RESUMO
Introduction: Rhodnius (Hemiptera: Reduviidae: Triatominae) species are made up of haematophagous insect vectors for Trypanosoma cruzi (Chagas' disease aetiological agent) and T. rangeli, an infective parasite that is not pathogenic for vertebrate hosts. The study of their salivary protein diversity enables the obtention of characteristic one-dimensional electrophoretic profiles of some triatomine species; however, few reports have dealt with Rhodnius species salivary proteins electrophoretic patterns. Objective: To compare R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus' salivary proteins one-dimensional electrophoretic profiles. Materials and methods: SDS-PAGE was used for obtaining electrophoretic profiles of saliva from the species under study. The unweighted pair group method with arithmetic mean (UPGMA) was used for constructing a phenogram. Results: Electrophoretic profiles of soluble saliva had protein bands ranging from 15 to 45 kDa, thereby enabling the five species studied to be differentiated. The phenogram revealed two main groups, one formed by the Pictipes and Prolixus cis-Andean groups and another consisting of the Pallescens trans-Andean group. Conclusion: Differences were revealed regarding R. colombiensis, R. pallescens, R. pictipes, R. prolixus, and R. robustus electrophoretic profiles of salivary proteins; their variability facilitated constructing a phenogram which was taxonomically congruent with the groups from the genus Rhodnius.
Introducción. Las especies Rhodnius (Hemiptera: Reduviidae: Triatominae) están conformadas por insectos hematófagos vectores de Trypanosoma cruzi, agente etiológico de la enfermedad de Chagas, y T. rangeli, parásito infectivo pero no patógeno para el vertebrado. El estudio de la diversidad proteica de la saliva de estos insectos permite la obtención de perfiles electroforéticos unidimensionales característicos de algunas especies de triatominos. Sin embargo, el reporte de los patrones electroforéticos de proteínas salivales de las especies de Rhodnius ha sido escaso. Objetivo. Hacer un análisis comparativo de los perfiles electroforéticos unidimensionales de las proteínas salivales de R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus. Materiales y métodos. Se obtuvieron los perfiles electroforéticos de la saliva de las especies en estudio mediante electroforesis en gel de poliacrilamida con dodecilsulfatosódico (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, SDS-PAGE) y se construyó un fenograma mediante el método UPGMA (Unweighted Pair Group Method Using Arithmetic Averages). Resultados. Los perfiles electroforéticos de las proteínas solubles de saliva presentaron bandas en un rango de masa aproximado de 15 a 45 kDa, los cuales permitieron diferenciar las cinco especies estudiadas. El fenograma reveló la existencia de dos grupos principales: uno conformado por los grupos cisandinos Pictipes y Prolixus y otro constituido por el grupo transandino Pallescens. Conclusiones. Existen diferencias en los perfiles electroforéticos de las proteínas salivales entre R. colombiensis, R. pallescens, R. pictipes, R. prolixus y R. robustus, cuya variabilidad permitió construir un fenograma congruente con los grupos del género Rhodnius.
Assuntos
Distribuição Animal , Proteínas de Insetos/análise , Insetos Vetores/classificação , Rhodnius/classificação , Proteínas e Peptídeos Salivares/análise , Animais , Colômbia , Eletroforese em Gel de Poliacrilamida , Variação Genética , Proteínas de Insetos/isolamento & purificação , Insetos Vetores/química , Peso Molecular , Filogenia , Rhodnius/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Especificidade da Espécie , Trypanosoma cruziRESUMO
Insect saliva induces significant antibody responses associated with the intensity of exposure to bites and the risk of disease in humans. Several salivary biomarkers have been characterized to determine exposure intensity to Old World Anopheles mosquito species. However, new tools are needed to quantify the intensity of human exposure to Anopheles bites and understand the risk of malaria in low-transmission areas in the Americas. To address this need, we conducted proteomic and bioinformatic analyses of immunogenic candidate proteins present in the saliva of uninfected Anopheles albimanus from two separate colonies-one originating from Central America (STECLA strain) and one originating from South America (Cartagena strain). A ~65 kDa band was identified by IgG antibodies in serum samples from healthy volunteers living in a malaria endemic area in Colombia, and a total of five peptides were designed from the sequences of two immunogenic candidate proteins that were shared by both strains. ELISA-based testing of human IgG antibody levels against the peptides revealed that the transferrin-derived peptides, TRANS-P1, TRANS-P2 and a salivary peroxidase peptide (PEROX-P3) were able to distinguish between malaria-infected and uninfected groups. Interestingly, IgG antibody levels against PEROX-P3 were significantly lower in people that have never experienced malaria, suggesting that it may be a good marker for mosquito bite exposure in naïve populations such as travelers and deployed military personnel. In addition, the strength of the differences in the IgG levels against the peptides varied according to location, suggesting that the peptides may able to detect differences in intensities of bite exposure according to the mosquito population density. Thus, the An. albimanus salivary peptides TRANS-P1, TRANS-P2, and PEROX-P3 are promising biomarkers that could be exploited in a quantitative immunoassay for determination of human-vector contact and calculation of disease risk.
