Phylogenetic variations in a novel family of hyperstable apple snail egg proteins: insights into structural stability and functional trends.

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.

Exaptation of two ancient immune proteins into a new dimeric pore-forming toxin in snails.

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.

Biosynthesis in the Albumen Gland-Capsule Gland Complex Limits Reproductive Effort in the Invasive Apple Snail Pomacea canaliculata.

High fecundity often contributes to successful invasives. In molluscs, this may be facilitated by the albumen gland-capsule gland complex, which in gastropods secretes the egg perivitelline fluid that nourishes and protects embryos. The biochemistry of the albumen gland-capsule gland complex and its relationship with fecundity remain largely unknown. We addressed these issues in Pomacea canaliculata (Lamarck, 1822), a highly invasive gastropod whose fecundity and reproductive effort exceed those of ecologically similar gastropods. We evaluated the dynamics of its major secretion compounds (calcium, polysaccharides, and total proteins) as well as the gene expression and stored levels of perivitellins during key moments of the reproductive cycle, that is, before and after first copulation and at low, medium, and high reproductive output. Copulation and first oviposition do not trigger the onset of albumen gland-capsule gland complex biosynthesis. On the contrary, soon after an intermediate reproductive effort, genes encoding perivitellins overexpressed. A high reproductive effort caused a decrease in all albumen gland-capsule gland complex secretion components. Right after a high reproductive output, the albumen gland-capsule gland complex restored the main secretion components, and calcium recovered baseline reserves; but proteins and polysaccharides did not. These metabolic changes in the albumen gland-capsule gland complex after multiple ovipositions were reflected in a reduction in egg mass but did not compromise egg quality. At the end of the cycle, egg dry weight almost doubled the initial albumen gland-capsule gland complex weight. Results indicate that albumen gland-capsule gland complex biosynthesis limits a constantly high reproductive output. Therefore, lowering fecundity by targeting biosynthesis could effectively reduce the rate of this species' spread.

The major egg reserve protein from the invasive apple snail Pomacea maculata is a complex carotenoprotein related to those of Pomacea canaliculata and Pomacea scalaris.

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.

Effect of crude oil petroleum hydrocarbons on protein expression of the prawn Macrobrachium borellii.

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.

Triacylglycerol catabolism in the prawn Macrobrachium borellii (Crustacea: Palaemoniade).

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.

Carbohydrates and glycoforms of the major egg perivitellins from Pomacea apple snails (Architaenioglossa: Ampullariidae).

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.

Isolation and characterization of a novel perivitellin from the eggs of Pomacea scalaris (Mollusca, Ampullariidae).

Perivitellins are important components of the perivitelline fluid (PVF) that surrounds gastropod embryos. The glyco-lipo-carotenoprotein ovorubin (OR) from eggs of the snail Pomacea canaliculata has been the most studied to date. Here we report the characterization of scalarin (SC), a glyco-lipo-carotenoprotein from the PVF of P. scalaris. SC was purified by ultracentrifugation and exclusion chromatography. It is the major egg protein, representing 64% of the total soluble protein. The particle has a hydration density of 1.26 g/ml, an apparent molecular mass of 380 kDa and it is an elongated compact protein as estimated by small angle X-ray scattering (SAXS). It is composed of three subunits of ca. 35, 28, and 24 kDa noncovalently bonded. SC is highly glycosylated (carbohydrate content 20.1%, by wt.), with a low lipid content (0.7%), being esterified sterols, pigments and polar lipids the most abundant lipid classes. HPTLC and spectrophotometric analysis of the carotenoid fraction revealed the presence of free astaxanthin (ASX; 62.0%), and an unidentified carotenoid (38.0%). The carotenoid-apoprotein interaction was studied by spectrophotometry. Carotenoids do not seem to affect the structural characteristics of the oligomer. However, the carotenoid-protein association protected ASX against oxidation. The cross-reactivity between SC and perivitellins of P. canaliculata was tested using polyclonal antibodies (PAb) against SC, OR, and perivitellin PV2. The PAbs failed to cross-react with any egg proteins of either the same or other species. SC, among other functional similarities with OR, would be an antioxidant carrier, protecting at the same time carotenoids from oxidation in the perivitellin fluid of the egg.

Egg carotenoproteins in neotropical Ampullariidae (Gastropoda: Arquitaenioglossa).

Carotenoid-binding proteins are commonly found in invertebrates. Their carotenoids form non-covalent complexes with proteins giving tissues a variety of colors. In molluscs they have been described in only a few species. In particular, the egg perivitellin fluid of those Ampullariid species which deposit eggs above the waterline is provided with carotenoproteins playing several roles ranging from photoprotection, antioxidant or antitrypsin actions to nutrient provision for development. These molecules form complex glyco-lipo-carotenoproteins of high molecular weight where either free astaxanthin (3,3'-dihydroxy-beta, beta'-carotene- 4,4'dione) or astaxanthin esterified with fatty acids, occur more frequently. This review compiles the current knowledge on the biochemical composition and biophysical data on the chemical and thermal stability of egg carotenoproteins in ampullariid. In addition, recent data on their metabolism, their cellular site of biosynthesis during perivitellogenesis, as well as their carotenoid binding properties are reviewed, highlighting the physiological significance of carotenoproteins in the context of the reproductive biology of these molluscs.

Pallial oviduct of Pomacea canaliculata (Gastropoda): ultrastructural studies of the parenchymal cellular types involved in the metabolism of perivitellins.

Seasonal variations in the morphology of the parenchymal mass and function of the albumen gland/capsule gland complex have been studied in Pomacea canaliculata, together with the cellular types involved in the synthesis and secretion of perivitellin fluid components. The two major parenchymal cell types, albumen secretory cells (AS) and labyrinthic cells (LC), undergo seasonal variations throughout the annual reproductive cycle, which is divided into three periods. Both cellular types show maximal development and structural complexity during the reproductive period (spring and summer). AS cells have a well-developed Golgi complex and rough endoplasmic reticulum and their secretory granules show electron-dense particles of about 20 nm (probably galactogen). These cells are uniquely involved in ovorubin and PV2 perivitellin synthesis and their secretory granules are the single storage site for these two major perivitellins, as revealed by immunoelectron microscopy. AS also possess calcium deposits that infiltrate the cytoplasmic matrix. The luminal surfaces of LC exhibit long cilia intermingled with sparce short microvilli. Basally, the plasma membrane shows deep irregular folds that extend through the cytoplasm up to the subapical region. Calcium deposits infiltrate the cytoplasm and accumulate in the extracellular space of the basal labyrinth. Nerve terminals seem to be involved in the regulation of parenchymal cell secretion. At the post-reproductive period, AS markedly change their aspect following the release of most of the secretory granules into the acinar lumen. LC decrease in volume, the number of their cilia decreases, their cytoplasmic folds are much thinner and their extracellular spaces lack calcium particles. At the pre-reproductive period (winter), AS and LC recover and prepare for the subsequent period.