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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 小鼠尿路凝集素(UP)Ia是尿路上皮尖表面的一种主要糖蛋白，已被证明可以作为1型菌毛大肠杆菌FimH凝集素粘附素的受体，但这种天然受体的糖链结构尚不清楚。我们用一种灵敏的方法，结合凝胶内糖苷酶和蛋白酶的消化，释放的糖链的全甲基化和质谱学，首次阐明了小鼠UPIA受体及其非结合同系物UPIb的天然糖形结构，并确定了糖基化位点的占有率。UPIA呈现高水平的末端暴露的甘露糖残基(位于Man6GlcNAc2到Man9GlcNAc2)，能够与FimH特异地相互作用。我们已经证明这一特性不仅在小鼠的尿板中是保守的，而且在牛中也是如此，更重要的是，在人类的UPIA中也是如此，从而建立了UPIA是人类和其他哺乳动物中甘露糖特异的FimH变体的主要尿路上皮受体的概念。相反，我们的结果表明，大多数末端暴露的小鼠UPIb糖链是非甘露糖残基，从而解释了FimH未能与该UPIb结合的原因。另一方面，在牛身上，复杂的碳水化合物仅占UPIB N-连接多糖的20%左右。人类UPIA只含有高甘露糖多糖，而人类UPIb只含有复杂的多聚糖。UPIA和UPIb蛋白是Tetraspanin家族中两个密切相关的成员，它们的碳水化合物加工方式截然不同，可能反映了它们在折叠和掩蔽方面的差异，这是因为它们分别与相关蛋白UPII和UPIIa相互作用。这项研究的结果揭示了尿路感染的分子发病机制，并可能有助于设计类似糖的抑制剂来预防和治疗这种疾病。

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.