RESUMO
l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 µM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.
Assuntos
Aranhas/enzimologia , alfa-L-Fucosidase/metabolismo , Animais , Feminino , Humanos , Filogenia , Aranhas/genética , Homologia Estrutural de Proteína , alfa-L-Fucosidase/genética , alfa-L-Fucosidase/isolamento & purificaçãoRESUMO
l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An alpha-L-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this alpha-L-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a K-m of 32 and 400 mu M for 4-methylumbelliferyl alpha-L-fucopyranoside and 4-nitrophenyl alpha-L-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata alpha-L-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.
RESUMO
BACKGROUND: Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput ("-omics") techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). RESULTS: We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. CONCLUSION: All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands.
Assuntos
Digestão , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteômica/métodos , Análise de Sequência de DNA/métodos , Aranhas/fisiologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Sistema Digestório/metabolismo , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Filogenia , Aranhas/genéticaRESUMO
Scorpions are among the oldest terrestrial arthropods and they have passed through small morphological changes during their evolutionary history on land. They are efficient predators capable of capturing and consuming large preys and due to envenomation these animals can become a human health challenge. Understanding the physiology of scorpions can not only lead to evolutionary insights but also is a crucial step in the development of control strategies. However, the digestive process in scorpions has been scarcely studied. In this work, we describe the combinatory use of next generation sequencing, proteomic analysis and biochemical assays in order to investigate the digestive process in the yellow scorpion Tityus serrulatus, mainly focusing in the initial protein digestion. The transcriptome generated database allowed the quantitative identification by mass spectrometry of different enzymes and proteins involved in digestion. All the results suggested that cysteine cathepsins play an important role in protein digestion. Two digestive cysteine cathepsins were isolated and characterized presenting acidic characteristics (pH optima and stability), zymogen conversion to the mature form after acidic activation and a cross-class inhibition by pepstatin. A more elucidative picture of the molecular mechanism of digestion in a scorpion was proposed based on our results from Tityus serrulatus. The midgut and midgut glands (MMG) are composed by secretory and digestive cells. In fasting animals, the secretory granules are ready for the next predation event, containing enzymes needed for alkaline extra-oral digestion which will compose the digestive fluid, such as trypsins, astacins and chitinase. The digestive vacuoles are filled with an acidic proteolytic cocktail to the intracellular digestion composed by cathepsins L, B, F, D and legumain. Other proteins as lipases, carbohydrases, ctenitoxins and a chitolectin with a perithrophin domain were also detected. Evolutionarily, a large gene duplication of cathepsin L occurred in Arachnida with the sequences from ticks being completely divergent from other arachnids probably due to the particular selective pressures over this group.
Assuntos
Proteínas de Artrópodes/genética , Catepsinas/genética , Digestão/genética , Proteoma/genética , Escorpiões/genética , Transcriptoma , Animais , Proteínas de Artrópodes/metabolismo , Evolução Biológica , Catepsinas/antagonistas & inibidores , Catepsinas/metabolismo , Quitinases/genética , Quitinases/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Estabilidade Enzimática , Feminino , Duplicação Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Concentração de Íons de Hidrogênio , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Anotação de Sequência Molecular , Pepstatinas/química , Inibidores de Proteases/química , Proteoma/metabolismo , Venenos de Escorpião/genética , Venenos de Escorpião/metabolismo , Escorpiões/classificação , Escorpiões/metabolismo , Tripsina/genética , Tripsina/metabolismoRESUMO
Cysteine cathepsins are widely spread on living organisms associated to protein degradation in lysosomes, but some groups of Arthropoda (Heteroptera, Coleoptera, Crustacea and Acari) present these enzymes related to digestion of the meal proteins. Although spiders combine a mechanism of extra-oral with intracellular digestion, the sporadic studies on this subject were mainly concerned with the digestive fluid (DF) analysis. Thus, a more complete scenario of the digestive process in spiders is still lacking in the literature. In this paper we describe the identification and characterization of cysteine cathepsins in the midgut diverticula (MD) and DF of the spider Nephilengys cruentata by using enzymological assays. Furthermore, qualitative and quantitative data from transcriptomic followed by proteomic experiments were used together with biochemical assays for results interpretation. Five cathepsins L, one cathepsin F and one cathepsin B were identified by mass spectrometry, with cathepsins L1 (NcCTSL1) and 2 (NcCTSL2) as the most abundant enzymes. The native cysteine cathepsins presented acidic characteristics such as pH optima of 5.5, pH stability in acidic range and zymogen conversion to the mature form after in vitro acidification. NcCTSL1 seems to be a lysosomal enzyme with its recombinant form displaying acidic characteristics as the native ones and being inhibited by pepstatin. Evolutionarily, arachnid cathepsin L may have acquired different roles but its use for digestion is a common feature to studied taxa. Now a more elucidative picture of the digestive process in spiders can be depicted, with trypsins and astacins acting extra-orally under alkaline conditions whereas cysteine cathepsins will act in an acidic environment, likely in the digestive vacuoles or lysosome-like vesicles.