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1.
Exp Parasitol ; 167: 94-102, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27240755

RESUMO

A gene encoding fatty acid- and retinoid-binding protein was isolated from the cereal cyst nematode Heterodera avenae and the biochemical function of the protein that it encodes was analysed. The full-length cDNA of the Ha-far-1 gene is 827 bp long and includes a 22- nucleotide trans-spliced leader sequence (SL1) at its 5-end. The genomic clone of Ha-far-1 consists of eight exons separated by seven introns, which range in size from 48 to 186 bp. The Ha-far-1 cDNA contains an open reading frame encoding a 191 amino acid protein, with a predicted secretory signal peptide. Sequence analysis showed that Ha-FAR-1 has highest similarity to the Gp-FAR-1 protein from the potato cyst nematode, Globodera pallida and that the protein was grouped with all homologues from other plant-parasitic nematodes in a phylogenetic analysis. Fluorescence-based ligand binding analysis confirmed that the recombinant Ha-FAR-1 protein was able to bind fatty acids and retinol. Spatial and temporal expression assays showed that the transcripts of Ha-far-1 accumulated mainly in the hypodermis and that the gene is most highly expressed in third-stage juveniles of H. avenae. Fluorescence immunolocalization showed that the Ha-FAR-1 protein was present on the surface of the infective second-stage juveniles of H. avenae. Nematodes treated with dsRNA corresponding to Ha-far-1 showed significantly reduced reproduction compared to nematodes exposed to dsRNA from a non-endogenous gene, suggesting that Ha-far-1 may be an effective target gene for control of H. avenae using an RNAi strategy.


Assuntos
Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Proteínas de Ligação ao Retinol/isolamento & purificação , Tylenchoidea/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA Complementar/química , DNA de Helmintos/isolamento & purificação , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Proteínas de Helminto/química , Proteínas de Helminto/genética , Hibridização In Situ , Ligantes , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Ligação ao Retinol/química , Proteínas de Ligação ao Retinol/genética , Alinhamento de Sequência , Transcrição Gênica , Triticum/parasitologia , Tylenchoidea/genética
2.
J Pept Sci ; 21(2): 105-13, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25557880

RESUMO

ß-Hairpin antimicrobial peptides are among the most potent peptide antibiotics of animal origin. Arenicins, isolated earlier from marine polychaeta lugworm Arenicola marina, belong to a family of ß-hairpin antimicrobial peptides and display a broad spectrum of biological activities. However, despite being potent antimicrobials, arenicins are partially unapplicable as therapeutics as a result of their relatively high cytotoxicity against mammalian cells. In this study, a template-based approach was used to create therapeutically valuable analogs of arenicin-1 and identify amino acid residues important for antibacterial and cytotoxic activities of the peptide. The plasmids encoding recombinant analogs were constructed by mutagenesis technique based on inverse PCR amplification of the whole arenicin-1 expression plasmid. The analogs were produced as a part of the fusion proteins in Escherichia coli. It was shown that an obvious reduction in hemolytic activity without lose of antimicrobial activity can be achieved by a single amino acid substitution in the non-polar face of the molecule with hydrophilic residues such as serine and arginine. As the result, the selective analog with 50-fold improved therapeutic index was developed. The circular dichroism spectra demonstrated that the secondary structure of the analog was similar to the natural arenicin-1 in water solution and sodium dodecyl sulfate micelles but significantly differed in the presence of dodecylphosphocholine micelles mimicking mammalian membranes. Similarly to arenicin-1, the designed analog killed bacteria via induction of the membrane damage, assessed using the fluorescent dye SYTOX Green uptake. Our results afford molecular insight into mechanism of antimicrobial action of the designed arenicin analogs and their possible clinical application.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Relação Dose-Resposta a Droga , Proteínas de Helminto/isolamento & purificação , Ensaios de Triagem em Larga Escala , Testes de Sensibilidade Microbiana , Relação Estrutura-Atividade
3.
PLoS One ; 8(6): e64984, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23762275

