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PubMed2005

Diversity of trypsins in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae), revealed by nucleic acid sequences and enzyme purification.

The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the optimisation of the use of protease inhibitors for pest control, and on the identification of endoprotease-mediated resistance to Bacillus thuringiensis Cry-toxins is discussed.

Amino Acid Sequence↗

PubMed1982

Functional modifications of alpha 2-macroglobulin by primary amines. Kinetics of inactivation of alpha 2-macroglobulin by methylamine, and formation of anomalous complexes with trypsin.

The unique steric inhibition of endopeptidases by human alpha(2)M (alpha(2)-macroglobulin) and the inactivation of the latter by methylamine were examined in relation to each other. Progressive binding of trypsin by alpha(2)M was closely correlated with the loss of the methylamine-reactive sites in alpha(2)M: for each trypsin molecule bound, two such sites were inactivated. The results further showed that, even at low proteinase/alpha(2)M ratios, no unaccounted loss of trypsin-binding capacity occurred. As alpha(2)M is bivalent for trypsin binding and no trypsin bound to electrophoretic slow-form alpha(2)M was observed, this indicates that the two sites must react (bind trypsin) in rapid succession. Reaction of [(14)C]methylamine with alpha(2)M was biphasic in time; in the initial rapid phase complex-formation with trypsin caused a largely increased incorporation of methylamine. In the subsequent slow phase trypsin had no such effect. These results prompted further studies on the kinetics of methylamine inactivation of alpha(2)M with time of methylamine treatment. It was found that conformational change of alpha(2)M and decrease in trypsin binding (activity resistant to soya-bean trypsin inhibitor) showed different kinetics. The latter decreased rapidly, following pseudo-first-order kinetics. Conformational change was much slower and followed complex kinetics. On the other hand, binding of (125)I-labelled trypsin to alpha(2)M did follow the same kinetics as the conformational change. This discrepancy between total binding ((125)I radioactivity) and trypsin-inhibitor-resistant binding of trypsin indicated formation of anomalous complexes, in which trypsin could still be inhibited by soya-bean trypsin inhibitor. Further examination confirmed that these complexes were proteolytically active towards haemoglobin and bound (125)I-labelled soya-bean trypsin inhibitor to the active site of trypsin. The inhibition by soya-bean trypsin inhibitor was slowed down as compared with reaction with free trypsin. The results are discussed in relation to the subunit structure of alpha(2)M and to the mechanism of formation of the complex.

Binding Sites↗

PubMed1989

Human cationic and anionic trypsins: differences of interaction with alpha 1-proteinase inhibitor.

Human cationic (trypsin 1) and anionic (trypsin 2) trypsins were obtained by controlled activation of purified trypsinogens 1 and 2, respectively. The interactions of trypsin 1 and trypsin 2 with human alpha 1-proteinase inhibitor (alpha 1PI) were analysed and compared by studies in vitro. The enzymatic activity and inhibitory capacity measurements were assessed using Glp-Gly-Arg-Nan as substrate. The association rate constants showed that the inhibition of trypsin 2 occurred more than 10 times faster than that of trypsin 1. The equimolar complexes obtained between either trypsin and alpha 1PI were visualized by electrophoresis followed by immunoblotting. The inhibition of the two trypsins was temporary i.e. the complexes trypsin 1-alpha 1PI and trypsin 2-alpha 1PI broke down with time yielding inactive alpha 1PI (Mr 50,000) and active enzyme. But the stability time for trypsin 1-alpha 1PI was much larger than that of trypsin 2-alpha 1PI. In vivo, alpha 1PI is not able to control the activity of trypsin 1 except when alpha 2-macroglobulin (alpha 2M) is already saturated. According to the delay times of inhibition calculated from normal concentrations in serum, alpha 1PI inhibits trypsin 2 as fast as alpha 2M inhibits trypsin 1. These results suggest that a significant role can be assigned to alpha 1PI in the inhibition of trypsin 2 in physiological conditions and of trypsin 1 in pathological ones.

