Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2 to induce inflammation and hyperalgesia

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased [Ca(2+)](i) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca(2+)](i) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.     

Trypsin IV, a novel agonist of protease-activated receptors 2 and 4

Certain serine proteases signal to cells by cleaving protease-activated receptors (PARs) and thereby regulate hemostasis, inflammation, pain and healing. However, in many tissues the proteases that activate PARs are unknown. Although pancreatic trypsin may be a physiological agonist of PAR(2) and PAR(4) in the small intestine and pancreas, these receptors are expressed by cells not normally exposed pancreatic trypsin. We investigated whether extrapancreatic forms of trypsin are PAR agonists. Epithelial cells lines from prostate, colon, and airway and human colonic mucosa expressed mRNA encoding PAR(2), trypsinogen IV, and enteropeptidase, which activates the zymogen. Immunoreactive trypsinogen IV was detected in vesicles in these cells. Trypsinogen IV was cloned from PC-3 cells and expressed in CHO cells, where it was also localized to cytoplasmic vesicles. We expressed trypsinogen IV with an N-terminal Igkappa signal peptide to direct constitutive secretion and allow enzymatic characterization. Treatment of conditioned medium with enteropeptidase reduced the apparent molecular mass of trypsinogen IV from 36 to 30 kDa and generated enzymatic activity, consistent with formation of trypsin IV. In contrast to pancreatic trypsin, trypsin IV was completely resistant to inhibition by polypeptide inhibitors. Exposure of cell lines expressing PAR(2) and PAR(4) to trypsin IV increased [Ca(2+)](i) and strongly desensitized cells to PAR agonists, whereas there were no responses in cells lacking these receptors. Thus, trypsin IV is a potential agonist of PAR(2) and PAR(4) in epithelial tissues where its resistance to endogenous trypsin inhibitors may permit prolonged signaling.     

Residue determinants and sequence analysis of cold-adapted trypsins

The digestive enzyme trypsin is among the most extensively studied proteins, and its structure has been reported from a large number of organisms. This article focuses on the trypsins from vertebrates adapted to life at low temperatures. Cold-adapted organisms seem to have compensated for the reduced reaction rates at low temperatures by evolving more active and less temperature-stable enzymes. We have analyzed 27 trypsin sequences from a variety of organisms to find unique attributes for the cold-adapted trypsins, comparing trypsins from salmon, Antarctic fish, cod, and pufferfish to other vertebrate trypsins. Both the "cold" and the "warm" active trypsins have about 50 amino acids that are unique and conserved within each class. The main unique features of the cold-adapted trypsins attributable to low-temperature adaptation seem to be (1) reduced hydrophobicity and packing density of the core, mainly because of a lower (Ile + Leu)/(Ile + Leu + Val) ratio, (2) reduced stability of the C-terminal, (3) lack of one warm trypsin conserved proline residue and one proline tyrosine stacking, (4) difference in charge and flexibility of loops extending the binding pocket, and (5) different conformation of the "autolysis" loop that is likely to be involved in substrate binding. Received: January 14, 1999 / Accepted: March 31, 1999     

Partial purification and characterization of pro-phospholipase A2 activating proteases from gill membranes of the red sea bream, Chrysophrys major

We previously reported that gill group IB secretory phospholipase A(2) (sPLA(2)) exists as an inactive pro-sPLA(2) with the dipeptide Ala-Arg, at the N-terminus of mature sPLA(2) in mucous cells. Pro-sPLA(2) should be activated after being secreted to the surface of gill epithelia by trypsin-like protease. To clarify the above hypothesis, we investigated the existence of pro-sPLA(2) activating protease (PAP) in the gills of the red sea bream, using gill pro-sPLA(2) as a substrate. PAP was solubilized from the membrane fraction of the gills with 2% sodium cholate and partially purified by benzamidine-Sepharose chromatography and reversed-phase HPLC. Partially purified proteases, PAP1 and PAP2 showed a high molecular mass of about 200 kDa by gelatin zymography. PAP1 and PAP2 had optimal pH from 7 to 9 and were inhibited by trypsin inhibitors. These properties of PAP1 and PAP2 suggest that both enzymes belong to the membrane-associated trypsin-like serine protease family, such as enteropeptidase and corin. This is the first report verifying the existence of the activating protease of group IB pro-sPLA(2) isoforms in a non-digestive tissue.     

