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W2068989897 abstract "Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K i app) of HK about 10-fold. Conversely, the catalytic efficiency of cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between cruzipain, HK, and heparan sulfate proteoglycans. Trypanosoma cruzi activates the kinin pathway through the activity of its major cysteine proteinase, cruzipain. Because kininogen molecules may be displayed on cell surfaces by binding to glycosaminoglycans, we examined whether the ability of cruzipain to release kinins from high molecular weight kininogen (HK) is modulated by heparan sulfate (HS). Kinetic assays show that HS reduces the cysteine proteinase inhibitory activity (K i app) of HK about 10-fold. Conversely, the catalytic efficiency of cruzipain on kinin-related synthetic fluorogenic substrates is enhanced up to 6-fold in the presence of HS. Analysis of the HK breakdown products generated by cruzipain indicated that HS changes the pattern of HK cleavage products. Direct measurements of bradykinin demonstrated an up to 35-fold increase in cruzipain-mediated kinin liberation in the presence of HS. Similarly, kinin release by living trypomastigotes increased up to 10-fold in the presence of HS. These studies suggest that the efficiency of T. cruzi to initiate kinin release is potently enhanced by the mutual interactions between cruzipain, HK, and heparan sulfate proteoglycans. The plasma kallikrein-kinin system is a paradigm of a tightly controlled pro-inflammatory proteolytic cascade activated by vascular injury (1Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar). Vasoactive peptides structurally related to bradykinin (generally termed as “kinins”) are derived from enzymatic excision from an internal segment (D4 domain) of kininogens. These peptides are implicated in a broad range of pathophysiological responses,e.g. edema formation, vasodilatation, and pain. Although the nonapeptide bradykinin is released by the action of plasma kallikrein on high molecular weight kininogen (HK), 1HKhigh molecular weight kininogenLKlow molecular weight kininogenGAGglycosaminoglycanHSheparan sulfaten-cruzipainnatural cruzipainr-cruzipainrecombinant cruzainDTTdithiothreitolCBZcarbobenzoxyAMCarginyl- 7-amido-4-methylcoumarinAbzO-aminobenzoylEDDnpethylenediamine 2,4-dinitrophenylE-64l-trans-epoxysuccinylleucylamido- (-4-guanidino)butane lysyl-bradykinin is liberated from extravascular low molecular weight kininogen (LK) or HK by the activity of tissue kallikreins (2de la Cadena R.A. Colman R.W. Trends Pharmacol. Sci. 1991; 12: 272-275Abstract Full Text PDF PubMed Scopus (39) Google Scholar). In inflammatory conditions, oxidized forms of kininogens may be cleaved by the concerted action of neutrophil elastase and mast cell tryptase, liberating Met-Lys-bradykinin (3Kozik A. Moore R.B. Potempa J. Imamura T. Rapala-Kozik M. Travis J. J. Biol. Chem. 1998; 273: 33224-33229Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Once liberated, kinins activate local endothelial or smooth muscle cells through the constitutively expressed B2 kinin receptor (4Farmer S.G. Burch R.M. Annu. Rev. Pharmacol. Toxicol. 1992; 32: 511-536Crossref PubMed Scopus (192) Google Scholar) or alternatively through the B1 kinin receptor that is up-regulated during inflammation (5Marceau F. Immunopharmacology. 1995; 30: 1-26Crossref PubMed Scopus (360) Google Scholar). The effect of kinin stimulation on its receptor(s) is tightly regulated by the action of kinin-degrading peptidases (kininases), such as the angiotensin-converting enzyme and neutral endopeptidase (1Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar). high molecular weight kininogen low molecular weight kininogen glycosaminoglycan heparan sulfate natural cruzipain recombinant cruzain dithiothreitol carbobenzoxy arginyl- 7-amido-4-methylcoumarin O-aminobenzoyl ethylenediamine 2,4-dinitrophenyl l-trans-epoxysuccinylleucylamido- (-4-guanidino)butane HK comprises six major domains, and the C-terminal domains (D5H and D6H) mediate plasma contact phase activation; they are not present in LK (6Kaufmann