Study of sialoside function using photocrosslinking sialic acid

利用光交联唾液酸研究唾液酸苷功能

• 批准号: 8988577
• 负责人: Jennifer J Kohler
• 金额: $ 36.57万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8988577
• 关键词: Affect; Affinity; Americas; Bacteria; Basic Science; Binding; Biological Assay; CD44 gene; Cell Line; Cells; Chloride Ion; Cholera; Cholera Toxin; Cholera Toxin Protomer B; Complex; Cultured Cells; Cyclic AMP; Data; Diarrhea; Diazomethane; Disease; Dominant-Negative Mutation; Endocytosis; Environment; Epithelial Cells; Event; Fluorescence Microscopy; Gangliosides; Gene Silencing; Glycoconjugates; Glycolipids; Glycoproteins; Grant; Haiti; Health; Human; ITGB4 gene; Immunoblotting; Infection; Intestines; Intoxication; Invaded; Ions; Label; Letters; Life; Link; Mass Spectrum Analysis; Measures; Mediating; Membrane Glycoproteins; Membrane Microdomains; Metabolic; Methods; Molecular; Morbidity - disease rate; Mucin 1 protein; Mucins; Oligosaccharides; Pathway interactions; Pattern; Play; Polysaccharides; Production; Protein Glycosylation; Proteins; Public Health; Reagent; Research; Role; Route; Sampling; Sialic Acids; Structure; Study models; Surface; Techniques; Technology; Testing; Tissues; Toxin; UV induced; Validation; Vibrio cholerae; World Health Organization; cell type; choleragen receptor; crosslink; fight against; genetic manipulation; glycosylation; immortalized cell; insight; instrumentation; novel; pathogen; pathogenic bacteria; receptor; research study; sugar; ultraviolet irradiation

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.

描述（由申请人提供）：唾液酸是许多糖蛋白和糖脂上发现的末端封端糖。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发挥着关键作用。特别是，唾液酸苷常常被病原菌产生的毒素识别。毒素-唾液酸苷结合是宿主细胞入侵和中毒的第一步。尽管唾液酸化分子具有许多重要作用，但由于识别事件的短暂性和低亲和力，识别它们的结合伴侣很困难。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中。将可渗透细胞的二氮丙啶修饰的唾液酸前体添加到培养的细胞中，细胞代谢该分子，引入光交联唾液酸代替正常唾液酸。紫外线诱导的二氮丙啶活化导致唾液酸化分子和结合伴侣之间发生共价交联；通过免疫印迹和/或质谱分析共价复合物以鉴定组分。使用这项技术，我们发现霍乱毒素提交 B (CTxB) 与肠上皮细胞表面显示的 O-连接糖蛋白交联，并且 不针对神经节苷脂 GM1a，其可接受的受体。此外，我们观察到 O 连接糖蛋白是肠上皮细胞系中主要的 CTxB 结合伴侣。功能分析表明，O-连接糖蛋白介导霍乱毒素 (CTx) 对宿主细胞的影响。最后，初步数据表明 CD44 是 CTxB 结合糖蛋白的有力候选者。在即将到来的资助期内，我们将定义 CTxB 与肠上皮细胞系结合的蛋白质和聚糖决定因素，并确定新型 CTx 结合配偶体的定位模式（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。额外的 CTxB 结合配偶体的存在提供了一种协调有关 CTxB 进入宿主细胞内吞机制的现有数据的方法。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来获得对宿主与病原体相互作用的新的和意想不到的见解。 O-连接糖蛋白与 CTxB 结合的发现具有重要意义，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批判性见解，（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 假设 CTxB 专门结合 GM1a。此外，光交联唾液酸的成功应用表明该试剂将得到广泛的应用 定义正常和病理生理识别事件。