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Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2016-09-27 , DOI: 10.1021/jacs.6b08161 Chengli Zong , Rongrong Huang , Eduard Condac , Yulun Chiu , Wenyuan Xiao , Xiuru Li , Weigang Lu , Mayumi Ishihara , Shuo Wang , Annapoorani Ramiah , Morgan Stickney , Parastoo Azadi , I Jonathan Amster , Kelley W Moremen , Lianchun Wang , Joshua S Sharp , Geert-Jan Boons 1
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2016-09-27 , DOI: 10.1021/jacs.6b08161 Chengli Zong , Rongrong Huang , Eduard Condac , Yulun Chiu , Wenyuan Xiao , Xiuru Li , Weigang Lu , Mayumi Ishihara , Shuo Wang , Annapoorani Ramiah , Morgan Stickney , Parastoo Azadi , I Jonathan Amster , Kelley W Moremen , Lianchun Wang , Joshua S Sharp , Geert-Jan Boons 1
Affiliation
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An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography-high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure-activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.
中文翻译：
确定 Robo1 硫酸乙酰肝素配体需求的综合方法
描述了一种综合方法，用于确定硫酸乙酰肝素 (HS) 结合蛋白的配体要求，该工作流程基于以下工作流程，其中通过部分酶促降解天然 HS 产生 HS 八糖，然后进行尺寸排阻纯化，使用固定化 HS 结合蛋白进行亲和富集兴趣，通过亲水相互作用色谱-高分辨率质谱平台确定分离化合物的推定结构，以及用于结构活性关系研究的明确的 HS 寡糖的化学合成。该方法用于确定人类 Roundabout 受体 1 (Robo1) 的配体需求，该受体参与许多发育过程。对起始八糖混合物和 Robo1 结合部分的质谱分析表明，Robo1 偏爱一组特定的结构。通过顺序全甲基化、脱硫和全十三十二乙酰化进行进一步分析，然后通过 MS/MS 进行在线分离和结构分析。可以从数据中推断出四糖的序列，并且通过结合组成和序列数据，可以提出假定的八糖配体（GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S）。采用模块化合成方法来制备目标化合物，并通过表面等离振子共振 (SPR) 结合研究证实其是 Robo1 的高亲和力配体。对多种四糖的进一步研究证实，C-6 处的硫酸酯对于结合至关重要，而 C-2 处的此类功能则大大减少了结合。高亲和力配体能够逆转 Slit2-Robo1 信号传导诱导的内皮细胞迁移减少。
更新日期：2016-09-27
中文翻译：
确定 Robo1 硫酸乙酰肝素配体需求的综合方法
描述了一种综合方法，用于确定硫酸乙酰肝素 (HS) 结合蛋白的配体要求，该工作流程基于以下工作流程，其中通过部分酶促降解天然 HS 产生 HS 八糖，然后进行尺寸排阻纯化，使用固定化 HS 结合蛋白进行亲和富集兴趣，通过亲水相互作用色谱-高分辨率质谱平台确定分离化合物的推定结构，以及用于结构活性关系研究的明确的 HS 寡糖的化学合成。该方法用于确定人类 Roundabout 受体 1 (Robo1) 的配体需求，该受体参与许多发育过程。对起始八糖混合物和 Robo1 结合部分的质谱分析表明，Robo1 偏爱一组特定的结构。通过顺序全甲基化、脱硫和全十三十二乙酰化进行进一步分析，然后通过 MS/MS 进行在线分离和结构分析。可以从数据中推断出四糖的序列，并且通过结合组成和序列数据，可以提出假定的八糖配体（GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S）。采用模块化合成方法来制备目标化合物，并通过表面等离振子共振 (SPR) 结合研究证实其是 Robo1 的高亲和力配体。对多种四糖的进一步研究证实，C-6 处的硫酸酯对于结合至关重要，而 C-2 处的此类功能则大大减少了结合。高亲和力配体能够逆转 Slit2-Robo1 信号传导诱导的内皮细胞迁移减少。
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