J., M. Haasemann S. Modrow S. Müller-Esterl W. J. Biol. Chem. 1993; 268: 9079-9091Abstract Full Text PDF PubMed Google Scholar). The other domains, D1–D4, are shared with LK. Domains 1–3 are structures homologous to the cysteine-proteinase inhibitors, cystatins (7Barrett A.J. Fritz H. Grubb A. Isemura S. Järvinen M. Katunuma N. Machleidt W. Müller-Esterl W. Sasaki M. Turk V. Biochem. J. 1986; 236: 312Crossref PubMed Scopus (277) Google Scholar), and the bradykinin-containing segment is domain 4. Recent efforts to define the structural basis of HK interaction with endothelial cells have focused on two binding sites. One site is represented by 27 amino acids located in the D3 domain (8Herwald H. Hasan A.K. Godovac-Zimmermann J. Schmaier A.H. Müller-Esterl W. J. Biol. Chem. 1995; 270: 14634-14642Abstract Full Text Full Text PDF PubMed Google Scholar), hence overlapping with one of the cystatin-like domains. The second binding site, located in the D5H domain of HK, is a highly basic region formed by clusters of histidine, lysine, and glycine (9Hasan A.A.K. Cines D.B. Herwald H. Schmaier A.H. Müller-Esterl N. J. Biol. Chem. 1995; 270: 19256-19261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). HK binds to a multi-protein receptor complex consisting of gC1q receptor, urokinase plasminogen activator receptor, and cytokeratin 1 (10Mahdi F. Shariat-Madar Z. Todd III, R.F. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar). Other studies demonstrated that heparan and chondroitin sulfate-type of proteoglycans are high affinity docking sites for HK accumulation on endothelial cells (11Renné T. Dedio J. David G. Müller-Esterl W. J. Biol. Chem. 2000; 275: 33688-33696Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar,12Renné T. Müller-Esterl W. FEBS Lett. 2001; 500: 36-40Crossref PubMed Scopus (36) Google Scholar). The assembly of HK molecules on human umbilical vein endothelial cells is required to prekallikrein activation, which modulates subsequent factors XI and XII activation (13Motta G. Rojkjaer R. Hassan A.A.K. Cines D.B. Schmaier A.H. Blood. 1998; 91: 516-528Crossref PubMed Google Scholar, 14Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 1999; 274: 7137-7145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 15Rojkjaer R. Hasan A.A.K. Motta G. Schousboe I. Schmaier A.H. Thromb. Haemostasis. 1998; 80: 74-81Crossref PubMed Scopus (86) Google Scholar). The kinin activation pathway was implicated in the spread of infection by several pathogens (16Herwald H. Collin M. Müller-Esterl W. Björck L. J. Exp. Med. 1996; 184: 665-673Crossref PubMed Scopus (156) Google Scholar, 17Imamura T. Pike R.N. Prochazka V. Kiefer M.C. Travis J. Barr P.J. J. Biol. Chem. 1995; 270: 1007-1010Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18Sakat Y. Akaike T. Suiga M. Ijri S. Ando M. Maeda H. Microbiol. Immunol. 1996; 40: 415-423Crossref PubMed Scopus (41) Google Scholar, 19Maeda H. Microbiol. Immunol. 1996; 40: 685-699Crossref PubMed Scopus (109) Google Scholar). Studies on Trypanosoma cruzi,the etiological agent of Chagas' heart disease, indicate that activation of bradykinin receptors by infective forms (trypomastigotes) potentiates cellular invasion (20Scharfstein J. Schmitz V. Morandi V. Capella M.A. Lima A.P.C.A. Morrot A. Juliano L. Müller-Esterl W. J. Exp. Med. 2000; 192: 1289-1299Crossref PubMed Scopus (191) Google Scholar). Bradykinin receptors were activated by bradykinin liberated from kininogen by the major cysteine proteinase of T. cruzi (21Del Nery E. Juliano M.A Lima A.P.C.A Scharfstein J. Juliano L. J. Biol. Chem. 1997; 272: 25713-25718Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), a papain-like enzyme conventionally designated as cruzipain (also known as, cruzain) (22Cazzulo J.J Cousi R. Raimondi A Wernstedt C. Hellman U. Mol. Biochem. Parasitol. 1989; 33: 33-42Crossref PubMed Scopus (122) Google Scholar, 23Murta A.C.M. Persechini P.M. Souto-Padron T. de Souza W. Guimarães J.A. Scharfstein J. Mol. Biochem. Parasitol. 