During organogenesis, immunosurveillance, and inflammation, chemokines selectively recruit leukocytes by activating seventransmembrane-spanning receptors. It has been suggested that an important component of this process is the formation of a haptotactic gradient by immobilization of chemokines on cell surface glycosaminoglycans (GAGs). However, this hypothesis has not been experimentally demonstrated in vivo. In the present study we investigated the effect of mutations in the GAG binding sites of three chemokines, monocyte chemoattractant protein-1͞CC chemokine ligand (CCL)2, macrophage-inflammatory protein-1␤͞ CCL4, and RANTES͞CCL5, on their ability to recruit cells in vivo. These mutant chemokines retain chemotactic activity in vitro, but they are unable to recruit cells when administered intraperitoneally. Additionally, monomeric variants, although fully active in vitro, are devoid of activity in vivo. These data demonstrate that both GAG binding and the ability to form higher-order oligomers are essential for the activity of particular chemokines in vivo, although they are not required for receptor activation in vitro. Thus, quaternary structure of chemokines and their interaction with GAGs may significantly contribute to the localization of leukocytes beyond migration patterns defined by chemokine receptor interactions.

Immune modulators such as cytokines and growth factors exert their biological activity through high-affinity interactions with cell-surface receptors, thereby activating specific signaling pathways. However, many of these molecules also participate in low-affinity interactions with another class of molecules, referred to as proteoglycans. Proteoglycans consist of a protein core to which glycosaminoglycan (GAG) chains are attached. The GAGs are long, linear, sulfated, and highly charged heterogeneous polysaccharides that are expressed throughout the body in different forms, depending on the developmental or pathological state of the organ/organism. They participate in many biological functions, including organogenesis and growth control, cell adhesion, signaling, inflammation, tumorigenesis, and interactions with pathogens. Recently, it was demonstrated that certain chemokines require interactions with GAGs for their in vivo function. The GAG interaction is thought to provide a mechanism for retaining chemokines on cell surfaces, facilitating the formation of chemokine gradients. These gradients serve as directional cues to guide the migration of the appropriate cells in the context of their inflammatory, developmental, and homeostatic functions. In this review, we discuss GAGs and their interaction with proteins, with a special emphasis on the chemokine system.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/ CCL2, RANTES/CCL5, and MIP-1␤/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAGinduced oligomerization beyond retention of chemokines at the site of production.