PROTEIN PURIFICATION FROM GASTROPODS

Gastropods are a class of invertebrates within the mollusks, commonly known as snails and slugs. Recently, scientific interest is directed on snails as pollution indicators, as destroyers of crops, but also as parasites carriers. Furthermore, some species of snails are considered as an important human food source in countries like France and Australia. In scientific research, snails are used as model animals especially in molecular biology and immunology. Some snails secrete purple matter with anti-cancer property; the snails use it to protect their eggs and scientists hope to create with it a weapon against breast cancer. In the light of this, the aim of the present work was to purify and characterize exoglycosidases, i.e. sugars-hydrolyzing enzymes, extracted from different species of snails and to study the glycosylation pattern of their tissues by using lectins as glycoprotein-specific antibodies. Purification work started with the screening of 7 different exoglycosidases (α-fucosidase, β-xylosidase, α-mannosidase, β-mannosidase, α-glucosidase, β-glucosidase, β- galactosidase and β-N-acetylglucosaminidase) in 8 different species of snails (Arion lusitanicus, Biomphalaria glabrata, Achatina fulica, Limax maximus, Cepaea hortensis, Lymnea stagnalis, Arianta arbustorum, Planorbarius corneus and egg from Achatina fulica). In order to create a purification scheme, many trials have been performed on the chosen enzyme, i.e. β-galactosidase from Arion lusitanicus. Ten snails were carefully washed after complete removal of the abdomen content to eliminate other sources of the enzyme. Snails were then homogenized and protein was precipitated by 1.2 M ammonium sulfate under cooling centrifugation. Then the precipitate was fractionated by the column hydrophobic interaction chromatography. Fractions exhibiting high enzyme activity were pooled, concentrated, desalted by ultrafiltration and then applied to affi gel blue. As the use of affi gel blue resulted in no binding between the enzyme and the gel beads, the unbound fractions were directly applied to anion exchange chromatography. As this procedure behaved as the same way of affi gel blue, the unbound fractions were pooled, concentrated to less than 2 ml and applied to size exclusion chromatography. Size exclusion chromatography exhibited high resolution purification, because a narrow peak was obtained, after testing the eluate for the enzyme activity. The fractions within this narrow peak were then applied to cation exchange chromatography, resulting in two new very sharp peaks. Fractions of the second peak from cation exchange chromatography were exposed to SDS-PAGE that indicated high quality purification, because just two proteins’ bands appeared. Finally, β-galactosidase specific affinity was the last purification step, where four different buffers were used (50mM sodium citrate pH 4.6, 1M NaCl in50 mM sodium citrate pH 4.6, 50 mM Tris/HCl pH 7.5 and 1M NaCl in 50 mM Tris/HCl pH 7.5) and the enzyme was eluted only with 50 mM Tris/HCl pH 7.5 buffer. Electrophoresis of this eluate resulted in a single protein band in one fraction, identified by LC-MS as galactocerebrosidase with molecular weight about 74 kDa. β-galactosidase characterization tests indicated that the optimal enzyme activity was at pH range 3.5 to 5.0 regardless to the used salt, i.e. the enzyme needs no special cations to be active, and indicated that the maximum activity of the enzyme is reached after 4 h incubation at 37 °C. β-galactosidase purification from species other than Arion lusitanicus resulted in different purified proteins but not the β-galactosidase. Moreover, as fucose and sialic acid are frequent and common modifications in snail glycans and they occur in a variety of different linkages and may therefore contribute to a number of recognition and adhesion processes, the glycosylation patterns of snails’ tissue were studied by different lectins, i.e. glycoproteins specific antibodies. We analysed eggs and adult land snails and water snails (Achatina fulica, Arion lusitanicus, Arianta arbustorum, Biomphalaria glabrata, Cepaea hortensis, Limax maximus, Lymnea stagnalis, Planorbarius corneus) for their N- and O-glycosylation pattern with a focus on their sialylation and fucosylation abilities. Their sialylation potential was investigated by Sambucus nigra agglutinin and Maackia amurensis agglutinin while their fucosylation potential was investigated by Aleuria aurantia lectin, Lens culinaris agglutin, Lotus tetragonolobus agglutinin and Ulex europaeus agglutinin before and after tissues’ digestion with glycopeptidase F and β-elimination, respectively. In conclusion, 1) β-galactosidase purification from Arion lusitanicus needs several purification steps and must be conducted with specific β-galactosidase affinity chromatography, otherwise several unwanted proteins will appear as co-purified proteins. 2) The same purification scheme of Arion lusitanicus, when adopted in other snails, did not result in purifying β-galactosidase. 3) From the lectin study, it was confirmed that fucose and sialic acid are frequent and common modifications in snail glycans.