1990; 43: 27-38Crossref PubMed Scopus (163) Google Scholar, 24Eakin A.E. Mills A.A. Harth G. McKerrow J.H. Craik C.S. J. Biol. Chem. 1992; 267: 7411-7420Abstract Full Text PDF PubMed Google Scholar). Because glycosaminoglycans (GAGs) modulate the catalytic activity of some papain-like enzymes (25Li Z. Hou W.S. Bromme D. Biochemistry. 2000; 39: 529-536Crossref PubMed Scopus (138) Google Scholar, 26Almeida P.C. Nantes I.L. Rizzi C.C.A. Júdice W.A.S. Chagas J.R. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 1999; 274: 30433-30438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 27Almeida P.C. Nantes I., L. Chagas J. Rizzi C. Faljoni-Alario A. Carmona E. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 2001; 276: 944-951Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), the effects of HS on the enzymatic activity of natural and/or recombinant cruzipain isoforms were investigated. These studies indicate that the kinin releasing efficiency of living trypomastigotes is dramatically enhanced by heparan sulfate through its interactions with HK and cruzipain. Natural cruzipain (n-cruzipain) was isolated from crude aqueous extracts of Dm28c epimastigotes as described (28Lima A.P.C.A. Scharfstein J. Storer A.C. Ménard R. Mol. Biochem. Parasitol. 1992; 56: 335-338Crossref PubMed Scopus (47) Google Scholar). Recombinant cruzain (kindly supplied by Dr. J. H. McKerrow from the University of California, San Francisco), henceforth designated as r-cruzipain 1, was expressedin Escherichia coli (24Eakin A.E. Mills A.A. Harth G. McKerrow J.H. Craik C.S. J. Biol. Chem. 1992; 267: 7411-7420Abstract Full Text PDF PubMed Google Scholar); r-cruzipain 2 (80% sequence similarity with r-cruzipain 1) was recombinantly expressed inSaccharomyces cerevisiae and purified as described elsewhere (29Lima A.P.C.A. Reis F.C.G. Serveau C. Lalmanach G. Juliano L. Ménard R. Vernet T. Thomas D.Y. Storer A.C. Scharfstein J. Mol. Biochem. Parasitol. 2001; 114: 41-52Crossref PubMed Scopus (71) Google Scholar). These recombinant proteases differ from their natural enzymes by: (i) having a truncated C terminus where residues 216–346 are deleted; (ii) glycosylation in yeast (r-cruzipain 2); and (iii) lack of glycosylation in E. coli(r-cruzipain 1). Purified HK was obtained from human plasma as described previously (9Hasan A.A.K. Cines D.B. Herwald H. Schmaier A.H. Müller-Esterl N. J. Biol. Chem. 1995; 270: 19256-19261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). HS from bovine lung (16,000 Da) was a generous gift from Dr. P. Bianchini (Opocrin Research Laboratories, Modena, Italy). The characterization of anti-HK monoclonal antibodies was reported in (6Kaufmann J., M. Haasemann S. Modrow S. Müller-Esterl W. J. Biol. Chem. 1993; 268: 9079-9091Abstract Full Text PDF PubMed Google Scholar). Briefly, MBK3 (IgG1) is directed to bradykinin, and HKH4 is directed to the D1 domain of HK. Antiserum to the light chain of HK was raised in goats and adsorbed with total kininogen-deficient plasma and purified LK (30Schmaier A.H. Zuckerberg A. Silverman C. Kuchibhotla J. Tuszynski G.P. Colman R.W. J. Clin. Invest. 1983; 71: 1477-1489Crossref PubMed Scopus (65) Google Scholar). HK (160 nm) was incubated with different concentrations of n-cruzipain (8, 16, 32, and 64 nm) in 50 mm sodium phosphate buffer, pH 6.5, 5 mm EDTA, 200 mm NaCl, 2.5 mmDTT for 1 h at 37 °C. In some experiments, the reactions were performed in the presence of 30 μm of HS, which was added to the HK solution 5 min prior to the addition of cruzipain. The reactions were stopped by the addition of SDS-PAGE sample buffer consisting of 200 mm Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.025% bromphenol blue (1:1 v/v) followed by boiling for 5 min. The samples were subjected to 9% SDS-PAGE, transferred to nitrocellulose, blocked with 9% nonfat dried milk in 10 mm Na2HPO4, pH 7.2, 150 mm NaCl (phosphate-buffered saline) containing 0.05% (v/v) Tween 20, and incubated with the primary antibody at a 1:1000 dilution in blocking buffer for 1 h at room temperature. After washing, the appropriate secondary antibodies were incubated, and the reactive bands were visualized upon the addition of 1.4 mm 3, 3′-diamino-benzidine (Sigma), 0.03% H2O2 in phosphate-buffered saline. The concentration of purified