Influence of N-Glycosylation and N-Glycan Trimming on the Activity and Intracellular Traffic of GD3 Synthase*
Influence of N-Glycosylation and N-Glycan Trimming on the Activity and Intracellular Traffic of GD3 Synthase*
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DOI：
10.1074/jbc.273.6.3725
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发表时间：
1998-02
期刊：
影响因子：
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通讯作者：
J. Martina;J. L. Daniotti;H. Maccioni
中科院分区：
文献类型：
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作者：
J. Martina;J. L. Daniotti;H. Maccioni
GD3 合酶 (ST8Sia I) 将 α-2→8 连接的唾液酸转移至 GM3 的唾液酸部分，形成神经节苷脂 GD3。 GD3 合酶的 cDNA 预测了几个假定的 N-糖基化位点。在这项工作中，我们研究了鸡 GD3 合酶中这些位点的占用情况以及它们如何影响其活性和细胞内流量。 COS-7 细胞用流感病毒血凝素 (HA) 表位标记形式的 GD3 合酶 (GD3 合酶-HA) 转染。细胞获得了 GD3 合酶活性、细胞表面 GD3 免疫表达以及高尔基复合体中 GD3 合酶-HA 免疫反应性。在蛋白质印迹中，检测到 47 kDa 的主要 GD3 合酶-HA 带，用 [2-3H] 甘露糖代谢标记后具有放射性。衣霉素阻止[2-3H]甘露糖掺入GD3合酶-HA中，阻断酶活性，并促进酶分子量降低6-7 kDa。 N-糖苷酶 F 的定时去糖基化表明 GD3 合酶-HA 的所有三个潜在 N-糖基化位点均被糖基化。去糖基化形式在酶学上比天然形式更不稳定。衣霉素处理细胞导致内质网 (ER) 中 GD3 合酶 -HA 免疫反应性的保留。栗精胺和脱氧野尻霉素是内质网加工酶 α-葡萄糖苷酶 I 和 II 的抑制剂，也阻止内质网排出，但基本上不影响酶的比活性。 1-脱氧甘露尻霉素和苦马豆素（甘露糖苷酶抑制剂）不影响酶活性或高尔基体定位。结果表明 (a)N-糖基化对于 GD3 合酶获得和维持催化活性折叠以及退出 ER 是必需的； (b) 内质网中的 N-聚糖修剪虽然对于酶活性不是必需的，但对于 GD3 合酶正确运输至高尔基复合体是必要的。
GD3 synthase (ST8Sia I) transfers a sialic acid in α-2→8 linkage to the sialic acid moiety of GM3 to form the ganglioside GD3. The cDNAs of GD3 synthases predict several putative N-glycosylation sites. In this work we have examined the occupancy of these sites in a chicken GD3 synthase and how they affect its activity and intracellular traffic. COS-7 cells were transfected with an influenza virus hemagglutinin (HA) epitope-tagged form of GD3 synthase (GD3 synthase-HA). Cells acquired GD3 synthase activity, cell surface GD3 immunoexpression, and GD3 synthase-HA immunoreactivity in the Golgi complex. In Western blots, a main GD3 synthase-HA band of 47 kDa was detected, which was radioactive upon metabolic labeling with [2-3H] mannose. Tunicamycin prevented the incorporation of [2-3H]mannose into GD3 synthase-HA, blocked the enzyme activity, and promoted a reduction of the enzyme molecular mass of 6–7 kDa. Timed deglycosylation with N-glycosidase F showed that all three potential N-glycosylation sites of GD3 synthase-HA were glycosylated. The deglycosylated forms were enzymatically more unstable than the native form. Tunicamycin treatment of cells led to retention of GD3 synthase-HA immunoreactivity in the endoplasmic reticulum (ER). Castanospermine and deoxynojirimycin, inhibitors of the ER-processing enzymes α-glucosidases I and II, also prevented the exit from the ER but did not essentially affect the enzyme specific activity. 1-Deoxymannojirimycin and swainsonine, inhibitors of mannosidases, did not affect either the enzyme activity or the Golgi localization. Results indicate that (a)N-glycosylation is necessary for GD3 synthase to attain and to maintain a catalytically active folding, and for exiting the ER; and (b) N-glycan trimming in the ER, while not required for enzyme activity, is necessary for proper trafficking of GD3 synthase to the Golgi complex.