基于吡唑啉酮类异二官能团试剂标记的肝癌糖蛋白O-糖链的非还原性释放、分离制备、结构解析及微阵列一体化分析研究

Hepatocarcinogenesis is accompanied by abnormal expression of glycoprotein glycans and the corresponding glycan-binding proteins (GBPs), about which little is known at present in terms of species and amounts. Glycan microarray represents one of the most efficient high-throughput analytical tools to study the interactions between glycans and GBPs. The analytical techniques associated with N-glycan microarray have been established and well studied in recent years. However, it is still a challenge to prepare purified O-glycans and perform O-glycan microarray analysis, due to the lack of reliable methods to generate reducing O-glycans from glycoproteins. In this program, we plan to develop an integrative technology that simultaneously enables one-pot release and labeling, separation, structural characterization and functional analysis of O-glycans, for the screening of abnormally expressed hepatoma GBPs specifically against O-glycans, based on our already-developed method for the one-pot nonreductive release and labeling of O-glycans, using pyrazolone-type heterobifunctional chromogenic reagents as the derivatization substrates and biological mass spectrometry (Bio-MS), high-performance liquid chromatography coupling with mass spectrometry (HPLC-MS) and microarray as the analytical tools. Additionally, we also plan to perform systematic, large-scale analysis of the differences of cell and serum O-glycan GBPs in species and expression level between hepatoma and healthy samples, based on this novel technology. Therefore, this program will provide a reliable platform to study the structure-function correlations of O-glycans and an important reference from the aspects of glycans and GBPs for the early diagnosis of liver cancer, having great significance for functional glycomics.

肝癌的发生伴随糖蛋白糖链的异常表达，与这些糖链特异相互作用的糖结合蛋白（GBP）的表达也会发生异常改变，目前对这些异常表达GBP的种类和数量知之甚少。微阵列(糖芯片）是研究糖与GBP相互作用最有效的高通量分析工具，现已建立N-糖链相关糖芯片分析技术，但O-糖链由于缺乏可靠的获取还原性糖链的方法，其制备纯化和糖芯片分析仍具有很大挑战性。本项目以筛选肝癌异常表达的O-糖链GBP为目的，拟在本实验室发展的O-糖链非还原性释放及同时衍生化新技术的基础上，以生物质谱、HPLC-MS和微阵列技术为主要分析检测手段，发展基于吡唑啉酮类异二官能团试剂的糖蛋白O-糖链释放及同时标记、分离制备、结构解析和功能研究一体化分析新方法， 系统化、规模化比较肝癌细胞和血清与正常样本在GBP表达种类和水平上的差异，为O-糖链结构功能关系研究提供可靠的技术平台，在糖链和糖结合蛋白两个层面上为肝癌的早期诊断提供参考。

糖芯片是研究糖与蛋白相互作用最有效的高通量分析工具，现已建立N-糖链相关糖芯片分析技术，但O-糖链由于缺乏可靠的获取还原性糖链的方法，其制备纯化和糖芯片分析仍具有很大挑战性。本项目主要取得了以下研究成果：①成功设计合成一种连接臂长度适中的结构稳定、化学选择性 强的吡唑啉酮类异二官能团荧光标记试剂：1-(4-乙腈基苯基)-3-甲基-5-吡唑啉酮（AEPMP)，该试剂结构稳定、化学选择性强，活性亚甲基可以用于标记糖链的还原端，通过发生克脑文盖尔缩合和迈克尔加成反应对糖进行高效衍生化，且在该条件下另一个官能团氰基耐受。。②建立了升级版“一锅法”释放糖蛋白O-糖链及同步标记氰基吡唑啉酮类异二官能团试剂标记ANPMP新方法；③建立了ANPMP衍生物寡糖单体的制备方法，获得了包括O-糖链衍生物单体在内的83中寡糖单体ANPMP衍生物，以上化合物均带有一个活性氨基，可高效地通过酰胺化与N-琥珀酰亚胺（NHS）功能化的固相载体反应将糖链固定，进而构建种类多样的天然糖芯片用于糖探针和糖结合蛋白的互作，为糖链的结构与功能关系研究提供有力的工具。④以AEPMP为Linker构建了包含83种糖链单体的鸟枪式天然糖芯片。⑤利用上述包含83种糖链的鸟枪式天然糖芯片研究了与植物凝集素结合互作作用及利用糖芯片技术研究了天然寡糖在早期肿瘤诊断中的应用。⑥发表SCI收录论文 7篇，还有一篇论文在审稿中(Integrated Analysis of Natural Glycans Using a Versatile Pyrazolone-type Heterobifunctional Tag ANPMP）；申请发明专利6件，其中5件已经授权。⑦ 1名博士后出站，现已入职西北大学食品科学与工程学院；2 名博士研究生、6 名硕士研究生已经毕业。