Assuntos
Anopheles/metabolismo , Malária/imunologia , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/isolamento & purificação , Animais , Formação de Anticorpos , Antígenos , Biomarcadores/sangue , Colômbia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Mordeduras e Picadas de Insetos , Proteínas de Insetos/imunologia , Mosquitos Vetores , Projetos Piloto , Proteômica , Saliva/químicaRESUMO
BACKGROUND: Leishmania parasites are transmitted in the presence of sand fly saliva. Together with the parasite, the sand fly injects salivary components that change the environment at the feeding site. Mice immunized with Phlebotomus papatasi salivary gland (SG) homogenate are protected against Leishmania major infection, while immunity to Lutzomyia intermedia SG homogenate exacerbated experimental Leishmania braziliensis infection. In humans, antibodies to Lu. intermedia saliva are associated with risk of acquiring L. braziliensis infection. Despite these important findings, there is no information regarding the repertoire of Lu. intermedia salivary proteins. METHODS AND FINDINGS: A cDNA library from the Salivary Glands (SGs) of wild-caught Lu. intermedia was constructed, sequenced, and complemented by a proteomic approach based on 1D SDS PAGE and mass/mass spectrometry to validate the transcripts present in this cDNA library. We identified the most abundant transcripts and proteins reported in other sand fly species as well as novel proteins such as neurotoxin-like proteins, peptides with ML domain, and three small peptides found so far only in this sand fly species. DNA plasmids coding for ten selected transcripts were constructed and used to immunize BALB/c mice to study their immunogenicity. Plasmid Linb-11--coding for a 4.5-kDa protein--induced a cellular immune response and conferred protection against L. braziliensis infection. This protection correlated with a decreased parasite load and an increased frequency of IFN-γ-producing cells. CONCLUSIONS: We identified the most abundant and novel proteins present in the SGs of Lu. intermedia, a vector of cutaneous leishmaniasis in the Americas. We also show for the first time that immunity to a single salivary protein from Lu. intermedia can protect against cutaneous leishmaniasis caused by L. braziliensis.
Assuntos
Perfilação da Expressão Gênica , Proteínas de Insetos/biossíntese , Leishmania braziliensis/imunologia , Psychodidae/genética , América , Animais , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Feminino , Biblioteca Gênica , Humanos , Imunidade Celular , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Proteínas de Insetos/isolamento & purificação , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/prevenção & controle , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Proteoma/análise , Psychodidae/imunologia , Psychodidae/parasitologia , Proteínas e Peptídeos Salivares/biossíntese , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/imunologia , Proteínas e Peptídeos Salivares/isolamento & purificaçãoRESUMO
BACKGROUND: Leishmania transmission occurs in the presence of insect saliva. Immunity to Phlebotomus papatasi or Lutzomyia longipalpis saliva or salivary components confers protection against an infection by Leishmania in the presence of the homologous saliva. However, immunization with Lutzomyia intermedia saliva did not protect mice against Leishmania braziliensis plus Lu. intermedia saliva. In the present study, we have studied whether the immunization with Lu. longipalpis saliva or a DNA plasmid coding for LJM19 salivary protein would be protective against L. braziliensis infection in the presence of Lu. intermedia saliva, the natural vector for L. braziliensis. METHODOLOGY/PRINCIPAL FINDINGS: Immunization with Lu. longipalpis saliva or with LJM19 DNA plasmid induced a Delayed-Type Hypersensitivity (DTH) response against Lu. longipalpis as well as against a Lu. intermedia saliva challenge. Immunized and unimmunized control hamsters were then intradermally infected in the ears with L. braziliensis in the presence of Lu. longipalpis or Lu. intermedia saliva. Animals immunized with Lu. longipalpis saliva exhibited smaller lesion sizes as well as reduced disease burdens both at lesion site and in the draining lymph nodes. These alterations were associated with a significant decrease in the expression levels of IL-10 and TGF-ß. Animals immunized with LJM19 DNA plasmid presented similar findings in protection and immune response and additionally increased IFN-γ expression. CONCLUSIONS/SIGNIFICANCE: Immunization with Lu. longipalpis saliva or with a DNA plasmid coding LJM19 salivary protein induced protection in hamsters challenged with L. braziliensis plus Lu. intermedia saliva. These findings point out an important role of immune response against saliva components, suggesting the possibility to develop a vaccine using a single component of Lu. longipalpis saliva to generate protection against different species of Leishmania, even those transmitted by a different vector.
