Structure and function of the glycosaminoglycan binding site of chemokine macrophage-inflammatory protein-1 beta.

The ability of chemokines to bind to glycosaminoglycans (GAGs) on cell surfaces and in the extracellular matrix is thought to play a crucial role in chemokine function. We investigated the structural basis for chemokine binding to GAGs by using in vitro mutagenesis to identify amino acids of chemokine macrophage-inflammatory protein-1 beta (MIP-1 beta) that contribute to its interaction with the model GAG heparin. Among six basic residues that are organized into a single basic domain in the folded MIP-1 beta monomer, three (R18, K45, and R46) were found to contribute significantly to heparin binding. Of these, R46 was found to play a dominant role, and proved essential for the interaction of MIP-1 beta with both heparin and heparan sulfate in physiological salt. The results of this mutational analysis have implications for the structure of the MIP-1 beta-heparin complex, and a comparison of these results with those obtained by mutational analysis of the MIP-1 alpha-heparin interaction suggests a possible structural difference between the MIP-1 beta-heparin and MIP-1 alpha-heparin complexes. To determine whether GAG binding plays an important role in receptor binding and cellular activation by MIP-1 beta, the activities of wild-type MIP-1 beta and R46-substituted MIP-1 beta were compared in assays of T lymphocyte chemotaxis. The two proteins proved equipotent in this assay, arguing that interaction of MIP-1 beta with GAGs is not intrinsically required for functional interaction of MIP-1 beta with its receptor.     

Structural and functional analysis of the RANTES-glycosaminoglycans interactions

Chemokines mediate their biological activity through activation of G protein coupled receptors, but most chemokines, including RANTES, are also able to bind glycosaminoglycans (GAGs). Here, we have investigated, by site-directed mutagenesis and chemical acetylation, the role of RANTES basic residues in the interaction with GAGs using surface plasmon resonance kinetic analysis. Our results indicate that (i) RANTES exhibited selectivity in GAGs binding with highest affinity (K(d) = 32.1 nM) for heparin, (ii) RANTES uses the side chains of residues R44, K45, and R47 for heparin binding, and blocking these residues in combination abolished heparin binding. The biological relevance of RANTES-GAGs interaction was investigated in CHO-K1 cells expressing CCR5, CCR1, or CCR3 and the various GAGs that bind RANTES. Our results indicate that the heparin binding site, defined as the 40s loop, is only marginally involved in CCR5 binding and activation, but largely overlaps the CCR1 and CCR3 binding and activation domain in RANTES. In addition, enzymatic removal of cell surface GAGs by glycosidases did not affect CCR5 binding and Ca(2+) response. Furthermore, addition of soluble GAGs inhibited both CCR5 binding and functional response, with a rank of potency similar to that found in surface plasmon resonance experiments. Thus, cell surface GAGs is not a prerequisite for receptor binding or signaling, but soluble GAGs can inhibit the binding and the functional response of RANTES to CCR5 expressing cells. However, the marked selectivity of RANTES for different GAGs may serve, in vivo, to control the concentration of specific chemokines in inflammatory situations and locations.     

Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses.

Chemokines selectively recruit and activate a variety of cells during inflammation. Interactions between cell surface glycosaminoglycans (GAGs) and chemokines drive the formation of haptotactic or immobilized gradients of chemokines at the site of inflammation, directing this recruitment. Chemokines bind to glycosaminoglycans on human umbilical vein endothelial cells (HUVECs) with affinities in the micromolar range: RANTES > MCP-1 > IL-8 > MIP-1alpha. This binding can be competed with by soluble glycosaminoglycans: heparin, heparin sulfate, chondroitin sulfate, and dermatan sulfate. RANTES binding showed the widest discrimination between glycosaminoglycans (700-fold), whereas MIP-1alpha was the least selective. Almost identical results were obtained in an assay using heparin sulfate beads as the source of immobilized glycosaminoglycan. The binding of chemokines to glycosaminoglycan fragments has a strong length dependence, and optimally requires both N- and O-sulfation. Isothermal titration calorimetry data confirm these results; IL-8 binds heparin fragments with a K(d) of 0.39-2.63 microM, and requires five saccharide units to bind each monomer of chemokine. In membranes from cells expressing the G-protein-coupled chemokine receptors CXCR1, CXCR2, and CCR1, soluble GAGs inhibit the binding of chemokine ligands to their receptors. Consistent with this, heparin and heparin sulfate could inhibit IL-8-induced neutrophil calcium flux. Chemokines can therefore form complexes with both cell surface and soluble GAGs; these interactions have different functions. Soluble GAG chemokines complexes are unable to bind the receptor, resulting in a block of the biological activity. Previously, we have shown that cell surface GAGs present chemokines to the G-protein-coupled receptors, by increasing the local concentration of protein. A model is presented which brings together all of these data. The selectivity in the chemokine-GAG interaction suggests selective disruption of the haptotactic gradient may be an achievable therapeutic approach in inflammatory disease.     

