Heterodimerization of CCR2 chemokines and regulation by glycosaminoglycan binding

Despite the wide range of sequence diversity among chemokines, their tertiary structures are remarkably similar. Furthermore, many chemokines form dimers or higher order oligomers, but all characterized oligomeric structures are based primarily on two dimerization motifs represented by CC-chemokine or CXC-chemokine dimer interfaces. These observations raise the possibility that some chemokines could form unique hetero-oligomers using the same oligomerization motifs. Such interactions could modulate the overall signaling response of the receptors, thereby providing a general mechanism for regulating chemokine function. For some chemokines, homo-oligomerization has also been shown to be coupled to glycosaminoglycan (GAG)-binding. However, the effect of GAG binding on chemokine hetero-oligomerization has not yet been demonstrated. In this report, we characterized the heterodimerization of the CCR2 ligands MCP-1 (CCL2), MCP-2 (CCL8), MCP-3 (CCL7), MCP-4 (CCL13), and eotaxin (CCL11), as well as the effects of GAG binding, using electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry. Strong heterodimerization was observed between CCL2 and CCL8 at the expense of homodimer formation. Using NMR, we showed that the heterodimer is predominant in solution and forms a specific CC chemokine-like dimer. By contrast, only moderate heterodimer formation was observed between CCL2.CCL13, CCL2.CCL11 and CCL8.CCL13, and no heterodimerization was observed when any other CCR2 ligand was added to CCL7. To investigate the effect of a highly sulfated GAG on the formation of heterodimers, each chemokine pair was mixed with the heparin pentasaccharide, Arixtra, and assayed by ESI-FTICR mass spectrometry. Although no CCL8.CCL11 heterodimer was observed in the absence of GAG, abundant ions corresponding to the ternary complex, CCL8.CCL11.Arixtra, were observed upon addition of Arixtra. Heterodimerization between CCL2 and CCL11 was also enhanced in the presence of Arixtra. In summary, these results indicate that some CCR2 ligands can form stable heterodimers in preference to homodimers and that these interactions, like those of homo-oligomers, can be influenced by some GAGs.     

Multiple Glycosaminoglycan-binding Epitopes of Monocyte Chemoattractant Protein-3/CCL7 Enable It to Function as a Non-oligomerizing Chemokine

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.     

CCR2 chemokines bind selectively to acetylated heparan sulfate octasaccharides

Chemokines participate in well documented interactions with glycosaminoglycans (GAGs). Although many chemokine amino acid residues involved in binding have been identified, much less is known about the bound regions of GAG. Heparan sulfate (HS) is the predominant cell surface GAG, and its heterogeneous nature offers proteins a variety of structural motifs with which to interact. In the present study, we describe the interactions of three CC chemokines, MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, with HS-derived oligosaccharides. To this end, we generated and characterized a complex HS octasaccharide library containing 17 different octasaccharide compositions based on acetyl and sulfate group content. Electrospray ionization mass spectrometry was used to detect chemokine-HS octasaccharide complexes in the bound state, and an affinity purification protocol was used to select and identify chemokine-binding octasaccharides from the complex mixture. The results indicate that HS octasaccharide sulfation is the foremost requirement for chemokine binding. However, within octasaccharides of constant charge density, acetylation is also observed to augment binding, suggesting that there may be as yet undiscovered specificity in the chemokine-HS interaction.     

Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.     

Chemokine-glycosaminoglycan binding: specificity for CCR2 ligand binding to highly sulfated oligosaccharides using FTICR mass spectrometry

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.     

Interference with Glycosaminoglycan-Chemokine Interactions with a Probe to Alter Leukocyte Recruitment and Inflammation In Vivo

In vivo leukocyte recruitment is not fully understood and may result from interactions of chemokines with glycosaminoglycans/GAGs. We previously showed that chlorite-oxidized oxyamylose/COAM binds the neutrophil chemokine GCP-2/CXCL6. Here, mouse chemokine binding by COAM was studied systematically and binding affinities of chemokines to COAM versus GAGs were compared. COAM and heparan sulphate bound the mouse CXC chemokines KC/CXCL1, MIP-2/CXCL2, IP-10/CXCL10 and I-TAC/CXCL11 and the CC chemokine RANTES/CCL5 with affinities in the nanomolar range, whereas no binding interactions were observed for mouse MCP-1/CCL2, MIP-1α/CCL3 and MIP-1β/CCL4. The affinities of COAM-interacting chemokines were similar to or higher than those observed for heparan sulphate. Although COAM did not display chemotactic activity by itself, its co-administration with mouse GCP-2/CXCL6 and MIP-2/CXCL2 or its binding of endogenous chemokines resulted in fast and cooperative peritoneal neutrophil recruitment and in extravasation into the cremaster muscle in vivo. These local GAG mimetic features by COAM within tissues superseded systemic effects and were sufficient and applicable to reduce LPS-induced liver-specific neutrophil recruitment and activation. COAM mimics glycosaminoglycans and is a nontoxic probe for the study of leukocyte recruitment and inflammation in vivo.     

