A fused lobes gene encodes the processing β-N-acetylglucosaminidase in Sf9 cells
A fused lobes gene encodes the processing β-N-acetylglucosaminidase in Sf9 cells
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DOI：
10.1074/jbc.m710279200
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发表时间：
2008-04-25
影响因子：
4.8
通讯作者：
Jarvis, Donald L.
中科院分区：
文献类型：
--
作者：
Geisler, Christoph;Aumiller, Jared J.;Jarvis, Donald L.
Man alpha 6（Man alpha 3）Man beta 4GlcNAc beta 4GlcNAc-R是昆虫细胞产生的主要加工蛋白N-聚糖的核心结构。最终，这种少甘露糖型结构由不寻常的β-N-乙酰葡糖胺糖苷酶产生，该酶从上游中间体Man α 6（GlcNAc β 2 Man α 3）Man β 4GlcNAc β 4GlcNAc-R去除末端N-乙酰葡糖胺残基。由于导致该中间体产生的N-聚糖加工途径在昆虫和高等真核生物中可能相同，因此存在或不存在该特异性加工β-N-乙酰氨基葡糖苷酶是区分这两种不同类型生物中加工途径的关键因素。最近的研究表明，融合叶（fdl）基因编码的特异性，加工β-N-乙酰氨基葡萄糖苷酶的果蝇。然而，在鳞翅目昆虫草地贪夜蛾中编码这种酶的基因的身份上有相互矛盾的报道。有人提出，一个基因或者命名为SfGlcNAcase-3或SfHex编码该功能，而另一个人提出，该基因编码在聚糖和几丁质降解中起作用的广谱β-N-乙酰葡糖胺糖苷酶。在这项研究中，我们通过分子克隆一个S。frugiperda fdl直系同源物（Sf-fdl），并证明其编码具有加工β-N-乙酰氨基葡糖苷酶预期的底物特异性的产物。此外，我们发现，在S.在一个实施方案中，本发明涉及经工程改造以表达源自Sf-fdl基因的双链RNA的草地害虫细胞。这些结果表明Sf-fdl编码S. frugiperda的，并验证了我们以前的建议，即由SfGlcNAcase-3/SfHex基因编码的广谱β-N-乙酰氨基葡糖苷酶更可能参与N-聚糖和/或几丁质降解。
Man alpha 6( Man alpha 3) Man beta 4GlcNAc beta 4GlcNAc-R is the core structure of the major processed protein N-glycans produced by insect cells. Ultimately, this paucimannose type structure is produced by an unusual beta-N-acetylglucosaminidase, which removes the terminal N-acetylglucosamine residue from the upstream intermediate, Man alpha 6(GlcNAc beta 2Man alpha 3) Man beta 4GlcNAc beta 4GlcNAc-R. Because the N-glycan processing pathways leading to the production of this intermediate are probably identical in insects and higher eukaryotes, the presence or absence of this specific, processing beta-N-acetylglucosaminidase is a key factor distinguishing the processing pathways in these two different types of organisms. Recent studies have shown that the fused lobes (fdl) gene encodes the specific, processing beta-N-acetylglucosaminidase of Drosophila melanogaster. However, there are conflicting reports on the identity of the gene encoding this enzyme in the lepidopteran insect, Spodoptera frugiperda. One has suggested that a gene alternatively designated SfGlcNAcase-3 or SfHex encodes this function, whereas another has suggested that this gene encodes a broad-spectrum beta-N-acetylglucosaminidase that functions in glycan and chitin degradation. In this study we resolved this conflict by molecularly cloning an S. frugiperda fdl ortholog (Sf-fdl) and demonstrating that it encodes a product with the substrate specificity expected of the processing beta-N-acetylglucosaminidase. Moreover, we showed that the endogenous levels of specific, processing beta-N-acetylglucosaminidase activity were significantly reduced in S. frugiperda cells engineered to express a double-stranded RNA derived from the Sf-fdl gene. These results indicate that Sf-fdl encodes the specific, processing beta-N-acetylglucosaminidase of S. frugiperda and validate our previous suggestion that the broad-spectrum beta-N-acetylglucosaminidase encoded by the SfGlcNAcase-3/SfHex gene is more likely to be involved in N-glycan and/or chitin degradation.