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: 8695999
• 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/8695999
• 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; 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; public health relevance; receptor; research study; sugar; ultraviolet irradiation

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.

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。特别是，委员会认为， 唾液酸糖苷经常被病原菌产生的毒素识别。毒素-唾液酸糖苷结合是 在宿主细胞的侵入和中毒中的步骤。尽管唾液酸化分子具有许多重要作用， 由于识别事件的短暂性和低亲和力，识别它们的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来引入二氮丙啶 光交联剂转化为细胞唾液酸残基。一种细胞可渗透的、二氮丙啶修饰的唾液酸前体， 添加到代谢该分子的培养细胞中，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的二氮丙啶之间的共价交联。 分子和结合配偶体;通过免疫印迹和/或质谱分析共价复合物， 标识组件。利用这种技术，我们发现霍乱毒素提交B（CTx B）交联到O- 在肠上皮细胞表面显示的连接糖蛋白，而不是神经节苷脂GM 1a， 接受受体此外，我们观察到O-连接的糖蛋白是CTxB的主要结合伴侣。 肠上皮细胞系。功能测定显示O-连接糖蛋白介导了 霍乱毒素（CTx）对宿主细胞的作用。最后，初步数据确定CD 44是CTxB的强有力候选者。 结合糖蛋白在即将到来的授权期间，我们将定义蛋白质和聚糖决定因素 CTxB与肠上皮细胞系的结合，并确定新CTxB的定位模式。 结合伴侣（Aim 1）。在目标2中，我们将通过以下方法测试CTxB结合糖蛋白的功能相关性： 测量其表达如何影响CTx内化、CTx诱导的cAMP产生和CTx诱导的cAMP生成。 氯离子分泌额外的结合CTxB结合伴侣的存在提供了一种调和 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 研究CTxB使用的内吞途径，并确定内吞机制是否依赖于 CTxB结合配偶体的身份。这里描述的实验利用了我们的光交联唾液酸 技术，以获得新的和意想不到的见解宿主-病原体相互作用。发现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）它有可能提供关键的洞察特征， 区分不同的内吞途径，和（3）它敦促谨慎解释荧光 由于这些实验依赖于CTxB结合的假设， GM 1a专用此外，光交联唾液酸的成功应用表明， 试剂将在定义正常和病理生理识别事件中找到广泛的应用。