Optimal Inhibition of X4 HIV Isolates by the CXC Chemokine Stromal Cell-derived Factor 1α Requires Interaction with Cell Surface Heparan Sulfate Proteoglycans
2001; Elsevier BV; Volume: 276; Issue: 28 Linguagem: Inglês
10.1074/jbc.m100411200
ISSN1083-351X
AutoresAgustı́n Valenzuela-Fernández, Tania Palanché, Ali Amara, Aude Magérus‐Chatinet, Ralf Altmeyer, Thierry Delaunay, Jean‐Louis Virelizier, Françoise Baleux, Jean‐Luc Galzi, Fernando Arenzana‐Seisdedos,
Tópico(s)HIV Research and Treatment
ResumoThe chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1α interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first β-strand of SDF-1α as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1α to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1α mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1α when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1α to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1α and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1α to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1α by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor. The chemokine stromal cell-derived factor 1 (SDF-1) is the natural ligand for CXC chemokine receptor 4 (CXCR4). SDF-1 inhibits infection of CD4+ cells by X4 (CXCR4-dependent) human immunodeficiency virus (HIV) strains. We previously showed that SDF-1α interacts specifically with heparin or heparan sulfates (HSs). Herein, we delimited the boundaries of the HS-binding domain located in the first β-strand of SDF-1α as the critical residues. We also provide evidence that binding to cell surface heparan sulfate proteoglycans (HSPGs) determines the capacity of SDF-1α to prevent the fusogenic activity of HIV-1 X4 isolates in leukocytes. Indeed, SDF-1α mutants lacking the capacity to interact with HSPGs showed a substantially reduced capacity to prevent cell-to-cell fusion mediated by X4 HIV envelope glycoproteins. Moreover, the enzymatic removal of cell surface HS diminishes the HIV-inhibitory capacity of the chemokine to the levels shown by the HS-binding-disabled mutant counterparts. The mechanisms underlying the optimal HIV-inhibitory activity of SDF-1α when attached to HSPGs were investigated. Combining fluorescence resonance energy transfer and laser confocal microscopy, we demonstrate the concomitant binding of SDF-1α to CXCR4 and HSPGs at the cell membrane. Using FRET between a Texas Red-labeled SDF-1α and an enhanced green fluorescent protein-tagged CXCR4, we show that binding of SDF-1α to cell surface HSPGs modifies neither the kinetics of occupancy nor activation in real time of CXCR4 by the chemokine. Moreover, attachment to HSPGs does not modify the potency of the chemokine to promote internalization of CXCR4. Attachment to cellular HSPGs may co-operate in the optimal anti-HIV activity of SDF-1α by increasing the local concentration of the chemokine in the surrounding environment of CXCR4, thus facilitating sustained occupancy and down-regulation of the HIV coreceptor. stromal cell-derived factor glycosaminoglycan heparan sulfate HS proteoglycan CXC chemokine receptor regulated on activation normal T cell expressed and secreted human immunodeficiency virus type 1 phosphate-buffered saline peripheral blood mononuclear cells fluorescence resonance energy transfer β-galactosidase 5- (and 6)-{[(4-chloromethyl)benzoyl]-amino} tetramethylrhodamine chondroitin sulfate fetal calf serum long terminal repeat enhanced green fluorescent protein Texas Red human embryo kidney counts per second The CXC chemokine stromal cell-derived factor 1 (SDF-1)1 stimulates intracellular calcium flux and chemotaxis in monocytes, T lymphocytes, and neutrophils (1Oberlin E. Amara A. Bachelerie F. Bessia C. 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Steinman R.M. J. Exp. Med. 1996; 184: 2433-2438Crossref PubMed Scopus (220) Google Scholar). HIV enters cells through sequential interaction of the viral envelope (Env) glycoprotein with the cell surface CD4 molecule and either CXCR4 or CCR5, another G-protein-coupled chemokine receptor (13Michael N.L. Moore J.P. Nat. Med. 2000; 5: 740-741Crossref Scopus (93) Google Scholar). HIV strains that infect macrophages and primary T cells use CCR5 (R5 viruses), whereas HIV strains that infect transformed CD4+ cell lines and primary T cells use CXCR4 (X4 virus). X4 HIV isolates, previously known as T lymphotropic, are characteristic of the late phases of infection and are frequently associated with a sharp decline on CD4 T cell counting and worsening of the clinical status (14Tersmette M. Lange J.M. de Goede R.E. de Wolf F. Eeftink- Schattenkerk J.K. Schellekens P.T. Coutinho R.A. Huisman J.G. Goudsmit J. Miedema F. 