RESUMO

BACKGROUND: Schistosomiasis is a disease caused by parasitic worms and more than 200 million people are infected worldwide. The emergence of resistance to the most commonly used drug, praziquantel (PZQ), makes the development of novel drugs an urgent task. 3-oxoacyl-ACP reductase (OAR), a key enzyme involved in the fatty acid synthesis pathway, has been identified as a potential drug target against many pathogenic organisms. However, no research on Schistosoma japonicum OAR (SjOAR) has been reported. The characterization of the SjOAR protein will provide new strategies for screening antischistosomal drugs that target SjOAR. METHODOLOGY/PRINCIPAL FINDINGS: After cloning the SjOAR gene, recombinant SjOAR protein was purified and assayed for enzymatic activity. The tertiary structure of SjOAR was obtained by homology modeling and 27 inhibitor candidates were identified from 14,400 compounds through molecular docking based on the structure. All of these compounds were confirmed to be able to bind to the SjOAR protein by BIAcore analysis. Two compounds exhibited strong antischistosomal activity and inhibitory effects on the enzymatic activity of SjOAR. In contrast, these two compounds showed relatively low toxicity towards host cells. CONCLUSIONS/SIGNIFICANCE: The work presented here shows the feasibility of isolation of new antischistosomal compounds using a combination of virtual screening and experimental validation. Based on this strategy, we successfully identified 2 compounds that target SjOAR with strong antischistosomal activity but relatively low cytotoxicity to host cells.


Assuntos
3-Oxoacil-(Proteína Carreadora de Acil) Redutase/isolamento & purificação , Anti-Helmínticos/farmacologia , Simulação por Computador , Descoberta de Drogas , Schistosoma japonicum/enzimologia , 3-Oxoacil-(Proteína Carreadora de Acil) Redutase/antagonistas & inibidores , 3-Oxoacil-(Proteína Carreadora de Acil) Redutase/genética , Animais , Morte Celular/efeitos dos fármacos , Clonagem Molecular , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Genes de Helmintos/genética , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Células Hep G2 , Humanos , Cinética , Schistosoma japonicum/citologia , Schistosoma japonicum/efeitos dos fármacos , Schistosoma japonicum/ultraestrutura , Relação Estrutura-Atividade , Análise de Sobrevida , Fatores de Tempo
4.
J Biol Chem ; 287(51): 42846-55, 2012 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-23115230

RESUMO

We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.


Assuntos
Líquidos Corporais/metabolismo , Quitina/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Helmintos/metabolismo , Análise de Sequência de Proteína , Sequência de Aminoácidos , Animais , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Glucosamina/metabolismo , Testes de Inibição da Hemaglutinação , Imuno-Histoquímica , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
5.
Parasitol Res ; 109(4): 1065-74, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21556690

RESUMO

Catecholamines, such as dopamine and noradrenaline, play important roles as neuromuscular transmitters and modulators in all parasitic helminthes, including Schistosoma japonicum. S. japonicum tyrosine hydroxylase (SjTH) was amplified by rapid amplification of cDNA ends polymerase chain reaction that shows strong homology to Schistosoma mansoni tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. The SjTH transcripts encoded the protein of 463 amino acids and a predicted size of 54 kDa. Purified recombinant SjTH as an N-terminal histidine fusion protein expressed in Escherichia coli showed catalytic activity that was confirmed with (3)H tyrosine uptake. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and similar sensitivity to be inhibited by high concentration of the substrate, tyrosine, as the mammalian enzyme. Also, purified SjTH showed characteristic inhibition by catecholamine products. The phosphorylated peptide from SjTH could interact with Sj14-3-3 signal protein. This evidence indicates that SjTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian hosts' enzyme, and its catalytic activity could be regulated by a phosphorylated or dephosphorylated form. This demonstration of SjTH further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.


