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: 7369229
• 负责人: Tung-Tien Sun
• 金额: $ 4.05万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369229
• 关键词: CHAR; GLYCANS; MOUSE; BOVINE; UROPLAKINS

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.

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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。两种与结构相关的糖蛋白，即尿粉蛋白（UP）IA和IB与UP II和III相互作用，形成16 nm颗粒，形成了六角形的16 nm颗粒，形成了2D晶体，这些晶体几乎覆盖了哺乳动物膀胱上皮的整个顶端表面。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。在这项研究中，已使用快速敏感的MS策略来确定聚糖和鉴定占据的糖基化位点。结果应有助于更好地理解尿路感染机制，并改善其诊断和治疗。 鼠和牛UPS IA和IB通过SDS-PAGE纯化。鼠24k-da UP IA和29k-da UP IB之间的出色分辨率与牛27-KDA UP IA和28K-DA UP IB之间的分辨率差形成对比。切除感兴趣的条带，并用PNGase F脱糖基化，并对提取的聚糖进行苄苄苄化化。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反射IV iv基质辅助解吸/离子化飞行时间（MALDI-TOF）质谱仪对肽和苄苄化寡糖进行表征，并使用QStar Pulsar i Qudrupole-Quadrupole-ostor-ottrol-ottrol-ottrol-ottrol-ottrole-ottrol-tof ）。通过MS/MS对碳水化合物和感兴趣的肽进行了测序。使用糖苷酶和蛋白酶内 - 凝胶消化，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UPS IA和IB中IB的单个糖基化位点的糖基型异质性的模式。鼠样品。发现牛UP IA/IB在UP IB的ASN131和UP IA的ASN170上含有一系列高甘露糖型的n型型N型聚糖。在Murine UP IA中，ASN169的N连接的聚糖人群被确定为一系列高甘露糖糖，而Mirine UP IB被发现包含一系列多抗菌综合体，高甘露糖和ASN131的混合N-链接Glycans 。这项研究中产生的苄氨基化聚糖池允许对聚糖成分的相对定量。使用MALDI-TOF MS进行了有关UP IA和IB中糖型分布的调查。发现鼠的主要糖基型是高甘露糖的高糖，而鼠UP IB的聚糖主要是复杂的聚糖（超过85％的糖型），以及少量的高甘露糖和高含量的高含量和混合聚糖。来自牛/Ib的冰甲基化聚糖的MALDI MS谱图表明，根据糖型的身份和分布，观察到的天然聚糖的观察到的天然聚糖谱与苄苄苄化后获得的结果非常吻合。这些结果与UPS IA和IB的电泳迁移率变化是一致的。我们的结果提供了一种生化解释，以观察到1型肌病大肠杆菌细菌与鼠泌尿紫红色1a的结合，但与密切相关的鼠泌尿紫红色IB结合。