RESUMO
This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar K(i) values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature.
Assuntos
Carboxipeptidases/metabolismo , Poliquetos/genética , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/genética , Sequência de Aminoácidos , Animais , Aprotinina/química , Aprotinina/genética , Aprotinina/farmacologia , Sequência de Bases , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Carboxipeptidases/antagonistas & inibidores , Bovinos , Clonagem Molecular , Relação Dose-Resposta a Droga , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Análise de Sequência de DNA , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologiaRESUMO
Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-L: -leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-α-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (K (m) = 0.18 ± 0.03 mM, k (cat) = 1.078 ± 0.055 s(-1), k (cat)/K (m) = 5.99 ± 0.57 s(-1) mM(-l)). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman's degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was suggested by molecular modeling.
Assuntos
Apocynaceae/metabolismo , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Látex/química , Sequência de Aminoácidos , Apocynaceae/enzimologia , Apocynaceae/genética , Sequência de Bases , Clonagem Molecular , Cisteína Proteases/genética , Cisteína Proteases/isolamento & purificação , DNA Complementar/genética , Frutas/enzimologia , Frutas/genética , Frutas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Ponto Isoelétrico , Cinética , Modelos Químicos , Dados de Sequência Molecular , Peso Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
A new proteolytic preparation from Vasconcellea quercifolia ("oak leaved papaya") latex containing several cysteine endopeptidases with high proteolytic activity has been obtained. The specific activity of the new enzymatic preparation (VQ) was higher than that of Carica papaya latex. VQ was able to coagulate milk and to hydrolyze caseins and then could be used to produce cheeses and/or casein hydrolysates. Ion exchange chromatography of VQ allowed the isolation of a new protease, named quercifoliain I, homogeneous when analyzed by SDS-PAGE, IEF and MALDI-TOF-MS. Molecular mass was 24195 Da, and its isoelectric point was >9.3. The N-terminal sequence was determined (YPESVDWRQ). Insulin B-chain cleavage showed higher specificity than that of papain and was restricted to glycyl and alanyl residues at P1' position. The tryptic peptide mass fingerprint of quercifoliain I analyzed with the MASCOT search tool did not find a match with papain or any other plant cysteine proteases.
Assuntos
Caricaceae/enzimologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Látex/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Biocatálise , Caricaceae/química , Caricaceae/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Estabilidade Enzimática , Insulina/química , Insulina/metabolismo , Ponto Isoelétrico , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por SubstratoRESUMO
Latices from Asclepias spp are used in wound healing and the treatment of some digestive disorders. These pharmacological actions have been attributed to the presence of cysteine proteases in these milky latices. Asclepias curassavica (Asclepiadaceae), "scarlet milkweed" is a perennial subshrub native to South America. In the current paper we report a new approach directed at the selective biochemical and molecular characterization of asclepain cI (acI) and asclepain cII (acII), the enzymes responsible for the proteolytic activity of the scarlet milkweed latex. SDS-PAGE spots of both purified peptidases were digested with trypsin and Peptide Mass Fingerprints (PMFs) obtained showed no equivalent peptides. No identification was possible by MASCOT search due to the paucity of information concerning Asclepiadaceae latex cysteine proteinases available in databases. From total RNA extracted from latex samples, cDNA of both peptidases was obtained by RT-PCR using degenerate primers encoding Asclepiadaceae cysteine peptidase conserved domains. Theoretical PMFs of partial polypeptide sequences obtained by cloning (186 and 185 amino acids) were compared with empirical PMFs, confirming that the sequences of 186 and 185 amino acids correspond to acI and acII, respectively. N-terminal sequences of acI and acII, characterized by Edman sequencing, were overlapped with those coming from the cDNA to obtain the full-length sequence of both mature peptidases (212 and 211 residues respectively). Alignment and phylogenetic analysis confirmed that acI and acII belong to the subfamily C1A forming a new group of papain-like cysteine peptidases together with asclepain f from Asclepias fruticosa. We conclude that PMF could be adopted as an excellent tool to differentiate, in a fast and unequivocal way, peptidases with very similar physicochemical and functional properties, with advantages over other conventional methods (for instance enzyme kinetics) that are time consuming and afford less reliable results.