Comparative study of gill neuroepithelial cells and their innervation in teleosts and Xenopus tadpoles

Peripheral O₂ chemoreceptors initiate adaptive cardiorespiratory responses to hypoxia in vertebrates. Morphological and physiological evidence suggests that, in fish, neuroepithelial cells (NECs) of the gill perform this role. We conducted a comparative examination in three species of teleosts (zebrafish, goldfish and trout) and larvae of the amphibian Xenopus laevis, using whole-mount gill preparations and confocal immunofluorescence, to elucidate the distribution, morphology and innervation of gill NECs. Nerve fibres were immunolabelled with the neuronal marker zn-12 and were associated with serotonin-immunoreactive NECs in the gills of all species tested. With the exception of trout, innervated NECs were present on all gill arches in the filaments and respiratory lamellae in fish and on homologous structures in Xenopus (i.e. gill “tufts”, including respiratory terminal branches). Thus, the distribution and innervation of NECs of the internal gills of amphibians and teleosts are relatively well conserved, suggesting an important role for gill NECs as O₂ chemoreceptors in aquatic vertebrates. Furthermore, the size and density of gill NECs is variable among teleosts and developmental stages of Xenopus larvae and may be dependent on general gill dimensions or environmental conditions. This report constitutes the first comparative study of gill NECs in fish and amphibians and highlights the significance of gill NECs as an evolutionary model for studying O₂ sensing in vertebrates. We acknowledge the Natural Sciences and Engineering Research Council (NSERC) of Canada for funding through an operating grant to C.A.N., and the NSERC and the Ontario Graduate Scholarship (OGS) program for postgraduate scholarships to M.G.J.     

Immunocytochemical Localization of Serotonin and Neuropeptides in the Neuroendocrine Paraneurons of Teleost and Lungfish Gills*

Neuroendocrine (NE) cells in the gills of six fish species were identified by serotonin and peptide immunohistochemistry. They occur either as single cells or as cell clusters within the filamental epithelium. Two populations of NE cells were identified that exhibit an immunoreactivity either for serotonin alone or for met-enkephalin and leu-enkephalin co-distributed in the same cells. We demonstrate for the first time the occurrence of open-type endocrine cells in the fish gill epithelium and confirm the presence of only serotonin-immunopositive cells of the closed type. The NE cells of the species studied do not react positively to other neuroendocrine markers occurring in the mammalian lung. Great differences probably exist in the expression of these epitopes on antigenic structures among various animal species. Although the physiological role of neuropeptides and serotonin in the fish gill NE cells is unknown, the presence of these molecular markers is of interest not only from an evolutionary aspect, but also in terms of a potential animal model to study the supposed functions of the airway neuroendocrine system in the vertebrates.     

Comparison of sea turtle thrombocyte aggregation to human platelet aggregation in whole blood

The endangered sea turtles are living "fossils" that afford us an opportunity to study the hemostatic process as it likely existed millions of years ago. There are essentially no data about turtle thrombocyte aggregation prior to our studies. Thrombocytes are nucleated cells that serve the same hemostatic functions as the anucleated mammalian platelet. Sea turtle thrombocytes aggregate in response to collagen and beta-thrombin. Ristocetin induces an agglutination/aggregation response indicating the presence of a von Willebrand-like receptor, GPIb, found in all mammalian platelets. Samples treated with alpha-thrombin plus gamma-thrombin followed by ristocetin results in a rapid, stronger response than ristocetin alone. These responses are inhibited by the RGDS peptide that blocks fibrinogen cross-linking of mammalian platelets via the fibrinogen receptor, GPIIb/IIIa. Three platelet-like proteins, GPIb, GPIIb/IIIa and P-selection are detected in sea turtle thrombocytes by fluorescence activated cell sorting. Turtle thrombocytes do not respond to ADP, epinephrine, serotonin, thromboxane A2 mimetic, U46619, trypsin, or alpha-thrombin and gamma-thrombin added alone. Comparison of hemostasis in sea turtles to other vertebrates could provide a framework for understanding the structure/function and evolution of these pathways and their individual components.     

