title: Enrichment of Two Isomeric Heparin Oligosaccharides Exhibiting Different Affinities toward Monocyte Chemoattractant Protein-1.
abstract: Chemokine-GAG interactions are crucial to facilitate chemokine immobilization, resulting in the formation of chemokine gradients that guide cell migration. Here we demonstrate chromatographic isolation and purification of two heparin hexasaccharide isomers that interact with the oligomeric chemokine Monocyte Chemoattractant Protein-1 (MCP-1)/CCL2 with different binding affinities. The sequences of these two hexasaccharides were deduced from unique MS/MS product ions and HPLC compositional analysis. Ion mobility mass spectrometry (IM-MS) showed that the two isolated oligosaccharides have different conformations and both displayed preferential binding for one of the two distinct conformations known for MCP-1 dimers. A significant shift in arrival time distribution of close to 70 Å2was observed, indicating a more compact protein:hexasaccharide conformation. Clear differences in the MS spectra between bound and unbound protein allowed calculation of Kdvalues from the resulting data. The structural difference between the two hexasaccharides was defined as the differential location of a single sulfate at either C-6 of glucosamine or C-2 of uronic acid in the reducing disaccharide, resulting in a 200-fold difference in binding affinity for MCP-1. These data indicate sequence specificity for high affinity binding, supporting the view that sulfate position, and not simply the number of sulfates, is important for heparan sulfate protein binding.
authors: Departments of Molecular and Cellular Biology and Chemistry, University of California , 1 Shields Drive, Davis, California 95616, United States.
authors: Rebecca L
authors: RL
authors: Miller
authors: Departments of Molecular and Cellular Biology and Chemistry, University of California , 1 Shields Drive, Davis, California 95616, United States.
authors: Andrew B
authors: AB
authors: Dykstra
authors: Departments of Molecular and Cellular Biology and Chemistry, University of California , 1 Shields Drive, Davis, California 95616, United States.
authors: Wei
authors: W
authors: Wei
authors: Departments of Molecular and Cellular Biology and Chemistry, University of California , 1 Shields Drive, Davis, California 95616, United States.
authors: Cynthia
authors: C
authors: Holsclaw
authors: Centre for Glycobiology, Department of Biochemistry, Institute of Integrative Biology, University of Liverpool , Crown Street, Liverpool, L69 7ZB, England.
authors: Jeremy E
authors: JE
authors: Turnbull
authors: Departments of Molecular and Cellular Biology and Chemistry, University of California , 1 Shields Drive, Davis, California 95616, United States.
authors: Julie A
authors: JA
authors: Leary
mesh_terms: D018932:Chemokine CCL2 / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D007536:Isomerism; D009844:Oligosaccharides / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D009844:Oligosaccharides; D006493:Heparin