RESUMO
The resistance of the opossum Didelphis aurita to Bothrops snake venoms is attributed to the opossum's antihemorrhagic (DM43) and antimyotoxic (DM64) acidic serum glycoproteins. The aim of this study was to characterize the N-glycosylation sites of these antiophidic proteins and to determine whether their glycans influence the biological activity measured by in vitro assays. Our experimental pipeline included the sequential enzymatic digestion of the inhibitors with two different proteinases (trypsin and endoproteinase Asp-N) and eventually with trypsin, peptide-N-glycosidase F (PNGase F) and endoproteinase Asp-N, used in that order. All of the peptide and protein samples were analyzed by MALDI-TOF/TOF MS. The results experimentally confirmed the putative N-glycosylation sites of DM43 (Asn23, Asn156, Asn160, and Asn175) and DM64 (Asn46, Asn179, Asn183, and Asn379). Following treatments with specific glycosidases, complex-type oligosaccharides containing galactose and sialic acid could be assigned to both proteins. The removal of these monosaccharide units by exoglycosidase digestion did not measurably affect the inhibitory activity. In contrast, partially deglycosylated DM43 treated with PNGase F under nondenaturing conditions was half as effective as native DM43. In conclusion, we have demonstrated that the contribution of the carbohydrate portion of these potentially therapeutic molecules, for their mechanism of action, should not be overlooked.
Assuntos
Bothrops/metabolismo , Venenos de Crotalídeos/antagonistas & inibidores , Didelphis/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Polissacarídeos/análise , Sequência de Aminoácidos , Animais , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Venenos de Crotalídeos/metabolismo , Glicosilação , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismoRESUMO
The accidental contact with Lonomia obliqua caterpillar causes local and systemic symptoms (such as fibrinogen depletion), leading, in some cases, to serious clinical complications (acute renal failure and intracranial haemorrhage). Fortunately, a successful therapeutical approach using anti-Lonomic serum, produced in horses against L. obliqua's bristle extract, has already been put in place. However, a global view of immunogenic toxins involved in the coagulation disorders could help to elucidate the envenoming process. In the present study, our aim was to identify bristle extract's immunogenic components, especially those related to the haemostasis, coupling proteomics and immunochemical approaches (bidimensional electrophoresis, mass spectrometry and immunoblotting). The bidimensional map of bristle extract showed a broad profile of 157 silver-stained spots, where at least 153 spots were immunochemically revealed. Twenty-four of these spots were submitted to sequencing by mass spectrometry and three different categories of proteins were identified: lipocalins, cuticle proteins and serpins. From these protein families, it was observed that the most abundant was the lipocalin family, specifically represented by different isoforms of Lopap (a prothrombin activator protein), reinforcing its relevance during envenoming. Peptide sequences of several other immunochemically revealed spots showed no correspondence to any known sequence and were classified as unknown proteins. These proteins could represent new immunogenic molecules and/or toxins. The sequences presented in this article can be used for oligonucleotide design aiming the amplification of cDNAs coding for new molecules using L. obliqua bristles' cDNA libraries or isolated RNAs as template.
Assuntos
Venenos de Artrópodes/toxicidade , Hemostasia/efeitos dos fármacos , Imunoquímica/métodos , Lepidópteros , Proteômica/métodos , Sequência de Aminoácidos , Animais , Hemostasia/fisiologia , Immunoblotting/métodos , Proteínas de Insetos/análise , Proteínas de Insetos/química , Lipocalinas/análise , Lipocalinas/química , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Mariposas , Serina Endopeptidases/análise , Serina Endopeptidases/química , Serpinas/análise , Serpinas/químicaRESUMO
Em países tropicais e subtropicais, o envenenamento por picadas de venenos de serpentes é um problema de saúde pública. A mortalidade global estimada é de 50 a 100 mil casos por ano. Entretanto, há um elevado número de subnotificações no que tange os índices de morbidade, seqüelas que incluem ulceração crônica, falência renal, dano neurológico e amputações. O tratamento atual é baseado na administração de antisoro de origem animal produzido contra as toxinas antigênicas presentes nos venenos. Contudo, o antiveneno é ineficaz no tratamento do dano tecidual local e sua administração pode provocar sérias reações adversas. Alguns animais apresentam resistência natural ao envenenamento por serpentes, principalmente devido à presença de fatores séricos neutralizantes. O marsupial Didelphis aurita é resistente ao veneno de várias serpentes da família Viperidae e do seu soro foram isoladas duas proteínas com atividades anti-hemorrágica (DM43) e antimiotóxica (DM64). DM43 e DM64 são glicoproteínas ácidas homodiméricas apresentando 21por cento e 15por cento de conteúdo de carboidrato por peso, respectivamente. O objetivo desse trabalho foi implementar e desenvolver diferentes técnicas analíticas para glicoproteínas, utilizando estes inibidores como modelo. Os diferentes peptídeos e glicopeptídeos gerados, após hidrólise enzimática, foram analisados por espectrometria de massas. As estruturas oligossacarídicas e a atividade biológica dos inibidores, após desglicosilação total ou parcial, também foram analisadas. Utilizando digestões seqüênciais com três diferentes proteases, com e sem digestão prévia com PNGase F, aliadas à análise por MALDI-TOF/TOF, quatro sítios de N-glicosilação foram confirmados em DM43 (Asn(elevado a 23), Asn(elevado a 156), Asn(elevado a 160) and Asn(elevado a 175)) e outros quatro determinados experimentalmente em DM64(Asn(elevado a 46), Asn(elevado a 179), Asn(elevado a 183) and Asn(elevado a 379)). Estruturas de oligossacarídeos...toxina.
Assuntos
Animais , Didelphis , Glicosilação , Imunidade Inata , Espectrometria de Massas , Venenos de SerpentesRESUMO
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.