Electrophoresis, Polyacrylamide Gel↗

PubMed1976

[Preparation and some properties of immobilized trypsin from the crayfish Cambarus affinis Say (author's transl)].

The anionic tryptic enzyme from the crayfish (crayfish trypsin) was adsorbed to DEAE-Sephadex A-50 and covalently coupled to BrCN-activated Sepharose 4B and porous glass loaded with isothiocyanate propyl groups (ITC-glass). The relative activities against p-tosylarginine methyl ester (TosArgOMe) were found to be 30 to 100% for DEAE-Sephadex crayfish trypsin, 20 to 53% for Sepharose crayfish trypsin, and 17 to 38% for ITC-glass crayfish trypsin. The relative activities rise with declining protein content of the enzyme matrix complexes. The highest relative proteinase activities (substrate: 1% casein) were obtained with Sepharose crayfish trypsin (74%), followed by DEAE-Sephadex crayfish trypsin (68%) and ITC-glass crayfish trypsin (45%). Similar results are obtained with protamine and native lactate dehydrogenase as substrates. In accordance with the Sepharose bovine trypsin complex the apparent Michaelis constant (Km(app)) of the Sepharose crayfish trypsin with TosArgOMe was found to be markedly higher than that of the native enzyme. The pH-activity profiles of the crayfish trypsin derivatives using TosArgOMe as substrate were shown to be displaced towards more alkaline pH values by 0.5 (ITC-glass crayfish trypsin) and 1 (Sepharose crayfish trypsin) pH units, respectively, or towards more acidic pH values (by 1.5 pH units) with the polycationic derivative (DEAE-Sephadex crayfish trypsin) as compared to the native enzyme (optimum pH 8.6). Concerning the temperature stability of the derivatives, Sepharose crayfish trypsin was more stabile, ITC-glass crayfish trypsin behaves like the native crayfish trypsin, and DEAE-Sephadex crayfish trypsin was more sensitive at elevated temperatures as compared to the soluble enzyme. The properties of the crayfish trypsin derivatives are compared with the properties of the bovine analogues.

Animals↗

PubMed1999

A molecular view of trypsin synthesis in the midgut of Aedes aegypti.

Ingestion of a blood meal induces two phases of trypsin synthesis in the midgut of Aedes aegypti females. The first phase, which encompasses the first 4-6 hours following a blood meal, is characterized by the presence of small amounts of early trypsin. The second phase, which occurs between 8 and 36 hours after blood feeding, is characterized by the presence of large amounts of late trypsin. A specific form of regulation of trypsin synthesis characterizes each phase: early trypsin synthesis is regulated at the translational level, while late trypsin synthesis is regulated at the transcriptional level.The enzymatic activity of early trypsin plays a unique and critical role in the regulation of late trypsin synthesis. Early trypsin acts like a "sensor". It carries out limited proteolysis of the ingested proteins and, somehow, the products of this limited proteolysis induce synthesis of late trypsin, which is the protease responsible for the majority of the endoproteolytic cleavage of the meal proteins.Transcription of the early trypsin gene starts a few hours after adult emergence and is under control of juvenile hormone. However, the early trypsin mRNA is stored in the midgut epithelium and remains untranslated until a blood meal is taken. The exact mechanism responsible for initiating translation is presently unknown, but an increase in the size of the amino acid pool in the midgut is sufficient to activate translation of early trypsin mRNA.The transcription of the late trypsin gene is regulated by uncharacterized proteolysis products generated by the action of early trypsin on the blood meal proteins. Once transcription has been activated, the rate of transcription of the late trypsin gene is proportional to the amount of protein present in the meal. In addition, the amount of late trypsin protein translation is controlled by the amount of amino acid released during digestion. Regulation at both transcriptional and translational levels allows the midgut to adjust the amount of late trypsin with remarkable flexibility in response to a particular meal.

Journal Article↗

PubMed1985

Binding and processing of trypsin by cultured bovine corneal endothelial cells.

