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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。两种结构相关的糖蛋白，尿斑蛋白 (UP) Ia 和 Ib，与 UP II 和 III 相互作用，形成六边形堆积的 16 nm 颗粒，形成几乎覆盖哺乳动物膀胱上皮整个顶面的 2D 晶体。有人提出，UP 的糖基化模式决定了引起尿路感染的细菌的结合效率。本研究采用快速、灵敏的 MS 策略来确定聚糖的结构并鉴定占用的糖基化位点。研究结果应有助于更好地了解尿路感染的机制并改进其诊断和治疗。 鼠和牛UPs Ia和Ib通过SDS-PAGE纯化。小鼠 24k-Da UP Ia 和 29k-Da UP Ib 之间具有出色的分辨率，而牛 27-kDa UP Ia 和 28k-Da UP Ib 之间的分辨率较差。切除感兴趣的条带并用 PNGase F 在凝胶中去糖基化，并对提取的聚糖进行全甲基化。 N-聚糖释放后，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) 质谱仪对肽和全甲基化寡糖进行表征，并使用 QSTAR Pulsar i 四极杆正交 TOF 质谱仪 (QoTOF MS) 进行进一步分析。通过 MS/MS 对感兴趣的碳水化合物和肽进行测序。结合使用糖苷酶和蛋白酶凝胶内消化、MALDI MS 和 ESI MS/MS，我们验证了蛋白质的氨基酸序列，并确定了牛和鼠样品中 UP Ia 和 Ib 中单个糖基化位点的糖型异质性模式。牛 UP Ia/Ib 被发现在 UP Ib 的 Asn131 和 UP Ia 的 Asn170 处含有一系列高甘露糖型 N 连接聚糖。小鼠 UP Ia 中 Asn169 处的 N 连接聚糖群体被确定为一系列高甘露糖聚糖，而小鼠 UP Ib 被发现在 Asn131 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库可以对聚糖成分进行相对定量。使用MALDI-TOF MS对UP Ia和Ib中的糖型分布进行了调查。发现鼠 UP Ia 的主要糖型是高甘露糖聚糖，而鼠 UP Ib 的主要糖型主要是复合聚糖（超过 85% 的糖型），以及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 图谱表明，就糖型的特性和分布而言，观察到的天然聚糖图谱与全甲基化后获得的结果非常一致。这些结果与 UP Ia 和 Ib 用内切 H 和 F 糖苷酶处理后的电泳迁移率变化一致。我们的结果为 1 型伞状尿路致病性大肠杆菌与小鼠尿斑蛋白 1a 结合，但不与密切相关的小鼠尿斑蛋白 Ib 结合的观察结果提供了生化解释。