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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。两种结构上相关的糖蛋白，尿斑蛋白（UP）Ia和Ib，与UP II和III相互作用，形成16 nm的颗粒，六角形包装，形成2D晶体，几乎覆盖整个哺乳动物膀胱上皮的顶面。已经提出，UPs的糖基化模式决定了引起尿路感染的细菌的结合效率。在本研究中采用了快速灵敏的MS策略，用于聚糖的结构测定和已占据糖基化位点的鉴定。结果有助于更好地了解尿路感染的机制，并提高其诊断和治疗。 通过SDS-PAGE纯化鼠和牛UPs Ia和Ib。鼠24 k-Da UP Ia和29 k-Da UP Ib之间的优异分离度与牛27-kDa UP Ia和28 k-Da UP Ib之间的差分离度形成对比。切下感兴趣的条带，并用PNGase F在凝胶中进行去糖基化，并对提取的聚糖进行全甲基化。在释放N-聚糖后，在凝胶中进行蛋白质的胰蛋白酶消化。使用Bruker Reflex IV基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱仪表征肽和全甲基化寡糖，并使用QSTAR Pulsar i四极杆正交TOF质谱仪（QoTOF MS）进一步分析。感兴趣的碳水化合物和肽通过MS/MS进行测序。使用糖苷酶和蛋白酶的凝胶内消化，MALDI MS和ESI MS/MS的组合，我们验证了蛋白质的氨基酸序列，并确定了来自牛和小鼠样品的UPs Ia和Ib中单个糖基化位点的糖型异质性模式。发现牛UP Ia/Ib在UP Ib的Asn 131和UP Ia的Asn 170处含有一系列高甘露糖型N-连接聚糖。在鼠UP Ia中Asn 169处的N-连接聚糖群被确定为一系列高甘露糖聚糖，而发现鼠UP Ib在Asn 131处含有一系列多触角复合物、高甘露糖和杂合N-连接聚糖。本研究中生成的全甲基化聚糖池允许对聚糖组分进行相对定量。利用MALDI-TOF MS对UP Ia和Ib中的糖型分布进行了调查。发现小鼠UP Ia的主要糖型是高甘露糖聚糖，而发现小鼠UP Ib的主要糖型是复杂聚糖（超过85%的糖型），沿着少量的高甘露糖和杂合聚糖。来自牛UP Ia/Ib的天然和全甲基化聚糖的MALDI MS谱表明，在糖型的身份和分布方面，观察到的天然聚糖谱与全甲基化后获得的结果非常一致。这些结果与UPs Ia和Ib经内切H和F糖苷酶处理后的电泳迁移率变化一致。我们的研究结果提供了一个生物化学解释的观察，1型菌毛，尿路致病性大肠杆菌。大肠杆菌与鼠尿斑蛋白1a结合，但不与密切相关的鼠尿斑蛋白Ib结合。

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. Two structurally related glycoproteins, the uroplakins (UP) Ia and Ib, interact with UP II and III, to form 16 nm particles hexagonally packed to form 2D crystals that cover almost the entire apical surface of mammalian bladder epithelium. It has been proposed that glycosylation patterns of the UPs determine the binding efficiency of bacteria that cause urinary tract infections. A rapid and sensitive MS strategy has been utilized in this study for the structural determination of the glycans and the identification of occupied glycosylation sites. The results should contribute to a better understanding of the mechanism of urinary tract infection and to improvements in its diagnosis and treatment. Murine and bovine UPs Ia and Ib were purified by SDS-PAGE. The excellent resolution between murine 24k-Da UP Ia and 29k-Da UP Ib contrasted with the poor resolution between bovine 27-kDa UP Ia and 28k-Da UP Ib. Bands of interest were excised and deglycosylated in-gel with PNGase F, and the extracted glycans were subjected to permethylation. Tryptic digestion of the proteins was performed in-gel, after release of the N-glycans. The peptides and the permethylated oligosaccharides were characterized using a Bruker Reflex IV matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, and further analyzed using a QSTAR Pulsar i quadrupole-orthogonal TOF mass spectrometer (QoTOF MS). The carbohydrates and peptides of interest were sequenced by MS/MS. Using a combination of glycosidase and protease in-gel digestion, MALDI MS, and ESI MS/MS, we verified the amino acid sequences of the proteins and determined the pattern of glycoform heterogeneity at the single glycosylation site in UPs Ia and Ib from bovine and murine samples. Bovine UP Ia/Ib were found to contain a series of high mannose type N-linked glycans at Asn131 of UP Ib and Asn170 of UP Ia. The N-linked glycan population at Asn169 in murine UP Ia was determined to be a series of high mannose glycans, while murine UP Ib was found to contain a series of multiple-antennary complex, high mannose, and hybrid N-linked glycans at Asn131. The permethylated glycan pool generated in this study allowed relative quantification of glycan constituents. The survey on the distribution of glycoforms in UP Ia and Ib was carried out using MALDI-TOF MS. The main glycoforms of murine UP Ia were found to be the high mannose glycans, while those of murine UP Ib were found to be mainly complex glycans (more than 85% of the glycoforms), along with small amounts of high mannose and hybrid glycans. MALDI MS profiles of the native and permethylated glycans from bovine UP Ia/Ib suggested that the observed profile of native glycans is in good agreement with the results obtained after permethylation, in terms of both the identities and distributions of glycoforms. These results are consistent with the electrophoretic mobility changes of UPs Ia and Ib after they are treated with endo H and F glycosidases. Our results provide a biochemical explanation for the observation that the type 1-fimbriated, uropathogenic E. coli bacteria bind to murine uroplakin 1a, but not to the closely related murine uroplakin Ib.