Bovine corneal endothelial cells in culture bind internalize and degrade [125I]-trypsin. Binding involves the active site of trypsin and increases as a function of [125I]-trypsin concentration. Saturation is observed at a concentration of 0.5-1.0 micrograms ml-1. The cell surface binding of [125I]-trypsin is specific: a seven-fold excess of unlabeled trypsin abolishes about 60% of the total cell surface-associated radioactivity. In addition, thrombin competes poorly with [125I]-trypsin cell surface binding and only 20% of the specific cell surface binding of [125I]-trypsin is subjected to competition with thrombin. This fraction of the cell surface-bound [125I]-trypsin which is accessible to competition with thrombin appears in a covalent complex of [125I]-trypsin X protease-nexin with a molecular weight of 64000 daltons. The cells, when incubated at 37 degrees C, appear to internalize the cell surface-bound [125I]-trypsin at a rate of 0.15-0.25 ng (10(6) cells)-1 min-1. Both the non-covalently cell surface-bound and the protease-nexin (PN) mediated-bound [125I]-trypsin are internalized by the cells, but the [125I]-trypsin X PN complexes contribute about 75% of the total amount of [125I]-trypsin internalized by the cells. The internalized [125I]-trypsin is degraded by the cultures at a rate of about 0.05 ng (10(6) cells)-1 min-1 and the degradation products are released by the cells into the incubation medium as a trichloroacetic acid non-precipitable material. Chloroquine inhibits about 60% of the internalization of [125I]-trypsin by the cells, and inhibits more than 80% of the degradation process of [125I]-trypsin, which indicates that the degradation of the ligand is taking place in lysosomes. Bovine corneal endothelial cells in culture have demonstrated the binding and metabolism of the serine protease trypsin. This described process may indicate the ability of corneal endothelial cells to control the activity of serine proteases in their microenvironment.

Amyloid beta-Protein Precursor↗

PubMed2005

Activity of recombinant trypsin isoforms on human proteinase-activated receptors (PAR): mesotrypsin cannot activate epithelial PAR-1, -2, but weakly activates brain PAR-1.

Trypsin-like serine proteinases trigger signal transduction pathways through proteolytic cleavage of proteinase-activated receptors (PARs) in many tissues. Three members, PAR-1, PAR-2 and PAR-4, are trypsin substrates, as trypsinolytic cleavage of the extracellular N terminus produces receptor activation. Here, the ability of the three human pancreatic trypsin isoforms (cationic trypsin, anionic trypsin and mesotrypsin (trypsin IV)) as recombinant proteins was tested on PARs. Using fura 2 [Ca(2+)](i) measurements, we analyzed three human epithelial cell lines, HBE (human bronchial epithelial), A549 (human pulmonary epithelial) and HEK (human embryonic kidney)-293 cells, which express functional PAR-1 and PAR-2. Human mesotrypsin failed to induce a PAR-mediated Ca(2+) response in human epithelial cells even at high concentrations. In addition, mesotrypsin did not affect the magnitude of PAR activation by subsequently added bovine trypsin. In HBE cells, which like A549 cells express high PAR-2 levels with negligible PAR-1 levels (<11%), half-maximal responses were seen for both cationic and anionic trypsins at about 5 nM. In the epithelial cells, mesotrypsin did not activate PAR-2 or PAR-1, whereas both anionic and cationic trypsins were comparable activators. We also investigated human astrocytoma 1321N1cells, which express PAR-1 and some PAR-3, but no PAR-2. High concentrations (>100 nM) of mesotrypsin produced a relatively weak Ca(2+) signal, apparently through PAR-1 activation. Half-maximal responses were observed at 60 nM mesotrypsin, and at 10-20 nM cationic and anionic trypsins. Using a desensitization assay with PAR-2-AP, we confirmed that both cationic and anionic trypsin isoforms cause [Ca(2+)](i) elevation in HBE cells mainly through PAR-2 activation. Desensitization of PAR-1 with thrombin receptor agonist peptide in 1321N1 cells demonstrated that all three recombinant trypsin isoforms act through PAR-1.Thus, the activity of human cationic and anionic trypsins on PARs was comparable to that of bovine pancreatic trypsin. Mesotrypsin (trypsin IV), in contrast to cationic and anionic trypsin, cannot activate or disable PARs in human epithelial cells, demonstrating that the receptors are no substrates for this isoenzyme. On the other hand, mesotrypsin activates PAR-1 in human astrocytoma cells. This might play a role in protection/degeneration or plasticity processes in the human brain.

