Characterization of the Chemokine CXCL11-Heparin Interaction Suggests Two Different Affinities for Glycosaminoglycans

Chemokines orchestrate the migration of leukocytes in the context of homeostasis and inflammation. In addition to interactions of chemokines with receptors on migrating cells, these processes require interactions of chemokines with glycosaminoglycans (GAGs) for cell surface localization. Most chemokines are basic proteins with Arg/Lys/His residue clusters functioning as recognition epitopes for GAGs. In this study we characterized the GAG-binding epitopes of the chemokine I-TAC/CXCL11. Four separate clusters of basic residues were mutated to alanine and tested for their ability to bind to GAGs in vitro and to activate the receptor, CXCR3. Mutation of a set of basic residues in the C-terminal helix (the 50s cluster, ⁵⁷KSKQAR⁶²) along with Lys¹⁷, significantly impaired heparin binding in vitro, identifying these residues as components of the dominant epitope. However, this GAG mutant retained nearly wild type receptor binding affinity, and its ability to induce cell migration in vitro was only mildly perturbed. Nevertheless, the mutant was unable to induce cell migration in vivo, establishing a requirement of CXCL11 for GAG binding for in vivo function. These studies also led to some interesting findings. First, CXCL11 exhibits conformational heterogeneity, as evidenced by the doubling of peaks in its HSQC spectra. Second, it exhibits more than one affinity state for both heparin and CXCR3, which may be related to its structural plasticity. Finally, although the binding affinities of chemokines for GAGs are typically weaker than interactions with receptors, the high affinity GAG binding state of CXCL11 is comparable with typical receptor binding affinities, suggesting some unique properties of this chemokine.     

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.     

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.     

A structural and dynamic model for the interaction of interleukin-8 and glycosaminoglycans: support from isothermal fluorescence titrations

Binding of interleukin-8 (IL-8) to glycosaminoglycans (GAGs) on the surface of endothelial cells is crucial for the recruitment of neutrophils to an inflammatory site. Deriving structural knowledge about this interaction from in silico docking experiments has proved difficult because of the high flexibility and the size of GAGs. Therefore, we developed a docking method that takes into account ligand and protein flexibility by running approximately 15,000 molecular dynamics simulations of the docking event with different initial orientations of the binding partners. The method was shown to successfully reproduce the residues of basic fibroblast growth factor involved in GAG binding. Docking of a heparin hexasaccharide to IL-8 gave an interaction interface involving the basic residues His18, Lys20, Arg60, Lys64, Lys67, and Arg68. By subjecting IL-8 single-site mutants, in which these amino acids were replaced by alanine, to isothermal fluorescence titrations, the affinities for heparin were determined to be wtIL-8 > IL-8(H18A) > IL-8(R68A) > IL-8(K67A) > IL-8(K20A) > IL-8(R60A) > IL-8(K64A). A comparison with the binding energies calculated from the model revealed high values for wtIL-8 and the H18A mutant and significantly lower but similar energies for the remaining mutants. Connecting the two fully sulfated hexasaccharides bound to each of the two IL-8 monomers in the dimeric chemokine by an N-acetylated dodecasaccharide gave a complex structure in which the GAG molecule aligned in a parallel fashion to the N-terminal alpha-helices of IL-8 like a horseshoe. A 5-ns molecular dynamics simulation of this complex confirmed its structural stability and revealed a reorientation in both binding sites where a disaccharide became the central binding unit. Isothermal fluorescence titration experiments using differently sulfated heparin disaccharides confirmed that a single disaccharide can indeed bind IL-8 with high affinity.     

CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10)

The chemokine IP-10 (interferon-inducible protein of 10 kDa, CXCL10) binds the G protein-coupled receptor CXCR3, which is found mainly on activated T cells and NK cells, and plays an important role in Th1-type inflammatory diseases. IP-10 also binds to glycosaminoglycans (GAGs), an interaction thought to be important for its sequestration on endothelial and other cells. In this study, we performed an extensive mutational analysis to identify the CXCR3 and heparin binding sites of murine IP-10. The mutants were characterized for heparin binding, CXCR3 binding, and the ability to induce chemotaxis, Ca(2+) flux, and CXCR3 internalization. Double mutations neutralizing adjacent basic residues at the C terminus did not lead to a significant reduction in heparin binding, indicating that the main heparin binding site of IP-10 is not along the C-terminal alpha helix. Alanine exchange of Arg-22 had the largest effect on heparin binding, with residues Arg-20, Ile-24, Lys-26, Lys-46, and Lys-47 further contributing to heparin binding. A charge change mutation of Arg-22 resulted in further reduction in heparin binding. The N-terminal residue Arg-8, preceding the first cysteine, was critical for CXCR3 signaling. Mutations of charged and uncharged residues in the loop regions of residues 20-24 and 46-47, which caused reduced heparin binding, also resulted in reduced CXCR3 binding and signaling. CXCR3 expressing GAG-deficient Chinese hamster ovary cells revealed that GAG binding was not required for IP-10 binding and signaling through CXCR3, which suggests that the CXCR3 and heparin binding sites of IP-10 are partially overlapping.     

