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Papers In Press, published online ahead of print July 20, 2005
J. Biol. Chem, 10.1074/jbc.M505738200
Submitted on May 26, 2005
Revised on July 15, 2005
Accepted on July 20, 2005

Chemokine-glycosaminoglycan binding: Specificity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR MS

Yonghao Yu, Matthew D. Sweeney, Ola M. Saad, Susan E. Crown, Tracy M. Handel, and Julie A. Leary

Genome center, University of California, Davis, Davis, CA 95616

Corresponding Author: jaleary{at}ucdavis.edu

Glycosaminoglycans (GAGs) have recently been demonstrated to be required for the in vivo activity of several chemokines. Minimally, the interaction is thought to provide a mechanism for retention at the site of secretion, and the formation of chemokine gradients that provide directional cues for receptor bearing cells, particularly in the presence of shear forces. Thus, a key issue will be to determine the sequence and structure of the GAGs that bind to specific chemokines. Herein, we describe a mass spectrometry assay that was developed to detect protein-oligosaccharide noncovalent complexes, in this case chemokine-GAG interactions, and to select for high-affinity GAGs. The process is facilitated by the ability of electrospray ionization to transfer the intact noncovalent complexes from solution into the gas phase. The elemental composition as well as the binding stoichiometry can be calculated from the mass of the complex. Ligands of the chemokine receptor, CCR2, (MCP-1/CCL2, MCP-2/CCL8, MCP-3/CCL7, MCP-4/CCL13 and Eotaxin/CCL11), and the CCR10 ligand CTACK/CCL27 were screened against a small, highly sulfated, heparin oligosaccharide library with limited structural variation. The results revealed heparin octasaccharides with 11 and 12 sulfates as binders. Oligomerization of some chemokines was observed upon GAG binding, whereas in other instances only the monomeric noncovalent complex was identified. The results indicate that in contrast to the apparent redundancy in the chemokine system, where several chemokines bind and activate the same receptor, these chemokines could be differentiated into two groups based on the stoichiometry of their complexes with the heparin oligosaccharides.


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