Astrocytoma↗

PubMed1997

Activation of type IV procollagenases by human tumor-associated trypsin-2.

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62-65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.

Amino Acid Sequence↗

PubMed2001

Association of trypsin expression with recurrence and poor prognosis in human esophageal squamous cell carcinoma.

BACKGROUND: Overexpression of the matrix serine proteinase (MSP) trypsin has been implicated in tumor growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathologic and prognostic significance of trypsin expression in esophageal squamous cell carcinomas (SCC). METHODS: Production of trypsin in tissue extracts was analyzed by immunoblotting and gelatin zymography. The authors analyzed the association between immunohistochemically detected trypsin expression in esophageal SCC and clinicopathologic characteristics, and they investigated whether trypsin is a predictor of recurrence and/or survival. RESULTS: Overproduction and activation of trypsin was observed in 6 of 10 tumor extracts. The trypsin immunoreactivities at the invasive front were more intense than those at the superficial layer. Sections with immunostaining signals in greater than 30% of carcinoma cells at the invasive front, which were observed in 52 (52%) cases, were judged to be positive for trypsin. Trypsin positivity was significantly correlated with the depth of invasion (P < 0.0001), lymph node metastasis (P = 0.0048), advanced pTNM classification (P = 0.0006), recurrence (P = 0.0003), and recurrence within the first postoperative year (P = 0.0005). Patients with trypsin positive carcinoma had significantly shorter disease free and overall survival times than did those with trypsin negative carcinoma (P < 0.0001 and P < 0.0001, respectively). Trypsin retained its significant predictive value for disease free and overall survival in multivariate analysis that included conventional clinicopathologic factors (P = 0.0029 and P = 0.0006, respectively). Patients with concomitant overexpression of trypsin and matrilysin at the invasive front, in which they often were colocalized, had the worst prognosis. CONCLUSIONS: The authors' results suggest that trypsin plays a key role in the progression of esophageal carcinoma. Detection of trypsin expression as well as matrilysin is useful for the prediction of recurrence and poor prognosis.

Carcinoma, Squamous Cell↗

PubMed2003

Association of trypsin expression with tumour progression and matrilysin expression in human colorectal cancer.

Overexpression of the matrix serine protease (MSP) trypsin has been implicated in tumour growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathological and prognostic significance of trypsin expression in colorectal cancer. This study analysed the association between immunohistochemically detected trypsin expression in colorectal cancer and clinicopathological characteristics, and investigated whether trypsin is a predictor of recurrence and/or survival. Trypsin immunoreactivity was more intense at the invasive front than in the superficial part of the tumour. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 48 cases (48%), were judged to be positive for trypsin. Trypsin positivity was significantly correlated with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, advanced pathological tumour-node-metastasis (TNM) stage, and recurrence. Patients with trypsin-positive carcinoma had significantly shorter overall and disease-free survival periods than did those with trypsin-negative carcinoma. Trypsin retained its significant predictive value for overall and disease-free survival in multivariate analysis that included conventional clinicopathological factors. It is well known that trypsin activates matrilysin (matrix metalloproteinase-7), which plays an important role in colorectal cancer progression. Patients with concordant overexpression of trypsin and matrilysin at the invasive front, in which they were often co-localized, had the worst prognosis. Trypsinogen-1-transfected HCT116 colon cancer cells showed not only trypsin activity, but also active matrilysin activity and were more invasive in vitro than mock-transfected HCT116 cells. These results suggest that trypsin plays a key role in the progression of colorectal cancer. Detection of trypsin expression as well as matrilysin is useful for the prediction of recurrence in and poor prognosis of colorectal cancer patients.

