RESUMO
African trypanosomes contain a cytosolic serine oligopeptidase, called OP-Tb, that is reversibly inhibited by the active principles of three of the five most commonly used trypanocidal drugs: pentamidine, diminazene and suramin. OP-Tb was inhibited by pentamidine in a competitive manner, and by suramin in a partial, non-competitive manner. The inhibition of OP-Tb by a variety of suramin analogues correlated with the trypanocidal efficacy of these analogues (P=0.03; by paired Student's t-test). Since intracellular (therapeutic) concentrations of pentamidine and suramin are reported to reach approximately 206Ki and 15Ki respectively, we suggest that these drugs may exert part of their trypanocidal activity through the inhibition of OP-Tb.
Assuntos
Diminazena/farmacologia , Pentamidina/farmacologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Suramina/análogos & derivados , Suramina/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma/enzimologia , Animais , Relação Estrutura-Atividade , Trypanosoma/efeitos dos fármacosRESUMO
Anti-peptide antibodies were raised against synthetic peptides selected from the sequences of human cathepsins B and L, porcine cathepsin D and human type IV collagenase. Sequences were selected from the active site clefts of the cathepsins in the expectation that these would elicit immunoinhibitory antibodies. In the case of type IV collagenase a sequence unique to this metalloproteinase subclass and suitable for immunoaffinity purification, was chosen. Antibodies against the chosen cathepsin B sequence were able to recognize the peptide but were apparently unable to recognise the whole enzyme. Antibodies against the chosen cathepsin L sequence were found to recognise and inhibit the native enzyme and were also able to discriminate between denatured cathepsins L and B on Western blots. Antibodies against the chosen cathepsin D sequence recognised native cathepsin D in a competition ELISA, but did not inhibit the enzyme. Native type IV collagenase was purified from human leukocytes by immuno-affinity purification with the corresponding anti-peptide antibodies.
Assuntos
Catepsinas/imunologia , Endopeptidases , Colagenase Microbiana/imunologia , Sequência de Aminoácidos , Formação de Anticorpos , Especificidade de Anticorpos , Western Blotting , Catepsina B/imunologia , Catepsina D/imunologia , Catepsina L , Cisteína Endopeptidases , Precursores Enzimáticos/imunologia , Ensaio de Imunoadsorção Enzimática , Metaloproteinase 9 da Matriz , Dados de Sequência MolecularRESUMO
Trypanosoma brucei contain a serine oligopeptidase (OP-Tb) that is released into (and remains active in) the blood of trypanosome-infected animals. Here a similar enzyme from Trypanosoma congolense is described. This oligopeptidase, called OP-Tc, was purified using three-phase partitioning, and ion-exchange and affinity chromatography. OP-Tc is inhibited by alkylating agents, by serine peptidase-specific inhibitors including 3,4-dichloroisocoumarin, 4-(2-aminoethyl)benzenesulfonylfluoride and diispropylfluoro-phosphate and by other peptidase inhibitors including leupeptin, antipain and peptidyl chloromethyl ketones. Reducing agents such as dithiothreitol enhanced activity as did heparin, spermine and spermidine. The enzyme has trypsin-like specificity since it cleaved fluorogenic peptides that have basic amino acid residues (Arg or Lys) in the P1 position. Potential substrates without a basic residue in P1 were not hydrolysed. Although OP-Tc has weak arginine aminopeptidase activity, the enzyme clearly preferred substrates that had amino acids in the P2 and P3 positions. Overall, OP-Tc appears to be less efficient than OP-Tb because it usually displayed lower k(cat)/Km values for the substrates tested. However, like OP-Tb, the best substrate for OP-Tc was Cbz-Arg-Arg-AMC (Km = 0.72 microM, k(cat) = 96 s(-1)). OP-Tc preference for amino acids in the P2 position was (Gly,Lys,Arg) > Phe > Leu > Pro. The results also suggest that the P3-binding site has hydrophobic characteristics. OP-Tc may not be a naturally immunodominant molecule because neither IgG nor IgM anti- OP-Tc antibodies were detected in the blood of experimentally infected cattle.
