CHAR OF GLYCANS FROM MOUSE & BOVINE UROPLAKINS IA & IB BY MASS SPECTROMETRY

来自小鼠的聚糖的字符

• 批准号: 7722979
• 负责人: Tung-Tien Sun
• 金额: $ 2.59万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7722979
• 关键词: Apical; Bacterial Adhesins; Binding; Bos taurus; Carbohydrates; Cattle; Complex; Computer Retrieval of Information on Scientific Projects Database; Digestion; Disease; Endopeptidases; Escherichia coli; Failure; Family; Funding; Gel; Glycoproteins; Glycoside Hydrolases; Grant; Hand; Homologous Gene; Human; Institution; Lectin; Light; Link; Mammals; Mannose; Masks; Mass Spectrum Analysis; Molecular; Mus; Organism; Pathogenesis; Peptide Hydrolases; Polysaccharides; Process; Property; Proteins; Research; Research Personnel; Resources; Site; Source; Structure; Surface; Time; United States National Institutes of Health; Urinary tract infection; Variant; concept; design; glycosylation; human PHEMX protein; inhibitor/antagonist; mannosyl(6)-N-acetyl(2)glucose; mannosyl(9)-N-acetylglucosamine2; member; mimetics; prevent; receptor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Although it has been shown that mouse uroplakin (UP) Ia, a major glycoprotein of urothelial apical surface, can serve as the receptor for the FimH lectin adhesin of type 1-fimbriated E. coli, the organism which causes a great majority of urinary tract infections (UTIs), the glycan structure of this native receptor was unknown. Using a sensitive approach that combines in-gel glycosidase and protease digestions, permethylation of released glycans and mass spectrometry, we have elucidated, for the first time, the native glycoform structures of the mouse UPIa receptor and those of its non-binding homolog, UPIb, and have determined the glycosylation site occupancy. UPIa presents a high level of terminally exposed mannose residues (located on Man6GlcNAc2 to Man9GlcNAc2) that are capable of specifically interacting with FimH. We have shown that this property is conserved, not only in the mouse uroplakins, but also in cattle and, even more importantly, in human UPIa, thus establishing the concept that UPIa is a major urothelial receptor in humans and other mammals for the mannose-specific FimH variant. In contrast, our results indicate that most terminally exposed glycans of mouse UPIb are non-mannose residues, thus explaining the failure of FimH to bind to this UPIb. In cattle, on the other hand, complex carbohydrates constituted only about 20% of the UPIb N-linked glycans. Human UPIa contained exclusively high-mannose glycans and human UPIb contained only complex glycans. The drastically different carbohydrate processing of the UPIa and UPIb proteins, two closely related members of the tetraspanin family, may reflect differences in their folding and masking due to their interactions with their associated proteins, UPII and UPIIIa, respectively. Results from this study shed light on the molecular pathogenesis of UTIs and may aid in the design of glyco-mimetic inhibitors for preventing and treating this disease.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 尽管已经表明，小鼠尿斑蛋白 (UP) Ia（尿路上皮顶端表面的一种主要糖蛋白）可以作为 1 型菌毛大肠杆菌（引起绝大多数尿路感染 (UTI) 的有机体）的 FimH 凝集素粘附素的受体，但这种天然受体的聚糖结构尚不清楚。使用结合凝胶内糖苷酶和蛋白酶消化、释放聚糖的全甲基化和质谱分析的灵敏方法，我们首次阐明了小鼠 UPIa 受体及其非结合同源物 UPIb 的天然糖型结构，并确定了糖基化位点占用。 UPIa 呈现高水平的末端暴露的甘露糖残基（位于 Man6GlcNAc2 至 Man9GlcNAc2 上），能够与 FimH 特异性相互作用。我们已经证明，这种特性不仅在小鼠尿斑蛋白中是保守的，而且在牛中也是保守的，更重要的是在人类 UPIa 中也如此，从而确立了 UPIa 是人类和其他哺乳动物中甘露糖特异性 FimH 变体的主要尿路上皮受体的概念。相反，我们的结果表明，小鼠 UPIb 的大多数末端暴露聚糖是非甘露糖残基，从而解释了 FimH 未能与该 UPIb 结合。另一方面，在牛中，复合碳水化合物仅占 UPIb N 连接聚糖的约 20%。人 UPIa 仅含有高甘露糖聚糖，而人 UPIb 仅含有复合聚糖。 UPIa 和 UPIb 蛋白是四跨膜蛋白家族的两个密切相关的成员，它们的碳水化合物加工过程截然不同，可能反映了它们由于分别与相关蛋白 UPII 和 UPIIIa 相互作用而导致的折叠和掩蔽方面的差异。这项研究的结果揭示了尿路感染的分子发病机制，并可能有助于设计用于预防和治疗这种疾病的糖模拟抑制剂。