Study of sialoside function using photocrosslinking sialic acid

描述（由申请人提供）：唾液酸是许多糖蛋白和糖脂中发现的末端覆盖糖。唾液化分子，也被称为唾液苷，在无数正常和病理识别事件中起着关键作用。特别是，唾液皂苷常常被致病菌产生的毒素所识别。毒素-唾液苷结合是入侵和毒害宿主细胞的第一步。尽管唾液化分子有许多重要的作用，但由于识别事件的短暂性和低亲和力，识别它们的结合伙伴是困难的。为了克服这一挑战，我们使用代谢低聚糖标记将重氮嘧啶光交联剂引入细胞唾液酸残基。将细胞渗透性的、重氮嘧啶修饰的唾液酸前体添加到培养细胞中，代谢分子，引入光交联唾液酸代替正常的唾液酸。紫外光诱导的重氮嘧啶活化导致唾液化分子与结合伙伴之间的共价交联；通过免疫印迹和/或质谱分析共价复合物以鉴定组分。利用这种技术，我们发现霍乱毒素将B （CTxB）交联到肠上皮细胞表面的O-糖蛋白(s)上

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.