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Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1
- Zong, Chengli, Huang, Rongrong, Condac, Eduard, Chiu, Yulun, Xiao, Wenyuan, Li, Xiuru, Lu, Weigang, Ishihara, Mayumi, Wang, Shuo, Ramiah, Annapoorani, Stickney, Morgan, Azadi, Parastoo, Amster, I. Jonathan, Moremen, Kelley
W., Wang, Lianchun, Sharp, Joshua S., Boons, Geert-Jan
- Journal of the American Chemical Society 2016 v.138 no.39 pp. 13059-13067
- binding proteins, cell movement, endothelial cells, esters, heparan sulfate, humans, ligands, mass spectrometry, sulfates, surface plasmon resonance
- 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.