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Murphy P.M. Farber J.M. Annu. Rev. Immunol. 1999; 17: 657-700Crossref PubMed Scopus (1890) Google Scholar). Occupancy of CXCR4 by SDF-1 prevents the interaction of X4 HIV Env with CXCR4, thus blocking the activation of the fusogenic capacity of gp41 and, ultimately, the entry of virions into the cell. The biological functions of chemokines are thought to be influenced by their association with cellular or matrix extracellular glycosaminoglycans (GAGs). Usually attached to a core protein to form proteoglycans (19Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar), the common GAGs (heparin, heparan sulfate (HS), dermatan sulfate, and chondroitin sulfate (CS)) are highly sulfated oligosaccharides. With the exception of CS, all of the above-mentioned GAGs are characterized by a high degree of structural heterogeneity (20Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1680) Google Scholar, 21Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1013) Google Scholar). Another common GAG is hyaluronic acid, but it is never attached to a core protein and is not a GAG sulfate (19Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1349) Google Scholar). We showed previously that SDF-1α, the best characterized isoform of the chemokine, interacts selectively with HS, with relatively high affinity in vitro (22Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). HSs are also responsible for the binding of SDF-1α to the membrane of CXCR4-negative epithelial or endothelial cells (22Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar), which have been identified among the few cell types producing the chemokine in human tissues (4Pablos J.L. Amara A. Bouloc A. Santiago B. Caruz A. Galindo M. Delaunay T. Virelizier J.L. Arenzana-Seisdedos F. Am. J. Pathol. 1999; 155: 1577-1586Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 5Coulomb-L'Herminé A. Amara A. Durand-Gasselin I. Foussat A. Schiff C. Ledee N. Galanaud P. Arenzana-Seisdedos F. Emilie D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8585-8590Crossref PubMed Scopus (93) Google Scholar). A cluster of basic residues (Lys24, His25, and Lys27) in the first β-strand of SDF-1α is necessary for the interaction of SDF-1α with HS both in vitro and in intact cells. Indeed, a SDF-1α derivative carrying three Ser substitutions in the Lys24, His25, Lys27 cluster (SDF-1 3/6) is unable to bind HS. Inactivation of the putative HS-binding site does not affect the capacity of SDF-1α to ligate CXCR4, suggesting that cellular HS proteoglycans (HSPGs) are dispensable for the HIV inhibitory effect of the chemokine. However, the contribution of cellular GAGs to the optimal, inhibitory activity of the CC chemokines RANTES or macrophage inflammatory protein 1β against HIV R5 is supported by an increasing body of evidence. Surprisingly, in some cases, the optimal inhibition of HIV-1 infection shown by RANTES·HS complexes contrasts with the impairment of the agonistic capacity of the complex as compared with the chemokine alone (23Burns J.M. Lewis G.K. DeVico A.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14499-14504Crossref PubMed Scopus (34) Google Scholar). Consequently, it can be hypothesized that despite the apparent preserved functionality of HS-binding disabled SDF-1α mutants, complexes of SDF-1α with cellular HSPGs may also be determinant for maintaining the optimal capacity of the chemokine to prevent entry of X4 HIV isolates. Analysis of the kinetics of SDF-1α/CXCR4 interactions investigated by fluorescence resonance energy transfer (FRET) and real time measurement of CXCR4 activation indicates that complexing with HS does not modify the intrinsic capacities of SDF-1α to interact with its cognate receptor. However, our findings indicate that selective interaction of SDF-1α with cell membrane HS is required for optimal inhibition of CXCR4 envelope-mediated cell-HIV fusion. Neither SDF-1α induced endocytosis nor recycling of CXCR4 is influenced by cell surface HSPGs. However, combining FRET and laser scanning confocal microscopy, we demonstrate the accumulation of large amounts of SDF-1α at the cell surface, which is largely independent of CXCR4 and relies on the presence of HS. We propose that attachment of SDF-1α to HSPGs increases the local concentration of the chemokine in the surrounding of CXCR4 thus facilitating sustained occupancy and down-regulation of the HIV coreceptor. SDF-1α bound to cell membrane HS may be a major constituent of a natural barrier limiting transmission and propagation of X4 HIV isolates in vivo. Soluble HS was obtained from Sigma (catalogue number H5393). Heparitinase I (EC 4.2.2.8) and chondroitinase ABC (EC 4.2.2.4) were purchased from Seikagaku Corp. K15C is an anti-SDF-1 monoclonal antibody (IgG2a κ) against the amino-terminal region of SDF-1 (22Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Q4120, an anti-CD4 monoclonal antibody (IgG1) that blocks the binding of gp120 to CD4, was obtained from the Medical Research Council AIDS Reagent Project. 