高亲和GlcNAc凝集素AAL2用于O-GlcNAc修饰蛋白质组学研究的方法学建立及机理

O-GlcNAc modification, which was discovered in 1984, is one of the most important post tanscriptional modifications. The cross talk between O-GlcNAcylation and phosphorylation was involved in many important physiological processes of cells and in the pathogenesis of many diseases. However, the identification of O- GlcNAcylated peptides has been the bottleneck for the research in this field. Our team has previously identified a novel lectin AAL2 (Biochemical Journal, 2012). The specificity and affinity of AAL2 toward N-actetylglucosamine (GlcNAc) has been found to be much higher than that of wheat germ agglutinin (WGA), the main reagent used in enrichment of O-GlcNAcylated peptides at the present. Further study shows that AAL2 also has high affinities toward O-GlcNAcylated peptides. Based on all these preliminary data, this application is proposed to perform the following two parts studies. The first part is to develop a new method to identify O-GlcNAcylated peptides by using lectin AAL2 affinity chromatography and mass spectrum. Synthetic O-GlcNAcylated peptides and other non O-GlcNAcylated peptdieds will be mixed and used as initial sample to test the efficiency of the AAL2 affinity chromatography. Then,the complex sample, the peptides of cytoplsamic and nuclear peptides prepared from tumor cells, pancreas and thymus, will be loaded on AAL2 affinity chromatography to enrich O-GlcNAcylated peptide and develop a new, high throughput method to identify the O-GlcNAcylated peptides as well as the modification sites. The second part is to investigate the mechanism of high affinity and specificity of AAL2 toward O-GlcNAc & O-GlcNAcylated peptides through crystallographic studies. The key binding sites will be investigated by the structural analysis and verified by point mutagenesis. All mutants will be tested for their affinities with O-GlcNAc by ITC or glucan array. The project will advance the O-GlcNAc proteom research and provide clues in lectin engineering of high affinity and specificity to intended glycans.

氧连N-乙酰葡萄糖胺（O-GlcNAc）修饰是非常重要的蛋白质翻译后修饰方式，与蛋白质磷酸化修饰互相调节，参与细胞重要生理反应和多种疾病发生过程。而O-GlcNAc修饰蛋白的鉴定是该领域发展的瓶颈问题。 本研究小组鉴定了一种新凝集素AAL2识别N-乙酰葡萄糖胺（GlcNAc），其特异性和亲和力远高于现用于富集O-GlcNAc修饰蛋白的麦胚凝集素（WGA）（BJ,2012）。进一步研究表明，AAL2对O-GlcNAc修饰肽段也具有高度亲和力。因此，提出本申请计划：1）用多种简单样品、复杂样品（细胞和组织）肽段检测，建立一种凝集素AAL2亲和层析富集、高通量鉴定O-GlcNAc修饰肽段的新方法。2）研究AAL2、AAL2+O-GlcNAc修饰肽段结晶结构，制备突变体，研究AAL2与O-GlcNAc修饰肽段高亲和和高特异性结合的机理。该项目将对O-GlcNAc修饰蛋白质组学研究有重要推动作用。

原申请的实验计划为：1)用多种简单样品、复杂样品（细胞和组织）肽段检测，建立一种凝集素AAL2 亲和层析富集、高通量鉴定O-GlcNAc修饰肽段的新方法。2)研究AAL2、AAL2+O-GlcNAc 修饰肽段结晶结构，制备突变体，研究AAL2 与O-GlcNAc 修饰肽段高亲和和高特异性结合的机理。该项目已顺利完成，用AAL2富集、鉴定了小鼠肝脏中的O-GlcNAc修饰肽段（文章已投稿Glycobiology）。完成AAL2和GlcNAc的糖共结晶分析（2015，PLOS ONE）。除此之外，对AAL2还有更深入的研究：（为超额完成的工作）1)建立了Dimer-AAL2(AAL2的二聚体)表达、纯化、糖结合特异性研究，并用Dimer-AAL2建立了富集和高通量鉴定O-GlcNAc修饰肽段的新方法（文章撰写中）。2）成功构建了AAL突变体AAL23和AAL26，得到表达和纯化，这两个突变体都表现出对GlcNAc更好的结合特异性，用AAL26建立了富集和高通量鉴定O-GlcNAc修饰肽段的新方法（文章撰写中）。3）用AAL2鉴定了早期肺癌病人血清中的特异表达的糖蛋白，作为肺癌早期诊断的生物标志物(2016, BJC)。