HK was determined by titration with papain (Sigma) that had been previously active site-titrated with E-64 (Sigma). Variable amounts of HK were incubated with 0.4 nm of papain in 50 mm sodium phosphate, pH 6.5, 200 mm NaCl, 5 mm EDTA, 2.5 mm DTT for 15 min at room temperature. The residual papain activity was detected by the hydrolysis of 10 μm of CBZ-FR-AMC (Sigma) in the same buffer containing 5% Me2SO for substrate solubility. Substrate hydrolysis was monitored in a F-4500 Hitachi spectrofluorometer at 380 nm excitation and 440 nm emission. The initial velocities were determined by linear regression of the substrate hydrolysis curves. The equation obtained from the linear regression of the plot V 0 × volume of HK was used to calculate the corresponding amount of HK (x) to which V 0 equals 0 (y), and the concentration of the stock HK was then calculated accordingly. Purified cruzipain was titrated upon incubation with various concentrations of recombinant cystatin C (a gift from Dr. M. Abrahamson, University of Lund, Lund, Sweden) as described before (29Lima A.P.C.A. Reis F.C.G. Serveau C. Lalmanach G. Juliano L. Ménard R. Vernet T. Thomas D.Y. Storer A.C. Scharfstein J. Mol. Biochem. Parasitol. 2001; 114: 41-52Crossref PubMed Scopus (71) Google Scholar). The effect of heparan sulfate on the inhibition of n-cruzipain by HK was verified by the determination of theK i app in the presence or absence of 50 μm of HS, according to Henderson (31Henderson P.J.F. Biochem. J. 1972; 127: 321-333Crossref PubMed Scopus (478) Google Scholar). Briefly, theK i app values were determined from the slope of a plot of [I]0/1 − a against 1/a, where a =V i/V 0. The initial velocities were measured using 10 μm of CBZ-FR-AMC as substrate at room temperature as described in Ref. 29Lima A.P.C.A. Reis F.C.G. Serveau C. Lalmanach G. Juliano L. Ménard R. Vernet T. Thomas D.Y. Storer A.C. Scharfstein J. Mol. Biochem. Parasitol. 2001; 114: 41-52Crossref PubMed Scopus (71) Google Scholar. The influence of glycosaminoglycans on the endopeptidase activity of n-cruzipain, r-cruzipain 1, and r-cruzipain 2 was determined spectrofluorometrically using the kinin-like fluorogenic substrate Abz-LGMISLMKRPQ-EDDnp as described previously (26Almeida P.C. Nantes I.L. Rizzi C.C.A. Júdice W.A.S. Chagas J.R. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 1999; 274: 30433-30438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 27Almeida P.C. Nantes I., L. Chagas J. Rizzi C. Faljoni-Alario A. Carmona E. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 2001; 276: 944-951Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Fluorescence intensity was monitored on a thermostatic Hitachi F-2000 spectrofluorometer with excitation and emission wavelengths set at 320 and 420 nm, respectively. The enzymes were activated by incubation for 5 min at 37 °C in 50 mmsodium phosphate, pH 6.4, containing 200 mm NaCl, 1 mm EDTA, and 2 mm DTT. The measurements were performed at 37 °C in the same buffer, and the kinetic parameters were determined by measuring the initial rate of hydrolysis at various substrate concentrations in the presence or absence of different concentrations of sulfated GAGs. The data were analyzed by nonlinear regression using the program GraFit 3.01 (Erithacus Software Ltd.) as described previously (26Almeida P.C. Nantes I.L. Rizzi C.C.A. Júdice W.A.S. Chagas J.R. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 1999; 274: 30433-30438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The kinetic model depicted in Equation 1describes the effect of HS on the hydrolysis of Abz-LGMISLMKRPQ-EDDnp by these cysteine proteinases, where S is Abz-GMISLMKRPQ-EDDnp; K S is the substrate dissociation constant; K H is the apparent HS dissociation constant; α is the parameter ofK S perturbation; and β is the parameter ofk cat perturbation. v=Vmax·[S]Km1+[HS]KH1+β·[HS]α·KH+[S]1+[HS]α·KH1+β·[HS]α·KHEquation 1 Human kininogen (40 μg) was incubated with 32 nm of cruzipain in 50 mm sodium phosphate buffer, pH 6.5, 5 mmEDTA, 200 mm NaCl, 2.5 mm DTT for 1 h at 37 °C. The reaction was stopped by the addition of SDS-PAGE sample