125I-SDF-1α (specific activity, 2200 Ci/mmol) was purchased from PerkinElmer Life Sciences. Fluorescent calcein-AM, Indo-1/AM, and 5- (and 6)-{[(4-chloromethyl)benzoyl]-amino} tetramethylrhodamine (CMTMR) probes were obtained from Molecular Probes. The HeLa P4.2 cell clone is stably transfected with a human CD4 cDNA and a HIV-LTR-driven β-gal reporter gene (24Clavel F. Charneau P. J. Virol. 1994; 68: 1179-1185Crossref PubMed Google Scholar). HeLa 243 cells, provided by Dr. M. Alizon, co-express both Tat and Env HIV-1 proteins from an HIV-LTR-driven vector derived from the X4 pLai proviral molecular clone (25Schwartz O. Alizon M. Heard J.M. Danos O. Virology. 1994; 198: 360-365Crossref PubMed Scopus (70) Google Scholar). Both cell cultures were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum (FCS), Glutamax, antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin, and 2 µm of Methotrexate. Baby hamster kidney cells (BHK-21) (ATCC CCL-10) were maintained in Glasgow's modified Eagle's medium (Life Technologies, Inc.) containing 5% FCS, 20 mm Hepes, and 10% tryptose phosphate broth. To express HIV-Env Lai BHK-21, cell monolayers were infected with a defective Semliki Forest virus encoding a full-length sequence of the HIV-1 LAI env gene at multiplicity of infection of 10 for 1 h. Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors using Ficoll (Amersham Pharmacia Biotech) density gradient centrifugation. PBMC were cultured in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% heat-inactivated FCS. PBMC were activated during 3 days with 1 µg/ml phytohemagglutinin (Murex Diagnostics S. A.). Adherent human embryo kidney (HEK) 293 cells were grown in minimum essential medium supplemented with 10% FCS. Wild type SDF-1α, SDF-1 3/6 (K24S/H25S/K27S), SDF-1 2/6 (K24S/K27S), and SDF-1α P2G were synthesized by the Merrifield solid phase method on a fully automated peptide synthesizer (Pioneer, Perspective Biosystems, and PerkinElmer Life Sciences). The procedure used for chemokine synthesis we described previously (22Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). The concentration of each chemokine was determined by amino acid analysis on a 6300 Beckman amino acid analyzer after hydrolysis for 20 h in 6 n HCl, 0.2% phenol in the presence of a known amount of norleucine as internal standard. All chemicals for the synthesis were purchased from Perspective Biosystems and PerkinElmer Life Sciences. CEM cells (2.5 × 106 cells/ml) were incubated in PBS with 0.25 nm iodinated SDF-1α (PerkinElmer Life Sciences, specific activity, 2200 Ci/mmol) and various concentrations of unlabeled SDF-1α or SDF-1 2/6 for 1 h at 4 °C in a final volume of 300 µl. Incubations were terminated by centrifugation at 4 °C. The cell pellets were washed twice in ice-cold PBS. Nonspecific binding was determined in the presence of 1 µm unlabeled SDF-1α. Cell pellet-associated radioactivity was counted using an LKB-Wallac microcomputer controlled 1272 CliniGamma counter. The binding data were analyzed using GraphPrad Prism 2.0 software. Adherent cells were plated 2 days before binding experiments. Cells were detached with 2 mm EDTA in PBS and washed twice with ice-cold binding buffer (RPMI 1640, 20 mm Hepes, 1% bovine serum albumin). 4 × 105 cells were incubated in the presence of the indicated concentration of chemokines in a total volume of 200 µl for 90 min at 4 °C with stirring. Unbound chemokine was removed by washing with binding buffer, and cell-bound SDF-1α was detected by incubation with the anti-SDF-1 monoclonal antibody K15C (15 µg/ml, diluted in PBS, 1% bovine serum albumin), which recognizes an epitope encompassing the amino-terminal end of the chemokine. After staining with phycoerythrin-conjugated anti-mouse immunoglobulins (Southern Biotechnology), cells were fixed in 1% formaldehyde buffer and analyzed by flow cytometry in a FACSCalibur (Becton Dickinson). HeLa 243 cells were co-incubated with HeLa P4.2 cells in a total volume of 250 µl in 96-well plates at a 1:1 ratio during 18 h. After 18 h of co-culture, cells were washed twice with PBS at 37 °C and lysed with 50 