Aged↗

PubMed1999

Isolation and characterization of two digestive trypsin-like proteinases from larvae of the stalk corn borer, Sesamia nonagrioides.

Two digestive trypsin-like proteinases from Sesamia nonagrioides Lef. (Lepidoptera: Noctuidae) larvae were purified by benzamidine-Sepharose affinity chromatography. The purified enzymes showed molecular size of 27 (trypsin-I) and 24 KDa (trypsin-II). Amino acid analysis and N-terminal sequencing confirmed their relationship with other trypsins from lepidopteran larvae. However, trypsin-I presented one lysine at position 11, being the first report of this amino acid in the sequence of a lepidopteran digestive trypsin. Trypsin-I had an isoelectric point of 6.0, and a Km of 2.2 x 10(-4) M and 3.9 x 10(-5) M for BApNa and BAEE, respectively. Trypsin-II presented an isoelectric point of 8.7, and Km values of 1.7 x 10(-4) M (BApNa) and 3.8 x 10(-5) M (BAEE). Both enzymes were differentially inhibited by some proteinase inhibitors. In particular, trypsin-I was inhibited by E-64 (ID50 = 6 microM) but not by lima bean trypsin inhibitor (LBI), whereas trypsin-II was inhibited by LBI (ID50 = 1 microM) and poorly by E-64 (ID50 = 85 microM). Changes in the susceptibility of the trypsin-like activity of midgut extracts from different larval instars to these inhibitors suggest that the relative proportion of these two enzymes varied through larval development, being predominant in early instars trypsin-I and in late instars trypsin-II.

Animals↗

PubMed2006

Trypsin in colorectal cancer: molecular biological mechanisms of proliferation, invasion, and metastasis.

Trypsin is involved in colorectal carcinogenesis and promotes proliferation, invasion, and metastasis. Although a well-known pancreatic digestive enzyme, trypsin has also been found in other tissues and various cancers, most importantly of the colorectum. Moreover, colorectal cancers with trypsin expression have a poor prognosis and shorter disease-free survival. Biological understanding of how trypsin causes cancer progression is emerging. It seems to act both directly and indirectly through a 'proteinase-antiproteinase-system', and by activation of other proteinase cascades. Invasion of the basal membrane by cancer cells may be promoted directly by trypsin digestion of type I collagen. Trypsin activates, and is co-expressed with matrix metalloproteinases (MMPs), which are known to facilitate invasion and metastasis. MMP-2, MMP-7, and MMP-9 are co-expressed together with trypsin and seem to be of particular importance in proliferation, progression, and invasion. MMPs may play a role in both conversion from adenoma to carcinoma, and in the initiation of invasion and metastasis. Co-segregation of trypsin and MMPs within the tumour environment is important for the activation of MMPs, and may explain the deleterious effect of trypsin on prognosis in colorectal cancer. Trypsin and proteinase-activated receptor 2 (PAR-2) act together in an autocrine loop that promotes proliferation, invasion, and metastasis through various mechanisms, of which prostaglandin synthesis is important. Stimulated by trypsin, both MMP and PAR-2 may activate the mitogenic MAPK-ERK pathway through activation of the epidermal growth factor receptor. Experimental trypsin inhibition is feasible but not very effective, and trypsin as a target for clinical therapy is unlikely to be successful owing to its universal distribution. However, as the pathways of trypsin and co-activated protein cascades emerge, biological understanding of colorectal carcinogenesis will be further illuminated and may pave the way for prognosticators, predictors, and novel targets of therapy.

Adenoma↗

PubMed1983

Cell association and degradation of alpha 2-macroglobulin-trypsin complexes in hepatocytes and adipocytes.

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.

Adipose Tissue↗

PubMed1989

Introduction of a cysteine protease active site into trypsin.