buffer and boiled for 5 min under reducing conditions. The fragments were separated by 9% SDS-PAGE, transferred to a polyvinylidine difluoride microporous membrane (Immobilon O, Millipore), stained with Coomassie Blue, destained, and washed extensively with distilled water. The major bands were excised and sequenced in a protein sequencer (Shimadzu Corporation, Tokyo, Japan; model PPSQ/23). Measurements of kinin peptides liberated by n-cruzipain, r-cruzipain 2, or living trypomastigotes were determined by a competitive enzyme-linked immunosorbent assay (Markit-M Bradykinin; Dainippon Pharmaceutical Co., Ltd., Osaka, Japan), according to the manufacturer's instructions (20Scharfstein J. Schmitz V. Morandi V. Capella M.A. Lima A.P.C.A. Morrot A. Juliano L. Müller-Esterl W. J. Exp. Med. 2000; 192: 1289-1299Crossref PubMed Scopus (191) Google Scholar). The kinin releasing reaction was performed by adding 5 nm of preactivated n-cruzipain or r-cruzipain 2 in 40 μl of 50 mmNa2HPO4, pH 6.5, 200 mm NaCl, 5 mm EDTA, 0.25 mm DTT containing 15 nm or 30 nm of HK. Dose-response dependence on HS (10–50 μm) was performed as described above. Enzyme specificity controls were carried out by pretreating activated n-cruzipain with 75 μm of the irreversible cysteine protease inhibitor E-64 (Sigma). After 1 h of incubation at 37 °C, the reaction was stopped by the addition of 100 μm E-64, 1 mg/ml bovine serum albumin, and 25 μm captopril. The samples were deproteinized with 20% trichloroacetic acid, and the kinin concentrations were estimated by enzyme-linked immunosorbent assay, using a standard curve prepared with synthetic bradykinin provided by the supplier. The values represent the means ± S.D. of three independent experiments. The kinin releasing activity of tissue culture trypomastigotes was measured by incubating the parasites (5 × 106 cells) with 15 nmHK in 200 μl of Ham's F-12 medium, 12.5 mm HEPES, pH 6.5, containing 25 μm captopril, and 1 mg/ml bovine serum albumin in the presence or absence of different HS concentrations for 1 h at 37 °C. The involvement of cysteine proteases in the kinin releasing activity of the parasites was examined by preincubating the cells with medium supplemented with 100 μm E-64. The parasites were centrifuged at 3000 × g for 10 min, and the supernatants were filtered through 0.2-μm Millipore filters and subsequently deproteinized as described above. The assays were carried out in duplicate. The values represent the means ± S.D. Because the catalytic efficiency of some papain-like proteases is modulated by GAGs (25Li Z. Hou W.S. Bromme D. Biochemistry. 2000; 39: 529-536Crossref PubMed Scopus (138) Google Scholar, 26Almeida P.C. Nantes I.L. Rizzi C.C.A. Júdice W.A.S. Chagas J.R. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 1999; 274: 30433-30438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 27Almeida P.C. Nantes I., L. Chagas J. Rizzi C. Faljoni-Alario A. Carmona E. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 2001; 276: 944-951Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), HS may likewise alter the kinetic properties of cruzipain, a member of the C1 peptidase family (32Barrett A.J. Rawlings N.D. Woessner J.F., Jr. Handbook of Proteolytic Enzymes. Academic Press, London1998Google Scholar), affecting its ability to function as a kininogenase (21Del Nery E. Juliano M.A Lima A.P.C.A Scharfstein J. Juliano L. J. Biol. Chem. 1997; 272: 25713-25718Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The effects of HS on the kinetic properties of cruzipain purified from epimastigote extracts (n-cruzipain) were compared with those from two genetically engineered isoforms, r-cruzipain 1 (cruzain) and r-cruzipain 2 (24Eakin A.E. Mills A.A. Harth G. McKerrow J.H. Craik C.S. J. Biol. Chem. 1992; 267: 7411-7420Abstract Full Text PDF PubMed Google Scholar, 29Lima A.P.C.A. Reis F.C.G. Serveau C. Lalmanach G. Juliano L. Ménard R. Vernet T. Thomas D.Y. Storer A.C. Scharfstein J. Mol. Biochem. Parasitol. 