Assuntos
Catecolaminas/metabolismo , Proteínas de Helminto/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma japonicum/enzimologia , Esquistossomose Japônica/parasitologia , Tirosina 3-Mono-Oxigenase/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Dados de Sequência Molecular , Fosforilação , Plasmídeos , Coelhos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schistosoma japonicum/genética , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Esquistossomose Japônica/metabolismo , Especificidade por Substrato , Transformação Bacteriana , Trítio/análise , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/isolamento & purificação
6.
Mol Biochem Parasitol ; 129(2): 167-77, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12850261

RESUMO

Hookworms, bloodfeeding intestinal nematodes, are a leading cause of iron deficiency anemia in the developing world. These parasites have evolved potent mechanisms of interfering with mammalian hemostasis, presumably for the purpose of facilitating bloodfeeding. Adult Ancylostoma caninum worm extracts contain an activity that inhibits platelet aggregation and adhesion by blocking the function of two cell surface integrin receptors, Glycoprotein IIb/IIIa and GPIa/IIa. Using rpHPLC, the hookworm platelet inhibitor activities have been purified from protein extracts of A. caninum. Because the two inhibitory activities co-purified through multiple chromatographic steps, have similar molecular masses and share identical N-terminal as well as internal amino acid sequence homology, it is likely that they represent a single gene product. A cDNA corresponding to the purified hookworm platelet inhibitor (HPI) protein has been cloned from adult A. caninum RNA, and the translated amino acid sequence shows significant homology to Neutrophil Inhibitory Factor and Ancylostoma Secreted Proteins, suggesting that these related hookworm proteins represent a novel class of integrin receptor antagonists. Polyclonal antibodies raised against the recombinant HPI protein recognize corresponding native proteins in A. caninum extracts and excretory/secretory products, and immunohistochemistry data have identified the cephalic glands as the major source of the inhibitor within the adult hookworm. These data suggest that HPI is secreted by the adult stage of the parasite at the site of intestinal attachment. As such, it may represent a viable target for a vaccine-based strategy aimed at interfering with hookworm-induced gastrointestinal hemorrhage and iron deficiency anemia.


Assuntos
Ancylostoma/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Inibidores da Agregação Plaquetária/isolamento & purificação , Sequência de Aminoácidos , Ancylostoma/genética , Animais , Anticorpos Anti-Helmínticos/imunologia , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Colágeno/metabolismo , DNA Complementar , Fibrinogênio/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Imunoglobulina G/imunologia , Imuno-Histoquímica , Integrina alfa2beta1/metabolismo , Dados de Sequência Molecular , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/imunologia , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray
7.
Mem. Inst. Oswaldo Cruz ; 96(suppl): 131-135, Sept. 2001. ilus, tab
Artigo em Inglês | LILACS, SES-SP | ID: lil-295892

RESUMO

Previous studies carried out with Sm14 in experimental vaccination against Schistosoma mansoni or Fasciola hepatica infections were performed with recombinant Sm14 (rSm14) produced in Escherichia coli by the pGEMEX system (Promega). The rSm14 was expressed as a 40 kDa fusion protein with the major bacteriophage T7 capsid protein. Vaccination experiments with this rSm14 in animal models resulted in consistent high protective activity against S. mansoni cercariae challenge and enabled rSm14 to be included among the vaccine antigens endorsed by the World Health Organization for phase I/II clinical trials. Since the preparation of pGEMEX based rSm14 is time consuming and results in low yield for large scale production, we have tested other E. coli expression systems which would be more suitable for scale up and downstream processing. We expressed two different 6XHis-tagged Sm14 fusion proteins in a T7 promoter based plasmids. The 6XHis-tag fusions allowed rapid purification of the recombinant proteins through a Ni+2-charged resin. The resulted recombinant 18 and 16 kDa proteins were recognized by anti-Sm14 antibodies and also by antiserum against adult S. mansoni soluble secreted/excreted proteins in Western-Blot. Both proteins were also protective against S. mansoni cercariae infection to the same extent as the rSm14 expressed by the pGEMEX system