Stromal cell-derived factor-1alpha associates with heparan sulfates through the first beta-strand of the chemokine.

Biological properties of chemokines are believed to be influenced by their association with glycosaminoglycans. Surface plasmon resonance kinetic analysis shows that the CXC chemokine stromal cell-derived factor-1alpha (SDF-1alpha), which binds the CXCR4 receptor, associates with heparin with an affinity constant of 38.4 nM (k(on) = 2.16 x 10(6) M(-1) s(-1) and k(off) = 0.083 x s(-1)). A modified SDF-1alpha (SDF-1 3/6) was generated by combined substitution of the basic cluster of residues Lys(24), His(25), and Lys(27) by Ser. SDF-1 3/6 conserves the global native structure and functional properties of SDF-1alpha, but it is unable to interact with sensor chip-immobilized heparin. The biological relevance of these in vitro findings was investigated. SDF-1alpha was unable to bind in a CXCR4-independent manner on epithelial cells that were treated with heparan sulfate (HS)-degrading enzymes or constitutively lack HS expression. The inability of SDF-1 3/6 to bind to cells underlines the importance of the identified basic cluster for the physiological interactions of SDF-1alpha with HS. Importantly, the amino-terminal domain of SDF-1alpha which is required for binding to, and activation of, CXCR4 remains exposed after binding to HS and is recognized by a neutralizing monoclonal antibody directed against the first residues of the chemokine. Overall, these findings indicate that the Lys(24), His(25), and Lys(27) cluster of residues forms, or is an essential part of, the HS-binding site which is distinct from that required for binding to, and signaling through, CXCR4.     

Dimeric peptides of the C-terminal region of CXCL14 function as CXCL12 inhibitors

We recently reported that CXCL14 binds to CXCR4 with high affinity and inhibits CXCL12-mediated chemotaxis. Here we found that the C-terminal 51–77 amino acid residues of CXCL14 are responsible for CXCR4 binding. A disulfide dimer peptide of CXCL14(51–77) bound to CXCR4 with comparable affinity to full length CXCL14, and exhibited CXCL12 inhibitor activity. CXCR4 was efficiently internalized upon binding of dimeric CXCL14(51–77), thereby being reduced on the cell surface. Substitution of 5 amino acid residues in combination with the use of an oxime linker for dimerization increased the solubility and chemical stability of the dimeric CXCL14(51–77).     

Glycosaminoglycans and syndecan-4 are involved in SDF-1/CXCL12-mediated invasion of human epitheloid carcinoma HeLa cells

Background

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 mediates its biological activities through activation of G protein-coupled receptor CXCR4 and binds to glycosaminoglycans (GAGs).

Methods

Using Bio-coat cell migration chambers, specific antagonists, flow cytometry and RNA interference, we evaluate the involvement of heparan sulfate proteoglycans (HSPG) in the SDF-1/CXCL12-induced invasion of human cervix epitheloid carcinoma HeLa cells.

Results

The SDF-1/CXCL12-induced cell invasion is dependent on CXCR4. Furthermore, Protein Kinase C delta (PKC δ) and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) are implicated in this event, but not extracellular signal-regulated kinase (ERK) 1/2. Moreover, the invasion of HeLa cells induced by SDF-1/CXCL12 was dependent on matrix metalloproteinase-9 (MMP-9). The pre-incubation of HeLa cells with heparin or with anti-heparan sulfate antibodies or with β-d-xyloside inhibited SDF-1/CXCL12-mediated cell invasion. Furthermore, the down-regulation of syndecan-4, a heparan sulfate proteoglycan, decreased SDF-1/CXCL12-mediated HeLa cell invasion. GAGs, probably on syndecan-4, are involved in SDF-1/CXCL12-mediated cell chemotaxis.

General significance

These data suggest that targeting the glycosaminoglycan/chemokine interaction could be a new therapeutic approach for carcinomas in which SDF-1/CXCL12 is involved.     

