RESUMO
The relationship between protein stability and function evolution has not been explored in proteins from natural sources. Here, we investigate the phylogenetic differences of Perivitellin-1 (PV1) a novel family of hyperstable egg carotenoproteins crucial to the reproductive success of Pomacea snails, as they have evolved clade-specific protective functions. We studied P. patula PV1 (PpaPV1) from Flagellata clade eggs, the most basal of Pomacea and compared it with PV1s orthologs from derived clades. PpaPV1 stands as the most stable, with longer unfolding half-life, resistance to detergent unfolding, and therefore higher kinetic stability than PV1s from derived clades. In fact, PpaPV1 is among the most hyperstable proteins described in nature. In addition, its spectral characteristics providing a pale egg coloration, mild lectin activity and glycan specificity are narrower than derived clades. Our results provide evidence indicating large structural and functional changes along the evolution of the genus. Notably, the lectin binding of PpaPV1 is less pronounced, and its glycan specificity is narrower compared to PV1s in the sister Bridgesii clade. Our findings underscore the phylogenetic disparities in terms of structural and kinetic stability, as well as defensive traits like a potent lectin activity affecting the gut morphology of potential predators within the Bridgesii clade or a conspicuous, likely warning coloration, within the Canaliculata clade. This work provides a comprehensive comparison of the structural attributes, stability profiles, and functional roles of apple snail egg PV1s from multiple species within a phylogenetic context. Furthermore, it proposes an evolutionary hypothesis suggesting a trade-off between structural stability and the functional aspects of apple snail's major egg defense protein.
RESUMO
The Membrane Attack Complex-Perforin (MACPF) family is ubiquitously found in all kingdoms. They have diverse cellular roles, however MACPFs with pore-forming toxic function in venoms and poisons are very rare in animals. Here we present the structure of PmPV2, a MACPF toxin from the poisonous apple snail eggs, that can affect the digestive and nervous systems of potential predators. We report the three-dimensional structure of PmPV2, at 17.2 Å resolution determined by negative-stain electron microscopy and its solution structure by small angle X-ray scattering (SAXS). We found that PV2s differ from nearly all MACPFs in two respects: it is a dimer in solution and protomers combine two immune proteins into an AB toxin. The MACPF chain is linked by a single disulfide bond to a tachylectin chain, and two heterodimers are arranged head-to-tail by non-covalent forces in the native protein. MACPF domain is fused with a putative new Ct-accessory domain exclusive to invertebrates. The tachylectin is a six-bladed ß-propeller, similar to animal tectonins. We experimentally validated the predicted functions of both subunits and demonstrated for the first time that PV2s are true pore-forming toxins. The tachylectin "B" delivery subunit would bind to target membranes, and then the MACPF "A" toxic subunit would disrupt lipid bilayers forming large pores altering the plasma membrane conductance. These results indicate that PV2s toxicity evolved by linking two immune proteins where their combined preexisting functions gave rise to a new toxic entity with a novel role in defense against predation. This structure is an unparalleled example of protein exaptation.
Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/ultraestrutura , Lectinas/ultraestrutura , Perforina/ultraestrutura , Conformação Proteica , Sequência de Aminoácidos/genética , Animais , Membrana Celular/química , Membrana Celular/ultraestrutura , Complexo de Ataque à Membrana do Sistema Complemento/química , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Cristalografia por Raios X , Dimerização , Lectinas/química , Lectinas/imunologia , Modelos Moleculares , Perforina/química , Perforina/imunologia , Subunidades Proteicas/genética , Espalhamento a Baixo Ângulo , Caramujos/ultraestrutura , Difração de Raios XRESUMO
Snails from the genus Pomacea lay conspicuous masses of brightly colored eggs above the water. Coloration is given by carotenoproteins that also which play important roles in protection against sun radiation, stabilizing and transporting antioxidant molecules and helping to protect embryos from desiccation and predators. They seem a key acquisition, but have been little studied. Here we report the characteristics of the major carotenoprotein from Pomacea maculata and the first comparison among these egg proteins. This particle, hereafter PmPV1, represents ~52% of perivitellin fluid protein. It is a glyco-lipo-carotenoprotein responsible for the bright reddish egg coloration. With VHDL characteristics, PmPV1 apparent molecular mass is 294kDa, composed of five non-covalently bound subunits of pI 4.7-9.8 and masses between 26 and 36kDa whose N-terminal sequences were obtained. It is a glyco-lipo-carotenoprotein scarcely lipidated (<1%) but highly glycosilated (13% by wt). Lipids include phospholipids, free fatty acids and carotenoids; mannose and galactose predominate over other monosaccharides. Main carotenoids are esterified and non-esterified astaxanthin (71 and 25%, respectively). Carotenoid removal does not seem to affect the structural characteristics of the oligomer, while deglycosilation reduces subunit number from five to a single one. The carotenoid-protein association protected the former against oxidation. PmPV1 cross reacts with polyclonal antibodies against the PcOvo, the major carotenoprotein from Pomacea canaliculata. The characterization of PmPV1 allows the first comparisons among snail carotenoproteins and further highlights the importance of these perivitellins in the reproductive strategy of Pomacea.