Assuntos
Peptídeo Hidrolases/isolamento & purificação , Peptídeo Hidrolases/metabolismo , Trypanosoma congolense/enzimologia , Animais , Anticorpos Antiprotozoários/sangue , Bovinos , Ativação Enzimática , Masculino , Peptídeo Hidrolases/imunologia , Ratos , Ratos Sprague-Dawley , Inibidores de Serina Proteinase/farmacologia , Especificidade por Substrato , Trypanosoma congolense/patogenicidade , Tripanossomíase Africana/imunologia , Tripanossomíase Africana/parasitologia , Tripanossomíase Africana/veterinária , Tripanossomíase Bovina/imunologia , Tripanossomíase Bovina/parasitologia , Tripsina/metabolismoRESUMO
Two groups of irreversible serine peptidase inhibitors, peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters, were examined for antitrypanosomal activity against the bloodstream form of Trypanosoma brucei brucei. Both peptidyl chloromethyl ketones and peptidyl phosphonate diphenyl esters inhibited trypsin-like peptidases of the parasites and exhibited antitrypanosomal activity at micromolar concentrations. In live T. b. brucei, labelled analogues of both of these groups of inhibitors primarily targeted an 80-kDa peptidase, possibly a serine oligopeptidase known as oligopeptidase B. In an in vivo mouse model of infection, one of these inhibitors, carbobenzyloxyglycyl-4-amidinophenylglycine phosphonate diphenyl ester, was curative at 5 mg kg(-1) day(-1) but appeared toxic at higher doses. There was no significant correlation between the inhibitory potency (as evaluated against purified T. b. brucei oligopeptidase B) and the in vitro antitrypanosomal efficacy of either group of inhibitors, suggesting that these inhibitors were acting on multiple targets within the parasites, or had different cell permeability properties. These findings suggest that serine peptidases may represent novel chemotherapeutic targets in African trypanosomes.
Assuntos
Alcanos/farmacologia , Ésteres/farmacologia , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Sítios de Ligação , Modelos Animais de Doenças , Progressão da Doença , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Peptídeo Hidrolases/efeitos dos fármacos , Inibidores de Proteases/uso terapêutico , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/tratamento farmacológico , Tripanossomíase Africana/enzimologiaRESUMO
Tubers of Callilepis laureola, a traditional remedy, contain an inhibitor of oxidative phosphorylation; atractyloside. A "competitive" ELISA was developed, using the antiserum produced to an atractyloside-protein conjugate. An ovalbumin-atractyloside conjugate was adsorbed to microtitre wells and plates incubated with sample (atractyloside or tuber extract) and antiserum. After successive incubation with secondary antibody-enzyme conjugate and substrate, the absorbance was read at 405 nm. Antibody working dilution was low, but results, confirmed by thin layer chromatography, indicate the immunoassay has diagnostic potential.
Assuntos
Atractilosídeo/análise , Plantas Medicinais/análise , Animais , Formação de Anticorpos , Cromatografia em Camada Fina , Ensaio de Imunoadsorção Enzimática , Coelhos/imunologia , Soroalbumina Bovina/imunologiaRESUMO
Cathepsin L was purified from the liver of a higher primate, the baboon (Papio ursinus), largely in a single-chain form and in the form of proteolytically active complexes with an endogenous cystatin. This mimics the situation found in both human and sheep livers. Both forms of cathepsin L were active at physiological pH. Physicochemical characterization and N-terminal amino sequencing of baboon cathepsin L showed a close relationship with the human enzyme. Cystatins with characteristics similar to those found for stefins A and B could also be purified from baboon livers. Proteolytically active, SDS-stable complexes could be shown to form in vitro with the molecules characterized as stefin B, but not with stefin A type cystatins. The non-inhibitory complexes could be shown to require less cysteine for activation than free cathepsin L and this, together with the above result, might indicate that a sulfhydryl interchange mechanism is responsible for the formation of covalent, non-inhibitory complexes.