Multiple charged and aromatic residues in CCR5 amino-terminal domain are involved in high affinity binding of both chemokines and HIV-1 Env protein

CCR5 is a functional receptor for MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major determinant for chemokine binding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residues in the CCR5 amino-terminal domain for chemokine binding, functional response to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the binding affinity for chemokines, which correlated with a similar drop in functional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak responses to high concentrations (500 nM) of RANTES or MIP-1beta. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env proteins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alanine-scanning mutagenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18) that played an important role in both chemokine and Env high affinity binding. The overlapping binding site of chemokines and gp120 on the CCR5 amino terminus, as well as the involvement of these residues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.     

Glycosaminoglycan disaccharide alters the dimer dissociation constant of the chemokine MIP-1 beta

Chemokines are immune system proteins that recruit and activate leukocytes to sites of infection. This recruitment is believed to involve the establishment of a chemokine concentration gradient by the binding of chemokines to glycosaminoglycans (GAGs). In previous studies, we elucidated the GAG binding site of the chemokine MIP-1beta and implicated the involvement of the chemokine dimer in GAG binding through residues across the dimer interface. In the present studies, nuclear magnetic resonance spectroscopy was used to investigate the effect of GAG binding on MIP-1beta dimerization. Using several dimerization-impaired variants of MIP-1beta (F13Y, F13L, L34W, and L34K), these studies indicate that the addition of disaccharide to the mutants increases their dimerization affinities. For MIP-1beta F13Y, the presence of the disaccharide increases the chemokine dimerization affinity about 9-fold as evidenced by a decrease in the dimer dissociation constant from 610 to 66 microM. Even more dramatically, the dimerization affinity of MIP-1beta L34W also increases upon addition of disaccharide, with the dimer dissociation constant decreasing from 97 to 6.5 microM. After this effect for the mutants of MIP-1beta was shown, similar experiments were conducted with the CC chemokine RANTES, and it was demonstrated that the presence of disaccharide increases its dimerization affinity by almost 7-fold. These findings provide further evidence of the importance of the dimer in chemokine function and provide the first quantitative investigation of the role of GAGs in the manipulation of the MIP-1beta quaternary structure.     

Characterization of the role of the N-loop of MIP-1 beta in CCR5 binding

MIP-1beta is a CC-chemokine that plays a role in inflammation and host defense mechanisms by interacting with its specific receptor CCR5. CCR5 is a major coreceptor for macrophage-tropic human immunodeficiency virus (HIV), and as a consequence, MIP-1beta can inhibit HIV entry. It is therefore of interest to understand how MIP-1beta and other CCR5 ligands bind to their receptor, as such understanding could lead to the rational design of more efficient HIV entry blockers. We have previously demonstrated the importance of Phe13, and of basic residues of the 40's loop, in mediating high-affinity binding of MIP-1beta to CCR5. We have now investigated further the relative contribution of other MIP-1beta residues in the interaction of the chemokine with CCR5, by studying the functional consequences of point mutations within the N-loop and the 3(10) turn of MIP-1beta, affecting the charge, size, and H-bonding properties of the side chains. Our data suggest that, in addition to Phe13, three amino acids of the N-loop and 3(10) turn (Arg18, Lys19, and Arg22) interact with CCR5 through their positive charge. We also found that Pro21 contributes to the CCR5 binding properties of MIP-1beta. Moreover, NMR spectroscopy has revealed that the presence of Tyr at position 15 is necessary for the proper folding of the chemokine. Our results therefore demonstrate that the binding determinants of MIP-1beta consist of residues arranged on one surface of the protein, including most of the basic residues in MIP-1beta, as well as two key hydrophobic groups. The good correlation observed between the potency of the mutants in a functional assay and their binding affinity strongly argues that basic residues Arg18, Lys19, and Arg22 of MIP-1beta are essential for its CCR5 binding properties, without a primary effect on CCR5 activation.     

