title: Multiple glycosaminoglycan-binding epitopes of monocyte chemoattractant protein-3/CCL7 enable it to function as a non-oligomerizing chemokine.
abstract: The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP-3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAG-binding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate that CCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces.
authors.affiliation: From the Skaggs School of Pharmacy and Pharmaceutical Science, University of California, San Diego, La Jolla, California 92093-0684 and.
authors.forename: Catherina L
authors.initials: CL
authors.lastname: Salanga
authors.affiliation: From the Skaggs School of Pharmacy and Pharmaceutical Science, University of California, San Diego, La Jolla, California 92093-0684 and.
authors.forename: Douglas P
authors.initials: DP
authors.lastname: Dyer
authors.affiliation: the Center for Proteomics and Bioinformatics and.
authors.forename: Janna G
authors.initials: JG
authors.lastname: Kiselar
authors.affiliation: the Center for Proteomics and Bioinformatics and Center for Synchrotron Biosciences, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Sayan
authors.initials: S
authors.lastname: Gupta
authors.affiliation: the Center for Proteomics and Bioinformatics and Center for Synchrotron Biosciences, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Chance
authors.affiliation: From the Skaggs School of Pharmacy and Pharmaceutical Science, University of California, San Diego, La Jolla, California 92093-0684 and thandel@ucsd.edu.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Handel
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D018932:Chemokine CCL2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054410:Chemokine CCL7 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000235:genetics / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance
chemical_list: D018932:Chemokine CCL2; D054410:Chemokine CCL7; D000939:Epitopes; D006025:Glycosaminoglycans
keywords: CCL2; CCL7; Chemokines; Glycosaminoglycan; Hydroxy Radical Footprinting; Mass Spectrometry (MS); Structural Biology; Surface Plasmon Resonance (SPR)
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