Assuntos
Catepsinas/isolamento & purificação , Endopeptidases , Fígado/enzimologia , Animais , Catepsina L , Catepsinas/química , Catepsinas/metabolismo , Fenômenos Químicos , Físico-Química , Cistatina B , Cistatinas/metabolismo , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Cinética , Papio , Fragmentos de Peptídeos/química , Análise de Sequência , OvinosRESUMO
Cathepsin L and stefin B were isolated from sheep liver, the cathepsin L being isolated by a low pH homogenisation method, which increases the proportion of the two-chain form of the enzyme, thus facilitating sequencing. The amino acid sequences of the isolated cathepsin L and stefin B were determined. The two-chain form of cathepsin L contains 217 amino acid residues and has an M(r) of 23,627. The sequence was obtained by sequencing the native active enzyme, the light and heavy chains and the peptides generated by cyanogen bromide cleavage. These peptides were aligned with peptides obtained by hydrolysis with endoproteinase Lys-C, glycyl endopeptidase and endoproteinase Glu-C. Sheep liver cathepsin L exhibits a high degree of sequence identity to human cathepsin L. Sheep stefin B consists of 98 amino acid residues and its calculated M(r) is 11,150. The inhibitor has its NH2-terminal amino acid residue blocked. Its amino acid sequence was determined by sequencing the peptides obtained by cleavage with cyanogen bromide and peptides obtained by hydrolysis with endoproteinase Glu-C and endoproteinase Lys-C. Sheep stefin B shows a high degree of sequence identity with bovine and human stefin B. The kinetics of the interaction between sheep cathepsin L and stefin B were determined, with the interaction of stefin B with papain used as a benchmark to compare with other published results. Despite the considerable homology between bovine and sheep stefin B, the kinetics of their interaction with papain and cathepsin L differed markedly, possibly due to the differences in the so-called "trunk" region of the cystatin molecule.
Assuntos
Catepsinas/antagonistas & inibidores , Catepsinas/química , Cistatinas/química , Endopeptidases , Sequência de Aminoácidos , Animais , Catepsina L , Bovinos , Cistatina B , Cistatinas/farmacologia , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase , Humanos , Cinética , Dados de Sequência Molecular , Papaína/química , Homologia de Sequência de Aminoácidos , OvinosRESUMO
Proteolytically active complexes of the proteinase cathepsin L, with an endogenous inhibitor of cysteine proteinases, were purified from sheep liver. The complexes were active against the synthetic substrate Z-Phe-Arg-NHMec and also the proteins azocasein and gelatin. The composition of the complexes was demonstrated by Western blotting, after reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with monospecific antibodies raised against purified sheep liver cathepsin L and purified sheep liver cysteine proteinase inhibitor (probably stefin B). Similar complexes could be formed in vitro, by coincubation of purified sheep liver cathepsin L with the purified sheep liver cystatin at a pH of 5.5 or higher.
Assuntos
Catepsinas/metabolismo , Cistatinas/metabolismo , Endopeptidases , Animais , Sítios de Ligação , Western Blotting , Catepsina L , Catepsinas/isolamento & purificação , Cromatografia em Gel , Cromatografia por Troca Iônica , Cistatinas/isolamento & purificação , Cisteína Endopeptidases , Eletroforese em Gel de Poliacrilamida , Cinética , Fígado/enzimologia , Substâncias Macromoleculares , Modelos Biológicos , Peso Molecular , Ligação Proteica , Ovinos , Especificidade por SubstratoRESUMO
Antibodies, raised in chickens (IgY) and rabbits (IgG) against the lysosomal proteinase cathepsin L, targeted the enzyme in an ELISA and Western blot. In contrast to the rabbit IgG, the chicken IgY was immunoinhibitory towards cathepsin L. An epitope that elicits immunoinhibitory antibodies has been localized to an active site-associated peptide sequence. The corresponding free peptide, coated down in an ELISA, is recognised by the chicken IgY, but not the rabbit IgG. This peptide was able to inhibit the immunoinhibition of cathepsin L by chicken anti-cathepsin L IgY, suggesting its complete or partial identity with an immunogenic epitope for chickens in whole cathepsin L.
Assuntos
Catepsinas/imunologia , Cisteína Endopeptidases/imunologia , Endopeptidases , Animais , Especificidade de Anticorpos , Sítios de Ligação , Catepsina L , Catepsinas/antagonistas & inibidores , Galinhas , Epitopos/isolamento & purificação , Imunoglobulina G , Imunoglobulinas , Peptídeos/imunologia , Coelhos , OvinosRESUMO
Anti-peptide antibodies were produced against the cysteine proteinase trypanopain-Tb from Trypanosoma brucei brucei and the effects of these antibodies on enzyme activity against carboxybenzoyl (Z)-Phe-Arg-aminomethylcoumarin (AMC) investigated. A peptide was synthesised corresponding to a region of the trypanopain-Tb active site around the active site histidine so that the resulting anti-peptide antibodies specifically targeted the active site of the enzyme. Such antibodies were considered more likely to modulate enzyme activity compared with antibodies directed against other regions of the enzyme. Trypanopain-Tb activity was modulated by rabbit and chicken antibodies produced against both the free and conjugated peptide. Rabbit anti-peptide antibodies enhanced trypanopain-Tb activity by up to 64% at 500 micrograms/ml relative to non-immune antibodies. Chicken antibodies on the other hand, both enhanced (by up to 176% at 500 mg/ml) and inhibited (by up to 85% at 250 mg/ml) trypanopain-Tb activity against Z-Phe-Arg-AMC. The nature of the antibody effect depended on the stage during the immunisation protocol at which the antibodies were produced. Chicken antibodies also modulated trypanopain-Tb activity in lysates of T.b. brucei, while rabbit antibodies were only effective against the purified enzyme. Anti-trypanopain-Tb peptide antibodies were thus shown to have the potential to affect trypanopain-Tb activity.
