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Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found.
The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.
For the successful production of Aeromonas sobria serine protease (ASP), open reading frame 2 (ORF2) protein, encoded at the 3' end of the protease operon, is required. In this study, we examined the action of ORF2 protein. The results showed that the protein associated with ASP in the periplasm and helped ASP to form an active structure.
The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.
Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica results in a spectrum of phenotypic changes that can include alterations in colony morphology and significant reductions in pigmentation, asexual sporulation, and virulence (hypovirulence). Deletion of 88% [Phe(25) to Pro(243)] of the virus-encoded papain-like protease, p29, in the context of an infectious cDNA clone of the prototypic hypovirus CHV1-EP713 (recombinant virus Deltap29) partially relieved virus-mediated suppression of pigmentation and sporulation without altering the level of hypovirulence. We now report mapping of the p29 symptom determinant domain to a region extending from Phe(25) through Gln(73) by a gain-of-function analysis following progressive repair of the Deltap29 deletion mutant. This domain was previously shown to share sequence similarity [including conserved cysteine residues Cys(38), Cys(48), Cys(70), and Cys(72)] with the N-terminal portion of the potyvirus-encoded helper component-proteinase (HC-Pro), a multifunctional protein implicated in aphid-mediated transmission, genome amplification, polyprotein processing, long-distance movement, and suppression of posttranscriptional silencing. Substitution of a glycine residue for either Cys(38) or Cys(48) resulted in no qualitative or quantitative changes in virus-mediated symptoms. Unexpectedly, mutation of Cys(70) resulted in a very severe phenotype that included significantly reduced mycelial growth and profoundly altered colony morphology. In contrast, substitution for Cys(72) resulted in a less severe symptom phenotype approaching that observed for Deltap29. The finding that p29-mediated symptom expression is influenced by two cysteine residues that are conserved in the potyvirus-encoded HC-Pro raises the possibility that these related viral-papain-like proteases function in their respective fungal and plant hosts by impacting ancestrally related regulatory pathways.
The acid protease produced by a strain of Mucor miehei isolated in Cuba was purified by column electrofocusing and partially characterized as to amino-acid composition, molecular weight, helical content, total carbohydrate content, and approximate isoelectric point; A detailed comparison of these results was reported previously for Mucor miehei protease (Ottesen, M. & Rickert, W;S. (1970) C.R. Trav. Labmcarlsberg 37, 301) suggested that the two enzymes are similar but not identicalmthis conclusion was reinforced by an analysis of circular-dichroism spectra.
Human airway trypsin-like protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20-55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [3H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular Ca2+ mobilization in HBF. The HAT-induced increase in Ca2+ was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts.
BACKGROUND: The phenotype of proliferated mast cells in Nippostrongylus brasiliensis-infected rat lung has been identified as mucosal mast cells (MMC) but not connective tissue mast cells (CTMC). However, a previous study of ours showed that the expression of rat mast cell tryptase (RMCT) mRNA, which has been reported to be confined to CTMC, significantly increased in rat lung 14 days after infection. METHODS: The expression of four mast cell proteases in rat lung during the course of infection with N. brasiliensis was examined by RNA blot analysis. Immunohistochemical analysis of RMCT and rat mast cell protease (RMCP) II, which has been reported to be confined to MMC was also performed. RESULTS: The number of lung mast cells did not change until 7 days after infection, then gradually increased until 21 days after infection. The expression of the RMCP II gene had increased 14 and 21 days after infection. In addition, the expression of the RMCP I and RMCT genes had also increased at the same time points, but RMCP III had not. By immunohistochemistry, most of the mast cells in infected lung were identified as RMCP II+/RMCT- (MMC), but both RMCP II+ and RMCT+ mast cells were also observed. CONCLUSIONS: The present results suggest that mast cell phenotype alteration or a distinct mast cell subset might be present in N. brasiliensis-infected rat lung, and therefore N. brasiliensis-infected rat lung may be a useful tool for studying the differentiation mechanism of mast cells.
Since recent studies suggest an imbalance between cathepsin B and its tissue protease inhibitors (PI) in the pathogenesis of acute and chronic diseases, we tested the hypothesis that release of activated cysteine proteases (P) such as cathepsins B, H, and L might play a role in the pathogenesis of gastric hemorrhagic mucosal lesions (HML) induced by ethanol (E) or ammonia (A). Anesthetized rats received 1 ml of 50% E or 1% A solution intragastrically for 1 min during in situ gastric luminal perfusion. Rapid activation and release of cathepsins B, L, and H into the luminal perfusate preceded the formation of HML quantified by planimetry. Mucosal presence and activity of cysteine PI and cathepsin B have also been investigated in the pathogenesis of chemically induced HML. We extracted and partially isolated acid and thermostable inhibitors of cathepsin B in the gastric mucosa, and found rapid inactivation of PI and activation of cathepsin B in the early phase of E- or A-induced HML. Negative correlations were found between P and PI activities by E or A solutions. Both the activation of cathepsins B, L, and H and the development of E-induced HML were prevented by pretreatment with the sulfhydryl alkylator N-ethylmaleimide. These results suggest that cysteine P may be activated in the rat stomach after E or A exposure, and cysteine P may have a role in the pathogenesis of E- or A-induced gastric HML. Endogenous PI may also participate in the mechanisms of gastric mucosal lesions and gastroprotection.