Dual-site recognition of different extracellular matrix components by anti-angiogenic/neurotrophic serpin,PEDF

Pigment epithelium-derived factor (PEDF), a member of the serine protease inhibitor (serpin) superfamily, possesses anti-angiogenic and neurotrophic activities. PEDF has been reported to bind to extracellular matrix (ECM) components such as collagens and glycosaminoglycans (GAGs). In this study, to determine the binding sites for collagens and GAGs, we analyzed the interaction of recombinant mouse PEDF (rPEDF) with collagen I and heparin. By utilizing residue-specific chemical modification and site-directed mutagenesis techniques, we revealed that the acidic amino acid residues on PEDF (Asp(255), Asp(257), and Asp(299)) are critical to collagen binding, and three clustered basic amino acid residues (Arg(145), Lys(146), and Arg(148)) are necessary for heparin binding. Mapping of these residues on the crystal structure of human PEDF (Simonovic, M., Gettins, P. G. W., and Volz, K. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11131-11135) demonstrated that the collagen-binding site is oriented toward the opposite side of the highly basic surface where the heparin-binding site is localized. These results indicate that PEDF possesses dual binding sites for different ECM components, and this unique localization of ECM-binding sites implies that the binding to ECM components could regulate PEDF activities.     

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.     

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.     

Structures of heparin-derived disaccharide bound to cobra cardiotoxins: context-dependent conformational change of heparin upon binding to the rigid core of the three-fingered toxin

Glycosaminoglycans (GAGs) have been suggested to be a potential target for cobra cardiotoxin (CTX) with high affinity and specificity via a cationic belt at the concave surface of the polypeptide. The interaction of GAGs, such as high-molecular weight heparin, with CTXs not only can induce aggregation of CTX molecules but also can enhance their penetration into membranes. The binding of short chain heparin, such as a heparin-derived disaccharide [DeltaUA2S(1-->4)-alpha-D-GlcNS6S], to CTX A3 from Taiwan cobra (Naja atra), however, will not induce aggregation and was, therefore, investigated by high-resolution (1)H NMR. A novel heparin binding site on the convex side of the CTX, near the rigid disulfide bond-tightened core region of Cys38, was identified due to the observation of intermolecular NOEs between the protein and carbohydrate. The derived carbohydrate conformation using complete relaxation and conformational exchange matrix analysis (CORCEMA) of NOEs indicated that the glycosidic linkage conformation and the ring conformation of the unsaturated uronic acid in the bound state depended significantly on the charge context of CTX molecules near the binding site. Specifically, comparative binding studies of several heparin disaccharide homologues with two CTX homologues (CTX Tgamma from Naja nigricollis and CTX A3) indicated that the electrostatic interaction of N-sulfate of glucosamine with NH(3)(+)zeta of Lys12 and of the 2-O-sulfate of the unsaturated uronic acid with NH(3)(+)zeta of Lys5 played an important role. These results also suggest a model on how the CTX-heparin interaction may regulate heparin-induced aggregation of the toxin via the second heparin binding site.     

CXCL12 is displayed by rheumatoid endothelial cells through its basic amino-terminal motif on heparan sulfate proteoglycans

The chemokine CXCL12 (also known as stromal cell-derived factor, SDF-1) is constitutively expressed by stromal resident cells and is involved in the homeostatic and inflammatory traffic of leukocytes. Binding of CXCL12 to glycosaminoglycans on endothelial cells (ECs) is supposed to be relevant to the regulation of leukocyte diapedesis and neoangiogenesis during inflammatory responses. To improve our understanding of the relevance of this process to rheumatoid arthritis (RA), we have studied the mechanisms of presentation of exogenous CXCL12 by cultured RA ECs. RA synovial tissues had higher levels of CXCL12 on the endothelium than osteoarthritis (OA) tissues; in both, CXCL12 colocalized to heparan sulfate proteoglycans (HSPGs) and high endothelial venules. In cultured RA ECs, exogenous CXCL12α was able to bind in a CXCR4-independent manner to surface HSPGs. Desulfation of RA EC HSPGs by pretreatment with sodium chlorate, or by replacing in a synthetic CXCL12α the residues Lys24 and Lys27 by Ser (CXCL12α-K2427S), decreased or abrogated the ability of the chemokine to bind to RA ECs. Ex vivo, synovial ECs from patients with either OA or RA displayed a higher CXCL12-binding capacity than human umbilical vein ECs (HUVECs), and in HUVECs the binding of CXCL12 was increased on exposure to tumor necrosis factor-α or lymphotoxin-α₁β₂. Our findings indicate that CXCL12 binds to HSPGs on ECs of RA synovium. The phenomenon relates to the interaction of HSPGs with a CXCL12 domain with net positive surface charge located in the first β strand, which encompasses a canonical BXBB HSPG-binding motif. Furthermore, we show that the attachment of CXCL12 to HSPGs is upregulated by inflammatory cytokines. Both the upregulation of a constitutive chemokine during chronic inflammation and the HSPG-dependent immobilization of CXCL12 in EC surfaces are potential sites for therapeutic intervention.     