Assuntos
Anticorpos Antiprotozoários/metabolismo , Cisteína Endopeptidases/imunologia , Trypanosoma brucei brucei/imunologia , Animais , Anticorpos Antiprotozoários/farmacologia , Formação de Anticorpos , Sítios de Ligação , Galinhas , Cumarínicos/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Dipeptídeos/metabolismo , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Coelhos , Relação Estrutura-Atividade , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologiaRESUMO
African trypanosomes are tsetse-transmitted protozoan parasites that cause sleeping sickness in humans and 'Nagana' in animals. A high relative molecular mass multicatalytic proteinase complex (MCP) was purified and biochemically characterized from the cytosolic fraction of Trypanosoma brucei brucei. The isolation procedure consisted of fractionation of the lysate by high speed centrifugation, chromatography on Q-sepharose molecular sieve filtration on Sephacryl S-300, chromatography on HA-Ultrogel and glycerol density gradient centrifugation (10-40%). The final enzyme preparation yielded a single protein band corresponding to a relative molecular mass of 630 kDa on a non-denaturing polyacrylamide gel. The enzyme hydrolyses a wide range of peptide substrates characteristic of chymotrypsin-like, trypsin-like, peptidylglutamylpeptide-hydrolysing activities determined by fluorogenic peptides, Z-Gly-Gly-Leu-NHMec, Z-Arg-Arg-NHMec and Z-Leu-Leu-Glu-beta NA, respectively. The enzyme was found to have a wide variation in pH optimal activity profile, with optimum activity against Z-Gly-Gly-Leu-NHMec at 7.8, Z-Arg-Arg-NHMec at pH 10.5 and Z-Leu-Leu-Glu-beta NA at pH 8.0, showing that the different activities are distinct. The enzyme hydrolysed oxidized proteins. In addition, the chymotryptic and trypsin-like activities were susceptible to inhibition by peptide aldehyde inhibitors with variable inhibition effects. The study demonstrates the presence of a non-lysosomal proteasome pathway of intracellular protein degradation in the bloodstream form of T. b. brucei. Further, the ability of the enzyme to hydrolyse most oxidized proteins, and the high immunogenicity exhibited suggests a possible involvement of the enzyme in pathogenesis of the disease.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Anticorpos Antiprotozoários/imunologia , Centrifugação com Gradiente de Concentração , Galinhas , Cromatografia em Gel , Quimotripsina/química , Quimotripsina/metabolismo , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/isolamento & purificação , Cisteína Endopeptidases/fisiologia , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Interações Hospedeiro-Parasita , Concentração de Íons de Hidrogênio , Hidrólise , Lisossomos/enzimologia , Peso Molecular , Complexos Multienzimáticos/imunologia , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/fisiologia , Oxirredução , Complexo de Endopeptidases do Proteassoma , Especificidade por Substrato , Trypanosoma brucei brucei/imunologia , Tripanossomíase Africana/enzimologia , Tripanossomíase Africana/parasitologia , Tripsina/química , Tripsina/metabolismoRESUMO
The activity of cathepsin L is affected by ionic strength, resulting in the measured pH optimum being higher in acetate-4-morpholineethane sulfonic acid (MES)-Tris buffers of constant ionic strength than in phosphate buffers of constant molarity (and hence varying ionic strength). In acetate-MES-Tris and phosphate buffers of constant ionic strength across the pH range, the catalytic constant, kcat, generally peaked at ca. pH 6.5 and essentially independently of ionic strength. Km values, of ca. 5 microM, manifested a slight rising trend with increasing ionic strength, with a sharp increase to 20-25 microM, specifically at pH 6.5 and I = 0.4. At physiological ionic strengths, the specific buffer ions present affected the activity of mature cathepsin L, kcat/Km declining above pH 6.5 in phosphate buffer, but only above pH 7 in acetate-MES-Tris buffer. In Hanks' balanced salt solution, a model of the extracellular fluid, measured values at pH 7.2 were kcat, 18.9 s-1; Km, 13.5 microM; and kcat/Km, 1.4 x 10(6) M-1 s-1. The stability of cathepsin L in the physiological pH range was also differentially affected by the specific buffer ions, generally in parallel with the enzyme activity. In Hanks' balanced salt solution, mature cathepsin L was substantially active and stable, having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumor pH).