Recently a novel serine protease in human plasma was described and was named PHBP, PHBSP or factor seven activating protease (FSAP), respectively, the latter according to the finding that it can support coagulation by factor VII activation. Later on FSAP was identified as a potent activator of single chain plasminogen activators, in particular of prourokinase, as well. The physiological role of FSAP is still speculative, but recent studies suggest a contribution to hemostasis. Due to its affinity to glycosaminoglycans a role in cell-associated or extracellular proteolytic events is also likely. The impact of a very recently uncovered frequent polymorphism impairing the prourokinase activation potential of FSAP needs to be investigated in more detail.
The fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N. et al., Biosci. Biotechnol. Biochem., 57, 1726-1730 (1993), 60, 293-300 (1996), and 63, 2031-2033 (1999)] were further characterized to exploit their catalytic functions. These enzymes are stable in solution for long periods at room temperature and strongly resistant to organic solvents, even toluene and n-hexane. The serine proteases can act on various protein substrates such as elastin and hemoglobin as well as fibrin, and also catalyzed the hydrolysis of esters such as ethyl acetate and a bioplastic, poly[(R)-3-hydroxybutyrate] film. The enzymes, in the absence of microbial degradation, contributed to the production of the earthworm autolysate possessing antioxidant ability and protease activity, whose components were similar to those of soy sauce. The extract of the earthworm autolysate could be used as a peptone substitute in media for the cultivation of microorganisms.
cDNAs were isolated that encode mouse mast cell protease-5 (MMCP-5), an approximately 30,000 Mr serine protease stored in the secretory granules of serosal mast cells (SMC) and Kirsten sarcoma virus-immortalized mast cells. Based on the deduced amino acid sequences of these cDNAs, MMCP-5 is synthesized as a 247-amino acid preproenzyme composed of a novel 19-residue hydrophobic signal peptide, a Gly-Glu activation peptide not present in other mast cell chymases, and a 226-amino acid protein that represents the mature enzyme. MMCP-5 possesses a unique Asn residue in the substrate binding cleft at residue 176 and is highly basically charged. The MMCP-5 gene was isolated, sequenced, and found to belong to a distinct subset of chymase genes. Allelic variations of the MMCP-5 gene were also detected. MMCP-5 is expressed in bone marrow-derived mast cells (BMMC), Kirsten sarcoma virus-immortalized mast cells, and SMC, but not in gastrointestinal mucosal mast cells of helminth-infected mice. The abundant levels of MMCP-5 mRNA in immature BMMC indicate that this chymase is expressed relatively early during the differentiation of mast cells. MMCP-5 is the first chymase to be molecularly cloned from progenitor mast cells and is also the first chymase shown to be expressed preferentially in the SMC subclass.
New type of protease-activated form of protein kinase C was generated from rat liver plasma membrane by action of endogenous trypsin-like protease. The molecular mass was estimated to be about 80,000 by immunoblot analysis which was slightly smaller (approximately 2,000) than that of native protein kinase C. The protein kinase activity was 2-times stimulated by Ca2+ and phospholipid and inhibited by the synthetic peptide derived from the pseudosubstrate region of protein kinase C. This type of activated kinase was produced in purified enzyme system in the absence of either Ca2+ or phospholipid or both. These results suggest that limited proteolysis generating the active form of Mr 80,000 may occur on the inactive form of protein kinase C.
The status of the blood kinin system, activity of serum inhibitors of proteinases (alpha 1-protease inhibitor and alpha 2-macroglobulin) as well as indices of activity of trypsin-like proteinases, elastolytic and fibrinolytic activity, free antitryptic activity and complete content of acid stable inhibitors were studied in the morning sputum and bronchoalveolar washesoff of 62 patients with intrathoracic sarcoidosis. It was shown that in active sarcoidosis proteinase-inhibitors balance got disturbed as a result of a decrease in the activity of serum inhibitors, suppression of local secretion of acid stable inhibitors and inactivation at the level of alpha 1-protease inhibitor alveoles. Excessive activation of the kinin system was noted in the blood flow and elevation of all types of proteolytic activity in the respiratory system. It was assumed that the above changes reflected disturbed self-control processes in the body and caused progression of disease and pneumofibrosis formation.