Serine proteases mediate inflammatory pain in acute pancreatitis

Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR(2)), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR(2) and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Injections of a subinflammatory dose of exogenous trypsin increased c-Fos immunoreactivity, indicative of spinal nociceptive activation, but did not cause inflammation, as assessed by measuring serum amylase and myeloperoxidase activity and by histology. The same dose of trypsin IV and P23 increased some inflammatory end points and caused a more robust effect on nociception, which was blocked by melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant trypsin isoforms. To determine the contribution of endogenous activation of trypsin and its minor isoforms, recombinant enterokinase (ENK), which activates trypsins in the duodenum, was administered into the pancreas. Intraductal ENK caused nociception and inflammation that were diminished by polypeptide inhibitors, including soybean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by melagatran. Finally, the secretagogue cerulein induced pancreatic nociceptive activation and nocifensive behavior that were reversed by melagatran. Thus trypsin and its minor isoforms mediate pancreatic pain and inflammation. In particular, the inhibitor-resistant isoforms trypsin IV and P23 may be important in mediating prolonged pancreatic inflammatory pain in pancreatitis. Our results suggest that inhibitors of these isoforms could be novel therapies for pancreatitis pain.     

The ultrastructure of the blood cells of the pike Esox lucius L.

The ultrastructure of pike peripheral blood cells, lymphocytes, thrombocytes, granulocytes, and monocytes is described. At present there are no reliable criteria for differentiating between round thrombocytes and small lymphocytes of fish on a routine basis. At the ultrastructural level thrombocytes could be clearly differentiated from lymphocytes by cytoplasmic canals and vesicles, marginal microtubules, and large glycogen deposits. Electron microscopic identification of thrombocytes was confirmed by examining the ultrastructural features of a purified thrombocyte fraction. In addition, a preliminary investigation of the structure of the haemopoietic cells in the thymus, anterior kidney, and spleen was carried out.     

Effect of phospholipase A on erythrocyte and thrombocyte aggregation]

A study was made of the effect of plasmin and trypsin on the phospholipase activation, and also of the action of phospholipase A (cobra venom) on the release reaction and the erythrocyte and thrombocyte aggregation. Trypsin and fibrinolysin proved to activate phospholipase, this being accompanied by the accumulation of nonesterified fatty acids in the blood serum. Phospholipase A caused a release of the thromboplastic factor from erythrocytes and thrombocytes and their aggregation. The later is inhibited by albumin and EDTA. It is suggested that the action of the proteolytic enzymes on the blood formed elements was realized through the phospholipase activation.     

First demonstration of the neuroepithelial cells and their chemical code in the accessory respiratory organ and the gill of the sharptooth catfish,Clarias gariepinus: A preliminary study

Available studies that have examined O₂ sensing in fish have indicated that oxygen-sensitive neuroepithelial cells (NECs) are O₂ sensors in the gills and initiate cardiorespiratory reflexes in aquatic vertebrates. This is the first study describing the occurrence of NECs in accessory respiratory organs in the air-breathing catfish Clarias gariepinus. Immunocytochemical stainings with specific neuronal markers such as nNOS, VAchT, 5-HT and TH have been shown to be very useful for location and distribution of these cells in the gill fans and suprabranchial chamber that take origin from the transformation of the gill tissue. But the response of these putative O₂ chemoreceptors, their role in the respiratory reflexes and their innervation await investigation.     