Assuntos
Carotenoides/química , Proteínas do Ovo/química , Caramujos/metabolismo , Animais , Carotenoides/metabolismo , Proteínas do Ovo/metabolismo , Espécies IntroduzidasRESUMO
Several agrochemicals like organophosphates are extensively used to control pests in agricultural practices but they also adversely affect non-target fauna. The effect of organophosphorous fenitrothion on the prawn Macrobrachium borellii was evaluated. The 96-h LC50 was determined. Activity of superoxide dismutase, catalase, glutathione-S-transferase and lipid oxidation levels, were evaluated in the hepatopancreas from adults exposed to sublethal fenitrothion concentrations for 1, 2, 4 and 7 days. In addition, superoxide dismutase mRNA expression, acetylcholinesterase inhibition and haemocyte DNA damage were determined. The 96-h LC50 was 4.24µg/l of fenitrothion. Prawn exposed to sublethal FS concentrations showed an increase of both catalase and superoxide dismutase activities, mainly after 2 and 4 days exposure and an increase of glutathione-S-transferase activity from day 2 to day 7 while lipid oxidation levels increased mainly on day 1. Superoxide dismutase transcripts were significantly higher in fenitrothion -treated prawns, indicating an induction mechanism. Hemolymph analysis showed that while acetylcholinesterase activity decreased after 2 days, haemocytes displayed most DNA damage after 7-day exposure to fenitrothion. These results indicate that prawn enzymes are highly sensitive to fenitrothion exposure, and these biological responses in M. borellii could be valuable biomarkers to monitor organophosphorous contamination in estuarine environments.
Assuntos
Fenitrotion/toxicidade , Palaemonidae/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/enzimologia , Dose Letal Mediana , Oxirredução , Palaemonidae/metabolismoRESUMO
Hydrocarbon pollution is a major environmental threat to ecosystems in marine and freshwater environments, but its toxicological effect on aquatic organisms remains little studied. A proteomic approach was used to analyze the effect of a freshwater oil spill on the prawn Macrobrachium borellii. To this aim, proteins were extracted from midgut gland (hepatopancreas) of male and female prawns exposed 7 days to a sublethal concentration (0.6 ppm) of water-soluble fraction of crude oil (WSF). Exposure to WSF induced responses at the protein expression level. Two-dimensional gel electrophoresis (2-DE) revealed 10 protein spots that were differentially expressed by WSF exposure. Seven proteins were identified using MS/MS and de novo sequencing. Nm23 oncoprotein, arginine methyltransferase, fatty aldehyde dehydrogenase and glutathione S-transferase were down-regulated, whereas two glyceraldehyde-3-phosphate dehydrogenase isoforms and a lipocalin-like crustacyanin (CTC) were up-regulated after WSF exposure. CTC mRNA levels were further analyzed by quantitative real-time PCR showing an increased expression after WSF exposure. The proteins identified are involved in carbohydrate and amino acid metabolism, detoxification, transport of hydrophobic molecules and cellular homeostasis among others. These results provide evidence for better understanding the toxic mechanisms of hydrocarbons. Moreover, some of these differentially expressed proteins would be employed as potential novel biomarkers.
