A non-glycosaminoglycan-binding variant of CC chemokine ligand 7 (monocyte chemoattractant protein-3) antagonizes chemokine-mediated inflammation.

A non-glycosaminoglycan (GAG)-binding variant of the pleiotropic chemokine CCL7 was generated by mutating to alanine the basic (B) amino acids within an identified (44)BXBXXB(49) GAG-binding motif. Unlike wild-type (wt) CCL7, the mutant sequence had no affinity for heparin. However, the mutant retained a normal affinity for CCR1, CCR2b, and CCR3, and produced a normal calcium flux in mononuclear leukocytes. Both the wt and mutant proteins elicited an equal leukocyte chemotactic response within a solute diffusion gradient but, unlike the wt protein, the mutant failed to stimulate cell migration across a model endothelium. The number of leukocytes recruited to murine air pouches by the mutant sequence was lower than that recruited by wt CCL7. Furthermore, the presence of a mixture of a mutant and wt CCL7 within the air pouch elicited no significant cell accumulation. Cell recruitment also failed using a receptor-sharing mixture of mutant CCL7 and wt CCL5 or a nonreceptor sharing mixture of mutant CCL7 and wt CXCL12. The potential of the mutant sequence to modulate inflammation was confirmed by demonstration of its ability to inhibit the chemotactic response generated in vitro by synovial fluid from patients with active rheumatoid arthritis. A further series of experiments suggested that the non-GAG-binding mutant protein could potentially induce receptor desensitization before, and at a site remote from, any physiological recognition of GAG-bound chemokines. These data demonstrate that GAG binding is required for chemokine-driven inflammation in vivo and also suggest that a non-GAG-binding chemokine receptor agonist can inhibit the normal vectorial leukocyte migration mediated by chemokines.

Rapid site-directed mutagenesis of chemokines and their purification from a bacterial expression system.

Chemokines are a family of small chemoattractant cytokines implicated in the recruitment and migration of leukocytes from the blood into tissues during disease and routine immune homeostasis. Although there are many similarities in the structure and function of certain chemokines, the importance of many residues in the function of these proteins is yet to be determined, and studies from related chemokines have shown that similar sequences may play different roles in each protein. The migration-inducing capacity of many chemokines is thought to involve the cell surface glycosaminoglycan (GAG), heparan sulphate (HS), which may assist in the formation of an immobilised chemokine gradient within inflamed tissues. To examine the heparan sulphate binding ability of the CC chemokine monocyte chemoattractant protein (MCP)-3 and its importance in chemotactic migration, we have identified and mutated conserved basic residues within the mature MCP-3 protein to the neutral amino acid alanine using a novel inverse polymerase chain reaction (I-PCR) method that rapidly generates essentially 100% mutational efficiency due to decreased requirements for template DNA and an alkaline denaturation step; this increased mutational efficiency reduces both screening time and sequencing costs. We also describe an optimised method for the expression of soluble, correctly folded MCP-3 in a bacterial system using nickel affinity columns and reverse-phase fast protein liquid chromatography (RP-FPLC), and achieve purified yields of up to 0.4 mg/l of initial culture medium after 5 h of induction. These optimised methods could work equally well for any small circular plasmid (< or =4.5 kb) incorporating a polyhistidine tag.