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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.