Assuntos
Transmissão de Doença Infecciosa/prevenção & controle , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/prevenção & controle , Vacinas Protozoárias/imunologia , Psychodidae/química , Proteínas e Peptídeos Salivares/imunologia , Vacinas de DNA/imunologia , Animais , Cricetinae , Feminino , Interferon gama/metabolismo , Interleucina-10/metabolismo , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Leishmaniose Cutânea/transmissão , Linfonodos/patologia , Masculino , Mesocricetus , Vacinas Protozoárias/administração & dosagem , Saliva/química , Saliva/imunologia , Proteínas e Peptídeos Salivares/isolamento & purificação , Pele/parasitologia , Pele/patologia , Vacinas de DNA/administração & dosagemRESUMO
The aim of this study was to evaluate the anti-tumor activity of Amblyomin-X, a serine protease Kunitz-type inhibitor. Amblyomin-X induced tumor mass regression and decreased number of metastatic events in a B16F10 murine melanoma model. Alterations on expression of several genes related to cell cycle were observed when two tumor cell lines were treated with Amblyomin-X. PSMB2, which encodes a proteasome subunit, was differentially expressed, in agreement to inhibition of proteasomal activity in both cell lines. In conclusion, our results indicate that Amblyomin-X selectively acts on tumor cells by inducing apoptotic cell death, possibly by targeting the ubiquitin-proteasome system.
Assuntos
Antineoplásicos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ixodidae/química , Melanoma Experimental/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas e Peptídeos Salivares/uso terapêutico , Inibidores de Serina Proteinase/uso terapêutico , Ubiquitina/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Proteínas de Artrópodes , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ixodidae/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/isolamento & purificaçãoRESUMO
This study identified and characterized hydrolytic enzymes in salivary gland products of Oestrus ovis larvae. Third instars were collected from the heads of slaughtered goats. Salivary glands were extracted, their products obtained by centrifugation and the enzymatic profile determined. Optimum pH, temperature of maximum proteolytic activity, thermal stability, and resistance of salivary gland products were determined on collagen and subclasses of proteases were identified using protease inhibitors. Zymograms were used to determine the molecular weight of proteases. Antigenic protein bands were revealed by immunoblotting using sera obtained from experimentally infested goats. Seven positive enzymatic activities were detected in salivary gland products: acid phosphatase, naphthol-AS-BI-phosphohydrolase, esterase (C4), esterase lipase (C8), leucine arylamidase, alpha-glucosidase and N-acetyl-beta-glucosaminidase. Optimum pH for proteolytic activity was 8.0; proteolytic activity increased with temperature (10-50 degrees C) then drastically decreased at 60 degrees C. Proteases in O. ovis salivary gland products belong to the serine subclass. In Zymograms, bands of proteolytic activity were detected in the 20-63 kDa range; the immunoblot showed three antigenic bands, one of them related to a protease band (63 kDa). Serine proteases in O. ovis salivary gland products are most likely involved in larval nutrition and host immuno-modulation.