µl of lysis buffer (from Roche β-gal reporter gene assay kit) for 20 min, and the enzymatic activity was evaluated according to the instructions provided by the manufacturer (β-gal reporter gene assay (chemiluminescent), Roche Diagnostics). This technique was modified from a procedure described previously by Puri et al. (26Puri A. Hug P. Jernigan K. Barchi J. Kim H.Y. Hamilton J. Wiels J. Murray G.J. Brady R.O. Blumenthal R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14435-14440Crossref PubMed Scopus (120) Google Scholar) and will be described in detail elsewhere. Briefly, BHK-21 expressing HIV-1 Lai Env and phytohemagglutinin-activated PBMC were loaded with aqueous calcein-AM and CMTMR, respectively. After for 15 h in culture, cell syncytia labeled with the two cytoplasmic dyes were analyzed by flow cytometry. When indicated, enzymatic cleavage of cell surface GAG was obtained by pretreating cells for 30 min before the onset of the culture with 5 milliunits/ml of either heparitinase I or chondroitinase ABC. GAG lytic enzymes were maintained in the cell cultures until the end of the experiment. HeLa P4.2 cells were incubated culture medium containing 5 µm Indo-1/AM for 30 min at 37 °C followed by a 15-min incubation in probe-free medium. Measurements were made at 37 °C in Hepes buffer (5.6 mm glucose, 10 mm Hepes, 0.4 mm NaH2PO4, 137.5 mmNaCl, 1.25 mm MgCl2, 1.25 mmCaCl2, 6 mm KCl, and 1% bovine serum albumin) supplemented with protease inhibitors (40 µg/ml bestatin, 40 µg/ml bacitracin, 50 µg/ml chymostatin, 20 µg/ml phosphoramidon, and 1 µg/ml leupeptin). Fluorescence emission was detected at 405 and 475 nm (excitation, 338 nm). Real time intracellular calcium measurements from cell suspensions were made on a Fluorolog (SPEX) spectrofluorometer equipped with a 450-W xenon lamp, a double grating excitation monochromator, and two single grating emission monochromators. Slits were set to 4 or 6 mm, yielding bandwidths of 7.2 or 10.8 nm at excitation and 14.4 and 21.6 nm at emission, respectively. Data were acquired with two photon counting photomultipliers. Real time measurements were carried out with an Applied Photophysics SK1E rapid mixing apparatus modified for mixing with living cells (final concentration, 106 cells/ml). The observation chamber was an Hellma 176.002 (100 µl) quartz circulation cuvette placed on the cuvette holder of the SPEX fluorolog spectrofluorometer. At a typical 12 bars air ram pressure, the flow rate was 4 ± 0.5 ml/s. Each measurement started after renewal of 7–8 times the volume of the observation cuvette. The dead volume (100 µl) and dead time of the apparatus (25 ± 5 ms) were experimentally determined. Time-based recordings were typically sampled every 20–100 ms. Actin polymerization was tested as described by Bleul et al. (27Bleul C.C. Fuhlbrigge R.C. Casasnovas J.M. Aiuti A. Springer T.A. J. Exp. Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1289) Google Scholar). Activated PBMC were incubated in RPMI medium supplemented with 10% heat-inactivated FCS, Glutamax, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) at 37 °C and induced with the chemokines as indicated. Forty-five s after induction, 100 µl of a stop solution containing 4 × 10−7m FITC-labeled phalloidin, 0.5 mg/ml 1-α-lysophosphatidylcholine (Sigma), and 15% formaldehyde in PBS were added to PBMC (in 400 µl). Analysis of FITC-phalloidin-labeled cells was performed by flow cytometry. Detection and quantification of association and dissociation kinetics of SDF-1α/CXCR4 interaction were measured by FRET, between enhanced green fluorescent protein (EGFP) and Texas Red (TR) chromophores on living cells. To this purpose, we engineered an EGFP-CXCR4 chimeric protein by fusing EGFP to the extracellular, amino-terminal end of CXCR4 as described (28Vollmer J.Y. Alix P. Chollet A. Takeda K. Galzi J.L. J. Biol. Chem. 1999; 274: 37915-37922Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) and synthesized a SDF-1α derivative carrying a single TR molecule bound to Lys68. A clone of HEK 293 cells stably expressing EGFP-CXCR4 was isolated. Upon excitation of EGFP-CXCR4 at 460–470 nm wavelength, association of the fluorescent ligand with the receptor was measured by monitoring the reduction of EGFP emission at 510 nm as described by Vollmer et al. (28Vollmer J.Y. Alix P. Chollet A. Takeda K. Galzi J.L. J. Biol. Chem. 1999; 274: 37915-37922Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Similarly, dissociation was measured as the increase of EGFP emission at 510 nm, when bound SDF-1α-TR was displaced by an excess of unlabeled SDF-1α. Real time fluorescence data (in counts per second (cps)) were stored using the DM3000 software provided with the fluorolog 2 spectrofluorometer and analyzed with Kaleidagraph (Synergy Software) by various analytical expressions, such as exponentials, sum of exponentials, or sum of exponentials and linear relationships (29Heidmann T. Changeux J.P. Eur. J. Biochem. 