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.     

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150–Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149–Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.     

Monoclonal antibodies to human pancreatic trypsin 1 inhibit the activation of human trypsinogens 1 and 2.

Two monoclonal antibodies (Mab) raised against human pancreatic trypsin 1, Mab G6 and A8, were previously isolated and characterized. The two Mab which recognize trypsinogen 1 are found to inhibit the activation of trypsinogen 1 by enterokinase. The inhibition of activation by the two Mab is concentration-dependent, rapid and virtually complete with Mab G6. Activation of trypsinogen 2 is totally inhibited by Mab G6, while Mab A8 has no effect on the activation of trypsinogen 2. The two monoclonal antibodies have opposite effects on the proteolytic activity of trypsin 1; Mab G6 increases proteolytic activity while Mab A8 inhibits trypsin activity by as much as 40%. This inhibition is concentration dependent but cannot account for the complete inhibition of activation of trypsinogen 1. Neither monoclonal antibody significantly inhibits the esterolytic activity of either form of human trypsin. Western-blot analysis of the reactivity of the two monoclonal antibodies with trypsinogens of various species shows that only Mab G6 cross-reacts with dog trypsinogen.     

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.     

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.     

Dose-related inhibition of gastric secretion by intraduodenal trypsinogen in dogs

It has been shown previously that trypsinogen and its activation peptide but not trypsin decreased gastric secretion. The purpose of this work was to study the dose-action relation between the intraduodenal infusion of trypsinogen and gastric secretion. Three dogs provided with gastric and duodenal Thomas fistulae were stimulated by continuous i.v. perfusion of porcine gastrin I-II (6 microgram kg-1 h-1). Pancreatic juice was diverted to the exterior and gastric secretion was collected. Upon reaching a gastric secretory plateau, porcine trypsinogen was infused intraduodenally at doses of 5, 10, 20, 40, 80 and 160 mg. Each test was continued for a further 60 min. Control was made with isotonic saline. There was a dose-related inhibition of the gastrin-stimulated gastric acid output. This inhibition reached a maximum of 50% with 40 mg of intraduodenal trypsinogen, showing no increase with higher doses.     

Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation

Mast cell tryptase is a secretory granule associated serine protease with trypsin-like specificity released extracellularly during mast cell degranulation. To determine the full primary structure of the catalytic domain and precursor forms of tryptase and to gain insight into its mode of activation, we cloned cDNAs coding for the complete amino acid sequence of dog mast cell tryptase and a second, possibly related, serine protease. Using RNA from dog mastocytoma cells, we constructed a cDNA library in lambda gt 10. Screening of the library with an oligonucleotide probe based on the N-terminal sequence of tryptase purified from the same cell source allowed us to isolate and sequence overlapping clones coding for dog mast cell tryptase. The tryptase sequence includes the essential residues of the catalytic triad and an aspartic acid at the base of the putative substrate binding pocket that confers P1 Arg and Lys specificity on tryptic serine proteases. The apparent N-terminal signal/activation peptide terminates in a glycine. A glycine in this position has not been observed previously in serine proteases and suggests a novel mode of activation. Additional screening of the library with a trypsinogen cDNA led to the isolation and sequencing of a full-length clone apparently coding for the complete sequence of a second tryptic serine protease (DMP) which is only 53.4% identical with the dog tryptase sequence but which contains an apparent signal/activation peptide also terminating in a glycine. Thus, the proteases encoded by these cloned cDNAs may share a common mode of activation from N-terminally extended precursors.     

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine. A possible means to regulate gene expression

Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.     

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.     

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.     

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).     

The tetra-aspartate motif in the activation peptide of human cationic trypsinogen is essential for autoactivation control but not for enteropeptidase recognition

The activation peptide of vertebrate trypsinogens contains a highly conserved tetra-aspartate sequence (Asp(19-22) in humans) preceding the Lys-Ile scissile bond. A large body of research has defined the primary role of this acidic motif as a specific recognition site for enteropeptidase, the physiological activator of trypsinogen. In addition, the acidic stretch was shown to contribute to the suppression of autoactivation. In the present study, we determined the relative importance of these two activation peptide functions in human cationic trypsinogen. Individual Ala replacements of Asp(19-22) had minimal or no effect on trypsinogen activation catalyzed by human enteropeptidase. Strikingly, a tetra-Ala(19-22) trypsinogen mutant devoid of acidic residues in the activation peptide was still a highly specific substrate for human, but not for bovine, enteropeptidase. In contrast, an intact Asp(19-22) motif was critical for autoactivation control. Thus, single Ala mutations of Asp(19), Asp(20) and Asp(21) resulted in 2-3-fold increased autoactivation, whereas the Asp(22) --> Ala mutant autoactivated at a 66-fold increased rate. These effects were multiplicative in the tri-Ala(19-21) and tetra-Ala(19-22) mutants. Structural modeling revealed that the conserved hydrophobic S2 subsite of trypsin and the unique Asp(218), which forms part of the S3-S4 subsite, participate in distinct inhibitory interactions with the activation peptide. Finally, mutagenesis studies confirmed the significance of the negative charge of Asp(218) in autoactivation control. The results demonstrate that in human cationic trypsinogen the Asp(19-22) motif per se is not required for enteropeptidase recognition, whereas it is essential for maximal suppression of autoactivation. The evolutionary selection of Asp(218), which is absent in the large majority of vertebrate trypsins, provides an additional mechanism of autoactivation control in the human pancreas.     

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.     