Assuntos
Animais , Feminino , Camundongos , Schistosoma mansoni/imunologia , Proteínas Recombinantes , Anticorpos Anti-Helmínticos/fisiologia , Proteínas de Helminto/fisiologia , Plasmídeos , Proteínas Recombinantes/isolamento & purificação , Proteínas de Transporte , Proteínas de Helminto/isolamento & purificação , Western Blotting , Sequência de Aminoácidos , Vacinação , DNA Complementar , Modelos Animais , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Ácidos Graxos
8.
J Neurochem ; 71(4): 1369-80, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9751167

RESUMO

Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni. We have cloned a novel S. mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Two subsets of SmTH transcripts were identified, one of which carries the S. mansoni spliced-leader (SL) sequence at its 5' end, whereas the other does not appear to be trans-spliced to the S. mansoni SL. The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa. Expression of SmTH as an N-terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified approximately 52-fold to apparent homogeneity and had a final specific activity of 0.78 micromol/min/mg. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme. Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme. This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation. This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.


Assuntos
Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Tirosina 3-Mono-Oxigenase/química , Tirosina 3-Mono-Oxigenase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Proteínas de Helminto/biossíntese , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Dados de Sequência Molecular , Schistosoma mansoni/química , Tirosina 3-Mono-Oxigenase/biossíntese , Tirosina 3-Mono-Oxigenase/isolamento & purificação
9.
Mol Plant Microbe Interact ; 9(1): 39-46, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8589421

RESUMO

Sodium dodecyl sulfate-extracted proteins from second-stage juveniles (J2) of the potato cyst nematode Globodera rostochiensis were fractionated by preparative continuous flow electrophoresis, and monoclonal antibodies (MAbs) were raised against the 38- to 40.5-kDa protein fraction. Screening of the hybridoma culture fluids by immunofluorescence microscopy of J2 resulted in the identification of 12 MAbs that bound specifically to the subventral esophageal glands. On Western blots of J2 these MAbs identified four protein bands with apparent molecular masses of 30, 31, 39, and 49 kDa. Immunoelectron microscopy with one of these MAbs showed an intense labeling of the electron dense core of the secretory granules in the subventral gland cells of J2. It is concluded that one or more of these proteins are localized within these secretory granules. Immunofluorescence microscopy of J2 from other plant parasitic nematode species showed that most of these MAbs also bind to the subventral glands of G. pallida and G. tabacum but not of Heterodera schachtii, H. glycines, Meloidogyne incognita, or M. hapla.


Assuntos
Anticorpos Anti-Helmínticos/imunologia , Grânulos Citoplasmáticos/química , Sistema Digestório/química , Proteínas de Helminto/isolamento & purificação , Nematoides/química , Animais , Anticorpos Monoclonais , Antígenos de Helmintos/isolamento & purificação , Western Blotting , Reações Cruzadas , Grânulos Citoplasmáticos/ultraestrutura , Sistema Digestório/ultraestrutura , Imunofluorescência , Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita , Microscopia Imunoeletrônica , Nematoides/crescimento & desenvolvimento , Nematoides/patogenicidade , Nematoides/ultraestrutura , Solanum tuberosum/parasitologia , Especificidade da Espécie , Virulência
10.
J Biol Chem ; 270(31): 18313-8, 1995 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-7629152

RESUMO

A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).


Assuntos
Brugia pahangi/enzimologia , Glutationa Peroxidase/metabolismo , Proteínas de Helminto/metabolismo , Selênio/metabolismo , Animais , Sequência de Bases , Brugia pahangi/genética , Células Cultivadas , Glutationa Peroxidase/genética , Glutationa Peroxidase/isolamento & purificação , Glicosilação , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Concentração de Íons de Hidrogênio , Ácidos Linolênicos/metabolismo , Peróxidos Lipídicos/metabolismo , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Fosfatidilcolinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Spodoptera/citologia , Especificidade por Substrato
11.
Mol Biochem Parasitol ; 70(1-2): 139-48, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7637694