2001; 114: 41-52Crossref PubMed Scopus (71) Google Scholar). Using a short dipeptidyl synthetic substrate,e.g. Z-Phe-Arg-MCA, HS reducedk cat values of n-cruzipain without inducing significant differences in the K m (data not shown). The kinetic parameters of hydrolysis for each protease were then determined using a longer kinin-like fluorogenic substrate, Abz-LGMISLMKRPQ-EDDnp, which spans the N-terminal flanking site of bradykinin. In the presence of HS, there is a significant alteration in the kinetic parameters of n-cruzipain for the hydrolysis of the kinin-like substrate (Fig. 1). These data are consistent with that reported for papain (26Almeida P.C. Nantes I.L. Rizzi C.C.A. Júdice W.A.S. Chagas J.R. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 1999; 274: 30433-30438Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and for mammalian cathepsin B (27Almeida P.C. Nantes I., L. Chagas J. Rizzi C. Faljoni-Alario A. Carmona E. Juliano L. Nader H.B. Tersariol I.L.S. J. Biol. Chem. 2001; 276: 944-951Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The k cat value of n-cruzipain for the hydrolysis of Abz-LGMISLMKRPQ-EDDnp increased significantly as a function of the HS concentration (Fig. 1 A). HS also caused a marked increase in the enzyme affinity for this substrate, evidenced by the decrease in the K m value (Fig.1 B). The effect of HS on the activity of n-cruzipain is described by a hyperbolic mixed type inhibition (Equation 1 under “Experimental Procedures”). The efficiency of hydrolysis of the synthetic substrate was estimated by changing eitherK m (parameter α) or k cat(parameter β). These data were fitted to Equation 1 using nonlinear regression, and the values for the constants were determined (TableI). These studies showed that HS bound free n-cruzipain (E) with a dissociation constant ofK H = 25 ± 1 μm, whereas the parameters obtained for the binding to enzyme-substrate complex was αK H = 11 ± 1 μm. The interaction of HS with n-cruzipain resulted in a 2.59-fold increase in the k cat of the enzyme (β = 2.59 ± 0.05) and also increased the affinity of the enzyme for the kinin-like synthetic substrate (α = 0.446 ± 0.006), resulting in an almost 6-fold increase in its catalytic activity (β/α). HS also increased the catalytic efficiency of r-cruzipain 1 (β/α = 3.0); however, it was at the expense of higherk cat (β) values, because it did not alter the affinity of the enzyme for the kinin-like substrate (dissociation constant α = 1). There also was a modest increase on thek cat (β = 1.5 ± 0.2) of r-cruzipain-2, but this value was too low to permit a precise estimation of the dissociation constant between this isoenzyme and HS.Table IInfluence of heparan sulfate on the kinetic parameters of hydrolysis of Abz-LGMISLMKRPQ-EDDnp by natural and recombinant cruzipain isoformsProteinaseK HαK HK SαK Sk catβk catβ/αμmμmμmμms−1s−1n-cruzipain25 ± 111 ± 10.12 ± 0.010.054 ± 0.0011.5 ± 0.13.9 ± 0.25.8r-cruzipain 115 ± 115 ± 10.80 ± 0.090.82 ± 0.080.40 ± 0.011.2 ± 0.23.0r-cruzipain 2NDND0.11 ± 0.010.10 ± 0.010.50 ± 0.060.75 ± 0.091.5K S is the substrate dissociation constant;K H is the apparent HS dissociation constant; α is the parameter of K S perturbation in presence of heparan sulfate; and β is the parameter of k catperturbation. ND, not detected. Open table in a new tab K S is the substrate dissociation constant;K H is the apparent HS dissociation constant; α is the parameter of K S perturbation in presence of heparan sulfate; and β is the parameter of k catperturbation. ND, not detected. The previous finding that cruzipain can liberate kinins from HK was unexpected because kininogens have two functionally active cystatin-like inhibitory domains (7Barrett A.J. Fritz H. Grubb A. Isemura S. Järvinen M. Katunuma N. Machleidt W. Müller-Esterl W. Sasaki M. Turk V. Biochem. J. 1986; 236: 312Crossref PubMed Scopus (277) Google Scholar) that exert potent inhibitory activity toward calpain (33Schmaier A.H. Bradford H. Silver L.D. Farber A. Scott C.F. Schutsky D. Colman R.W. J. Clin. Invest. 