Structural determinants of chemokine binding by an Ectromelia virus-encoded decoy receptor

The EVM1 protein encoded by Ectromelia virus is a member of a highly conserved family of poxvirus chemokine binding proteins that interfere with host immune surveillance processes. EVM1 is abundantly expressed early during mousepox infection and is able to selectively bind CC chemokines and inhibit their interactions with host receptors. Here, we characterize the interaction between EVM1 and the human and murine chemokines CCL3 (MIP-1alpha), CCL2 (MCP-1), and CCL5 (RANTES). Each of these CC chemokines binds EVM1 with 1:1 stoichiometry and equilibrium dissociation constants ranging from 29 pM to 20 nM. The interactions are characterized by rapid-association kinetics between acidic EVM1 and generally basic chemokines with half-lives enduring up to 30 min. The 2.6-A crystal structure of EVM1 reveals a globular beta sandwich with a large, sequence-conserved surface patch encircled by acidic residues on one face of the protein. To determine whether this conserved cluster of residues is involved in chemokine engagement, a structure-based mutational analysis of EVM1 was employed. Mapping of the mutational results onto the surface of EVM1 reveals that a cluster of five residues (I173, S171, S134, N136, and Y69) emanating from one beta sheet is critical for CCL2 and CCL3 sequestration. Additionally, we find that the extended beta2-beta4 loop flanking this conserved cluster is also essential for high-affinity, lasting interactions with chemokines. This analysis provides insight into the mechanism of CC-chemokine inhibition employed by the poxvirus family of chemokine decoy receptors.     

CXC and CC chemokines form mixed heterodimers: association free energies from molecular dynamics simulations and experimental correlations

CXC and CC chemokines are involved in numerous biological processes, and their function in situ may be significantly influenced by heterodimer formation, as was recently reported, for example, for CXC chemokines CXCL4/PF4 and CXCL8/IL8 that interact to form heterodimers that modulate chemotactic and cell proliferation activities. Here we used molecular dynamics simulations to determine relative association free energies (overall average and per residue) for homo- and heterodimer pairs of CXC (CXCL4/PF4, CXCL8/IL8, CXCL1/Gro-alpha, and CXCL7/NAP-2) and CC (CCL5/RANTES, CCL2/MCP-1, and CCL8/MCP-2) chemokines. Even though structural homology among monomer folds of all CXC and CC chemokines permits heterodimer assembly, our calculated association free energies depend upon the particular pair of chemokines in terms of the net electrostatic and nonelectrostatic forces involved, as well as (for CC/CXC mixed chemokines) the selection of dimer type (CC or CXC). These relative free energies indicate that association of some pairs of chemokines is more favorable than others. Our approach is validated by correlation of calculated and experimentally determined free energies. Results are discussed in terms of CXC and CC chemokine function and have significant biological implications.     

The pH-dependent stability of wild-type and mutant transthyretin oligomers

A reduction in pH is known to induce the disassociation of the tetrameric form of transthyretin and favor the formation of amyloid fibers. Using continuum electrostatic techniques, we calculate the titration curves and the stability of dimer and tetramer formation of transthyretin as a function of pH. We find that the tetramer and the dimer become less stable than the monomer as the pH is lowered. The free energy difference is 13.8 kcal/mol for dimer formation and 27 kcal/mol for tetramer formation, from the monomers, when the pH is lowered from 7 to 3.9. Similar behavior is observed for both the wild-type and the mutant protein. Certain residues (namely Glu-72, His-88, His-90, Glu-92, and Tyr-116), play an important role in the binding process, as seen by the considerable pK(1/2) change of these residues upon dimer formation.     