Assuntos
Dípteros/enzimologia , Peptídeo Hidrolases/metabolismo , Proteínas e Peptídeos Salivares/química , Animais , Antígenos/metabolismo , Doenças das Cabras/imunologia , Doenças das Cabras/parasitologia , Cabras , Concentração de Íons de Hidrogênio , Immunoblotting , Larva/efeitos dos fármacos , Larva/enzimologia , Miíase/parasitologia , Miíase/veterinária , Inibidores de Proteases/farmacologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/enzimologia , Proteínas e Peptídeos Salivares/isolamento & purificação , TemperaturaRESUMO
The saliva of the sand fly Lutzomyia longipalpis, a major vector of Leishmania, exhibits pharmacological and immunomodulatory activities that may facilitate entry and establishment of parasites into the vertebrate host. Salivary gland components of the sand fly are, therefore, potential candidates in the development of a vaccine against human leishmaniasis. With the objective of identifying sand fly saliva proteins that could be used to immunise animals against canine visceral leishmaniasis, we have evaluated anti-saliva antibody reactivity using serum samples collected from dogs naturally infected with Leishmania chagasi. Two proteins with molecular weights of 28.6 and 47.3 kDa were recognised by dog antibodies in Western blot assays. Protein bands were excised from an SDS-PAGE gel and the sequences determined by mass spectrometry. The proteins were identified as LuLo-D7 and Lulo YELLOW, respectively. The significance of these findings in the context of the development of multi-component vaccination experiments is discussed.
Assuntos
Anticorpos/imunologia , Doenças do Cão/imunologia , Proteínas de Insetos/imunologia , Leishmaniose Visceral/veterinária , Psychodidae/imunologia , Saliva/química , Proteínas e Peptídeos Salivares/imunologia , Animais , Anticorpos/sangue , Doenças do Cão/sangue , Cães , Feminino , Proteínas de Insetos/isolamento & purificação , Leishmaniose Visceral/imunologia , Saliva/imunologia , Proteínas e Peptídeos Salivares/análise , Proteínas e Peptídeos Salivares/isolamento & purificaçãoRESUMO
The salivary complex of leeches contains many components able to modulate physiological mechanisms, such as coagulation and fibrinolysis, and it is composed by the salivary glands and proboscis, encompassing two different proteomes. The bidimensional electrophoretic pattern of the salivary complex from the Haementeria depressa leech revealed a total of 352 spots, 103 in common with the muscular tissue and 249 exclusive from the salivary complex as detected by silver staining; these spots showed isoelectric points from 3.5 to 9.5 and covered an apparent molecular weight range from 10 to 105 kDa. The following isoforms of proteins were identified by mass spectrometry analysis: antiplatelet protein, myohemerythrin and carbonic anhydrase. Since the leeches were not fed for about 2-3 months to stimulate the secretion of proteins that facilitates the blood metabolism, these most abundant proteins in the salivary complex excised from leeches, are expected to play a role during feeding and might have some anti-hemostatic properties. Furthermore, by zymography, a gelatinolytic and a fibrinolytic protein were identified.
Assuntos
Sanguessugas/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Sequência de Aminoácidos , Animais , Anidrases Carbônicas/genética , Anidrases Carbônicas/isolamento & purificação , Eletroforese em Gel Bidimensional , Hemeritrina/genética , Hemeritrina/isolamento & purificação , Sanguessugas/genética , Sanguessugas/fisiologia , Dados de Sequência Molecular , Mapeamento de Peptídeos , Glândulas Salivares/química , Glândulas Salivares/fisiologia , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/fisiologia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A alta especificidade das proteases da coagulação tem sido atribuída não somente aos resíduos que cercam o sítio ativo, mas também a outros domínios de superfície que estão envolvidos no reconhecimento e interação com substratos macromoleculares e inibidores. Inibidores específicos da coagulação sanguínea obtidos de fontes exógenas como glândulas salivares de animais hematófagos e venenos de serpentes têm sido identificados. Alguns desses inibidores interagem com os exosítios das enzimas da coagulação. Dois exemplos são discutidos nesta curta revisão. A Botrojaracina é uma proteína derivada de veneno de serpente que se liga aos exosítios 1 e 2 da trombina. A formação do complexo impede várias atividades da trombina dependentes do exosítio 1 incluindo a clivagem do fibrinogênio e a ativação plaquetária. A Botrojaracina também interage com o proexosítio 1 da protrombina diminuindo a ativação do zimogênio pelo complexo protrombinase (FXa/FVa). O ixolaris é um inibidor com dois domínios Kunitz obtido da glândula salivar de carrapato, homólogo ao inibidor da via do fator tecidual. Recentemente foi demonstrado que o ixolaris se liga ao exosítio de ligação à heparina do FXa, impedindo o reconhecimento da protrombina pela enzima. Além disso, o ixolaris interage com o FX, possivelmente através do proexosítio de ligação à heparina. Diferentemente do FX, o complexo ixolaris-FX não é reconhecido como substrato pelo complexo tenase intrínseco (FIXa/FVIIIa). Nós concluimos que esses inibidores podem servir como ferramentas para o estudo dos exosítios da coagulação, assim como protótipos para novas drogas anticoagulantes.