The core domain of chemokines binds CCR5 extracellular domains while their amino terminus interacts with the transmembrane helix bundle

CCR5 is a functional receptor for various inflammatory CC-chemokines, including macrophage inflammatory protein (MIP)-1alpha and RANTES (regulated on activation normal T cell expressed and secreted), and is the main coreceptor of human immunodeficiency viruses. The second extracellular loop and amino-terminal domain of CCR5 are critical for chemokine binding, whereas the transmembrane helix bundle is involved in receptor activation. Chemokine domains and residues important for CCR5 binding and/or activation have also been identified. However, the precise way by which chemokines interact with and activate CCR5 is presently unknown. In this study, we have compared the binding and functional properties of chemokine variants onto wild-type CCR5 and CCR5 point mutants. Several mutations in CCR5 extracellular domains (E172A, R168A, K191A, and D276A) strongly affected MIP-1alpha binding but had little effect on RANTES binding. However, a MIP/RANTES chimera, containing the MIP-1alpha N terminus and the RANTES core, bound to these mutants with an affinity similar to that of RANTES. Several CCR5 mutants affecting transmembrane helices 2 and 3 (L104F, L104F/F109H/F112Y, F85L/L104F) reduced the potency of MIP-1alpha by 10-100 fold with little effect on activation by RANTES. However, the MIP/RANTES chimera activated these mutants with a potency similar to that of MIP-1alpha. In contrast, LD78beta, a natural MIP-1alpha variant, which, like RANTES, contains a proline at position 2, activated these mutants as well as RANTES. Altogether, these results suggest that the core domains of MIP-1alpha and RANTES bind distinct residues in CCR5 extracellular domains, whereas the N terminus of chemokines mediates receptor activation by interacting with the transmembrane helix bundle.     

Examination of the function of RANTES, MIP-1alpha, and MIP-1beta following interaction with heparin-like glycosaminoglycans

Chemokines are a group of small proteins that have a variety of functions, including the activation and recruitment of immune cells during episodes of inflammation. In common with many cytokines, it has been observed that chemokines have the potential to bind heparin-like glycosaminoglycan molecules, which are normally expressed on proteoglycan components of the cell surface and extracellular matrix. The significance of this interaction for chemokine activity remains a subject of debate. In this study, Chinese hamster ovary cells were transfected separately with the human chemokine receptors CCR1 and CCR5, and these receptors were shown to induce an intracytoplasmic Ca(2+) flux and cellular chemotaxis following stimulation with the natural CC chemokine ligands (MIP-1alpha, RANTES (regulated on activation normal T cell expressed), and MIP-1beta). In further experiments, mutant CHO cells, with a defect in normal glycosaminoglycan (GAG) expression, were also transfected with, and shown to express similar levels of, CCR1 and CCR5. Although these receptors were functional, it was found that the mutant cells required exposure to higher concentrations of ligands than the wild-type cells in order to produce the same intracytoplasmic Ca(2+) flux. Radioligand binding experiments demonstrated that specific chemokine receptors expressed by wild-type cells had a significantly greater affinity for MIP-1alpha than similar receptors expressed by GAG-deficient mutants. However, there was no significant difference between these cells in their affinity for RANTES or MIP-1beta. In conclusion, it has been demonstrated clearly that GAG expression is not necessary for the biological activity of the chemokines MIP-1alpha, RANTES, or MIP-1beta. However, the presence of cell surface GAGs does enhance the activity of low concentrations of these chemokines by a mechanism that appears to involve sequestration onto the cell surface.     

