The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing
The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing
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DOI：
10.1074/jbc.m511023200
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发表时间：
2006-02-24
影响因子：
4.8
通讯作者：
Altmann, F
中科院分区：
文献类型：
--
作者：
Léonard, R;Rendic, D;Altmann, F
大多数处理，E。G.昆虫糖蛋白上岩藻糖基化的N-聚糖终止于甘露糖，但相关的修饰酶需要N-乙酰葡糖胺基转移酶I的优先作用。这导致了氨基己糖苷酶在N-聚糖成熟过程中起作用的假设。为了确定黑腹果蝇基因组是否真的编码这种酶，克隆了一个对应于融合叶（fdl）的cDNA，一种假定的具有潜在跨膜结构域的β-N-乙酰氨基葡萄糖苷酶。当在巴斯德毕赤酵母中表达时，该酶表现出与先前描述的来自Sf-9细胞的氨基己糖苷酶活性相似的底物特异性，即它仅水解与N-聚糖的核心五糖的α 1，3-连接的甘露糖连接的GlcNAc残基。它也水解对硝基苯基-N-乙酰基-β-氨基葡萄糖苷，但不水解壳寡糖;相反，在毕赤酵母中表达的果蝇HEXO 1和HEXO 2切割这两种底物，但不切割N-聚糖。用免疫电镜对果蝇S2细胞中标记有绿色荧光蛋白的重组FDL的定位表明，该酶通过高尔基体转运，存在于质膜和多泡体中，并被分泌。最后，通过质谱和反相高效液相色谱分析了两个品系的fdl突变果蝇的N-聚糖。具有末端GlcNAc的结构与不具有末端GlcNAc的结构（即少甘露糖苷N-聚糖）的结构的比率在fdl缺陷型果蝇中急剧增加。因此，我们得出结论，fdl基因编码一种新的氨基己糖苷酶负责在果蝇中发生的少甘露糖苷N-聚糖。
Most processed, e. g. fucosylated, N-glycans on insect glycoproteins terminate in mannose, yet the relevant modifying enzymes require the prior action of N-acetylglucosaminyltransferase I. This led to the hypothesis that a hexosaminidase acts during the course of N-glycan maturation. To determine whether the Drosophila melanogaster genome indeed encodes such an enzyme, a cDNAcorresponding to fused lobes (fdl), a putative beta-N-acetylglucosaminidase with a potential transmembrane domain, was cloned. When expressed in Pichia pastoris, the enzyme exhibited a substrate specificity similar to that previously described for a hexosaminidase activity from Sf-9 cells, i.e. it hydrolyzed exclusively the GlcNAc residue attached to the alpha 1,3-linked mannose of the core pentasaccharide of N-glycans. It also hydrolyzed p-nitrophenyl-N-acetyl-beta-glucosaminide, but not chitooligosaccharides; in contrast, Drosophila HEXO1 and HEXO2 expressed in Pichia cleaved both these substrates but not N-glycans. The localization of recombinant FDL tagged with green fluorescent protein in Drosophila S2 cells by immunoelectron microscopy showed that this enzyme transits through the Golgi, is present on the plasma membrane and in multivesicular bodies, and is secreted. Finally, the N-glycans of two lines of fdl mutant flies were analyzed by mass spectrometry and reversed-phase high-performance liquid chromatography. The ratio of structures with terminal GlcNAc over those without (i.e. paucimannosidic N-glycans) was drastically increased in the fdl-deficient flies. Therefore, we conclude that the fdl gene encodes a novel hexosaminidase responsible for the occurrence of paucimannosidic N-glycans in Drosophila.