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

Abstract

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 β-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 β-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.

Keywords: Crassostrea gigas; Glycosyltransferase; GnT-I; Mollusca; N-glycosylation; UDP-N-acetylglucosamine:α-1,3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I.

Fig. 1

Sequence comparison of GnT-I from different species (H. sapiens: P26572.2; C. elegans: NP_509566.1; D. melanogaster: Q60GL7; B. glabrata: A0A2C9M4W7 and C. gigas: XP_034321804.1). The transmembrane domains are depicted in yellow boxes, the 7 binding sites (substrate and Mn²⁺ binding) in red boxes and disulfide bridges by green bars. Putative N-glycosylation sites (shown solely for C. gigas) are indicated by red arrows

Fig. 2

MALDI-TOF analysis of enzyme activity of truncated GnT-I with C-terminal His-tag. a Man5-PA substrate; b Man5-PA incubated with GnT-I from C. gigas; Structures were created using bioRENDER

Fig. 3

MALDI-TOF analysis of enzyme activity of truncated GnT-I with C-terminal His-tag. a MM-PA substrate; b MM-PA incubated with GnT-I from C. gigas; c GnM-PA substrate; d GnM-PA incubated with GnT-I from C. gigas. Structures were created using bioRENDER

Fig. 4

Effect of temperature on GnT-I from C. gigas. a Optimal storage temperature, b Optimal reaction temperature. Data points represent mean values of two biological replica with corresponding standard deviation

Fig. 5

Biochemical properties of GnT-I from C. gigas. a Stability of the enzyme in presence of chemicals (methanol, acetonitrile, imidazole, glycerol). The red line represents a control without the addition of chemicals. b Inhibition studies by adding Gal, Man, GlcNAc, Glc, UMP, UDP or UTP to the assay to a final concentration of 0.1% (w/v). The red line represents a control without the addition of inhibitors. Data points represent mean values of two biological replica with corresponding standard deviation

Fig. 6

Biochemical properties of GnT-I from C. gigas. a pH-curve using different buffer salts, b Cation requirement and c Optimal Mn²⁺ concentration. Data points represent mean values of two biological replica with corresponding standard deviation