1986; 77: 1565-1573Crossref PubMed Scopus (59) Google Scholar) and papain-like enzymes, such as cruzipain itself (34Stoka V. Nycander M. Lenarcic B. Labriola C. Cazzulo J.J. Björk I. Turk V. FEBS Lett. 1995; 370: 101-104Crossref PubMed Scopus (87) Google Scholar). Kinetic analysis in the presence of HS revealed that this GAG interfered with the cysteine-protease inhibitory capacity of HK. The apparent inhibition constants of HK over n-cruzipain were determined in the presence or absence of 100 μm of HS. The inhibitory activity of HK (K i app = 0.007 nm) decreased about 10-fold in the presence of HS (K i app = 0.07 nm). These results suggested that the HS interaction with HK and/or n-cruzipain significantly reduced the binding affinity of the cystatin-like domains of HK for the parasite proteinase. Because HS impaired the cysteine protease inhibitory activity of HK and enhanced the catalytic activity of n-cruzipain in assays performed with a kinin-like synthetic substrate, we examined whether this GAG changed the proteolysis of HK by n-cruzipain. Assays performed at variable molar ratios of HK/cruzipain ranging from 20:1 to 2:1 were performed in the absence or presence of a molar excess of HS (30 μm), and the HK breakdown products were characterized by immunoblotting and N-terminal sequencing of the cleavage fragments. Heavy chain fragments were defined by using a monoclonal antibody (HKH4) directed to the N-terminal D1 domain of HK. The assays performed with this antibody revealed the presence of two major breakdown products, referred to as H67 and H63 (Fig.2 A, lanes 2–4), in reaction mixtures that did not contain HS. Edman degradation of H67 and H63 did not reveal any sequences, indicating that these heavy chain fragments contain an intact N terminus, which is blocked in the native HK by a pyro-Glu residue (35Kellermann J. Lottspeich F. Henschen A. Müller-Esterl W. Eur. J. Biochem. 1986; 154: 471-478Crossref PubMed Scopus (61) Google Scholar). Unlike H67, the smaller fragment H63 was not detected by MBK3, an antibody to the bradykinin epitope; consistent with this result, H63 co-migrates with the kinin-free heavy chain fragment (∼63 kDa) of HK generated by tissue kallikrein (data not shown). The presence of HS almost completely prevented the formation of H67 in assays performed with relatively low concentrations of n-cruzipain, whereas the H63 form was abundant (Fig. 2 A,lanes 6–8). These results indicate that HS redirects cruzipain to more N-terminally located cleavage site(s). Next we did N-terminal sequencing of the breakdown products identified by the anti-light chain antibodies (Fig. 2 B). L55 and L51, the two major fragments detected polyclonal antibodies directed to the light chain of HK, displayed the sequences NAEVY and APAQ, respectively, at their N terminus (Fig. 2 B). This indicates that L55 and L51 are generated by n-cruzipain cleavage of sites that are located at the N-terminal and C-terminal flanking regions of the kinin domain D4 (see Fig. 3 for schematic representation). In the absence of HS, L51 is the major light chain product of HK released by n-cruzipain (Fig. 2 B, lanes 2–4), whereas L55 is the principal fragment formed in the presence of the GAG (Fig.2 B, lanes 6 and 7); these data are consistent with the corresponding pattern of the heavy chain fragments,i.e. H67 (−HS) and H63 (+HS). Because HS binds to the histidine-rich region of D5H (11Renné T. Dedio J. David G. Müller-Esterl W. J. Biol. Chem. 2000; 275: 33688-33696Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar), the data suggested that this interaction prevented access of the parasite protease to an otherwise susceptible cleavage site on HK, thereby precluding the formation of L51. The finding that H67, the heavy chain fragment complementary to L51, was not generated in appreciable amounts in the presence of HS (Fig. 2 A, lanes 6–8) further suggests that the GAG protects the cleavage site in D" @default.
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W2068989897 date "2002-02-01" @default.
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W2068989897 title "Heparan Sulfate Modulates Kinin Release by Trypanosoma cruzi through the Activity of Cruzipain" @default.
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W2068989897 doi "https://doi.org/10.1074/jbc.m108518200" @default.
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