Study of sialoside function using photocrosslinking sialic acid

唾液酸是末端，覆盖了许多糖蛋白和糖脂上的糖。唾液酸化分子， 也被称为唾液酸苷，在无数正常和病理识别事件中发挥关键作用。特别是， 唾液酸苷通常被病原菌产生的毒素所识别。毒素-唾液酸苷结合是最初的 参与宿主细胞的入侵和中毒。尽管唾液酸化分子有许多重要作用， 由于识别事件的瞬时性和低亲和力，识别它们的结合伙伴是困难的。 为了克服这一挑战，我们使用代谢寡糖标记来引入二氮嗪 光交联剂转化为细胞唾液酸残基。一种细胞透过性的、二氮杂氮改性的唾液酸前体 添加到培养的细胞中，使分子代谢，引入光交联唾液酸来代替 正常唾液酸。紫外光诱导二氮杂环的活化导致唾液酸基之间的共价交联 分子和结合伙伴；通过免疫印迹和/或质谱学分析共价化合物以 确定组件。利用这一技术，我们证明了霍乱毒素B(CTxB)与O- 连接糖蛋白(S)表达于肠上皮细胞表面，而不与神经节苷脂GM1a结合，其 已接受的受体。此外，我们观察到O-连接的糖蛋白是CTxB的主要结合伙伴 肠上皮细胞系。功能分析表明，O-连接糖蛋白(S)介导的作用 霍乱毒素(CTX)对宿主细胞的影响。最后，初步数据表明CD44是CTxB的有力候选者- 结合糖蛋白。在即将到来的授权期内，我们将确定蛋白质和多糖的决定因素 CTxB与肠上皮细胞的结合并确定新型CTxB的定位模式 结合伙伴(目标1)。在目标2中，我们将通过以下方式测试CTxB结合糖蛋白的功能相关性 检测其表达如何影响CTX内化、CTX诱导的cAMP产生和CTX诱导的 氯离子分泌物。另一个结合CTxB的结合伙伴的存在提供了一种协调的方法 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 调查CTxB使用的内吞途径并确定内吞机制是否依赖于 CTxB绑定伙伴的身份。这里描述的实验利用了我们的光交联唾液酸 技术，以获得对宿主-病原体相互作用的新的和意想不到的见解。发现一种O- 连接的糖蛋白与CTxB结合是重要的，因为：(1)它改变了我们对CTxB的基本理解 霍乱中毒的机制，(2)它有可能提供对以下特征的关键洞察 区分不同的内吞途径，以及(3)在解释荧光时应谨慎。 显示脂筏的显微镜实验，因为这些实验依赖于CTxB结合的假设 GM1a独家提供。此外，光交联唾液酸的成功应用表明这一点 试剂将在定义正常和病理生理识别事件方面得到广泛应用。

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 of 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 CTxB 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 critical insight into the features that distinguish different endocytic pathways, and (3) it urges caution in 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.