Léonard Renaud, Rendic Dubravko, Rabouille Catherine, Wilson Iain B H, Préat Thomas, Altmann Friedrich
Glycobiology Group, Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria.
J Biol Chem. 2006 Feb 24;281(8):4867-75. doi: 10.1074/jbc.M511023200. Epub 2005 Dec 8.
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 cDNA corresponding 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 alpha1,3-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.
大多数经过加工的昆虫糖蛋白上的N -聚糖，如岩藻糖基化的N -聚糖，都以甘露糖结尾，但相关的修饰酶需要N -乙酰葡糖胺转移酶I的先于其的作用。这引发了一个假说，即一种己糖胺酶在N -聚糖成熟过程中发挥作用。为了确定果蝇基因组是否真的编码这样一种酶，克隆了一个与融合叶（fdl）对应的cDNA，fdl是一种具有潜在跨膜结构域的假定β - N -乙酰葡糖胺酶。当在毕赤酵母中表达时，该酶表现出与先前描述的来自Sf - 9细胞的己糖胺酶活性相似的底物特异性，即它仅水解连接到N -聚糖核心五糖的α1,3 -连接甘露糖上的GlcNAc残基。它还能水解对硝基苯基 - N -乙酰 - β - 葡糖胺，但不能水解壳寡糖；相比之下，在毕赤酵母中表达的果蝇HEXO1和HEXO2能切割这两种底物，但不能切割N -聚糖。通过免疫电子显微镜对果蝇S2细胞中绿色荧光蛋白标记的重组FDL进行定位显示，这种酶通过高尔基体转运，存在于质膜和多囊泡体中，并被分泌。最后，通过质谱和反相高效液相色谱对两株fdl突变果蝇品系的N -聚糖进行了分析。在fdl缺陷果蝇中，具有末端GlcNAc的结构与没有末端GlcNAc的结构（即寡甘露糖型N -聚糖）的比例急剧增加。因此，我们得出结论，fdl基因编码一种新型己糖胺酶，它负责果蝇中寡甘露糖型N -聚糖的出现。
