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.

Graphical abstract

Illustration of GnT-I activity. (a) Transfer of GlcNAc to Man5-PA, creating Man5GlcNAc3-PA. (b) Transfer of GlcNAc to MM-PA, creating MGn-PA. (c) Transfer of GlcNAc to GnM-PA, creating GnGn-PA. Blue squares represent N-acetylglucosamine, green cycles depict mannose. Graphic illustration of N-glycans were created using bioRENDER.

中文翻译：

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太平洋牡蛎巨牡蛎中 UDP-N-乙酰氨基葡萄糖：α-1,3-D-甘露糖苷 β-1,2-N-乙酰氨基葡萄糖转移酶 I (GnT-I) 的测定、表达和表征

软体动物是多种寄生虫的中间宿主。逃避宿主免疫反应所需的识别过程依赖于碳水化合物。因此，软体动物糖基化能力的研究对于了解寄生虫与其宿主的相互作用具有很高的相关性。 UDP-N-乙酰氨基葡萄糖:α-1,3-D-甘露糖苷 β-1,2-N-乙酰氨基葡萄糖转移酶 I (GnT-I) 是杂合和复合型 N-聚糖生物合成的关键酶，催化 N 的转移-乙酰葡糖胺从UDP-N-乙酰葡糖胺到Man ₅ GlcNAc ₂的α-1,3 Man天线。由此，酶产生其他酶的合适底物，例如α-甘露糖苷酶II、GlcNAc-转移酶II、半乳糖基转移酶或岩藻糖基转移酶。以相应的人酶为模板，通过同源搜索获得来自太平洋牡蛎（ Crassostrea gigas）的GnT-I序列。获得的基因编码445个氨基酸长的II型跨膜糖蛋白，并与其他物种的酶共享典型的结构元件。该酶在昆虫细胞中表达，并使用与单克隆 His 标签抗体连接的 Protein A/G-plus 琼脂糖珠通过免疫沉淀进行纯化。 GnT-I 活性是针对底物 Man5-PA、MM-PA 和 GnM-PA 测定的。使用 Man5-PA 作为底物，该酶在 pH 7.0 和 30 °C 时表现出最高活性。二价阳离子对于该酶是不可或缺的，在40 mM Mn ²⁺时具有最高活性，而添加EDTA或Cu ^{2+ 则}完全消除该活性。添加 UDP、UTP 或半乳糖也会降低活性。在本研究中，我们展示了来自太平洋牡蛎的第一个软体动物 UDP-N-乙酰氨基葡萄糖：α-1,3-D-甘露糖苷 β-1,2-N-乙酰氨基葡萄糖转移酶 I (GnT-I) 的鉴定、表达和生化特征巨牡蛎。

图形概要

GnT-I 活性的图示。 (a) 将 GlcNAc 转移至 Man5-PA，产生 Man5GlcNAc3-PA。 (b) 将 GlcNAc 转移至 MM-PA，产生 MGn-PA。 (c) 将 GlcNAc 转移至 GnM-PA，产生 GnGn-PA。蓝色方块代表 N-乙酰氨基葡萄糖，绿色循环代表甘露糖。 N-聚糖的图示是使用bioRENDER 创建的。