1979; 94: 255-279Crossref PubMed Scopus (152) Google Scholar, 30Neubig R.R. Sklar L.A. Mol. Pharmacol. 1993; 43: 734-740PubMed Google Scholar). The uniqueness of the fit was checked by repeated calculations performed with distinct experimental data points (from several experiments) or with distinct values for initiation of the fitting procedure. For a bimolecular reaction scheme,R+L↔RL(Eq. 1) describing ligand-receptor interactions, fluorescence traces were analyzed using the following relationship,y=λexp(−kapp×t)(Eq. 2) where λ is the amplitude of the exponential fluorescence decline, t is the recording time, andk app is the apparent rate constant of the reaction. The apparent rate constant k app is equal to k app = k 1 ×L + k –1, where L is the concentration of ligand, k 1 is the forward rate constant, and k –1 is the backward rate constant of Equation 1. HEK 293 cells expressing EGFP-CXCR4 were grown on glass coverslips and incubated in Ringer solution (140 mm NaCl, 5 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 10 mm Hepes, 11 mm glucose, pH 7.3) at 19 °C to avoid CXCR4 endocytosis. Images were taken on an inverted microscope (Nikon Eclipse TE300) equipped with a confocal imaging system (Bio-Rad MRC 1024 ES) using a Plan Apo × 40 oil immersion objective (Nikon). Excitation light (488 nm) was obtained using 1 or 3% power from a 30-mW krypton-argon laser. Emitted fluorescence was recorded at 522 ± 5 and 605 ± 22 nm and was color-coded (256 gray levels). Images were acquired from a section located 3–6 µm above the glass coverslip at 2–4 Hz and analyzed using Bio-Rad time course software. Ligands were applied in the bath using a perfusion pipette. We have previously identified a cluster of basic residues (Lys24, His25, and Lys27) in the first β-strand of SDF-1α necessary for interaction with HS, bothin vitro and in intact cells (22Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Indeed, a mutant SDF-1α carrying the substitutions K24S/H25S/K27S (SDF-1 3/6) selectively failed to bind HS. To further delimit the boundaries of the HS-binding site, new SDF-1α mutants were engineered by replacing each amino acid of the basic cluster with Ser. Binding of SDF-1α derivatives to cell surface HS was assessed using an anti-SDF-1α monoclonal antibody (K15C) that recognizes an epitope in the amino-terminal end of the chemokine. Each single-point mutant (K24S, H25S, or K27S) SDF-1α induced CXCR4-dependent actin polymerization and bound to cell surface HS with efficiency and affinity comparable to wild type SDF-1α (data not shown). In contrast, a mutant combining K24S/K27S substitutions (SDF-1 2/6) failed, like SDF-1 3/6, to bind to cell surface HS at concentrations up to 1 µm (Fig.1 b). Gel chromatography and plasmon resonance spectroscopy confirmed that SDF-1 2/6 lost, to the same extent as SDF-1 3/6, the affinity for immobilized heparin. Like SDF-1 3/6, the SDF-1 2/6 mutant bound (Fig. 1 a) and induced CXCR4 activation with affinity and efficiency comparable to the wild type counterpart (Fig. 1 c). These results reinforce our conviction (22Amara A. Lorthioir O. Valenzuela A. Magerus A. Thelen M. Montes M. Virelizier J.L. Delepierre M. Baleux F. Lortat-Jacob H. Arenzana-Seisdedos F. J. Biol. Chem. 1999; 274: 23916-23925Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar) that amino acid substitutions inactivating the putative HS-binding site of SDF-1α preserve the overall three-dimensional conformation of the protein. Moreover, our findings confirm that the HS-binding site on SDF-1α is indeed outside the amino-terminal domain involved in occupancy and signaling of CXCR4. However, they do not exclude the possibility that other residues, such as Arg41and Lys43, contribute to forming an accessible, positively charged surface for interaction with HS. In any case, Lys24and Lys27 appear to be the critical residues, which either make physical contact with or critically shape the surface for attachment of HS. The conserved ability of HS-binding-disabled derivatives of SDF-1α to bind and activate CXCR4 prompted us to investigate whether attachment of SDF-1α to cell surface HSPGs contributes to the HIV inhibitory activity of the chemokine. For this purpose, an HIV-Env-mediated cell-ce
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