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.     

Mass spectrometric detection of tyrosine sulfation in human pancreatic trypsinogens, but not in tumor-associated trypsinogen

Trypsinogen-1 and -2 are well-characterized enzymes that are expressed in the pancreas and also in several other tissues. Many cancers produce trypsinogen isoenzymes that differ from the pancreatic ones with respect to substrate specificity and isoelectric point. These tumor-associated trypsinogens play a pivotal role in cancer progression and metastasis. The differences between these and the pancreatic isoenzymes have been suggested to be caused by post-translational modification, either sulfation or phosphorylation of a tyrosine residue. We aimed to elucidate the cause of these differences. We isolated trypsinogens from pancreatic juice and conditioned medium from a colon carcinoma cell line. Intact proteins, and tryptic and chymotryptic peptides were characterized by electrospray ionization mass spectrometry. We also used immunoblotting with antibody against phosphotyrosine and N-terminal sequencing. The results show that pancreatic trypsinogen-1 and -2 are sulfated at Tyr154, whereas tumor-associated trypsinogen-2 is not. Detachment of a labile sulfogroup could be demonstrated by both in-source dissociation and low-energy collision-induced dissociation in a tandem mass spectrometer. Tyrosine sulfation is an ubiquitous protein modification occurring in the secretory pathway, but its significance is often underestimated due to difficulties in its analysis. Sulfation is an almost irreversible modification that is thought to regulate protein-protein interactions and the activity of proteolytic enzymes. We conclude that the previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are probably caused by sulfation of Tyr154 in pancreatic trypsinogens.     

Stability of acetylated and superguanidinated chymotrypsinogens

Conversion of lysine residues to homoarginine led to protein stabilization as determined earlier by hydrogen isotope exchange (P. Cupo W. El-Deiry, P. L. Whitney and W. M. Awad, Jr., 1980, J. Biol. Chem.255, 10828–10833). In order to see if neutralization of charges on lysine residues affected stability, a homogeneous derivative of chymotrypsinogen was prepared wherein all amino groups were acetylated. Hydrogen isotope exchange studies indicated that the derivative was less stable than the native protein. In addition, highly guanidinated chymotrypsinogen was prepared by first coupling ethylenediamine to carboxyl groups of guanidinated chymotrypsinogen. Thereafter the protein was treated with O-methylisourea to form guanidinoethylamido groups at the ends of carboxyl residues. Acrylamide gel electrophoresis indicated that two products were formed. Hydrogen isotope exchange studies demonstrated that superguanidinated chymotrypsinogen is even less stable than the acetylated derivative. Thus guanidination of residues in addition to lysine does not lead to protein stabilization. The possibility is that such a highly cationic protein causes backbone fluctuations because of repulsion of surface charges.     

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.     

Identification of two major proteins of bovine pancreatic stones as immunoreactive forms of trypsinogens.

Two major proteins have been identified in sodium citrate extracts of bovine pancreatic stones from 15 glands with lithiasis. They were found to have a molecular weight of about 24 000 and were further characterized by a variety of methods, including polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, isoelectric focusing, two-dimensional electrophoresis, immunodiffusion, immunoelectrophoresis and determination of N-terminal residues. These two immunologically and electrophoretically different proteins were definitely shown to be immunoreactive forms of anionic and cationic trypsinogens, which are normal components of pancreatic juice. However, in contrast with both secretory trypsinogens, the stone proteins displayed an important charge heterogeneity under isoelectric-focusing conditions. A possible role for both secretory trypsinogens in pancreatic lithogenesis is suggested by the reproducibility of the data. Finally, two minor proteins with a lower molecular weight (about 11 000--13 000) have also been found to be present in all extracts, but have not yet been identified.     

The two human trypsinogens. Evidence of complex formation with basic pancreatic trypsin inhibitor-proteolytic activity.

