FT IR SPECTROMETRIC ANALYSIS OF CARBOHYDRATE COMPOSITION

碳水化合物成分的 FT IR 光谱分析

• 批准号: 6653540
• 负责人: ROBERTA K MERKLE
• 金额: $ 28.8万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6653540
• 关键词: biological products; biomedical resource; carbohydrates; nuclear magnetic resonance spectroscopy; technology /technique

This project studies the interaction between galectin-1, a soluble galactose-binding lectin, and a variety of N-acetyllactosamine-containing oligosaccharides. A modified form of Chinese hamster ovary (CHO) cell galectin-1 has been generated and expressed in large quantities in E. coli. The initial goal was to attempt to assign all of the 1H resonances of the protein and to obtain binding constants for the ligands from a study of chemical shift and line width changes. Inter-proton distance information obtained from nuclear Overhauser effect (NOE) measurements will be used to try to identify contact areas between galectin-1 and its ligand. Another goal of this project is to generate isotope-enriched forms of the lectin for detailed NMR studies. To date, approximately 10 mg of [15N]-galectin-1 and 10 mg of [13C,15N]-doubly labeled galectin-1 have been isolated, and 1H-15N correlated spectra have been obtained to test the feasibility of using this sample to assign the 1H and 15N resonances. With the successful preparation of [13C,15N]-doubly isotope-labeled galectin-1, work began on optimizing 3D triple-resonance experiments for proton, carbon, and nitrogen assignments. Due to the high molecular weight of the homodimer, versions of the standard experiments (e.g., HNCO, HNCA, CBCACONH, HNCACB) were adopted that minimize signal loss due to relaxation. In addition, the buffer conditions were varied in order to promote solubilization and reduce aggregation. An alternate form of the galectin-1 protein has also been obtained by generating a mutant galectin-1 protein that at high concentrations forms an active monomer. Since NMR protein structure studies require protein concentrations in the millimolar range, we have generated mutations in order to form a monomer that is stable at high concentrations. The isolated monomer lectin was found to be both inactive and abnormally folded. The lectin did not agglutinate erythrocytes, it bound very poorly to immobilized asialofetuin, and it did not compete with agglutination by the dimeric C2S lectin. In addition, the protein displayed an altered circular dichroism spectrum indicating that the protein was assuming an alternate folding pattern. These data indicate that the nature of the truncation mutations that were introduced into the molecule were too severe to maintain folding of the binding domain of the monomeric lectin. Polylactosamine ligands for the lectin have been generated, including the complete series of lacto-N-neotetraose and lactosamine oligomers with up to eight sugars. NMR techniques applied to the galectin-oligosaccharide mixtures include line width analysis, magnetization transfer, and transferred NOE studies. For the ligands analyzed so far, the data are compatible with predominant binding to the terminal lactosamine unit. When no terminal galactosyl is available, the oligosaccharide slides into the binding groove such that a galactosyl residue can occupy the main binding site. It is not yet known how far into the binding groove longer oligosaccharides may extend. Differentiation between identical residues and determination of the location on the polylactosamine chain where interactions are occurring requires specific labeling of targeted residues. This work was begun by synthesizing a tetra-N-acetyllactosamine containing a terminal [1-13C]-galactosyl residue. The next synthetic step is to extend the length of the polylactosamine structure and generate a series of oligosaccharides, each with a uniquely labeled galactosyl residue at each position within the polylactosamine chain. We can then confirm whether the galectin always prefers the terminal residues when presented with a longer repeating poly-N-acetyllactosamine. A paper has been submitted for publication.

该项目研究半乳糖凝集素-1（一种可溶性半乳糖凝集素）之间的相互作用 半乳糖结合凝集素和多种 含N-乙酰基乳糖胺的寡糖。 的修改形式 中国仓鼠卵巢（CHO）细胞半乳糖凝集素-1已生成并 在大肠杆菌中大量表达。 最初的目标是 尝试分配蛋白质的所有 1H 共振并 从化学研究中获得配体的结合常数 移位和线宽变化。 质子间距离信息 从核奥弗豪瑟效应（NOE）测量中获得的结果将是 用于尝试识别 galectin-1 与其 配体。 该项目的另一个目标是产生富含同位素的 用于详细 NMR 研究的凝集素形式。 迄今为止，大约 10 mg [15N]-galectin-1 和 10 mg [13C,15N]-双标记 已分离出galectin-1，并绘制了1H-15N相关谱图 测试使用该样本分配 1H 的可行性 和 15N 共振。 随着筹备的成功 [13C,15N]-双同位素标记半乳糖凝集素-1，开始优化工作 质子、碳和氮的 3D 三重共振实验 作业。 由于同二聚体的高分子量， 标准实验的版本（例如，HNCO、HNCA、CBCACONH、 采用 HNCACB）可最大限度地减少因弛豫而造成的信号损失。 在 此外，改变缓冲条件以促进 增溶并减少聚集。 另一种形式是 半乳糖凝集素-1蛋白也已通过产生突变体获得 半乳糖凝集素-1 (galectin-1) 蛋白在高浓度时形成活性物质 单体。 由于 NMR 蛋白质结构研究需要蛋白质 浓度在毫摩尔范围内，我们已经产生了突变 以形成在高浓度下稳定的单体。 这 发现分离的单体凝集素既无活性又异常 折叠。 凝集素不凝集红细胞，它结合非常 去唾液酸胎球蛋白的固定化效果很差，并且它不与 由二聚体 C2S 凝集素引起的凝集。 此外，蛋白质 显示改变的圆二色光谱表明 蛋白质呈现出另一种折叠模式。 这些数据 表明截断突变的性质 引入分子太严重而无法维持折叠 单体凝集素的结合域。 聚乳糖胺配体 已生成凝集素，包括完整的系列 乳糖-N-新四糖和乳糖胺低聚物，最多含有八种糖。 核磁共振技术应用于半乳糖低聚糖混合物 包括线宽分析、磁化转移和转移 NOE 研究。 对于迄今为止分析的配体，数据是兼容的 主要与末端乳糖胺单元结合。 当没有 末端半乳糖基可用，寡糖滑入 结合槽使得半乳糖基残基可以占据主要 结合位点。 目前尚不清楚进入绑定槽有多深 更长的寡糖可以延伸。 相同之间的区别 聚乳糖胺上的残留物和位置的测定 发生相互作用的链需要特定的标记 目标残留物。 这项工作是通过合成 含有末端[1-13C]-半乳糖基的四-N-乙酰基乳糖胺 残留物。 下一步的合成步骤是延长 聚乳糖胺结构并生成一系列寡糖， 每个在每个位置都有一个独特标记的半乳糖基残基 在聚乳糖胺链内。 然后我们可以确认是否 当与半乳糖凝集素一起出现时，半乳糖凝集素总是更喜欢末端残基 更长重复的聚-N-乙酰基乳糖胺。 论文已提交 供出版。