Assuntos
Catepsinas/metabolismo , Endopeptidases , Espaço Extracelular , Acetatos , Ácidos Alcanossulfônicos , Soluções Tampão , Catepsina L , Cisteína Endopeptidases , Estabilidade Enzimática , Humanos , Concentração de Íons de Hidrogênio , Soluções Isotônicas , Rim/enzimologia , Morfolinas , Concentração Osmolar , Fosfatos , TrometaminaRESUMO
In vitro, living bloodstream forms of Trypanosoma congolense were shown to hydrolyse p-nitrophenyl phosphate, a substrate for phosphatases. This activity appears to be from an acid phosphatase because it was enhanced at low pH values, was inhibited by the acid phosphatase inhibitor sodium fluoride, and was not inhibited by the alkaline phosphatase inhibitor tetramisole. The activity did not appear to be secreted into the surrounding medium by the living parasites although phosphatase activity could be detected in the surrounding medium when dead or dying parasites were present. Studies at various temperatures indicated that at least some of this acid phosphatase activity may be associated with the surface of the parasites, rather than with endocytic or intracellular systems. This was supported by subcellular fractionation of radiolabelled parasites which showed some cosedimentation of acid phosphatase activity with radiolabelled iodine. Histochemical studies of the parasites also supported this conclusion. Electron microscopical examination of trypanosomes incubated with lead nitrate and p-nitrophenyl phosphate showed lead phosphate deposits on the surface of the parasites in addition to the expected localisation in the flagellar pocket. We conclude that Trypanosoma congolense possesses a surface-bound acid phosphatase.
Assuntos
Fosfatase Ácida/análise , Trypanosoma congolense/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Animais , Centrifugação com Gradiente de Concentração , Microanálise por Sonda Eletrônica , Inibidores Enzimáticos/farmacologia , Histocitoquímica , Concentração de Íons de Hidrogênio , Hidrólise , Indicadores e Reagentes/metabolismo , Microscopia Eletrônica , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fluoreto de Sódio/farmacologia , Tetramizol/farmacologia , Trypanosoma congolense/ultraestruturaRESUMO
Adhesins from oral bacteria perform an important function in colonizing target tissues within the dentogingival cavity. In Porphyromonas gingivalis certain of these adhesion proteins exist as a complex with either of two major proteinases referred to as gingipain R (arginine-specific gingipain) and gingipain K (lysine-specific gingipain) (R. N. Pike, W. T. McGraw, J. Potempa, and J. Travis, J. Biol. Chem. 269:406-411, 1994). With specific proteinase inhibitors, it was shown that hemagglutination by either proteinase-adhesin complex could occur independently of proteinase activity. Significantly, low concentrations of fibrinogen, fibronectin, and laminin inhibited hemagglutination, indicating that adherence to these proteins and not the hemagglutination activity was a primary property of the adhesin activity component of complexes. Binding studies with gingipain K and gingipain R suggest that interaction with fibrinogen is a major function of the adhesin domain, with dissociation constants for binding to fibrinogen being 4 and 8.5 nM, respectively. Specific association with fibronectin and laminin was also found. All bound proteins were degraded by the functional proteinase domain, with gingipain R being more active on laminin and fibronectin and gingipain K being more effective in the digestion of fibrinogen. Cumulatively, these data suggest that gingipain R and gingipain K, acting as proteinase-adhesin complexes, progressively attach to, degrade, and detach from target proteins. Since such complexes appear to be present on the surfaces of both vesicles and membranes of P. gingivalis, they may play an important role in the attachment of this bacterium to host cell surfaces.