Active site serine 195 of rat anionic trypsin was replaced with a cysteine by site-specific mutagenesis in order to determine if a thiol group could function as the catalytic nucleophile in serine protease active site environment. Two genetically modified rat thiol trypsins were generated; the first variant contained a single substitution of Ser195 with Cys (trypsin S195C) while the second variant contained the Ser195 to Cys as well as an Asp102 to Asn substitution (trypsin D102N,S195C) that more fully mimics the putative catalytic triad of papain. Both variants were expressed as his J signal peptide-trypsin fusion proteins to high levels under the control of the tac promoter. The mature forms of both variants were secreted into the periplasmic space of Escherichia coli. Trypsin S195C shows a low level of activity toward the activated ester substrate Z-Lys-pNP, while both trypsin S195C and trypsin D102N,S195C were active toward the fluorogenic tripeptide substrate Z-GPR-AMC. Esterase and peptidase activities of both thiol trypsin variants were inhibited by known Cys protease inhibitors as well as by specific trypsin inhibitors. The kcat of trypsin S195C was reduced by a factor of 6.4 x 10(5) relative to that of trypsin while the kcat of trypsin D102N,S195C was lowered by a factor of 3.4 x 10(7) with Z-GPR-AMC as substrate. Km values were unaffected. The loss of activity of trypsin D102N,S195C was partially attributed to an inappropriate Asn102-His57 interaction that precludes the formation of the catalytically competent His57-Cys195 ion pair although loss of the negative charge of D102 at the active site probably contributes to diminished activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1996

Thrombin and trypsin-induced Ca(2+) mobilization in human T cell lines through interaction with different protease-activated receptors.

This study was conducted to determine whether serine proteinases may induce [Ca(2+)]i mobilization in different hematopoietic cell lines and to analyze their mechanisms of action. We show that in addition to thrombin and thrombin receptor agonist peptide (TRP, SFLLRN), trypsin induced [Ca(2+)]i mobilization in a highly thrombin-sensitive Jurkat T cell clone. Thrombin, TRP, and trypsin were found to induce [Ca(2+)]i release in three different Jurkat T cell clones differing in the level of T cell receptor expression. Similar results were obtained with a prothymocytic leukemic cell line, HPB.ALL, although these cells were much more responsive to trypsin than to thrombin and TRP. Other cell types such as THP1, a myelomonocytic cell line, or CEM, a CD4(+) positive leukemic cell line, were unresponsive to thrombin, TRP, and trypsin. The effect of trypsin was mimicked by SLIGRL, a peptide corresponding to the cleaved amino-terminal sequence of the recently characterized murine trypsin-activated receptor (PAR2). At suboptimal concentrations, the effects of SFLLRN and SLIGRL were additive, whereas saturating doses of peptides did not further increase [Ca(2+)]i mobilization in Jurkat T cells, indicating that both peptides were able to mobilize the same pool of calcium. Northern blot analysis of mRNAs from different leukemic cell lines indicated a remarkable correlation between PAR2 expression in different cell lines and SLIGRL or trypsin responses in the same cells. The expression of the "trypsin receptor" was also confirmed by polymerase chain reaction analysis. Moreover, a 24 h treatment of Jurkat cells by an anti-CD3 monoclonal antibody, a condition known to down-regulate thrombin receptor expression, induced loss of thrombin and TRP responses but only partially affected trypsin stimulation of [Ca(2+)]i release. Finally, after a first stimulation with either thrombin or trypsin, Jurkat cells were still able to respond to trypsin or thrombin, respectively, demonstrating that thrombin and trypsin essentially activated their own receptors. Our data provided evidence that 1) the human T leukemic cell line Jurkat and other T cell lines express at least two different functional protease-activated receptors, the thrombin receptor and a highly sensitive trypsin receptor, likely the human counterpart of the murine PAR2, and 2) at variance with the commonly accepted model, trypsin exerts most of its effect in T leukemic cell lines by thrombin receptor-independent mechanisms.

Animals↗

PubMed1975

The two human trypsinogens. Inhibition spectra of the two human trypsins derived from their purified zymogens.