Localization of the heparin binding exosite of factor IXa

Factor IXa (FIXa) is known to have a binding site for heparin that has not been mapped by a mutagenesis study. By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin. These residues, Lys(98), Lys(126), Arg(165), Arg(170), Lys(173), Lys(230), Arg(233), and Lys(239) (chymotrypsin numbering) were substituted with Ala in separate constructs in Gla-domainless forms. Following activation, it was found that all FIXa derivatives cleaved the chromogenic substrate CBS 31.39 with near normal catalytic efficiencies. Similarly, antithrombin inactivated FIXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of a full-length heparin, however, k(2) values were dramatically impaired with certain mutants. Direct binding studies revealed that the same mutants lost their affinities for binding to heparin-Sepharose. Both kinetic and direct binding data indicated that five basic residues of FIXa in the following order of importance, Arg(233) > Arg(165) > Lys(230) > Lys(126) > Arg(170) are critical for binding to heparin. Consistent with these results, examination of the crystal structure of the catalytic domain of FIXa indicated that all five basic residues are spatially aligned in a manner optimal for interaction with heparin.     

Cysteine residues are critical for chemokine receptor CXCR2 functional properties

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.     

Matrix metalloproteinase processing of CXCL11/I-TAC results in loss of chemoattractant activity and altered glycosaminoglycan binding

The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5-73), -(5-63), and -(5-58) forms. NH2-terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5-73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5-73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.     

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.     

Monomeric and Dimeric CXCL8 Are Both Essential for In Vivo Neutrophil Recruitment

Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist in vivo, and if so, the relevance of each form for in vivo function is not known. In this study, using a ‘trapped’ non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease.     

Structural determinants in streptococcal unsaturated glucuronyl hydrolase for recognition of glycosaminoglycan sulfate groups

Pathogenic Streptococcus agalactiae produces polysaccharide lyases and unsaturated glucuronyl hydrolase (UGL), which are prerequisite for complete degradation of mammalian extracellular matrices, including glycosaminoglycans such as chondroitin and hyaluronan. Unlike the Bacillus enzyme, streptococcal UGLs prefer sulfated glycosaminoglycans. Here, we show the loop flexibility for substrate binding and structural determinants for recognition of glycosaminoglycan sulfate groups in S. agalactiae UGL (SagUGL). UGL also degraded unsaturated heparin disaccharides; this indicates that the enzyme released unsaturated iduronic and glucuronic acids from substrates. We determined the crystal structures of SagUGL wild-type enzyme and both substrate-free and substrate-bound D175N mutants by x-ray crystallography and noted that the loop over the active cleft exhibits flexible motion for substrate binding. Several residues in the active cleft bind to the substrate, unsaturated chondroitin disaccharide with a sulfate group at the C-6 position of GalNAc residue. The sulfate group is hydrogen-bonded to Ser-365 and Ser-368 and close to Lys-370. As compared with wild-type enzyme, S365H, S368G, and K370I mutants exhibited higher Michaelis constants toward the substrate. The conversion of SagUGL to Bacillus sp. GL1 UGL-like enzyme via site-directed mutagenesis demonstrated that Ser-365 and Lys-370 are essential for direct binding and for electrostatic interaction, respectively, for recognition of the sulfate group by SagUGL. Molecular conversion was also achieved in SagUGL Arg-236 with an affinity for the sulfate group at the C-4 position of the GalNAc residue. These residues binding to sulfate groups are frequently conserved in pathogenic bacterial UGLs, suggesting that the motif "R-//-SXX(S)XK" (where the hyphen and slash marks in the motif indicate the presence of over 100 residues in the enzyme and parentheses indicate that Ser-368 makes little contribution to enzyme activity) is crucial for degradation of sulfated glycosaminoglycans.     

Profiling heparin-chemokine interactions using synthetic tools.

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.