Insight into the regulation of glycan synthesis in Drosophila chaoptin based on mass spectrometry.
Insight into the regulation of glycan synthesis in Drosophila chaoptin based on mass spectrometry.
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DOI：
10.1371/journal.pone.0005434
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发表时间：
2009
期刊：
影响因子：
3.7
通讯作者：
Kanie O
中科院分区：
文献类型：
--
作者：
Kanie Y;Yamamoto-Hino M;Karino Y;Yokozawa H;Nishihara S;Ueda R;Goto S;Kanie O
蛋白质上存在多种N-糖链，它们与许多重要的生物学功能有关。内质网(ER)和高尔基体定位的酶负责这种不依赖模板的糖的合成，从而在每个天冬酰胺残基上形成糖形式。这种糖合成的调节机制在很大程度上仍不清楚。为了研究糖结构与蛋白质构象的关系，我们分析了果蝇的一种糖蛋白Chaoptin(CHP)，它定位于光感受器细胞中，并通过糖基磷脂酰肌醇锚点与细胞膜结合。基于质谱学的详细分析揭示了13个N-糖基化位点的存在以及每个位点的糖形式的组成。从观察到的糖链结构和每个N-糖基化位点的组成推测了多糖的合成途径，并提出了新的合成路线。CHP多肽上糖形式的分布表明，在高尔基体中，各种加工酶作用于CHP的外部，尽管实际上没有任何酶能进入马蹄形支架的内部，因此解释了内部存在较长的糖聚糖。此外，对一个影响内质网中N-糖链合成的突变体(针对α-d-甘露糖基转移酶的RNA干扰)的CHP分析表明，截短的糖链结构被加工了。结果，只有高甘露糖型多糖的糖型分布受到影响，而其他类型的多糖与在对照和野生型中观察到的相似。这些结果表明，多糖的加工在很大程度上依赖于母体多肽的骨架结构。我们获得的信息可以应用于LRR家族的其他成员。
A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate α-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.
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