The formation of complexes between human trypsinogens and the basic pancreatic trypsin inhibitor is demonstrated by using affinity chromatography on Sepharose coupled to basic pancreatic trypsin inhibitor. This interaction indicates the pre-existence of the active site in human trypsinogens. This active site induces the proteolytic activity of the two zymogens which activate spontaneously at pH 5.6 and pH 8.0 before and after affinity chromatography. The effect of affinity-chromatography on trypsinogen spontaneous activation is not the same on trypsinogens 1 and 2. A striking difference appears between the activation of the two trypsinogens. In all cases, trypsinogen 1 autoactivates more rapidly than trypsinogen 2, except at pH 5.6 in the presence of 10 mM Ca2+, which inhibits the autoactivation of trypsinogen 1. The effect of inherent proteolytic activity of human trypsinogens is discussed in relation to pathological conditions of enterokinase deficiency and acute pancreatitis.     

Characterization of trypsinogens 1 and 2 in two human pancreatic adenocarcinoma cell lines; CFPAC-1 and CAPAN-1.

Proteins with trypsin-like immunoreactivity (first detected by a specific immunoenzymatic assay) were isolated from CAPAN-1 and CFPAC-1 cell culture-conditioned media by chromatography on an immunoadsorbent prepared with a polyclonal antibody directed against trypsin 1. The adsorbed proteins were devoid of free trypsin activity but trypsin activity was present after enterokinase activation demonstrating that the immunoreactive trypsin present in cell supernatants corresponds to trypsinogens. When characterised by Western blotting using a monoclonal antibody directed against human trypsin 1 two protein bands corresponding to trypsinogen 1 (23 kDa) and trypsinogen 2 (25 kDa) gave a positive reaction. These results demonstrate the presence of trypsinogens 1 and 2 in CAPAN-1 and CFPAC-1 cells and in their culture-conditioned media.     

Structural difference of chymotrypsinogens forming chymotrypsin variants in Japanese quail

A chymotrypsinogen showing the phenotype AA of chymotrypsin was purified from quail pancreas, and its chromatographic behavior, molecular weight, and electrophoretic mobility were very similar to those of another chymotrypsinogen, showing the aa phenotype of chymotrypsin. However, after activation, this chymotrypsinogen from AA showed band 5 of chymotrypsin, while the other chymotrypsinogen from aa did not. Peptide mapping demonstrated that the molecular structures of the two chymotrypsinogens are different. It was recognized that chymotrypsin variants result from activated products of different molecular structures of chymotrypsinogens.     

Studies on the activation of canine trypsinogens in vitro.

The activation of canine anionic and cationic trypsinogen by enterokinase, trypsin, thrombin, plasmin and extracts from canine granulocytes were studied in vitro. Enterokinase activates both trypsinogens about 1000 times faster than trypsin. The enterokinase-catalyzed activation is not inhibited by the main serum protease inhibitors, alpha-macroglobulin and alpha 1-antitrypsin. alpha-Macroglobulin cannot inhibit the activation of the trypsinogens by trypsin but this reaction is completely inhibited by alpha 1-antitrypsin. The results are discussed in relation to the pathogenesis of acute pancreatitis.     

Limited mitochondrial permeabilization is an early manifestation of palmitate-induced lipotoxicity in pancreatic beta-cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.     

Two human trypsinogens. Purification, molecular properties, and N-terminal sequences

The two human trypsinogens have been isolated from human pancreatic juice in a sufficient amount to study molecular and structural properties. The purification procedure included filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The two trypsinogens represent 19% of total proteins of pancreatic juice. Trypsinogen 1, the major form, is present in a quantity twice that of trypsinogen 2, which is the most anionic protein in human pancreatic juice. The two proteins have partial immunological identity, close molecular weights (23 438 and 25 006 for trypsinogens 1 and 2, respectively) and similar amino acid compositions. The N-terminal sequences are the same for the first 9 residues: Ala-Pro-Phe-Asp4-Lys-Ile. The two proteins differ in the activation peptides released during the transformation to trypsins. Trypsinogen 2 liberates one octapeptide Ala-Pro-Phe-Asp4-Lys while trypsinogen 1 liberates two peptides, the same octapeptide and the pentapeptide (Asp)4-Lys.