The identification of neotrypsinogens in samples of bovine trypsinogen

Isoelectric focusing of commercial samples of bovine trypsinogen detected a component with a lower isoelectric pH than that of trypsinogen. The isoelectric pH was 8.75 compared to 9.3 for trypsinogen, and the amount of the component varied from 16 to 41% of the total protein. The protein (24,000 Da) was converted to fragments of 13,800 and 10,500 Da on reduction with dithioerythritol, showing that the component was a modified form of trypsinogen containing a cleaved peptide bond. The cleavage site was established from the study of four polypeptide fragments which were isolated from the fully reduced and S-carboxymethylated trypsinogen. The molecular weights, amino acid compositions, and amino-terminal sequences of these fragments identified a cleavage of Lys 131-Ser 132, namely from a Ser-neotrypsinogen, or at Arg 105-Val 106, from a Val-neotrypsinogen. Val-neotrypsinogen was the more abundant of the two and was approximately 71% of the total neotrypsinogen in the trypsinogen sample. Both neotrypsinogens were converted to active trypsin molecules in high yields, showing that the zymogens closely resembled the conformation of intact trypsinogen. Presumably, the neotrypsinogens were produced during the isolation of the zymogen when pancreatic tissue was partly autolyzed and active trypsin was present.     

Purification, characterization and cDNA cloning of a trypsin from the hepatopancreas of snakehead (Channa argus)

A trypsin was purified from the hepatopancreas of snakehead (Channa argus) by ammonium sulfate fractionation and a series of column chromatographies including DEAE-Sepharose, Sephacryl S-200 HR and Hi-Trap Capto-Q. The molecular mass of the purified trypsin was about 22 kDa, as estimated by SDS-PAGE. The optimum pH and temperature of the purified trypsin were 9.0 and 40 °C, respectively. The trypsin was stable in the pH range of 7.5-9.5 and below 45 °C. The enzymatic activity was strongly inhibited by serine proteinase inhibitors, such as MBTI, Pefabloc SC, PMSF, LBTI and benzamidine. Peptide mass fingerprinting (PMF) of the purified protein obtained 2 peptide fragments with 25 amino acid residues and were 100% identical to the trypsinogen from pufferfish (Takifugu rubripes). The activation energy (Ea) of this enzyme was 24.65 kJ·M^− 1. Apparent K_m was 1.02 μM and k_cat was 148 S^− 1 for fluorogenic substrate Boc-Phe-Ser-Arg-MCA. A trypsinogen gene encoding 247 amino acid residues was further cloned on the basis of the sequence obtained from PMF and the conserved site peptide of trypsinogen together with 5′-RACE and 3′-RACE. The deduced amino acid sequence contains a signal peptide of 15 residues and an activation peptide of 9 amino acid residues with a mature protein of 223 residues. The catalytic triad His-64, Asp-107, Ser-201 and 12 Cys residues which may form 6 disulfide bonds were conserved. Compared with the PMF data, only 2 amino acid residues difference were identified, suggesting the cloned trypsinogen is quite possibly the precursor of the purified trypsin.     

Carbon-13 nuclear magnetic resonance studies of the selectively isotope-labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin

A previously characterized modification of the basic pancreatic trypsin inhibitor (BPTI), with the carbonyl carbon atom of Lys-15 selectively enriched in 13C, the peptide bond Arg-39--Ala-40 cleaved, and Arg-39 removed, was used for 13C NMR studies of the reactive site peptide bond Lys-15--Ala-16 in the complexes with trypsin, trypsinogen, and anhydrotrypsin. The chemical shift of [1-13C]Lys-15 was 175.7 ppm in the free inhibitor, 176.4 ppm in the complexes with trypsin and anhydrotrypsin and the ternary complex with trypsinogen and H-Ile-Val-OH, and 175.7 ppm in a neutral solution containing the inhibitor and trypsinogen. These data show that the trypsin--BPTI complex does not contain a covalent tetrahedral carbon atom in the position of the reactive site peptide carbonyl of the inhibitor. They would be consistent with the formation of a noncovalent complex but cannot at present be used to further characterize the degree of a possible pyramidalization of the carbonyl carbon of Lys-15 in such a complex. The identical chemical shifts in the complexes with trypsin and anhydrotrypsin indicate that the gamma-hydroxyl group of Ser-195 of trypsin does not have an important role in the binding of the inhibitor. The previously described [Perkins, S. J. & Wüthrich, K. (1980) J. Mol. Biol. 138, 43--64] stepwise transition from the trypsinogen conformation to an intermediate conformational state in the trypsinogen--BPTI complex and a trypsin-like conformation in the ternary complex trypsinogen--BPTI--H-Ile-Val-OH appears to be manifested also in the chemical shift of [1-13C]Lys-15 of labeled BPTI.     

Sepharose-bound trypsin and activated sepharose-bound trypsinogen. Studies with small and large substrates

Several enzymic and physical properties of Sepharose-bound trypsin and activated Sepharose-bound trypsinogen have been compared to those of the soluble enzyme. Sepharose-bound trypsinogen could be activated to the same extent as soluble trypsinogen; the release of the activation peptide and formation of the active site occurred as expected in the presence of catalytic amounts of trypsin. With synthetic substrates, the relative activity and pH dependence of both immobilized trypsin preparations were essentially identical and nearly the same as the soluble enzyme. Sepharose-trypsin also formed an inactive complex with soybean trypsin inhibitor, with 85% of the active sites participating. In contrast, the activity of Sepharose-trypsin with chymotrypsinogen and with trypsinogen as substrates was only 40% that of soluble trypsin. There is evidence for some catalytic heterogeneity of active sites of bound trypsin; probably those sites buried within the gel have a limited catalytic efficiency with macromolecular substrates. The immobilized enzyme is more stable than the soluble enzyme at elevated temperatures and to concentrated urea, and denaturation by urea at pH 8 is fully reversible since the loss of molecules by autolysis is eliminated.     

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.     

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.