Proteolysis of the exodomain of recombinant protease-activated receptors: prediction of receptor activation or inactivation by MALDI mass spectrometry

Protease-activated receptors (PARs) mediate cell activation after proteolytic cleavage of their extracellular amino terminus. Thrombin selectively cleaves PAR1, PAR3, and PAR4 to induce activation of platelets and vascular cells, while PAR2 is preferentially cleaved by trypsin. In pathological situations, other proteolytic enzymes may be generated in the circulation and could modify the responses of PARs by cleaving their extracellular domains. To assess the ability of such proteases to activate or inactivate PARs, we designed a strategy for locating cleavage sites on the exofacial NH(2)-terminal fragments of the receptors. The first extracellular segments of PAR1 (PAR1E) and PAR2 (PAR2E) expressed as recombinant proteins in Escherichia coli were incubated with a series of proteases likely to be encountered in the circulation during thrombosis or inflammation. Kinetic and dose-response studies were performed, and the cleavage products were analyzed by MALDI-TOF mass spectrometry. Thrombin cleaved PAR1E at the Arg41-Ser42 activation site at concentrations known to induce cellular activation, supporting a native conformation of the recombinant polypeptide. Plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3 cleaved at multiple sites and would be expected to disable PAR1 by cleaving COOH-terminal to the activation site. Cleavage specificities were further confirmed using activation site defective PAR1E S42P mutant polypeptides. Surface plasmon resonance studies on immobilized PAR1E or PAR1E S42P were consistent with cleavage results obtained in solution and allowed us to determine affinities of PAR1E-thrombin binding. FACS analyses of intact platelets confirmed the cleavage of PAR1 downstream of the Arg41-Ser42 site. Mass spectrometry studies of PAR2E predicted activation of PAR2 by trypsin through cleavage at the Arg36-Ser37 site, no effect of thrombin, and inactivation of the receptor by plasmin, calpain and leukocyte elastase, cathepsin G, and proteinase 3. The inhibitory effect of elastase was confirmed on native PAR1 and PAR2 on the basis of Ca(2+) signaling studies in endothelial cells. It was concluded that none of the main proteases generated during fibrinolysis or inflammation appears to be able to signal through PAR1 or PAR2. This strategy provides results which can be extended to the native receptor to predict its activation or inactivation, and it could likewise be used to study other PARs or protease-dependent processes.     

Localization of group IB phospholipase A(2) isoform in the gills of the red sea bream,Pagrus (Chrysophrys) major

We previously reported that PLA(2) activity in the gills is higher than that in other tissues in red sea bream and purified PLA(2) from the gills belongs to the group IB PLA(2) as well as other red sea bream PLA(2)s. In this study, we reconfirmed that the level of PLA(2) activity is extremely high in the gills compared with other tissues, and gill PLA(2) was detected only in the gills by immunoblotting and inhibition test using anti-gill PLA(2) monoclonal antibody. The level of PLA(2) activity and protein expression in the gills are well correlated. Fish can be roughly divided into high and low groups based on the level of PLA(2) activity. Gill PLA(2) was detected in the gills of the high group, but not the low group by immunoblotting. In the gills of the high group, gill PLA(2) was detected in the mucous cells and pavement cells located on the surface of gill epithelia by immunohistochemistry. On the other hand, positive signals were observed only in the mucous cells by in situ hybridization. We also isolated inactive proPLA(2), having AR propeptide, preceding the mature enzyme from the gill extract. These results suggest that gill PLA(2) is synthesized as an inactive proPLA(2) in the mucous cells and is secreted to the surface of gill epithelia.     

Trypsin induces activation and inflammatory mediator release from human eosinophils through protease-activated receptor-2.

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50-70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.     

