Proteomic analysis of N-glycosylation in mosquito dopachrome conversion enzyme.
Proteomic analysis of N-glycosylation in mosquito dopachrome conversion enzyme.
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蚊子多巴色素转化酶 N-糖基化的蛋白质组学分析。
DOI：
10.1002/pmic.200601053
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发表时间：
2007
期刊：
影响因子：
3.4
通讯作者：
Li,Jianyong
中科院分区：
文献类型：
--
作者：
Li,JunsuoS;Vavricka,ChristopherJ;Christensen,BruceM;Li,Jianyong
多巴色素转化酶（DCE）是一种存在于昆虫体内的新型色素转化酶，参与昆虫的黑化过程. DCE与从细菌到人类的任何非昆虫物种都没有序列同源性。已经有几个DCE序列，但酶的结构和催化机制尚不清楚。本研究涉及DCE PTM，特别是糖基化。纯化了一种蚊子DCE，并测定了其单糖组成、N-糖基化位点和寡糖结构。结果表明，蚊DCE中主要单糖为N-乙酰氨基葡萄糖和D-甘露糖，次要单糖为L-岩藻糖、D-木糖和D-阿拉伯糖。根据胰蛋白酶消化的DCE糖肽的MS和MS/MS光谱解析了糖基化位点和寡糖结构。在DCE中鉴定了单个N-糖基化位点（Asn 285-Glu-Thr），并证明其完全糖基化。Man 3（Fuc）1- 2GlcNAc 2及其截短结构是主要的寡糖。此外，还鉴定了高甘露糖型结构（Man 4 -7（Fuc）GlcNAc 2）。用肽N-糖苷酶（PNGase F）去除DCEN-寡糖降低了其活性和热稳定性。然而，用α-甘露糖苷酶或α-岩藻糖苷酶进行部分DCE去糖基化在一定程度上刺激了其活性并改善了其热稳定性。在DCE糖肽的质谱分析期间，其CID模式非常有趣，因为一些糖肽在CID期间经历C末端重排和二聚体结构的形成。这项研究的结果提供了一个有趣的例子，在糖肽CID片段化模式的潜在复杂性。
A novel dopachrome conversion enzyme (DCE) is present in insects and involved in their melanization pathway. DCE shares no sequence homology with any noninsect species from bacteria to humans. Several DCE sequences have been available, but enzyme structure and catalytic mechanism are unclear. This study concerns DCE PTMs, especially glycosylation. A mosquito DCE was purified and its monosaccharide composition,N‐glycosylation site, and oligosaccharide structures were determined. Results showed thatN‐acetylD‐glucosamine andD‐mannose are the major monosaccharides andL‐fucose,D‐xylose, andD‐arabinose are the minor ones in mosquito DCE. Glycosylation site and oligosaccharide structures were elucidated from MS and MS/MS spectra of trypsin‐digested DCE glycopeptides. A singleN‐glycosylation site (Asn285‐Glu‐Thr) was identified in DCE and was proven to be fully glycosylated. Man3GlcNAc2, Man3(Fuc)1–2GlcNAc2, and their truncated structures were the dominant oligosaccharides. In addition, high mannose‐type structures (Man4–7(Fuc)GlcNAc2) were also identified. Removal of DCEN‐oligosaccharides with peptideN‐glycosidase (PNGase F) decreased its activity and thermal stability. However, partial DCE deglycosylation with α‐mannosidase or α‐fucosidase somewhat stimulated its activity and improved its thermal stability. During mass spectrometric analysis of DCE glycopeptides, their CID patterns were highly intriguing, in that some glycopeptides underwent both C‐terminal rearrangement and formation of dimeric structures during CID. Results of this study provide an interesting example in terms of potential complexity of the glycopeptide CID fragmentation pattern.