PDF(Pubmed)
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.
摘要：
软体动物是几种寄生虫的中间宿主。识别过程，逃避宿主的免疫反应，依赖于碳水化合物。因此,软体动物糖基化能力的研究对于理解寄生虫与其宿主的相互作用具有高度的相关性。UDP-N-乙酰葡糖胺：α-1,3-D-甘露糖苷β-1,2-N-乙酰葡糖胺基转移酶I（GnT-I）是生物合成杂合和复合型N-聚糖的关键酶，催化N-乙酰葡糖胺从UDP-N-乙酰葡糖胺转移到Man5GlcNAc2的α-1,3曼角。因此，该酶为其他酶产生合适的底物，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。来自太平洋牡蛎的GnT-I序列，Crassostreagigas,通过使用相应的人酶作为模板的同源性搜索获得。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标签抗体连接的蛋白A/G加琼脂糖珠通过免疫沉淀纯化。GnT-I对底物Man5-PA的活性测定,MM-PA和GnM-PA。该酶在pH7.0和30°C时表现出最高的活性，使用Man5-PA作为底物。二价阳离子是酶不可缺少的，在40mMMn2+时活性最高，而添加EDTA或Cu2完全取消了活性。添加UDP也降低了活性，UTP或半乳糖。在这项研究中，我们提出了鉴定，第一个软体动物UDP-N-乙酰葡糖胺的表达和生化表征：α-1,3-D-甘露糖苷β-1,2-N-乙酰葡糖胺基转移酶I，GnT-I,来自太平洋牡蛎Crassostreagigas。
