Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.
Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.
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DOI：
10.1021/jacs.6b08161
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发表时间：
2016-10-05
影响因子：
15
通讯作者：
Boons GJ
中科院分区：
文献类型：
--
作者：
Zong C;Huang R;Condac E;Chiu Y;Xiao W;Li X;Lu W;Ishihara M;Wang S;Ramiah A;Stickney M;Azadi P;Amster IJ;Moremen KW;Wang L;Sharp JS;Boons GJ
描述了一种集成方法，用于建立硫酸乙酰肝素（HS）结合蛋白的配体要求，该方法基于以下工作流程：通过天然HS的部分酶促降解产生HS八糖，然后进行尺寸排阻纯化，使用固定的HS结合感兴趣蛋白进行亲和富集，通过亲水相互作用色谱-高分辨率质谱平台对分离的化合物进行推定结构测定，和化学合成定义明确的HS寡糖，用于构效关系研究。该方法被用来建立人的Roundabout受体1（Robo 1），这是参与了一些发展过程的配体要求。质谱分析的起始八糖混合物和Robo 1结合的馏分表明，Robo 1具有一组特定的结构的偏好。通过顺序的全甲基化、双甲基化和全三氘代乙酰化进行进一步的分析，然后通过MS/MS进行在线分离和结构分析。可以从数据中推导出四糖的序列，并且通过组合组成和序列数据，可以提出推定的八糖配体（GlA-GlcNS 6S-IdoA-GlcNS-IdoA 2S-GlcNS 6S-IdoA-GlcNAc 6S）。采用模块化合成方法制备目标化合物，通过表面等离子体共振（SPR）的结合研究证实它是Robo 1的高亲和力配体。对许多四糖的进一步研究证实，C-6位的硫酸酯对于结合至关重要，而C-2位的此类官能度大大降低了结合。高亲和力配体能够逆转Slit 2-Robo 1信号转导诱导的内皮细胞迁移减少。
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.
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DOI：
10.1074/jbc.m800688200
发表时间：
2008-06-06
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Fukuhara N;Howitt JA;Hussain SA;Hohenester E
通讯作者：
Hohenester E
DOI：
10.1021/acs.joc.5b02172
发表时间：
2015-12-18
期刊：
The Journal of organic chemistry
影响因子：
--
作者：
Dulaney SB;Xu Y;Wang P;Tiruchinapally G;Wang Z;Kathawa J;El-Dakdouki MH;Yang B;Liu J;Huang X
通讯作者：
Huang X
DOI：
10.1016/b978-0-12-396527-1.00003-6
发表时间：
2012
影响因子：
--
作者：
Dulaney, Steven B.;Huang, Xuefei
通讯作者：
Huang, Xuefei
影响因子：
7
作者：
Chiu, Yulun;Huang, Rongrong;Sharp, Joshua S.
通讯作者：
Sharp, Joshua S.
影响因子：
15.9
作者：
Esko, JD;Lindahl, U
通讯作者：
Lindahl, U