基于结构糖组学的生姜糖蛋白糖基化位点与糖链空间构象解析

With regard to the complexity of the ginger-derived glycoproteins and their diversity in sequences, as well as their unevenly micro-distribution, the glycosylation site and glycan structure of ginger glycoprotein has not been clarified. Based on our previous preliminary results, we propose to continue the purification of ginger-derived glycoprotein, and elucidate its structure in light of its glycosylation site, molecular mass, the linkage mode of glycopeptide bond, the monosaccharide composition, and the deduction of the linkage type of glycosidic bond, with aid of the latest technique of structural glycomics. Firstly, the ginger derived glycoprotein will be collected and enriched , based on the lectin affinity technique of structural glycomics, which is specified for its carbohydrate binding property; Secondly, in combination of tandem MS technique, proteinase and glycosidase, the proteolytic fragments, or the glycopeptides in the protein location, will be investigated, in order to clarify the glycosylation sites; Finally, by the utilization of ESI and MALDI-TOF of glycomics MSn spectrometry techniques, structure of ginger glycoprotein will be clarified according to the MS/MS and GLYCOSCIENCE database..The proposal should provide a novel research approach for the enrichment and characterization of ginger-derived glycoprotein. Moreover, this research is significantly important to characterize glycoproteins from fruits and vegetables.

鉴于生姜糖蛋白样品的复杂性及其存在着微观不均一性，目前生姜糖蛋白的糖基化位点及糖链空间构象信息尚不明确，制约糖蛋白结构表征及糖链功能研究。本项目在前期基础上拟利用结构糖组学探讨生姜糖蛋白的有效富集途径并阐明其糖基化位点与特定糖型的糖链空间结构，研究中运用糖组学的凝集素亲和技术探索富集特定糖型生姜糖蛋白的有效策略；借鉴糖组学的生物质谱技术并协同蛋白质酶和糖苷酶裂解手段，通过明确糖蛋白酶解片段糖肽的相对分子质量和肽段在蛋白质中的位置，阐明糖基化位点；利用糖组学中多级质谱技术，综合利用ESI和MALDI-TOF的各自优势及碰撞机理，根据MS/MS及GLYCOSCIENCE数据库进行检索和比对，实现阐明生姜糖蛋白的糖基化位点和糖链结构信息的目的。本研究可为生姜糖蛋白糖链的结构解析提供新的思路和参考，同时对丰富果蔬糖蛋白的基础理论具有重要意义。

糖蛋白是由寡糖链与蛋白质共价相连而成的分子,而糖链是糖蛋白的重要组成部分，对生命体起着重要的作用。本研究以生姜糖蛋白为研究对象，利用凝集素磁性微粒技术富集生姜糖蛋白，并在糖苷酶F和糖苷酶A 酶解下释放糖链，结合生物质谱技术深入分析了生姜糖蛋白的糖链结构和糖基化位点，并结合KEGG通路分析生姜糖蛋白参与的重要生理过程或信号通路，以期为生姜糖蛋白的生理功能的研究提供参考。所取得的主要研究结果如下：利用凝集素芯片技术和磁性微粒筛选并富集生姜糖蛋白，Con A、LCA、LEL、VVA、STL可以与生姜糖蛋白特异性结合，推测生姜糖蛋白中含有Man，GlcNAc，Fuc以及末端葡萄糖结构。MALDI-TOF-MS分析生姜糖蛋白总糖链以及Con A、STL分别富集后的生姜糖链结构，3种样品糖链结构中复杂型糖链结构居多，且3种样品中Man，N-GlcNAc，Fuc，Xyl是常见糖型，其中Fuc修饰糖链数量最多。LC-MS/MS分析生姜糖肽的糖基化位点，结合KEGG通路搜索糖肽中蛋白部分所参与的生理功能。本项目已鉴定生姜糖蛋白10个N-糖基化位点，其中6个位点的肽段序列通过数据库搜索发现原始蛋白，结合KEGG通路发现可以调控植物昼夜节律途径的CK2α蛋白激酶，对糖蛋白在高尔基体中修饰加工有重要作用的COG复合体，以及影响DNA复制，转录等生命活动的Topo2。本项目为糖蛋白糖基化位点的分析以及深入理解糖蛋白结构与功能的关系提供依据。