Structural insight for the roles of fas death domain binding to fadd and oligomerization degree of the fas–fadd complex in the death‐inducing signaling complex formation: A computational study

Fas binding to Fas‐associated death domain (FADD) activates FADD–caspase‐8 binding to form death‐inducing signaling complex (DISC) that triggers apoptosis. The Fas–Fas association exists primarily as dimer in the Fas–FADD complex, and the Fas–FADD tetramer complexes have the tendency to form higher order oligomer. The importance of the oligomerized Fas–FADD complex in DISC formation has been confirmed. This study sought to provide structural insight for the roles of Fas death domain (Fas DD) binding to FADD and the oligomerization of Fas DD–FADD complex in activating FADD–procaspase‐8 binding. Results show Fas DD binding to FADD stabilized the FADD conformation, including the increased stability of the critical residues in FADD death effector domain (FADD DED) for FADD–procaspase‐8 binding. Fas DD binding to FADD resulted in the decreased degree of both correlated and anticorrelated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain (FADD DD). The exposure of procaspase‐8 binding residues in FADD that allows FADD to interact with procaspase‐8 was observed with Fas DD binding to FADD. We also observed different degrees of conformational and motion changes of FADD in the Fas DD–FADD complex with different degrees of oligomerization. The increased conformational stability and the decreased degree of correlated motion of the residues in FADD in Fas DD–FADD tetramer complex were observed compared to those in Fas DD–FADD dimer complex. This study provides structural evidence for the roles of Fas DD binding to FADD and the oligomerization degree of Fas DD–FADD complex in DISC formation to signal apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP. Ornithine and IMP function as activators, whereas UMP is an inhibitor. CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors. Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer. The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units. Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains. There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units. When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP. In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors. These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains. Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed. The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer, whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer. In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain. No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another.     

Chemokines bind to sulfatides as revealed by surface plasmon resonance

Chemokines bind to sulfated cell surface glycosaminoglycans and thereby modulate signaling mediated by G-protein-coupled seven-transmembrane domain chemokine receptors. Similar to glycosaminoglycans, sulfated oligosaccharides are also exposed on the cell surface by sulfatides, a class of glycosphingolipids. We have now identified sulfated glycosphingolipids (sulfatides) as novel binding partners for chemokines. Using surface plasmon resonance (SPR), the binding of proinflammatory and homeostatic chemokines to glycosphingolipids, in particular sulfatides, was investigated. Chemokines were immobilized while glycosphingolipids or additional phospholipids incorporated into liposomes were applied as soluble analytes. A specific affinity of the chemokines MCP-1/CCL2, IL-8/CXCL8, SDF-1alpha/CXCL12, MIP-1alpha/CCL3 and MIP-1beta/CCL4 to the sulfatides SM4s, SM3, SM2a and SB2, SB1a was detected. No significant interactions with the chemokines were observed for gangliosides, neutral glycosphingolipids or phospholipids. Chemokine receptors have been associated with the detergent-insoluble fraction supposed to contain 'rafts', i.e., glycosphingolipid enriched microdomains of the cell surface. Accordingly, the data suggest that early chemokine receptor signaling may take place in the vicinity of sulfated glycosphingolipids on the cell surface, whereby these sulfatides could modulate the chemokine receptor-mediated cell activation signal.     

Inhibitor of inflammation,peptide fragment (65–76) of monocyte chemotactic protein-1 (MCP-1), inhibits binding of MCP-1 to heparin

Monocyte chemotactic protein-1 (MCP-1, CCL2) is one of the most important chemokines involved in inflammation. MCP-1 stimulates migration of monocytes and certain lymphocyte populations to the affected area, in particular to the sites of atherosclerotic plaque formation. Development of drugs inhibiting MCP-1 is now a topical task. We earlier designed and synthesized a dodecapeptide from C-terminal domain of MCP-1 (65–76, peptide X) that possessed an anti-inflammatory activity. The mechanism of action of chemokines (in particular, of MCP-1) in vivo is based on activation of CCR2 receptor on target cells and binding to glycosaminoglycans (GAGs) on the cell surface of and extracellular matrix. Peptide X did not affect the MCP-1-CCR2 interaction. Thus, we hypothesized that peptide X could impair MCP-1 binding to GAGs. Here we studied the effect of peptide X on the MCP-1 binding to heparin using the label-free biosensing device Picoscope®, enzyme-linked immunoassay (ELISA), and the intrinsic fluorescence method. According to the data obtained, peptide X interfered with the MCP-1-heparin binding, which may be due to the competition of peptide X with MCP-1 for heparin binding sites. Probably, this effect determines the anti-inflammatory activity of peptide X in vivo.     

Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer

Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (K_d = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.     

Cystatin forms a tetramer through structural rearrangement of domain-swapped dimers prior to amyloidogenesis

The cystatins were the first amyloidogenic proteins to be shown to oligomerize through a 3D domain swapping mechanism. Here we show that, under conditions leading to the formation of amyloid deposits, the domain-swapped dimer of chicken cystatin further oligomerizes to a tetramer, prior to fibrillization. The tetramer has a very similar circular dichroism and fluorescence signature to the folded monomer and dimer structures, but exhibits some loss of dispersion in the 1H-NMR spectrum. 8-Anilino-1-naphthalene sulfonate fluorescence enhancement indicates an increase in the degree of disorder. While the dimerization reaction is bimolecular and most likely limited by the availability of a predominantly unfolded form of the monomer, the tetramerization reaction is first-order. The tetramer is formed slowly (t(1/2)=six days at 85 degrees C), dimeric cystatin is the precursor to tetramer formation, and thus the rate is limited by structural rearrangement within the dimer. Some higher-order oligomerization events parallel tetramer formation while others follow from the tetrameric form. Thus, the tetramer is a transient intermediate within the pathway of large-scale oligomerization.     

Crystal structure of viral macrophage inflammatory protein I encoded by Kaposi's sarcoma-associated herpesvirus at 1.7A

Viral macrophage inflammatory protein I (vMIP-I) is a chemokine encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) that selectively activates the CC chemokine receptor 8 (CCR8), for which the endogenous ligand is CCL1. The crystal structure of vMIP-I was determined at 1.7A for comparison with other chemokines, especially those that bind CCR8, such as vMIP-II from KSHV, a CCR8 antagonist and the closest homolog (40% identical). vMIP-I has a typical chemokine fold consisting of an extended N-terminal loop, followed by a three-stranded antiparallel beta-sheet and a C-terminal alpha-helix. The four molecules in the asymmetric unit comprise two MIP-1beta-like dimers. Electrostatic surface representations of CCR8-binding chemokines reveal only minor areas of correlating surface potential, which must be reconciled with promiscuity in receptor and glycosaminoglycan (GAG) binding. In addition, the biological relevance of chemokine oligomerization is examined by comparing the oligomeric states of all chemokine structures deposited to date in the RCSB PDB.     

Heparin Oligosaccharides Inhibit Chemokine (CXC Motif) Ligand 12 (CXCL12) Cardioprotection by Binding Orthogonal to the Dimerization Interface,Promoting Oligomerization,and Competing with the Chemokine (CXC Motif) Receptor 4 (CXCR4) N Terminus

The ability to interact with cell surface glycosaminoglycans (GAGs) is essential to the cell migration properties of chemokines, but association with soluble GAGs induces the oligomerization of most chemokines including CXCL12. Monomeric CXCL12, but not dimeric CXCL12, is cardioprotective in a number of experimental models of cardiac ischemia. We found that co-administration of heparin, a common treatment for myocardial infarction, abrogated the protective effect of CXCL12 in an ex vivo rat heart model for myocardial infarction. The interaction between CXCL12 and heparin oligosaccharides has previously been analyzed through mutagenesis, in vitro binding assays, and molecular modeling. However, complications from heparin-induced CXCL12 oligomerization and studies using very short oligosaccharides have led to inconsistent conclusions as to the residues involved, the orientation of the binding site, and whether it overlaps with the CXCR4 N-terminal site. We used a constitutively dimeric variant to simplify the NMR analysis of CXCL12-binding heparin oligosaccharides of varying length. Biophysical and mutagenic analyses reveal a CXCL12/heparin interaction surface that lies perpendicular to the dimer interface, does not involve the chemokine N terminus, and partially overlaps with the CXCR4-binding site. We further demonstrate that heparin-mediated enzymatic protection results from the promotion of dimerization rather than direct heparin binding to the CXCL12 N terminus. These results clarify the structural basis for GAG recognition by CXCL12 and lend insight into the development of CXCL12-based therapeutics.     

Chemokines and cytokines in patients with an occult Onchocerca volvulus infection

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness.     

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.