Autoproteolytic stability of a trypsin from the marine crab Cancer pagurus

Autoproteolytic stability is a crucial factor for the application of proteases in biotechnology. In contrast to vertebrate enzymes, trypsins from shrimp and crayfish are known to be resistant against autolysis. We show by characterisation of a novel trypsin from the gastric fluid of the marine crab Cancer pagurus that this property might be assigned to the entire class of crustaceans. The isolated and cloned crab trypsin (C.p.TryIII) exhibits all characteristic properties of crustacean trypsins. However, its overall sequence identity to other trypsins of this systematic class is comparatively low. The high resistance against autoproteolysis was determined by mass spectrometry, which revealed a low susceptibility of the N-terminal domain towards autolysis. By homology modelling of the tertiary structure, the elevated stability was attributed to the distinctly different pattern of autolytic cleavage sites, which is conserved in all known crustacean trypsin sequences.     

The midgut trypsins of shrimp (Penaeus monodon). High efficiency toward native protein substrates including collagens

Two shrimp trypsins have been purified from the midguts of Penaeid shrimps by various chromatographies and HPLC. The molecular masses of them are 27 and 29 kDa, respectively. They show the typical specificity of trypsin for substrates and inhibitors, and their N-terminal amino-acid sequences are homologous to those of other trypsins. The shrimp enzymes are very acidic (pI less than or equal to 2.4), and show distinctively low Km for the synthetic amide substrates. They also hydrolyse various native proteins more efficiently than bovine trypsin in vitro. However, the anionic shrimp trypsins do not have special preference for basic protein substrates over the acidic one. Collagenolytic activity of the midgut extract was mainly due to serine proteases. The collagenolytic activity of the purified shrimp trypsin was confirmed by assays with either soluble or insoluble native type I collagens. In comparison with the other trypsins from the Crustacean decapods, the shrimp enzymes have four pairs of disulfide bonds, intermediary between the crayfish trypsin (three pairs) and the crab trypsin (five pairs), and are immunochemically different from them.     

A trypsin homolog in amphioxus: expression, enzymatic activity and evolution

Trypsin has been documented in a variety of species including both vertebrates and invertebrates, but little is known about it in amphioxus, a model organism for insights into the origin and evolution of vertebrates. Here we identified a trypsin gene in Branchiostoma japonicum. The cDNA was 978 bp long with an ORF encoding a deduced protein of 272 amino acids. The deduced protein had an N-terminal signal peptide of 15 amino acids, a 16 activation peptide with the typical cleavage site Arg/Ile, a Tryp_SPc domain with the catalytic triad His⁷²-Asp¹¹⁸-Ser²¹⁵ and the S1 substrate binding residue Asp²⁰⁹, which are all characteristic of trypsinogens. The recombinant trypsin protein was able to hydrolyse the trypsin prototypic substrate BAEE, which was inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. Both northern blotting and tissue-section in situ hybridization demonstrated that trypsin gene was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum, mid-gut and ovary. And the whole mount in situ hybridization showed that it began to express in the middle third of the full-length primitive gut in 2-day larvae, where the hepatic caecum will form later during development. Phylogenetic analysis indicated that both amphioxus and ascidian trypsins are more closer to each other than to vertebrate trypsins, suggesting a continuous evolutionary divergence of vertebrate trypsins after split from protochordate/vertebrate common ancestor.     

Atlantic Cod Trypsins: From Basic Research to Practical Applications

Atlantic cod trypsin I is an appropriate representative of the traditionally classified cold-adapted group I trypsins, and the recombinant form of cod trypsin Y is the only biochemically characterized member of the novel group III trypsins. Trypsin Y is adapted to lower temperatures than all other presently known trypsins. This review describes the basic characteristics of and practical uses for trypsins of Atlantic cod, as well as those of other organisms. Overexpression of the recombinant forms of cod trypsins I and Y in microorganisms is explained as well as the advantages of using site-directed mutagenesis to increase their stability toward autolysis and thermal inactivation. Trypsins appear to play a key role in the nutrition and development of marine fish. We discuss the potential use of cod trypsins as biomarkers to evaluate the nutritional status of cod larvae and describe the industrial applications of cod trypsin I and other trypsins.