A Model of GAG/MIP-2/CXCR2 Interfaces and Its Functional Effects

MIP-2/CXCL2 is a murine chemokine related to human chemokines that possesses the Glu-Leu-Arg (ELR) activation motif and activates CXCR2 for neutrophil chemotaxis. We determined the structure of MIP-2 to 1.9 ? resolution and created a model with its murine receptor CXCR2 based on the coordinates of human CXCR4. Chemokine-induced migration of cells through specific G-protein coupled receptors is regulated by glycosaminoglycans (GAGs) that oligomerize chemokines. MIP-2 GAG-binding residues were identified that interact with heparin disaccharide I-S by NMR spectroscopy. A model GAG/MIP-2/CXCR2 complex that supports a 2:2 complex between chemokine and receptor was created. Mutants of these disaccharide-binding residues were made and tested for heparin binding, in vitro neutrophil chemotaxis, and in vivo neutrophil recruitment to the mouse peritoneum and lung. The mutants have a 10-fold decrease in neutrophil chemotaxis in vitro. There is no difference in neutrophil recruitment between wild-type MIP-2 and mutants in the peritoneum, but all activity of the mutants is lost in the lung, supporting the concept that GAG regulation of chemokines is tissue-dependent.     

The binding surface and affinity of monomeric and dimeric chemokine macrophage inflammatory protein 1 beta for various glycosaminoglycan disaccharides

Chemokines comprise a family of proteins that function in the immune response to recruit leukocytes to sites of infection. This recruitment is believed to be carried out by the establishment of a chemokine gradient by the binding of chemokines to sulfated polysaccharides known as glycosaminoglycans (GAGs) located on the extracellular surface of endothelial cells. In the present studies, multidimensional NMR spectroscopy was used to study the interaction of monomeric and dimeric chemokine macrophage inflammatory protein (MIP)-1 beta variants with a series of differentially sulfated disaccharides. The data define a GAG binding surface, including both basic and uncharged residues such as Arg(18), Asn(23), Val(25), Thr(44), Lys(45), Arg(46), and Ser(47). Dissociation constants determined from these NMR studies consistently show for each disaccharide that dimeric wild type MIP-1 beta binds more tightly than monomeric MIP(9). Furthermore, analysis of the binding surface suggests that participation in the dimer of residues Met(3), Gly(4), and Ser(5) may be responsible for this higher affinity. These studies also indicate that the specificity of MIP-1 beta for particular GAG disaccharides is directly related not only to the degree of disaccharide sulfation but also to the position of the sulfate moiety, with O-sulfation at position 2 of the hexuronic acid unit and position 6 of the D-glucosamine being major determinants for binding.     

CC chemokine MIP-1 beta can function as a monomer and depends on Phe13 for receptor binding

The reported structures of many CC chemokines show a conserved dimer interface along their N-terminal region, raising the possibility that the quaternary arrangement of these small immune proteins might influence their function. We have produced and analyzed several mutants of MIP-1 beta having a range of dimer K(d) values in order to determine the significance of dimerization in receptor binding and cellular activation. NMR and analytical ultracentrifugation were used to analyze the oligomeric state of the mutants. Functional relevance was determined by receptor binding affinity and the ability to invoke intracellular calcium release from CHO cells transfected with the MIP-1 beta receptor CCR5. The monomeric N-terminally truncated mutant MIP(9) was able to bind the CCR5 receptor with a K(i) of 600 pM but displayed weak agonistic properties, while the monomeric mutant P8A still retained the ability to tightly bind (K(i) = 480 pM) and to activate (EC(50) = 12 nM) the receptor. These data suggest that the MIP-1 beta dimer is not required for CCR5 binding or activation. In addition, we identified Phe13, the residue immediately following the conserved CC motif in MIP-1 beta, as a key determinant for binding to CCR5. Replacement of Phe13 by Tyr, Leu, Lys, and Ala showed the aromatic side chain to be important for both binding to CCR5 and chemokine dimerization.     

