Agglutinating Activity and Structural Characterization of Scalarin, the Major Egg Protein of the Snail Pomacea scalaris (d’Orbigny, 1832)

Abstract

Apple snail perivitellins are emerging as ecologically important reproductive proteins. To elucidate if the protective functions of the egg proteins of Pomacea canaliculata (Caenogastropoda, Ampullariidae), involved in embryo defenses, are present in other Pomacea species we studied scalarin (PsSC), the major perivitellin of Pomacea scalaris. Using small angle X-ray scattering, fluorescence and absorption spectroscopy and biochemical methods, we analyzed PsSC structural stability, agglutinating activity, sugar specificity and protease resistance. PsSC aggluttinated rabbit, and, to a lesser extent, human B and A erythrocytes independently of divalent metals Ca2+ and Mg2+ were strongly inhibited by galactosamine and glucosamine. The protein was structurally stable between pH 2.0 to 10.0, though agglutination occurred only between pH 4.0 to 8.0 (maximum activity at pH 7.0). The agglutinating activity was conserved up to 60°C and completely lost above 80°C, in agreement with the structural thermal stability of the protein (up to 60°C). PsSC was able to withstand in vitro gastrointestinal digestion, and showed no trypsin inhibition activity. The presence of lectin activity has been reported in eggs of other Pomacea snails, but here we link for the first time, this activity to an apple snail multifunctional perivitellin. This novel role for a snail egg storage protein is different from closely related P.canaliculata defensive proteins.

Novel Animal Defenses against Predation: A Snail Egg Neurotoxin Combining Lectin and Pore-Forming Chains That Resembles Plant Defense and Bacteria Attack Toxins

“…This procedure was repeated several times until the supernatant remained clear. Hemagglutinating activity was assayed in microtiter U plates (Greiner Bio One, Germany) by incubating a two-fold serial dilution of PcPV2 (1.6 g/L) with 1% erythrocyte suspension in phosphate buffer at 37°C for 2 h. Results were expressed as the inverse of the last dilution showing visible hemagglutinating activity at naked eye [32]. Hemolytic activity was followed measuring the release of hemoglobin spectrophotometrically at 412 nm in the supernatant of the wells [33].…”

Insights from an Integrated View of the Biology of Apple Snails (Caenogastropoda: Ampullariidae)

“…In spite of the central role of perivitellins in reproduction and development, there is little information on their structural features in invertebrates. Studies in molluscs are limited essentially to the perivitellins of Pomacea, mostly in P. canaliculata but also P. scalaris and P. maculata (Ituarte et al, 2012;Pasquevich et al, 2014).…”

“…The perivitelline fluid of P. scalaris contains two major perivitellins, of which only the pigmented one, PsSC, which is structurally similar to PcOvo, has been characterized (Ituarte et al, , 2012. The perivitelline fluid of P. maculata is also similar, with two major perivitellins, PmPV1 and PmPV2 (Pasquevich et al, 2014).…”

“…However, unlike PcOvo, this perivitellin is a carbohydrate binding protein, which agglutinates erythrocytes, showing a high specificity for galactosamine and glucosamine containing glycans (Ituarte et al, 2012). Carbohydrate binding proteins may have an immune function, agglutinating bacteria or other pathogens.…”

A highly stable, nondigestible lectin fromPomacea diffusaunveils clade-related protection systems in apple snail eggs

“…Extensive studies of the most abundant perivitellins in Pomacea species, collectively called PV1s, have shown that they are all oligomeric carotenoproteins of high molecular weight, representing ~70% of the PVF soluble protein (Dreon, Heras and Pollero, 2003; Ituarte et al ., 2008; Pasquevich, Dreon and Heras, 2014). These carotenoproteins are highly stable in a wide range of pH and temperatures (Dreon, Ceolín and Heras, 2007; Dreon et al ., 2008; Sun et al ., 2012; Pasquevich et al ., 2017) and withstand gastrointestinal digestion (Dreon, Ituarte and Heras, 2010; Ituarte et al ., 2012; Dreon et al ., 2013; Pasquevich et al ., 2017). Nevertheless, some differences emerged: Although the PV1 carotenoprotein of the non-invasive species P. scalaris (PsSC) is a lectin (carbohydrate binding protein) with a remarkably strong activity (Ituarte et al ., 2012, 2018), its orthologues from the invasive species P. canaliculata and P. maculata (PcOvo and PmPV1, respectively) lack lectin activity.…”