The two human anionic trypsinogens 1 and 2 were purified from human pancreatic juice by gel filtration on Sephadex G-100 and by chromatography on DEAE-cellulose. After activation of their respective zymogens by porcine enterokinase, human trypsins 1 and 2 were studied for their reaction with a wide variety of proteinase inhibitors. Kunitz pancreatic trypsin inhibitor and human pancreatic secretory trypsin inhibitor completely inhibited both human trypsins at a stoichiometric inhibitor-to-enzyme ratio of one to one. In contrast, bovine pancreatic secretory trypsin inhibitor (Kazal's inhibitor) failed to inhibit either human trypsin. The inhibition of both human trypsins by porcine pancreatic secretory trypsin inhibitor was demonstrated. The reactions of the trypsins with chicken ovomucoid, Ascaris lumbricoides (type suis), human sperm and blood plasma trypsin inhibitors were studied. The most striking difference between the two human trypsins was the reaction with soybean trypsin inhibitor (Kunitz). Trypsin 2 was completely inhibited in a one-to-one molar ratio while trypsin 1 was poorly inhibited. The presence of a prekallikrein in human pancreatic juice is discussed.

Animals↗

PubMed1986

The effect of trypsinization on the plasma membrane binding and action of 3,5,3'-triiodothyronine in rat thymocytes.

We have previously demonstrated that in the rat thymocyte T3 produces a prompt, extranuclear effect to increase cellular cAMP concentration and 2-deoxyglucose (2-DG) uptake. We have also demonstrated the presence of specific receptors for T3 in the rat thymocyte plasma membrane. From these and other observations we have suggested that T3 initiates these actions by binding to its receptors on the surface of this cell. To test this hypothesis further we have now examined whether a decrease in the binding of T3 to the thymocyte plasma membrane, induced by mild trypsinization, attenuates the effect of T3 on thymocyte cAMP concentration and 2-DG uptake. Mild trypsin treatment of rat thymocytes in cell suspension did not cause appreciable nonspecific damage to the cells, since it did not change the ability of the cells to exclude either trypan blue or mannitol, a marker of extracellular water. Further, treatment with trypsin did not alter the ability of thymocytes to bind concanavalin A. It did produce, however, a dose-related decrease in the binding of T3 to its plasma membrane binding sites owing to a decrease in their number, but not their affinity. This was associated with a progressive decrease in the T3-induced increase in both 2-DG uptake and cAMP concentration. Thus, low concentrations of trypsin had no significant effect on 2-DG uptake in the absence of T3, but decreased significantly the increase in sugar uptake induced by T3, whereas a higher concentration of trypsin (5 mg/ml) decreased basal 2-DG uptake by only 23%, but abolished the response to T3 altogether. T3, epinephrine, and trypsin alone induced dose-related increases in cellular cAMP concentration, the response to trypsin exceeding that to T3 or epinephrine. Trypsinization also resulted in a progressive decrease in the response of cellular cAMP to both hormones, so that at a trypsin concentration of 5 mg/ml no further increase was produced by either hormone. The possibility that a maximum activation of adenylate cyclase by trypsin could explain these results was rendered unlikely by experiments with prostaglandin E1 (PGE1). The increase in cellular cAMP concentration induced by this agent was vastly greater than that produced by trypsin alone. Although the response to PGE1 was greatly inhibited by trypsin, cellular cAMP concentration in thymocytes incubated with trypsin (5 mg/ml) plus PGE1 was much higher than that in cells treated with trypsin alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1995

Molecular cloning and characterization of anionic and cationic variants of trypsin from Atlantic salmon.

Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were constructed and screened with salmon trypsin-specific probes. Five clones containing near full-length transcripts were selected for further characterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substrate-binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP-I, SalTRP-IA and SalTRP-IB) possessed very similar sequences and may represent allelic variants encoded by the same gene focus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other trypsin clones (SalTRP-II and SalTRP-III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation of fish during evolution. A structural model based on X-ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge between anionic and cationic trypsins were located at exposed regions of the proteins.

Amino Acid Sequence↗