The size of androgen receptors from rat ventral and dorsal prostate, dorsal prostate (Dunning) tumor, testis, epididymis, and seminal vesicle was determined using Sephadex G-200 chromatogrpahy and sucrose gradient centrifugation. The protease inhibitor diisopropyl fluorophosphate (DFP) was used to minimize receptor breakdown. An 8-9 S, 85 to 106 A receptor (Mr = 280,000 to 365,000; f/fo = 1.9 to 2.4) observed in unfractionated cytosol prepared in low ionic strength buffer with or without DFP is in equilibrium with a 4.5-5 S, 58 A form (Mr = 117,000; f/fo = 1.8) observed at salt concentrations greater than 0.1 M KCl. Receptor partially purified using (NH4)2SO4 or phosphocellulose chromatography in the absence of DFP was present as smaller fragments of 3.6 S, 37 A and 3.0 S, 23 A. Similar fragments could be generated from the 4.5 S or 8 S receptor by mild trypsin treatment. In addition, ventral prostate contains a DFP-insensitive enzyme which specifically converts the 4.5 S, 58 A receptor to the 3.6 S 37 A fragment. The DFP-insensitive enzyme is partially inhibited by rabbit bile and appears similar to the enzyme seminin, a secretory protein of human prostate. Androgen receptor isolated in the presence of DFP from nuclei labeled in vivo is predominantly 4.5 S, 58 A, with smaller forms (37 and 23 A) appearing in the absence of DFP. The 4.5 S, 58 A nuclear receptors were also in equilibrium with a large 8 S form. Receptor breakdown by DFP-insensitive and sensitive proteases appears to be an in vitro phenomenon. Furthermore, the size of the androgen receptor is not significantly changed during receptor migration from cytoplasm to nucleus.
The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.
In previous work the primary structure of a previously unknown protease was deduced from the sequence of a dog mastocytoma cDNA. The predicted preproprotein shares some features with mast cell tryptases but is no more than 49% identical in sequence to known trypsin-like enzymes, including dog tryptase. This study explores the expression of this protein, termed dog mast cell protease-3 (dMCP-3). A polyclonal Ab was raised to a peptide corresponding to residues 166-181 of the deduced sequence. Anti-dMCP-3(166-181) Ig recognizes dMCP-3 expressed as a CheY fusion protein in Escherichia coli and binds to a approximately 36-kDa protein in extracts of dog mastocytomas. The Ab does not recognize dog tryptase, dMCP-3s closest known relative in mastocytoma cells. When used with fluorescein-conjugated and alkaline phosphatase-conjugated secondary Abs, anti-dMCP-3(166-181) Ig yields punctate cytoplasmic staining in mastocytoma cells, suggesting localization to intracellular granules. Staining is greatly reduced by preincubation with synthetic dMCP-3 peptide, supporting the specificity of the Ab. Immunohistochemical staining of normal dog tissues reveals scattered dMCP-3 reactive cells in skin, intestine, trachea, and lung parenchyma. Double staining with Ab and methylene blue shows that anti-dMCP-3(166-188) Ig recognizes extravascular mononuclear tissue cells with metachromatic granules. In addition, cytoplasmic staining is seen in polymorphonuclear leukocytes within vessels in tissue sections and in leukocytes harvested from blood. Hybridization of dMCP-3 cDNA to dog skin RNA provides further evidence of dMCP-3 gene transcription in normal tissue. Thus, this study provides immunochemical evidence of dMCP-3 expression in dog mast cell tumors, normal tissue mast cells, and neutrophils.
The expression of a cellular receptor for the blood-clotting protease factor Xa, designated effector cell protease receptor-1 (EPR-1), was investigated in lymphoma. Immunohistochemical analysis demonstrated prominent reactivity of monoclonal antibodies to EPR-1 with Reed-Sternberg cells in 30 of 35 cases of nodular-sclerosis, lymphocyte-depletion, and mixed-cellularity Hodgkin's disease (HD). In contrast, several non-Hodgkin's lymphomas, or the nonneoplastic cellular components of HD, did not react with anti-EPR-1 monoclonal antibodies. A single molecular species of approximately 62 kD, consistent with the size and structural organization of EPR-1, was immunoblotted by an anti-EPR-1 monoclonal antibody from tissue samples of HD, but not from normal lymph nodes. Expression of EPR-1 transcripts in Reed-Sternberg cells was demonstrated by in situ hybridization with an antisense EPR-1 riboprobe, and by amplification of reverse-transcribed HD RNA with EPR-1-specific primers. These findings identify the factor Xa receptor, EPR-1, as a novel marker of Reed-Sternberg cells, and suggest its potential role in the histopathogenesis of HD.