Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.
Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry.
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使用设计方法和质谱法通过糖组微阵列揭示葡聚糖识别系统。
DOI：
10.1074/mcp.m115.048272
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发表时间：
2015-04
期刊：
影响因子：
--
通讯作者：
Chai W
中科院分区：
文献类型：
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作者：
Palma AS;Liu Y;Zhang H;Zhang Y;McCleary BV;Yu G;Huang Q;Guidolin LS;Ciocchini AE;Torosantucci A;Wang D;Carvalho AL;Fontes CM;Mulloy B;Childs RA;Feizi T;Chai W
葡聚糖是具有线性或分支序列中不同键的d-葡萄糖的聚合物。它们是微生物和植物细胞壁的组成部分，参与重要的生物识别过程，包括免疫调节、抗癌活性、病原菌毒力和植物细胞壁的生物降解。医学和生物技术领域的这些活动有相当大的翻译可能性。需要高通量的微量方法来筛选识别特定葡聚糖序列的蛋白质，以此作为结构功能研究和开发的基础。我们描述了第一个序列定义的葡糖规模微阵列的构建，使用目标配体葡聚糖结合一种新的高灵敏度质谱测序方法作为筛选工具来指定葡聚糖识别基序。葡聚糖微阵列由153个高纯度的寡糖探针组成，代表了葡聚糖的主要序列。采用碰撞诱导解离负离子电喷雾串联质谱法，对直链、杂链和支链序列中的低聚糖进行了全连锁分析。该系统使用已知在不同生物环境中针对α-或β-葡聚糖的抗体和碳水化合物结合模块进行验证，扩展了对其特异性的了解，并应用于揭示免疫系统的两个信号分子对病原体识别葡聚糖的新信息：DECIN-1和DC-SIGN。利用MS方法对葡聚糖低聚糖进行测序，并在芯片上对其进行查询，提供了葡聚糖识别蛋白的连接、序列和链长要求的详细信息，是揭示多糖中意外序列的敏感手段。
Glucans are polymers of d-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes, including immunomodulation, anticancer activities, pathogen virulence, and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure–function studies and their exploitation. We describe construction of a “glucome” microarray, the first sequence-defined glycome-scale microarray, using a “designer” approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. Negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear “homo” and “hetero” and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signaling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.