase. An interaction was also observed when purified amylase was used, whereas no interaction with recombinant or purified cationic trypsinogen was seen. ZG29p could also be cross-linked to three membrane proteins with molecular masses of 40, 18, and 16 kDa. The binding of ZG29p to amylase and to the membrane proteins was inhibited in the presence of synthetic peptides matching the consensus sequence of proline-rich SH3 binding sites present in ZG29p. The synthetic peptides could be cross-linked to amylase and to three yet unidentified acidic content proteins with molecular masses of about 30 kDa. The peptides also interacted with purified or recombinant amylase, but not with recombinant or purified cationic trypsinogen. In a condensation-sorting assay, the binding (sorting) of zymogen complexes to the granule membrane was reduced in the presence of the peptides. Our results indicate that the interaction of ZG29p with amylase is mediated by SH3 binding domains and that these domains are involved in the sorting of amylase to the granule membrane.     

Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family.

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855-nucleotide mRNA contains a short 5' noncoding region of 8 nucleotides and a long 3' noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N-terminal 15-amino-acid signal peptide followed by an 8-amino-acid activation peptide. The zymogen (232 residues) contains an additional C-terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73-85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.     

Molecular pathology and evolutionary and physiological implications of pancreatitis-associated cationic trypsinogen mutations

Since the identification in 1996 of a "gain of function" missense mutation, R122H, in the cationic trypsinogen gene (PRSS1) as a cause of hereditary pancreatitis, continued screening of this gene in both hereditary and sporadic pancreatitis has found more disease-associated missense mutations than expected. In addition, functional analysis has yielded interesting findings regarding their underlying mechanisms resulting in a gain of trypsin. A critical review of these data, in the context of the complicated biogenesis and complex autoactivation and autolysis of trypsin(ogen), highlights that PRSS1 mutations cause the disease by various mechanisms depending on which biochemical process they affect. The discovery of these mutations also modifies the classical perception of the disease and, more importantly, reveals fascinating new aspects of the molecular evolution and normal physiology of trypsinogen. First, activation peptide of trypsinogen is under strong selection pressure to minimize autoactivation in higher vertebrates. Second, the R122 primary autolysis site has further evolved in mammalian trypsinogens. Third, evolutionary divergence from threonine to asparagine at residue 29 in human cationic trypsinogen provides additional advantage. Accordingly, we tentatively assign, in human cationic trypsinogen, the strongly selected activation peptide as the first-line and the R122 autolysis site as the second-line of the built-in defensive mechanisms against premature trypsin activation within the pancreas, respectively, and the positively selected asparagine at residue 29 as an "amplifier" to the R122 "fail-safe" mechanism.     

Immunoreactive trypsins in sera from dogs before and after induction of experimental pancreatitis

Radioimmunoassays for anionic and cationic dog trypsins are described. Characterization of the immunoreactivities in sera from fasting dogs demonstrated the presence of the two proenzymes only. Fasting sera from 10 dogs contained anionic and cationic trypsinogen in concentrations between 17-110 micrograms/l and 7-19 micrograms/l, respectively. Induction of experimental pancreatitis in dogs was accompanied by a large increase of immunoreactive anionic and cationic trypsins in the circulation. During the progress of the pancreatitis, immunoreactive trypsin with larger molecular weight than trypsinogen appeared. This high molecular weight immunoreactive trypsin was not seen in serum after intravenous injection of pancreatic juice in dogs. The high molecular weight immunoreactive trypsin probably consists of trypsin in complex with protease inhibitors. In vitro studies showed that the immunoreactivity of trypsin decreased considerably after binding to alpha 1-antitrypsin or alpha-macroglobulins.     

Gain-of-function mutations associated with hereditary pancreatitis enhance autoactivation of human cationic trypsinogen

Hereditary pancreatitis (HP), an autosomal dominant disorder, has been associated with mutations in the cationic trypsinogen gene. Here we demonstrate that the two most frequent HP mutations, Arg117 --> His and Asn21 --> Ile, significantly enhance autoactivation of human cationic trypsinogen in vitro, in a manner that correlates with the severity of clinical symptoms in HP. In addition, mutation Arg117 --> His inhibits autocatalytic inactivation of trypsin, while mutation Asn21 --> Ile has no such effect. The findings strongly argue that increased trypsinogen activation in the pancreas is the common initiating step in both forms of HP, whereas trypsin stabilization might also contribute to HP associated with the Arg117 --> His mutation.     

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.     

Evolution of trypsinogen activation peptides

The activation peptide of mammalian trypsinogens contains a highly conserved tetra-aspartate sequence (D19-D20-D21-D22) preceding the K23-I24 scissile peptide bond, which is hydrolyzed as the first step in the activation process. Here, we examined the evolution and function of trypsinogen activation peptides through integrating functional characterization of disease-associated mutations with comparative genomic analysis. Activation properties of three chronic pancreatitis-associated activation peptide mutants (the novel D19A and the previously reported D22G and K23R) were simultaneously analyzed, for the first time, in the context of recombinant human cationic trypsinogen. A dramatic increase in autoactivation of cationic trypsinogen was observed in all three mutants, with D22G and K23R exhibiting the most marked increases. The physiological activator enteropeptidase activated the D19A mutant normally, activated the D22G mutant very poorly, and stimulated activation of the K23R mutant. The biochemical and structural data, taken together with a comprehensive sequence comparison, indicates that the tetra-aspartate sequence in mammalian trypsinogen activation peptides has evolved not only for optimal enteropeptidase recognition in the duodenum but also for efficient inhibition of trypsinogen autoactivation within the pancreas. Moreover, the use of lysine instead of arginine at the P1 position of activation peptides also has an advantageous effect against trypsinogen autoactivation. Finally, fixed substitutions in the key residues of the trypsinogen activation peptide may suggest the evolution of new functions unrelated to digestion, as found in the group III trypsinogens of cold-adapted fishes.     

Trypsinogen stabilization by mutation Arg117-->His: a unifying pathomechanism for hereditary pancreatitis?

Mutations Arg117-->His and Asn21-->Ile of the human cationic trypsinogen have been recently identified in patients affected by hereditary pancreatitis (HP). The Arg117-->His substitution is believed to cause pancreatitis by eliminating an essential autolytic cleavage site in trypsin, thereby rendering the protease resistant to inactivation through autolysis. Here we demonstrate that the Arg117-->His mutation also significantly inhibits autocatalytic trypsinogen breakdown under Ca(2+)-free conditions and stabilizes the zymogen form of rat trypsin. Taken together with recent findings demonstrating that the Asn21-->Ile mutation stabilizes rat trypsinogen against autoactivation and consequent autocatalytic degradation, the observations suggest a unifying molecular pathomechanism for HP in which zymogen stabilization plays a central role.