Assuntos
Asclepias/enzimologia , Cisteína Endopeptidases/metabolismo , Látex/análise , Clonagem Molecular , Cisteína Proteases/metabolismo , Inibidores de Cisteína Proteinase , Hidrólise , Isoenzimas/genética , Látex/química , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
In the search for new metal-based drugs for the treatment of tumoral and parasitic diseases a vanadyl complex, [V(IV)O(SO(4))(H2O)(2)(dppz)].2H(2)O, that includes the bidentate polypyridyl DNA intercalator dipyrido[3,2-a:2',3'-c]phenazine (dppz), was synthesized, characterized by a combination of techniques, and in vitro evaluated on the human acute promyelocytic leukemia cell line HL-60 and against Dm28c strain epimastigotes of the parasite Trypanosoma cruzi, causative agent of Chagas' disease. EPR spectroscopy suggests a distorted octahedral geometry for the complex with the dppz ligand acting as bidentate, binding through both nitrogen donor atoms in an axial-equatorial mode. An oxo group, two water molecules and a sulphate donor occupy the remainder coordination positions. The complex, as well as the anti-trypanosomal reference drug Nifurtimox, showed IC(50) values in the muM range against T. cruzi Dm28c strain. In addition the complex exhibited excellent in vitro anti-tumor activity against leukemia (HL-60 cell line) comparable to that of cisplatin, inducing cell death by apoptosis with IC(50) values in the micromolar range. Data from gel electrophoresis and atomic force microscopy indicate that the complex interacts with DNA, suggesting that its mechanism of action may include DNA as a target. EPR and (51)V NMR experiments were also carried out with aged aerated solutions of the complex to get insight into the stability of the complex in solution and the species responsible for the in vitro activities observed.
Assuntos
Antineoplásicos/farmacologia , Antiprotozoários/farmacologia , Doença de Chagas/tratamento farmacológico , Substâncias Intercalantes/farmacologia , Neoplasias/tratamento farmacológico , Fenazinas/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimento , Vanadatos/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antiprotozoários/síntese química , Antiprotozoários/química , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HL-60 , Humanos , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/química , Fenazinas/síntese química , Fenazinas/química , Vanadatos/síntese química , Vanadatos/químicaRESUMO
After screening 25 marine invertebrates, a novel metallocarboxypeptidase (SmCP) has been identified by activity and MS analytical approaches, and isolated from the marine annelid Sabellastarte magnifica. The enzyme, which is a minor component of the molecularly complex animal body, as shown by 2D gel electrophoresis, has been purified from crude extracts to homogeneity by affinity chromatography on potato carboxypeptidase inhibitor and by ion exchange chromatography. SmCP is a protease of 33792 Da, displaying N-terminal and internal sequence homologies with M14 metallocarboxypeptidase-like enzymes, as determined by MS and automated Edman degradation. The enzyme contains one atom of Zn per molecule, is activated by Ca2+ and is drastically inhibited by the metal chelator 1,10-phenanthroline, as well as by excess Zn2+ or Cu2+, but moderately so by EDTA. SmCP is also strongly inhibited by specific inhibitors of metallocarboxypeptidases, such as benzylsuccinic acid and the protein inhibitors found in potato and leech (i.e. recombinant forms, both at nanomolar levels). The enzyme displays high peptidase efficiency towards pancreatic carboxypeptidase-A synthetic substrates, such as those with hydrophobic residues at the C-terminus but, remarkably, also towards the acidic ones. This property, previously described as for carboxypeptidase O-like activity, has been shown on long peptide substrates by MS. The results obtained in the present study indicate that SmCP is a novel member of the M14 metallocarboxypeptidases family (assignable to the M14A or pancreatic-like subfamily) with a wider specificity that has not been described previously.