RESUMO

A muscle-specific gene of Echinococcus granulosus has been identified and characterized. A lambda gt11 clone (10P1), containing an incomplete copy of the gene, was originally isolated from a larval E. granulosus cDNA library by serum antibodies from dogs infected with the parasite. The full-length cDNA sequence was obtained by PCR amplification of cDNA from an adult E. granulosus lambda gt22A library. Southern blot analysis indicated the presence of the gene as a single copy in the genome of E. granulosus and also detected homologous genes in genomic DNA of E. multilocularis and Taenia saginata. The 21.2-kDa protein deduced from the complete cDNA sequence contains two regions of 12 amino acids with similarity to the EF-hand motif of calcium binding proteins. Antibodies raised against the purified 10P1-GST fusion protein detected a 22-kDa antigen in the E. granulosus developmental stages examined. Immunoelectron microscopy localized the native protein in the muscle of the parasite. The amino-acid sequence of the E. granulosus protein shows significant homology to the muscle proteins mp20 of Drosophila melanogaster, chicken SM22 alpha and mammalian calponin, and also to the neuronal protein NP25 of rats. A conserved carboxy-terminal motif of 17 amino acids is present in all the homologous proteins and is proposed to be the characteristic feature of a novel protein family. The term myophilin is proposed for the E. granulosus protein due to its localization and homology to other muscle proteins.


Assuntos
Antígenos de Helmintos , Echinococcus/imunologia , Genes de Helmintos , Proteínas de Helminto/isolamento & purificação , Proteínas Musculares/isolamento & purificação , Sequência de Aminoácidos , Animais , Animais Selvagens/parasitologia , Sequência de Bases , Proteínas de Ligação ao Cálcio/química , Clonagem Molecular , Sequência Consenso , DNA Complementar/genética , Cães , Equinococose/parasitologia , Equinococose/veterinária , Echinococcus/genética , Echinococcus/crescimento & desenvolvimento , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Larva , Proteínas dos Microfilamentos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/imunologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/imunologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Calponinas
12.
Exp Parasitol ; 75(3): 308-22, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1426133

RESUMO

We recently purified a 16-kDa cytosolic Cu/Zn superoxide dismutase (CT Cu/Zn-SOD) from Schistosoma mansoni, a human parasite. Three peptide sequences were obtained, one from the unblocked N-terminal and two from internal peptides which were generated by digestions with trypsin and cyanogen bromide. These sequences were aligned to the corresponding sequences of 19 cytosolic Cu/Zn-SODs from various species. Degenerate oligonucleotides were then designed according to the sequence and the position of each peptide. The oligonucleotides were used to amplify a complete cDNA using the polymerase chain reaction with either adult schistosome total RNA or a cercariae lambda gt11 phage cDNA library as the template. The protein encoded by the cDNA has 153 amino acids with a calculated molecular weight of 15,693. It also has 60-65% homology to 19 cytosolic Cu/Zn-SOD from various species. All of the copper/zinc binding sites and SOD activity sites are conserved. Computer analysis predicts that the Cu/Zn-SOD has a pI value of 6.6, which is very close to the experimental results of IEF analysis (6.0 and 6.3). The entire coding sequence from the cDNA was cloned into a bacterial alkaline phosphatase cytosolic expression vector and a large amount of soluble product was expressed and purified to homogeneity. We compared the bacterially expressed Cu/Zn-SOD with the native enzyme derived from schistosomes and found that they are identical by the following criteria: (1) They focus at the same positions on IEF gels; (2) they form dimers in solution as measured by gel filtration; (3) they have the same unblocked N-terminal sequence; (4) they both are enzymatically active with comparable specific activities. The specific activity of the bacterially derived enzyme was increased somewhat (approximately 10%) by incubation with copper and zinc ions.


Assuntos
Genes de Helmintos/genética , Proteínas de Helminto/genética , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Superóxido Dismutase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Compartimento Celular , Clonagem Molecular , Citosol/enzimologia , Escherichia coli/genética , Proteínas de Helminto/biossíntese , Proteínas de Helminto/isolamento & purificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/biossíntese , Superóxido Dismutase/isolamento & purificação , Transcrição Gênica
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