Assuntos
Exposição Ambiental/análise , Hidrocarbonetos/efeitos adversos , Palaemonidae/efeitos dos fármacos , Petróleo/efeitos adversos , Proteoma/análise , Animais , Biomarcadores/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Palaemonidae/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteoma/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Testes de Toxicidade , Poluentes Químicos da Água/efeitos adversosRESUMO
While invertebrates store neutral lipids as their major energy source, little is known about triacylglycerol (TAG) class composition and their differential catabolism in aquatic arthropods. This study focuses on the composition of the main energy source and its catabolism by lipase from the midgut gland (hepatopancreas) of the crustacean Macrobrachium borellii. Silver-ion thin-layer chromatography of prawn large TAG deposit (80% of total lipids) and its subsequent fatty acid analysis by gas chromatography allowed the identification of 4 major fractions. These are composed of fatty acids of decreasing unsaturation and carbon chain length, the predominant being 18:1n-9. Fraction I, the most unsaturated one, contained mainly 20:5n-3; fraction II 18:2n-6; fraction III 18:1n-9 while the most saturated fraction contained mostly 16:0. Hepatopancreas main lipase (Mr 72 kDa) cross-reacted with polyclonal antibodies against insect lipase, was not dependent on the presence of Ca(2+) and had an optimum activity at 40°C and pH 8.0. Kinetic analysis showed a Michaelis-Menten behavior. A substrate competition assay evidenced lipase specificity following the order: 18:1n-9-TAG>PUFA-enriched-TAG>16:0-TAG different from that in vertebrates. These data indicate there is a reasonable correspondence between the fatty acid composition of TAG and the substrate specificity of lipase, which may be an important factor in determining which fatty acids are mobilized during lipolysis for oxidation in crustaceans.
Assuntos
Crustáceos/metabolismo , Lipase/metabolismo , Metabolismo dos Lipídeos/fisiologia , Palaemonidae/metabolismo , Triglicerídeos/metabolismo , Animais , Crustáceos/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/metabolismo , Hepatopâncreas/química , Hepatopâncreas/enzimologia , Hepatopâncreas/metabolismo , Cinética , Lipase/química , Metabolismo , Palaemonidae/química , Sensibilidade e Especificidade , Especificidade por Substrato , Triglicerídeos/químicaRESUMO
The aim of the present work was to evaluate the effect of the water soluble fraction of hydrocarbons (WSF) on the antioxidant status of the freshwater prawn Macrobrachium borellii. First, seasonal variations were studied in a non-polluted area. Hepatopancreas and gills showed season-related fluctuations in catalase (CAT), glutathione-S-transferase (GST) activities and in lipid peroxidation levels (LPO), but not in superoxide dismutase (SOD). Then, adults were exposed semi-statically to sublethal doses for 7days. CAT, SOD, GST, and glutathione peroxidase (GPx) activities and LPO, reduced glutathione (GSH) and protein oxidation (PO) levels were determined. Exposed individuals showed significant increases in CAT, SOD, and GST activities in hepatopancreas and CAT activity in gills. GPx activity did not vary in either tissues. While LPO levels increased, GSH levels decreased significantly in hepatopancreas of exposed animals, but PO levels showed no variation. Induction of SOD was also assessed by Real-time PCR mRNA expression in hepatopancreas. The non-enzymatic antioxidant activity was also tested; ABTS 2,2'-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) was higher in hemolymph of treated-prawns compared to controls, but ferric reducing activity of plasma assay (FRAP) values did not change. Taken together, the present results indicated that the antioxidant defenses of M. borellii, mainly in hepatopancreas, were significantly affected by aquatic hydrocarbon contamination, regardless of the season.
Assuntos
Antioxidantes/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo , Oxirredutases/metabolismo , Palaemonidae/efeitos dos fármacos , Petróleo/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Biomarcadores/metabolismo , Catalase/genética , Catalase/metabolismo , Água Doce/química , Regulação da Expressão Gênica/efeitos dos fármacos , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Glutationa/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/metabolismo , Oxirredução , Oxirredutases/genética , Palaemonidae/crescimento & desenvolvimento , Palaemonidae/metabolismo , Petróleo/análise , RNA Mensageiro/metabolismo , Estações do Ano , Solubilidade , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Poluentes Químicos da Água/químicaRESUMO
To better understand how glycans contribute to the multiple roles of perivitellins in embryo development, the carbohydrate moieties and glycoforms of the carotenoglycoproteins ovorubin and scalarin from the eggs of Pomaceacanaliculata (Lamarck, 1822) and Pomaceascalaris (d'Orbigny, 1835) were studied. All subunits of both proteins are glycosylated and appear to be glycoforms with isoelectric points ranging from approximately 5.3 to approximately 9.1. Complete deglycosylation reduced subunit heterogeneity to spots of similar molecular weight (approximately 27 and approximately 25 kDa in scalarin and ovorubin, respectively) but with varying IP. Serine phosphorylation, present in both perivitellins, explains part of the isoforms. Glycosylation patterns of scalarin were determined using biotinylated lectins, PNGaseF treatment and selective chemical deglycosylation, which revealed the presence of hybrid and oligomannose N-linked glycans in all subunits. Scalarin has terminal sialic acid residues possibly resistant to neuraminidase and O-linked residues derived from the T- and Tn antigens. Ovorubin displayed predominantly the same glycans, though in different amounts. Capillary gas chromatography (GC) showed galactose and mannose as the major monosaccharides followed by GlcNAc and fucose. An interesting feature was the important amount of sialylated and fucosylated structures found in both perivitellins determined by GC, spectroscopy and lectins. This is the first report of these structures in gastropods other than heterobranchs.