Assuntos
Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Hemaglutininas/metabolismo , Porphyromonas gingivalis/metabolismo , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Cisteína Endopeptidases Gingipaínas , Hemaglutinação/efeitos dos fármacos , Laminina/metabolismo , Dados de Sequência Molecular , Ligação ProteicaRESUMO
An unusual bacterium causing respiratory disease in chickens emerged in South Africa in February 1989. The disease resembled infectious coryza but the organism differed from typical Haemophilus paragallinarum especially in that it did not require V-factor for growth. It has been termed an NAD-independent H. paragallinarum. A study of avian haemophili isolated from diseased chickens in Kwazulu-Natal over the past five years revealed the presence of typical H. paragallinarum, NAD-independent H. paragallinarum and H. avium (now transferred to the genus Pasteurella). Before the end of 1989 the NAD-independent H. paragallinarum had become the predominate isolate and thereafter was isolated from commercial chickens in other regions of South Africa. The disease affected all strains of chickens in an overall age range of 14 days to 64 weeks. The organism was responsible for upper respiratory disease of broilers and layers and implicated in lower respiratory disease of broilers. It was commonly isolated from diseased adult birds previously vaccinated against typical H. paragallinarum. Broilers were most commonly infected from 3 weeks of age and layers within the placement to peak production period. Whole cell protein profiles of NAD-independent H. paragallinarum isolates from five different commercial poultry units were identical but differed from that of a typical isolate.
RESUMO
A trypsin-like serine peptidase activity, levels of which correlate with blood parasitemia levels, is present in the plasma of rats acutely infected with Trypanosoma brucei brucei. Antibodies to a trypanosome peptidase with a trypsin-like substrate specificity (oligopeptidase B [OP-Tb]) cross-reacted with a protein in the plasma of trypanosome-infected rats on a Western blot. These antibodies also abolished 80% of the activity in the plasma of trypanosome-infected rats, suggesting that the activity may be attributable to a parasite-derived peptidase. We purified the enzyme responsible for the bulk of this activity from parasite-free T. b. brucei-infected rat plasma and confirmed its identity by protein sequencing. We show that live trypanosomes do not release OP-Tb in vitro and propose that disrupted parasites release it into the host circulation, where it is unregulated and retains full catalytic activity and may thus play a role in the pathogenesis of African trypanosomiasis.
Assuntos
Proteínas de Protozoários/sangue , Serina Endopeptidases/sangue , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/sangue , Animais , Anticorpos Antiprotozoários/imunologia , Western Blotting , Catálise , Reações Cruzadas , Masculino , Ratos , Ratos Sprague-Dawley , Serina Endopeptidases/imunologia , Serina Endopeptidases/isolamento & purificaçãoRESUMO
African trypanosomes contain proteases that may be released into the bloodstream of their infected hosts. This paper describes a novel, combined isolation of a cysteine proteinase (called trypanopain-Tb) and a serine oligopeptidase (which we call oligopeptidase-Tb) from Trypanosoma brucei brucei, as well as a comparison of the activities of these two enzymes against several host regulatory molecules. The enzymes differed in various respects. Firstly, purified trypanopain-Tb was shown to readily cleave proteins such as gelatin maximally at acidic pH. In contrast, oligopeptidase-Tb, which is optimally active at alkaline pH, did not hydrolyse proteins larger than 4 kDa. However, it readily hydrolysed various polypeptides, including neurotensin and atrial natriuretic factor. The interaction of the two enzymes with mammalian protease inhibitors also differed. Cystatins and alpha2-macroglobulin effectively inhibited trypanopain-Tb, with the Ki values for cystatin C and low-molecular-mass kininogen (approximately 10(-11) M) predicting, that trypanopain-Tb is likely to be effectively controlled by these inhibitors if released into the host bloodstream. In contrast, oligopeptidase-Tb was not inhibited by serpins or (a2-macroglobulin, suggesting that it may remain active if released into the host bloodstream. In support of these in vitro results, the blood of trypanosome-infected rats displayed no trypanopain-Tb-like activity, but exhibited high oligopeptidase-Tb-like activity. Thus, while trypanopain-Tb seems likely to be confined to an intracellular role within the parasite, oligopeptidase-Tb has the potential to remain active in the host bloodstream and so contribute directly to pathogenesis.
