Abstract
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.
Highlights
Chemokines are small secreted proteins that are critically involved in many biological processes, including routine immunosurveillance, inflammation, and development
Some specific chemokine receptors provide the portals by which human immunodeficiency virus gets into cells, whereas others have been implicated in inflammatory diseases associated with inappropriate cell migration [1, 2]
Oligomerization has been shown to be required for some chemokines as oligomerization-deficient mutants that function like wild type (WT) in vitro, have been shown to be nonfunctional in vivo [19]
Summary
Chemokines are small secreted proteins that are critically involved in many biological processes, including routine immunosurveillance, inflammation, and development. Chemokines are thought to form a concentration gradient that controls the direction of the leukocyte cell migration This process is mediated by the interactions between the chemokines and G protein-coupled seven transmembrane receptors on leukocytes; the binding event subsequently triggers downstream signaling pathways that lead to cell migration and activation [3]. GAGs are linear, highly sulfated, and heterogeneous polysaccharides that are often covalently linked to core proteins residing on the membrane of cells or within the extracellular matrix They have been classified into several major families, primarily heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan, and keratan sulfate [11,12,13]. The objectives of this study are to develop methods for selecting and sequencing GAG ligands of a given chemokine, to determine the extent of their sequence diversity, and to establish if the “redundant” chemokines can be distinguished by their GAG-binding properties
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