Characterization of binding sites for chemokines MCP-1 and MIP-1alpha on human brain microvessels

The presence of binding sites for the beta chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) has recently been identified on human brain microvessels. We extend these findings in this report to reveal that such sites exemplify characteristics of the recognized major receptors for MCP-1 and MIP-1alpha: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP-1 binding to isolated brain microvessels was inhibited by unlabeled MCP-1 and MCP-3, the latter another CCR2 ligand, but not by MIP-1alpha. Inhibition of labeled MIP-1alpha binding was achieved with unlabeled MIP-1alpha and RANTES, the latter a beta chemokine that binds to both CCR1 and CCR5, but not by MCP-1. Labeled MIP-1alpha binding was also antagonized by unlabeled MCP-3, which is also recognized by CCR1, and MIP-1beta, which is a ligand for CCR5. Labeled MCP-1 and MIP-1alpha were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the microvascular surface at 37 degrees C. Additionally, exposure of microvessels to unlabeled MCP-1 or MIP-1alpha was accompanied by the initial loss and subsequent recovery of surface binding sites for these chemokines, which occurred on a time scale consistent with ligand-induced endocytosis and recycling. These collective features bear striking similarity to those that characterize interactions of MCP-1 and MIP-1alpha with their receptors on leukocytes and underscore the concept of cognate chemokine receptors on brain microvascular endothelium.     

Identification of human macrophage inflammatory proteins 1alpha and 1beta as a native secreted heterodimer

Chemokines are secreted proteins that function as chemoattractants for leukocytes. The chemokines macrophage inflammatory protein 1alpha and 1beta (MIP-1alpha and MIP-1beta) now have been shown to be secreted from activated human monocytes and peripheral blood lymphocytes (PBLs) as a heterodimer. Immunoprecipitation and immunoblot analysis revealed that antibodies to either MIP-1alpha or MIP-1beta precipitated a protein complex containing both MIP-1alpha and MIP-1beta under normal conditions from culture supernatants and lysates of these cells. Mass spectrometry of the complexes, precipitated from the culture supernatants of monocytes and PBLs, revealed the presence of NH(2)-terminal truncated MIP-1alpha (residues 5-70) together with either intact MIP-1beta or NH(2)-terminal truncated MIP-1beta (residues 3-69), respectively. The secreted MIP-1alpha/beta heterodimers were dissociated into their component monomers under acidic conditions. Exposure of monocytes or PBLs to monensin induced the accumulation of heterodimers composed of NH(2)-terminal truncated MIP-1alpha and full-length MIP-1beta in the Golgi complex. The mixing of recombinant chemokines in vitro demonstrated that heterodimerization of MIP-1alpha and MIP-1beta is specific and that it occurs at physiological conditions, pH 7.4, and in the range of nanomolar concentrations. The data presented here provide the first biochemical evidence for the existence of chemokine heterodimers under natural conditions. Formation of heterodimers of MIP-1alpha/beta may have an impact on intracellular signaling events that contribute to CCR5 and possibly to other chemokine receptor functions.     

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.     

Reduced chemokine and chemokine receptor expression in spinal cords of TCR BV8S2 transgenic mice protected against experimental autoimmune encephalomyelitis with BV8S2 protein

The perivascular transmigration and accumulation of macrophages and T lymphocytes in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) may be partly regulated by low m.w. chemotactic cytokines. Using the RNase protection assay and ELISA, we quantified expression of chemokines and chemokine receptors in the spinal cord (SC), brain, and lymph nodes of BV8S2 transgenic mice that developed or were protected from EAE by vaccination with BV8S2 protein. In paralyzed control mice, the SC had increased cellular infiltration and strong expression of the chemokines RANTES, IFN-inducible 10-kDa protein, and monocyte chemoattractant protein-1 and the cognate chemokine receptors CCR1, CCR2, and CCR5, with lower expression of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2; whereas brain had less infiltration and a lower expression of a different pattern of chemokines and receptors. In TCR-protected mice, there was a decrease in the number of inflammatory cells in both SC and brain. In SC, the reduced cellular infiltrate afforded by TCR vaccination was commensurate with profoundly reduced expression of chemokines and their cognate chemokine receptors. In brain, however, TCR vaccination did not produce significant changes in chemokine expression but resulted in an increased expression of CCR3 and CCR4 usually associated with Th2 cells. In contrast to CNS, lymph nodes of protected mice had a significant increase in expression of MIP-2 and MIP-1beta but no change in expression of chemokine receptors. These results demonstrate that TCR vaccination results in selective reduction of inflammatory chemokines and chemokine receptors in SC, the target organ most affected during EAE.     

Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function

The chemokine receptor CCR5 is the major coreceptor for R5 human immunodeficiency virus type-1 strains. We mapped the epitope specificities of 18 CCR5 monoclonal antibodies (mAbs) to identify domains of CCR5 required for chemokine binding, gp120 binding, and for inducing conformational changes in Env that lead to membrane fusion. We identified mAbs that bound to N-terminal epitopes, extracellular loop 2 (ECL2) epitopes, and multidomain (MD) epitopes composed of more than one single extracellular domain. N-terminal mAbs recognized specific residues that span the first 13 amino acids of CCR5, while nearly all ECL2 mAbs recognized residues Tyr-184 to Phe-189. In addition, all MD epitopes involved ECL2, including at least residues Lys-171 and Glu-172. We found that ECL2-specific mAbs were more efficient than NH2- or MD-antibodies in blocking RANTES or MIP-1beta binding. By contrast, N-terminal mAbs blocked gp120-CCR5 binding more effectively than ECL2 mAbs. Surprisingly, ECL2 mAbs were more potent inhibitors of viral infection than N-terminal mAbs. Thus, the ability to block virus infection did not correlate with the ability to block gp120 binding. Together, these results imply that chemokines and Env bind to distinct but overlapping sites in CCR5, and suggest that the N-terminal domain of CCR5 is more important for gp120 binding while the extracellular loops are more important for inducing conformational changes in Env that lead to membrane fusion and virus infection. Measurements of individual antibody affinities coupled with kinetic analysis of equilibrium binding states also suggested that there are multiple conformational states of CCR5. A previously described mAb, 2D7, was unique in its ability to effectively block both chemokine and Env binding as well as coreceptor activity. 2D7 bound to a unique antigenic determinant in the first half of ECL2 and recognized a far greater proportion of cell surface CCR5 molecules than the other mAbs examined. Thus, the epitope recognized by 2D7 may represent a particularly attractive target for CCR5 antagonists.     

The human CC chemokine MIP-1beta dimer is not competent to bind to the CCR5 receptor

Chemokine dimerization has been the subject of much interest in recent years as evidence has accumulated that different quaternary states of chemokines play different biological roles; the monomer is believed to be the receptor-binding unit, whereas the dimer has been implicated in binding cell surface glycosaminoglycans. However, although several studies have provided evidence for this paradigm by making monomeric chemokine variants or dimer-impaired chemokines, few have provided direct evidence of the receptor function of a chemokine dimer. We have produced a covalent dimer of the CC chemokine macrophage inflammatory protein-1beta (MIP-1beta) by placing a disulfide bond at the center of its dimer interface through a single amino acid substitution (MIP-1beta-A10C). This variant was shown to be a nondissociating dimer by SDS-PAGE and analytical ultracentrifugation. NMR reveals a structure largely the same as the wild type protein. In studies of glycosaminoglycan binding, MIP-1beta-A10C binds to a heparin-Sepharose column as tightly as the wild type protein and more tightly than monomeric variants. However, MIP-1beta-A10C neither binds nor activates the MIP-1beta receptor CCR5. It was found that the ability to activate CCR5 was recovered upon reduction of the intermolecular disulfide cross-link by incubation with 1 mm dithiothreitol. This work provides the first definitive evidence that the CC chemokine MIP-1beta dimer is not able to bind or activate its receptor and implicates the CC chemokine monomer as the sole receptor-interacting unit.     

Novel chicken CXC and CC chemokines

Upon stimulation with lipopolysaccharide (LPS) the chicken macrophage cell line HD-11 secretes factors with cytokine activity. To characterize these molecules, representational difference analysis with RNA of LPS-induced and uninduced HD-11 cells was performed. Two cDNA clones were isolated that code for polypeptides with structural features of chemokines. cDNA K60 codes for a novel CXC chemokine of 104 residues including a putative signal peptide of 20 amino acids at the N-terminus. It is 67% identical to the previously cloned chicken chemokine 9E3/CEF4. K60 exhibits a similar degree of sequence identity to human interleukin 8 and other related CXC chemokines (about 50%), rendering straight-forward predictions of its biological properties difficult. cDNA K203 codes for a novel CC chemokine of 89 amino acids including a putative N-terminal signal peptide of 21 residues. It is 43% identical to a previously characterized chicken protein with homology to mammalian macrophage inflammatory protein 1beta (MIP-1beta). K203 exhibits about 50% sequence identity to human MIP-1beta and other related CC chemokines.     

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.