GLYCAN PHENOTYPE ANALYSIS OF ORGAN-SPECIFIC HEPARAN SULFATE

器官特异性硫酸乙酰肝素的聚糖表型分析

• 批准号: 8170929
• 负责人: JOSEPH ZAIA
• 金额: $ 2.33万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170929
• 关键词: Acetylglucosamine; Amides; Aorta; Area; Binding; Biological; Biological Process; Blood capillaries; Capillary Electrophoresis; Cattle; Computer Retrieval of Information on Scientific Projects Database; Custom; Databases; Derivation procedure; Digestion; Disaccharides; Electrons; Funding; Glucuronic Acids; Goals; Grant; Heparin Lyase; Heparitin Sulfate; Heterogeneity; Housing; Iduronic Acid; Inorganic Sulfates; Institution; Intestines; Kidney; Knowledge; Ligands; Lung; Lyase; Oligosaccharides; Organ; Phenotype; Plant Resins; Play; Polysaccharides; Positioning Attribute; Protein-Carbohydrate Interaction; Research; Research Personnel; Resources; Role; Running; Sampling; Source; Structure; System; Tissues; United States National Institutes of Health; Unspecified or Sulfate Ion Sulfates; Work; base; capillary; dimer; epimerization; extracellular; liquid chromatography mass spectrometry; macromolecule; mass spectrometer; stable isotope; sulfation

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. Heparan sulfate (HS) is a polyanionic, linear polysaccharide that plays a role in many biological functions by modulating the binding activity of extracellular ligands. It is comprised of alternating glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) units and has the general formula [GlcA¿(1,4)GlcNAc¿(1,4)]n. This structure may be modified by epimerization of GlcA to iduronic acid and by sulfation of this residue at the 2O- position. The structure can then be 3O-, 6O-, or N-sulfated on GlcN. With a total of 48 possible disaccharide structures, HS represents the most highly variable biological macromolecule. Structural analysis of HS chains is complicated due to the inherent heterogeneity of this compound class. The work presented herein extends the knowledge of HS domain structure through the analysis of tissue-derived HS oligosaccharides by LC/MS. Commercially available bovine kidney HS (Sigma) as well as purified bovine HS from aorta, lung, intestine, and kidney was completely depolymerized with heparin lyases. The resultant disaccharides were derivatized with 2-aminoacridone and analyzed by capillary electrophoresis (CE). For oligosaccharide analysis, HS samples were digested to partial completion. The products of each digestion were reductively aminated with custom tetraplex stable isotope tags to facilitate analysis of three sample replicates in one LC/MS run. Samples were injected onto a 250 ¿m x 15 cm capillary column packed in-house with Amide-80 resin (Tosoh) connected online to an Applied Biosystems/MDS Sciex QSTAR mass spectrometer or a Thermo Electron Corporation LTQ Orbitrap mass spectrometer. Glycan compositions were assigned using a Microsoft Excel based database and abundances calculated based on integrated peak area in the MS mode. The goal of this work was to determine how HS structures vary as a function of organ derivation. From CE analysis of exhaustive lyase digests, it was found that HS from different bovine organs shows distinct differences in disaccharide composition. Specifically, the degree to which the HS chains are sulfated or acetylated differs between organ types. While important, disaccharide analysis only begins to provide a structural understanding of the role of HS in protein-carbohydrate interactions. Larger oligosaccharides must be generated in order to investigate intact HS domain structures, which consist of N-sulfated and N-acetylated regions. In order to better understand this domain organization and to optimize conditions for analysis, multiple lyase digestions of bovine kidney HS were produced (including 20, 40, 60, 80, and 100% digestion completion). Oligosaccharides ranging from dimers to octamers could be simultaneously analyzed by the amide LC/MS system. Based on this analysis, an appropriate partial digest will be selected in order to analyze oligosaccharides derived from the various available bovine organ HS samples. The analysis will provide an HS oligosaccharide profile that is a function of organ type.

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。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 中心，不一定是研究者的机构。 硫酸乙酰肝素 (HS) 是一种聚阴离子、线性多糖，通过调节细胞外配体的结合活性在许多生物功能中发挥作用，它由交替的葡萄糖醛酸 (GlcA) 和 N-乙酰氨基葡萄糖 (GlcNAc) 单元组成，具有通用性。式[GlcA¿ (1,4)GlcNAc¿ (1,4)]n 可以通过 GlcA 差向异构化为艾杜糖醛酸并通过该残基在 2O-位置的硫酸化来修饰，然后该结构可以在 GlcN 上进行 3O-、6O-或 N-硫酸化。 HS 共有 48 种可能的二糖结构，是变异性最高的生物大分子。由于该化合物固有的异质性，HS 链的结构分析非常复杂。这里介绍的工作通过 LC/MS 分析组织来源的 HS 寡糖扩展了 HS 结构域结构的知识。 使用肝素裂解酶将市售的牛肾 HS (Sigma) 以及来自主动脉、肺、肠和肾的纯化牛 HS 完全解聚，用 2-氨基吖啶酮衍生化所得二糖，并通过毛细管电泳 (CE) 进行寡糖分析。分析中，HS 样品消化至部分完成，每次消化的产物均用定制的四重稳定剂还原胺化。同位素标签，便于在一次 LC/MS 运行中分析三个样品重复样品被注射到 250 ¿内部填充有 Amide-80 树脂 (Tosoh) 的 m x 15 cm 毛细管柱，在线连接到 Applied Biosystems/MDS Sciex QSTAR 质谱仪或 Thermo Electron Corporation LTQ Orbitrap 质谱仪，并使用基于 Microsoft Excel 的数据库分配聚糖成分。丰度基于 MS 计算模式中的积分峰面积。 这项工作的目标是确定 HS 结构如何随着器官来源的变化而变化。通过对详尽裂解酶消化物的 CE 分析，发现来自不同牛器官的 HS 在二糖组成方面表现出明显的差异。 HS 链的硫酸化或乙酰化程度因器官类型而异，虽然很重要，但二糖分析只是开始提供对 HS 在蛋白质-碳水化合物相互作用中的作用的结构理解。必须生成寡糖才能获得完整的 HS 结构域结构，该结构由 N-硫酸化和 N-乙酰化区域组成。为了更好地了解该结构域组织并优化分析条件，对牛肾 HS 进行了多重裂解酶消化（包括）。 20%、40%、60%、80% 和 100% 消化完成）可以通过酰胺 LC/MS 同时分析从二聚体到八聚体的寡糖。基于该分析，将选择适当的部分消化以分析源自各种可用牛器官 HS 样品的寡糖。该分析将提供作为器官类型的函数的 HS 寡糖谱。