Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas.

Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host's immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of Man5GlcNAc2. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn2+, while the addition of EDTA or Cu2+ abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.

Expression and Characterization of a ß-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

ß-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal ß-D-galactose residues in ß1,3-, ß1,4- or ß1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first ß-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the ß-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal ß1,3- and ß1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the ß-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed ß-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of ß-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

Identification, Characterization, and Expression of a ß-Galactosidase from Arion Species (Mollusca).

ß-Galactosidases (ß-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing ß-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a ß-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a putative ß-galactosidase from Arion vulgaris (A0A0B7AQJ9) in Sf9 cells. The entire gene codes for a protein consisting of 661 amino acids, comprising a putative signal peptide and an active domain. Specificity studies show exo- and endo-cleavage activity for galactose ß1,4-linkages. Both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, display similar biochemical parameters. Both ß-galactosidases are most active in acidic environments ranging from pH 3.5 to 4.5, and do not depend on metal ions. The ideal reaction temperature is 50 °C. Long-term storage is possible up to +4 °C for the A. vulgaris enzyme, and up to +20 °C for the A. lusitanicus enzyme. This is the first report of the expression and characterization of a mollusk exoglycosidase.