Assuntos
Animais , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/farmacologia , Proteínas e Peptídeos Salivares/farmacologia , Trombina/efeitos dos fármacos , Anticoagulantes/isolamento & purificação , Bothrops , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Fator X/efeitos dos fármacos , Fator Xa/efeitos dos fármacos , Proteínas e Peptídeos Salivares/isolamento & purificaçãoRESUMO
The high specificity of blood coagulation proteases has been attributed not only to residues surrounding the active site but also to other surface domains that are involved in recognizing and interacting with macromolecular substrates and inhibitors. Specific blood coagulation inhibitors obtained from exogenous sources such as blood sucking salivary glands and snake venoms have been identified. Some of these inhibitors interact with exosites on coagulation enzymes. Two examples are discussed in this short revision. Bothrojaracin is a snake venom-derived protein that binds to thrombin exosites 1 and 2. Complex formation impairs several exosite-dependent activities of thrombin including fibrinogen cleavage and platelet activation. Bothrojaracin also interacts with proexosite 1 on prothrombin thus decreasing the zymogen activation by the prothrombinase complex (FXa/FVa). Ixolaris is a two Kunitz tick salivary gland inhibitor, that is homologous to tissue factor pathway inhibitor. Recently it was demonstrated that ixolaris binds to heparin-binding exosite of FXa, thus impairing the recognition of prothrombin by the enzyme. In addition, ixolaris interacts with FX possibly through the heparin-binding proexosite. Differently from FX, the ixolaris-FX complex is not recognized as substrate by the intrinsic tenase complex (FIXa/FVIIIa). We conclude that these inhibitors may serve as tools for the study of coagulation exosites as well as prototypes for new anticoagulant drugs.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/enzimologia , Venenos de Crotalídeos/farmacologia , Proteínas e Peptídeos Salivares/farmacologia , Trombina/efeitos dos fármacos , Animais , Anticoagulantes/isolamento & purificação , Bothrops , Venenos de Crotalídeos/química , Venenos de Crotalídeos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Fator X/efeitos dos fármacos , Fator Xa/efeitos dos fármacos , Proteínas e Peptídeos Salivares/isolamento & purificação , Serina Endopeptidases/efeitos dos fármacosRESUMO
We have characterized a pore-forming lytic protein from the saliva of the hematophagous insect Triatoma infestans, a vector of Chagas disease. This protein, named trialysin, has 22 kDa and is present in the saliva at about 200 microg/ml. Purified trialysin forms voltage-dependent channels in planar lipid bilayers with conductance of 880 +/- 40 pS. It lyses protozoan parasites and bacteria indicating that it has a role in the control of microorganism growth in the salivary glands. At higher concentrations, but below those found in saliva, trialysin can also permeabilize and lyse mammalian cells, suggesting that it might also facilitate insect blood feeding by interfering with the cell response of the host. The translated cDNA sequence of trialysin shows a basic, lysine-rich protein in which the N-terminal region is predicted to form an amphipathic alpha-helical structure with positive charges on one side and hydrophobic amino acids on the opposite side. A synthetic peptide corresponding to this cationic amphipathic alpha-helix induces protozoan lysis and mammalian cell permeabilization, showing that this region is involved in lytic activity. However, the lytic peptide G6V32 is 10-fold less efficient than trialysin in lysing parasites and 100-fold less efficient in permeabilizing mammalian cells. Trialysin activity is about 10-fold reduced in salivary gland homogenates prepared in the presence of an irreversible serine-protease inhibitor. Since trialysin precursor contains an anionic pro-sequence of 33 amino acids contiguous to the cationic amphipathic putative alpha-helix, we propose that removal of the acidic pro-sequence by limited proteolysis activates trialysin by exposing this lytic basic amphipathic motif.
