Multimerization of monocyte chemoattractant protein-1 is not required for glycosaminoglycan-dependent transendothelial chemotaxis

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Abstract
Chemokines interact with specific G-protein-coupled cell-surface receptors and with glycosaminoglycans (GAGs), such as heparan sulphate. Although chemokines often form multimers in solution, this process may be enhanced following interaction with GAGs on the cell surface, or within the extracellular matrix. However, the significance of multimerization for chemokine function remains controversial. In the present study, a fusion protein was prepared between the prototypical human CC chemokine, monocyte chemoattractant protein-1 (MCP-1; also known as CCL-2) and a large secreted placental alkaline phosphatase (SEAP) moiety. This fusion protein (MCP-1-SEAP) remained monomeric under conditions that promote oligomerization of the native chemokine. Radioligand binding showed that both native MCP-1 and MCP-1-SEAP competed for the same site on the surface of HEK-293 cells expressing the CCR2b chemokine receptor. The interaction between either chemokine species and endothelial cell surface GAGs was antagonized by the addition of the heparan sulphate-like molecule, heparin. Both MCP-1 and MCP-1-SEAP induced a Ca(2+)-flux in the THP-1 monocytic cell line, and were equally effective at promoting transendothelial chemotaxis of mononuclear immune cells, with maximal migration being produced by treatment with 12 nM of either species. In each case this chemotactic response was almost completely antagonized by the addition of heparin. The importance of interaction between either native MCP-1 or MCP-1-SEAP and cell-surface GAGs for transcellular migration was demonstrated by the almost complete absence of leucocyte chemotaxis across monolayers of GAG-deficient mutant cells. In summary, this study shows that multimerization is neither necessary for, nor potentiates, the biological activity of MCP-1. However, the results do clearly demonstrate the importance of the interaction between MCP-1 and cell-surface heparan sulphate for transmonolayer leucocyte chemotaxis.