Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.
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.
Chengli
C
Zong
Rongrong
R
Huang
Eduard
E
Condac
Yulun
Y
Chiu
Wenyuan
W
Xiao
Xiuru
X
Li
Weigang
W
Lu
Mayumi
M
Ishihara
Shuo
S
Wang
Annapoorani
A
Ramiah
Morgan
M
Stickney
Parastoo
P
Azadi
I Jonathan
IJ
Amster
Kelley W
KW
Moremen
Lianchun
L
Wang
Joshua S
JS
Sharp
Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, and Bijvoet Center for Biomolecular Research, Utrecht University , Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
Geert-Jan
GJ
Boons
D002465:Cell Movement; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D011971:Receptors, Immunologic / Q000378:metabolism*; D000097905:Roundabout Proteins
D006497:Heparan Sulfate; D008024:Ligands; D009419:Nerve Tissue Proteins; D011971:Receptors, Immunologic; D000097905:Roundabout Proteins