Assuntos
Polissacarídeos/química , Caramujos , Vitelinas/química , Animais , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilação , Lectinas/química , Lectinas/metabolismo , Monossacarídeos/química , Monossacarídeos/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Oviposição , Polissacarídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Caramujos/enzimologia , Caramujos/fisiologia , Vitelinas/metabolismoRESUMO
Lipovitellin (LV) is essential in crustacean eggs for embryo viability and development. Two LV were isolated from eggs of Macrobrachium borellii. corresponding to early (LVe ) and late (LVl) embryo developing stages. They differ in lipid composition but not in lipid/protein ratio or apoprotein composition. Structural information was obtained by fluorescence spectroscopy, far-UV circular dichroism, partial trypsinolysis and electron microscopy applied to LVe and LVl and two partially delipidated forms of LVe generated by phospholipase A2 (LVp) or Triton X-100 (LVt) treatment. All LV forms contained two apoprotein subunits of 94 and 112 kDa, being the 112k Da subunit more accessible to trypsinolysis in all. Only in LVp, different cleavage sites appeared. Secondary structure was similar in LVe and LVl, but LVp and LVt showed a small increase in beta-sheet at expense of alpha-helix. Electron microscopy revealed a spheroidal morphology in all LV and a decreased size in LVp. Delipidated LVs were more resistant to denaturation with guanidinium-HCl. Acrylamide quenching of tryptophan fluorescence was more efficient in delipidated LVs, probably due to apolipoprotein rearrangement, as reinforced by fluorescence anisotropy. It is concluded that LV stability, shape, and apoprotein conformation is not largely affected by the changes in lipid composition that take place during embryogenesis.
Assuntos
Proteínas do Ovo/química , Lipídeos/análise , Lipídeos/química , Palaemonidae/química , Animais , Dicroísmo Circular , Proteínas do Ovo/metabolismo , Eletroforese em Gel de Poliacrilamida , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Feminino , Guanidina/farmacologia , Metabolismo dos Lipídeos , Microscopia Eletrônica , Octoxinol/metabolismo , Palaemonidae/embriologia , Fosfolipases A2/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Estabilidade Proteica , Solventes/química , Espectrofotometria Ultravioleta , Tripsina/metabolismoRESUMO
The prawn Macrobrachium borellii has lecithotrophic eggs with highly-abbreviated development. The major yolk component is lipovitellin (LV), a lipoprotein with 30% lipids (by weight). LV consumption during embryogenesis was followed by ELISA and Western blot analysis using an anti-LV polyclonal antibody. No cross-reacting proteins were observed and LV-like lipoproteins were strongly recognized by the antibody in hemolymph (vitellogenin), yolk (LV) and embryos (LVe), as determined by Western Blot analysis. LV decreased significantly along development from 9.4 to 1.1 microg/mg egg. Consumption rate of LV was slow in early embryogenesis, followed by a rapid utilization in late embryonic stages. Significant LVe amounts were still present at hatching. LV apolipoproteins were selectively degraded during embryo development, being the highest molecular weight subunit the most affected. Comparison among in vitro, in vivo and theoretical proteolysis suggested that trypsin may be involved in LV degradation during late embryogenesis. Embryo lipoprotein (HDLe) synthesis was first detected at stage 6. HDLe shared the same density, MW and subunit composition as adult hemolymph HDL(1) and did not cross-react with LV-like lipoproteins. Though expressed at low concentration, it fulfilled embryo needs for lipid transport among organs.