Assuntos
Cisteína Endopeptidases/química , Inibidores Enzimáticos/farmacologia , Peptídeo Hidrolases/química , Serina Endopeptidases/química , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/enzimologia , Sequência de Aminoácidos , Animais , Cistatina A , Cistatina B , Cistatina C , Cistatinas/farmacologia , Cisteína Endopeptidases/sangue , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Cininogênios/farmacologia , Dados de Sequência Molecular , Peptídeo Hidrolases/sangue , Peptídeo Hidrolases/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Ratos , Serina Endopeptidases/sangue , Serina Endopeptidases/metabolismo , Trypanosoma brucei brucei/químicaRESUMO
Trypanosoma brucei brucei is a causative agent of bovine trypanosomiasis (nagana), a disease of considerable economic significance in much of Africa. Here we report investigations on the effects of various irreversible cysteine proteinase inhibitors, including vinyl sulfones (VS), peptidyl chloromethylketones (CMK), diazomethylketones, and fluoromethyl ketones, on the major lysosomal cysteine proteinase (trypanopain-Tb) of T. b. brucei and on in vitro-cultured bloodstream forms of the parasite. Many of the tested inhibitors were trypanocidal at low micromolar concentrations. Methylpiperazine urea-Phe-homoPhe-VS was the most effective trypanocidal agent, killing 50% of test populations at a work ing concentration of 0.11 microM, while carbobenzoxy-Phe-Phe-CMK was the most trypanocidal of the methylketones with an IC50 of 3.6 microM. Labelling of live and lysed T. b. brucei with biotinylated inhibitor derivatives suggests that trypanopain-Tb is the likely intracellular target for these inhibitors. Kinetic analysis of the inhibition of purified trypanopain-Tb by the inhibitors showed that most had kass values in the 10(6) M-1 s-1 range. We conclude that cysteine proteinase inhibitors have potential as trypanocidal agents and that a major target of these compounds is the lysosomal enzyme trypanopain-Tb.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Animais , Western Blotting , Bovinos , Cisteína Endopeptidases/química , Cisteína Endopeptidases/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , Tripanossomíase Africana/parasitologia , Tripanossomíase Bovina/parasitologiaRESUMO
BACKGROUND: Protozoan parasites of the genus Trypanosoma cause disease in a wide range of mammalian hosts. Trypanosoma brucei brucei, transmitted by tsetse fly to cattle, causes a disease (Nagana) of great economic importance in parts of Africa. T. b. brucei also serves as a model for related Trypanosoma species, which cause human sleeping sickness. MATERIALS AND METHODS: Chalcone and acyl hydrazide derivatives are known to retard the growth of Plasmodium falciparum in vitro and inhibit the malarial cysteine proteinase, falcipain. We tested the effects of these compounds on the growth of bloodstream forms of T. b. brucei in cell culture and in a murine trypanosomiasis model, and investigated their ability to inhibit trypanopain-Tb, the major cysteine proteinase of T. b. brucei. RESULTS: Several related chalcones, acyl hydrazides, and amides killed cultured bloodstream forms of T. b. brucei, with the most effective compound reducing parasite numbers by 50% relative to control populations at a concentration of 240 nM. The most effective inhibitors protected mice from an otherwise lethal T. b. brucei infection in an in vivo model of acute parasite infection. Many of the compounds also inhibited trypanopain-Tb, with the most effective inhibitor having a Ki value of 27 nM. Ki values for trypanopain-Tb inhibition were up to 50- to 100-fold lower than for inhibition of mammalian cathepsin L, suggesting the possibility of selective inhibition of the parasite enzyme. CONCLUSIONS: Chalcones, acyl hydrazides, and amides show promise as antitrypanosomal chemotherapeutic agents, with trypanopain-Tb possibly being one of their in vivo targets.
Assuntos
Amidas/farmacologia , Chalcona/farmacologia , Endopeptidases , Hidrazinas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Amidas/química , Amidas/uso terapêutico , Animais , Catepsina L , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Chalcona/química , Chalcona/uso terapêutico , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico , Modelos Animais de Doenças , Humanos , Hidrazinas/química , Hidrazinas/uso terapêutico , Concentração Inibidora 50 , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Ovinos , Tripanossomicidas/química , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Tripanossomíase/tratamento farmacológico , Tripanossomíase/parasitologiaRESUMO
Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P(1) and P(2) (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k(cat)/K(m) = 529 s(-1) microM(-1)). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.