Assuntos
Carboxipeptidases/metabolismo , Poliquetos/enzimologia , Animais , Cálcio/metabolismo , Carboxipeptidases/antagonistas & inibidores , Carboxipeptidases/química , Cátions Bivalentes , Quelantes/química , Ácido Edético/química , Ativação Enzimática , Fenantrolinas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Zinco/metabolismoRESUMO
Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.
Assuntos
Asclepias/enzimologia , Asclepias/genética , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Cisteína Endopeptidases/química , DNA Complementar/genética , Modelos Moleculares , Dados de Sequência Molecular , Pichia/genética , Pichia/metabolismo , Proteínas de Plantas/química , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Trypanosoma cruzi is the aetiological agent of Chagas' disease, a chronic infection that affects millions in Central and South America. Proteolytic enzymes are involved in the development and progression of this disease and two metallocarboxypeptidases, isolated from T. cruzi CL Brener clone, have recently been characterized: TcMCP-1 and TcMCP-2. Although both are cytosolic and closely related in sequence, they display different temporary expression patterns and substrate preferences. TcMCP-1 removes basic C-terminal residues, whereas TcMCP-2 prefers hydrophobic/aromatic residues. Here we report the three-dimensional structure of TcMCP-1. It resembles an elongated cowry, with a long, deep, narrow active-site cleft mimicking the aperture. It has an N-terminal dimerization subdomain, involved in a homodimeric catalytically active quaternary structure arrangement, and a proteolytic subdomain partitioned by the cleft into an upper and a lower moiety. The cleft accommodates a catalytic metal ion, most likely a cobalt, which is co-ordinated by residues included in a characteristic zinc-binding sequence, HEXXH and a downstream glutamate. The structure of TcMCP-1 shows strong topological similarity with archaeal, bacterial and mammalian metallopeptidases including angiotensin-converting enzyme, neurolysin and thimet oligopeptidase. A crucial residue for shaping the S(1') pocket in TcMCP-1, Met-304, was mutated to the respective residue in TcMCP-2, an arginine, leading to a TcMCP-1 variant with TcMCP-2 specificity. The present studies pave the way for a better understanding of a potential target in Chagas' disease at the molecular level and provide a template for the design of novel therapeutic approaches.
Assuntos
Carboxipeptidases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carboxipeptidases/genética , Domínio Catalítico , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Mutação Puntual , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Trypanosoma cruzi/genética , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
In this work we report the isolation, purification and characterization of a new protease from latex of Asclepias curassavica L. Crude extract (CE) was obtained by gathering latex on 0.1 M citric-phosphate buffer with EDTA and cysteine with subsequent ultracentrifugation. Proteolytic assays were made on casein or azocasein as substrates. Caseinolytic activity was completely inhibited by E-64. Stability at different temperatures, optimum pH and ionic strength were evaluated by measuring the residual caseinolytic activity at different times after the incubation. CE showed the highest caseinolytic activity at pH 8.5 in the presence of 12 mM cysteine. CE was purified by cation exchange chromatography (FPLC). Two active fractions, homogeneous by SDS-PAGE, were isolated. The major purified protease (asclepain cI) showed a molecular mass of 23.2 kDa by mass spectrometry and a pI higher than 9.3. The N-terminal sequence showed a high similarity with those of other plant cysteine proteinases. When assayed on N-alpha-CBZ-aminoacid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative. Determinations of kinetic parameter (km and Kcat) were performed with PFLNA.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Látex/metabolismo , Sequência de Aminoácidos , Asclepias , Bioquímica/métodos , Caseínas/química , Cátions , Cromatografia por Troca Iônica , Cisteína/química , Relação Dose-Resposta a Droga , Ácido Edético/farmacologia , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Temperatura Alta , Concentração de Íons de Hidrogênio , Íons , Cinética , Látex/química , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Fatores de Tempo , UltracentrifugaçãoRESUMO
A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.