Assuntos
Proteínas de Insetos/fisiologia , Saliva/fisiologia , Proteínas e Peptídeos Salivares/metabolismo , Triatoma/fisiologia , Sequência de Aminoácidos , Animais , Eritrócitos/efeitos dos fármacos , Eucariotos/efeitos dos fármacos , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacologia , Bicamadas Lipídicas , Dados de Sequência Molecular , Conformação Proteica , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Proteínas e Peptídeos Salivares/farmacologia , Proteínas e Peptídeos Salivares/fisiologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
The salivary gland proteins of adult female Anopheles darlingi were fractionated by reverse-phase HPLC and the five major peaks were submitted for amino-terminal sequencing using automated Edman degradation. The amino acid sequence of one of the purified salivary gland proteins showed similarity with the D7r3 protein of An. gambiae. Cloning and sequencing of two cDNAs allowed the prediction of the complete sequence of the An. darlingi D7 protein. The D7r3 protein is present specifically in adult female salivary glands of An. darlingi and despite being one of the major salivary gland proteins its function is not known. Predictions of secondary and tertiary structures revealed the similarity of the An. darlingi D7 protein to insect odorant binding proteins. This suggests that D7 proteins may act as carriers of hydrophobic molecules in mosquito saliva.
Assuntos
Anopheles/fisiologia , Proteínas de Insetos/química , Proteínas e Peptídeos Salivares/química , Sequência de Aminoácidos , Animais , Anopheles/química , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Feminino , Concentração de Íons de Hidrogênio , Proteínas de Insetos/genética , Proteínas de Insetos/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Solubilidade , TenebrioRESUMO
The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent K(I) of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the triatoma Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.
Assuntos
Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Fator Xa , Proteínas de Helminto/isolamento & purificação , Sanguessugas/química , Saliva/química , Proteínas e Peptídeos Salivares/isolamento & purificação , Inibidores de Serina Proteinase/isolamento & purificação , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Compostos Cromogênicos/metabolismo , Proteínas de Helminto/farmacologia , Hemeproteínas/química , Hemeproteínas/farmacologia , Hemeritrina/análogos & derivados , Hemeritrina/química , Humanos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/farmacologia , Especificidade da Espécie , Especificidade por Substrato , Tromboplastina/antagonistas & inibidoresRESUMO
1. The effects of the saliva of the aquatic hemipteran Belostoma anurum were investigated on the isolated guinea-pig heart, perfused with Locke solution by the Langendorff's method. 2. The electrocardiogram (bipolar lead), the contractile force (g) and coronary flow (ml/min) were simultaneously recorded. 3. The total protein present in the saliva of each belostomatid was 11.17 +/- 2.39 micrograms (N = 10), and SDS-PAGE of the saliva showed a complex protein composition (N = 10). 4. Bolus injections of 0.2 ml saliva obtained from 10 belostomatids elicited complex effects which were divided into two phases (N = 8): an initial phase characterized by sinus bradycardia and/or A-V block associated with a decrease in contractile force and an increase in coronary flow followed by a decrease and a second phase, 0.5-5 min after saliva injection, characterized by a progressive increase in resting tension (ventricular contracture). 5. Since pretreatment of the preparation with atropine or verapamil did not prevent the initial and the late effects (N = 12), we conclude that these effects are not explained either by a muscarinic effect or by a stimulation of calcium channels. 6. Injection of saliva previously mixed with heparin (1000 IU/ml, N = 7), evoked the first but not the second phase (ventricular contracture). 7. Injection of saliva from two belostomatids into the isolated hearts of guinea pigs, not previously treated with heparin, elicited dramatic effects, such as ventricular contracture (N = 6). 8. We suggest that the formation of an acid-base complex (heparin-saliva) would prevent, in part, the toxic effects of the saliva on the heart.