CONFORMATIONS & DYNAMICS OF CARBOHYDRATES, FREE IN SOLUTION & BOUND TO LECTINS

构象

• 批准号: 6122184
• 负责人: KELLEY W. MOREMEN
• 金额: $ 14.48万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6122184
• 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.

本项目研究了半乳糖凝集素-1与可溶性 半乳糖结合凝集素，以及各种 含N-乙酰乳糖胺的寡糖。一种修改形式的 中国仓鼠卵巢(CHO)细胞Galectin-1已获得成功 在大肠杆菌中大量表达。最初的目标是 尝试指定蛋白质的所有1H共振峰，并 从化学研究中获得配体的结合常数 移位和线宽发生变化。质子间距离信息 从核Overhauser效应(NOE)测量获得的将是 用于识别Galectin-1与其之间的接触区域 莱兰德。该项目的另一个目标是生产富含同位素的 用于详细的核磁共振研究的凝集素的形式。到目前为止，大约 10毫克[15N]-Galectin-1和10 mg[13C，15N]-双标记物 半乳糖凝集素-1已被分离，并获得了~1H-15N相关谱 以测试使用此样本分配1H的可行性 和15N共振。随着成功地准备了 [13C，15N]-双同位素标记Galectin-1，优化工作开始 质子、碳和氮的三维三重共振实验 任务。由于同源二聚体的高分子量， 标准实验的版本(例如，HNCO，HNCA，CBCACONH， HNCACB)，将松弛引起的信号损失降至最低。在……里面 此外，还改变了缓冲条件，以促进 增溶和减少聚集。的另一种形式 Galectin-1蛋白也是通过产生一个突变体获得的 Galectin-1蛋白在高浓度时形成活性 单体。由于核磁共振蛋白质结构研究需要蛋白质 浓度在毫摩尔范围内，我们产生了突变 以形成在高浓度下稳定的单体。这个 分离的单体凝集素被发现既不活跃又异常 折好了。凝集素不能凝集红细胞，它与红细胞的结合非常紧密。 对去唾液酸酶的固定化很差，它没有与之竞争 二聚体C2S凝集素的凝集作用。此外，这种蛋白质 显示了一个改变的圆二色谱，表明 蛋白质呈现一种交替的折叠模式。这些数据 表明截断突变的性质是 被引入到分子中，太严重了，不能保持折叠 单体凝集素的结合域。聚乳糖胺配体 已经产生了凝集素，包括完整的系列 乳糖-N-新四糖和乳糖胺低聚物，最多含八种糖。 核磁共振技术在半乳糖-低聚糖混合物中的应用 包括线宽分析、磁化转移和转移 没有学习。对于到目前为止所分析的配体，数据是兼容的 主要结合在末端的乳糖胺单元上。当不是时 末端半乳糖可用，低聚糖滑入 结合槽，使得半乳糖残基可以占据主要 结合部位。目前还不知道结合槽深入到什么程度 较长的寡糖可能会延长。完全相同之间的区别 聚乳糖胺的残留及其位置测定 发生交互的链需要特定的标签 靶向残留物。这项工作是从合成一个 含有末端[1-13C]-半乳糖基的四-N-乙酰乳糖胺 残留物。下一个合成步骤是延长 聚乳糖胺结构并产生一系列低聚糖， 每个在每个位置都有唯一标记的半乳糖残基 在多聚乳糖胺链中。然后我们可以确认是否 半乳糖凝集素总是偏爱末端残基 重复较长的多-N-乙酰乳糖胺。已经提交了一篇论文 以供出版。