Study of sialoside function using photocrosslinking sialic acid

描述（由申请人提供）：唾液酸是在许多糖蛋白和糖脂上发现的末端加帽糖。唾液酸化分子，也称为唾液酸苷，在无数正常和病理识别事件中起关键作用。特别地，唾液酸糖苷通常被病原菌产生的毒素识别。毒素-唾液酸糖苷结合是宿主细胞侵入和中毒的初始步骤。尽管唾液酸化分子具有许多重要作用，但由于识别事件的瞬时性和低亲和力，识别其结合配偶体是困难的。为了克服这一挑战，我们使用代谢寡糖标记，将二氮丙啶光交联剂引入细胞唾液酸残基。将细胞可渗透的、二氮丙啶修饰的唾液酸前体添加到培养的细胞中，其代谢该分子，引入光交联唾液酸代替正常唾液酸。UV诱导的二氮丙啶活化导致唾液酸化分子和结合伴侣之间的共价交联;通过免疫印迹和/或质谱法分析共价复合物以鉴定组分。利用这种技术，我们发现霍乱毒素提交B（CTx B）交联到肠上皮细胞表面的O-连接糖蛋白上， 而不是神经节苷脂GM 1a，它的受体。此外，我们观察到O-连接的糖蛋白是肠上皮细胞系中主要的CTxB结合伴侣。功能测定揭示O-连接糖蛋白介导霍乱毒素（CTx）对宿主细胞的作用。最后，初步的数据确定CD 44作为CTxB结合糖蛋白的强有力的候选者。在即将到来的授权期间，我们将确定CTxB与肠上皮细胞系结合的蛋白质和聚糖决定簇，并确定新型CTx结合伴侣的定位模式（目标1）。在目标2中，我们将通过测量CTxB结合糖蛋白的表达如何影响CTx内化、CTx诱导的cAMP产生和CTx诱导的氯离子分泌来测试CTxB结合糖蛋白的功能相关性。另外的结合CTxB结合配偶体的存在提供了一种方法来调和关于CTxB进入宿主细胞的内吞机制的现有数据。因此，在目标3中，我们将研究CTxB使用的内吞途径，并确定内吞机制是否依赖于CTxB结合伴侣的身份。这里描述的实验利用我们的光交联唾液酸技术，以获得新的和意想不到的见解宿主-病原体相互作用。O-连接糖蛋白与CTxB结合的发现是重要的，因为：（1）它改变了我们对霍乱中毒机制的基本理解，（2）它有可能为区分不同内吞途径的特征提供关键的见解，以及（3）它敦促谨慎解释荧光显微镜实验以可视化脂筏，因为这些实验依赖于CTxB仅结合GM 1a的假设。此外，光交联唾液酸的成功应用表明，该试剂具有广阔的应用前景 在定义正常和病理生理识别事件。

DESCRIPTION (provided by applicant): Sialic acid is the terminal, capping sugar found on many glycoproteins and glycolipids. Sialylated molecules, also known as sialosides, play critical roles in myriad normal and pathological recognition events. In particular, sialosides are often recognized by toxins produced by pathogenic bacteria. Toxin-sialoside binding is the initial step in invasion and intoxication of host cells. Despite the many essential roles of sialylated molecules, identifying their binding partners is difficult, due to the transience and low affinity f the recognition events. To surmount this challenge, we use metabolic oligosaccharide labeling to introduce the diazirine photocrosslinker into cellular sialic acid residues. A cell-permeable, diazirine-modified sialic acid precursor is added to cultured cells, which metabolize the molecule, introducing photocrosslinking sialic acid in place of normal sialic acid. UV-induced activation of the diazirine leads to covalent crosslinking between sialylated molecules and binding partners; covalent complexes are analyzed by immunoblot and/or mass spectrometry to identify components. Using this technique, we showed that cholera toxin submit B (CTxB) crosslinks to O- linked glycoprotein(s) displayed on the surface of intestinal epithelial cells and not to ganglioside GM1a, its accepted receptor. Further, we observe that O-linked glycoproteins are the primary CTxB binding partner in intestinal epithelial cell lines. Functional assays reveal that O-linked glycoprotein(s) mediate the effects of cholera toxin (CTx) on host cells. Finally, preliminary data identify CD44 as a strong candidate for the CTxB- binding glycoprotein. During the upcoming granting period, we will define the protein and glycan determinants of CTxB binding to intestinal epithelial cell lines and determine the localization pattern of the novel CTx binding partner (Aim 1). In Aim 2, we will test the functional relevance of the CTxB-binding glycoprotein by measuring how its expression affects CTx internalization, CTx-induced cAMP production, and CTx-induced chloride ion secretion. The existence of an additional binding CTxB binding partner offers a way to reconcile existing data regarding the endocytic mechanism by which CTxB enters host cells. Thus, in Aim 3, we will investigate the endocytic route used by CTxB and determine whether endocytic mechanism is dependent on the identity of the CTxB binding partner. Experiments described here exploit our photocrosslinking sialic acid technology to obtain new and unanticipated insights into host-pathogen interactions. The discovery that an O- linked glycoprotein binds to CTxB is significant because: (1) it alters our fundamental understanding of the mechanism of cholera intoxication, (2) it has the potential to provide critica insight into the features that distinguish different endocytic pathways, and (3) it urges caution i the interpretation of fluorescence microscopy experiments to visualize lipids rafts, since these experiments rely the assumption that CTxB binds GM1a exclusively. Furthermore, successful application of photocrosslinking sialic acid suggests that this reagent will find broad application in defining normal and pathophysiological recognition events.