3 Laboratoire de Biologie Cellulaire, Biothérapies Bénéfices et Risques, UPRES 3410, UFR-SMBH, Université Paris XIII, 74, rue Marcel Cachin, 93017, Bobigny, France
4 Laboratoire de Ciblage Fonctionnel des Tumeurs Solides, UPRES 2360, UFR-SMBH, Université Paris XIII, 74, rue Marcel Cachin, 93017, Bobigny, France and Hôpital Jean Verdier, 93017, Bondy, France
5 CEA, Saclay, Département d'Ingénierie et d'Etudes des Protéines, 91191 Gif-sur-Yvette, France
Received on January 20, 2003; revised on May 20, 2003; accepted on May 20, 2003
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Abstract |
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Key words: CCR5 / HIV / RANTES / syndecans
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Introduction |
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It was recently suggested that cell surface heparan sulfate (HS) proteoglycans (HSPGs) act as HIV-1 attachment receptors on specific target cells. It was also shown that soluble polyanions inhibit HIV infection (Callahan et al., 1991; Carre et al., 1995
; Oravecz et al., 1997
). It has recently been shown that a family of HSPGs, the syndecans (SDs), mediates HIV-1 attachment on human primary macrophages (Saphire et al., 2001
). Cell surface HSPGs are anchored in the cell membrane either via a transmembrane domain (the SDs) or by glycosylphosphoinositol linkage (glypicans) (Saphire et al., 2001
). The aim of this study was to determine whether RANTES binds PGs expressed on the plasma membrane of cells, expressing or lacking RANTES GPCRs, CCR5.
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Results |
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Migrations of SD-1 and -4 from whole cell lysates were more heterogeneous than those of the PGs from the plasma membrane of the cells, solubilized in the presence of the same detergent; therefore, differences in the associations of these PGs with cellular constituents may occur according to their localization. In parallel, heparitinases I- and III-, and chondroitinase ABC-treated PGs were prepared from CCR5+/CCR5 HeLa cells lysates containing Triton X100 and urea. Their analysis revealed 3132-kDa, 34-kDa, 45-kDa, 6062-kDa, and 90-kDa proteins, all immunoreactive with the anti-stub 3G10 mAb but not with the isotype. The 3132-kDa proteins were also immunoreactive with anti-SD-4 mAb 5G9, the 45-kDa and 90-kDa proteins with anti-SD-1 mAb DL-101, and the 34-kDa proteins with anti-SD-2 Abs (Figure 2B and data not shown). Therefore, the core protein of SD-4 migrates as 3132-kDa species, whereas that of SD-1 as 90-kDa and 45-kDa species. The 90-kDa species may represent a dimeric form of the 45-kDa one. The apparent molecular masses of these core proteins are in agreement with those previously observed (Simons and Horowitz, 2001). They are, however, higher than the predicted ones (Rosenberg et al., 1997
). They may result, according to others (Oh et al., 1997
; Zimmermann and David, 1999
), from noncovalently linked sodium dodecyl sulfateresistant homo- or hetero-oligomerization.
Binding of RANTES to HeLa cells expressing or lacking CCR5
Because RANTES aggregates at µM concentrations (Hoogewerf et al., 1997), the cells were incubated in this study with low nM concentrations of RANTES. The binding of 125I-RANTES to HeLa cells expressing or lacking CCR5 was dose-dependent and saturable. It was significantly inhibited by unlabeled RANTES. Maximum 125I-RANTES binding (B/T) to HeLa cells expressing CCR5, determined in the absence of unlabeled RANTES, was 13 ± 0.3%. Minimum 125I-RANTES binding (B/T) to these cells, determined in the presence of 10.4 nM unlabeled RANTES, was 9 ± 1%. Scatchard analysis (Figure 3B) of the displacement curve of binding of 125I-RANTES (7.5 pM) to these cells (Figure 3A) revealed two classes of binding sites: one with 1500 ± 690 binding sites per cell and a 0.27 ± 0.17 nM Kd and the other with 34,500 ± 690 binding sites per cell and a 4.3 ± 1.45 nM Kd. Therefore, RANTES binds to high- and low-affinity binding sites of HeLa cells expressing CCR5.
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Colocalization experiments between RANTES and PGs
Fluorescently labeled biotinylated 1-RANTES partly colocalized with SD-1 and -4 on the plasma membranes of HeLa cells expressing or lacking CCR5 as assessed by indirect immunofluorescence: the yellow (red-green colocalization) staining strongly suggests the formation of a complex between the chemokine and respectively, SD-1 or -4 (Figure 8). Preparations, incubated with or without RANTES and with the isotypes, were not stained (data not shown). This further shows an association of RANTES and SD-1 or -4.
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Effects of RANTES concentrations on its binding to CCR5 and PGs
In the presence of RANTES, from 2.5 nM up to 250 nM (which represents, respectively, the physiological seric concentrations of RANTES and the pathological ones in inflammatory diseases; Gattegno et al., unpublished data), no changes in bound CCR5 from the CCR5-positive HeLa cells was detected (Figure 7, lanes 13). After incubation of these cells with 2.5 nM RANTES, an increased amount of the SD-1 and -4 molecules bound by the chemokine and immunoreactive with the anti-SD antibodies was observed, as compared with the results observed in the presence of 250 nM RANTES (Figure 7, lanes 911 and 57). This suggests that SD-1 and -4, expressed on the CCR5-positive HeLa cells surface, may sequester RANTES if the chemokine is present at low nM concentrations. This may facilitate the subsequent interaction of RANTES with CCR5. On the contrary, some events, such as RANTES aggregation at high concentration (250 nM), may decrease RANTES binding to the ectodomains of SD-1 and -4 expressed on the cells. If the CCR5-positive HeLa cells were incubated for 2 h at 37°C with RANTES (from 2.5 nM to 250 nM), no changes in the labeling of the ectodomains of SD-1 or -4 by anti-SD-1 DL-101 or anti-SD-4 5G9 mAbs was detected (data not shown). This suggests that RANTES has no effect on the shedding of these molecules. We nevertheless currently observe that the ectodomains of SD-1 and -4, which are constitutively shed from the HeLa cells, form a trimolecular complex with RANTES (Gattegno et al., unpublished data).
Effect of BCD on RANTES associations with CCR5 and SD-1
We next examined whether RANTES associations with SD-1 and -4 may take place in membrane rafts. Pretreatment of the CCR5-positive HeLa cells with hydroxypropyl-ß-cyclodextrin (BCD), which is known to extract membrane cholesterol (Nguyen and Taub, 2002), strongly decreased, as expected (Nguyen and Taub, 2002
), the binding of the two anti-CCR5 mAbs tested here (2D7 and 182, both specific for ECL2) as well as that of anti-CXCR4 mAb 12G5, to these cells; the control, anti-CD4 mAb Q4120, did not show any change in mean fluorescence intensity (Table I). BCD treatment of these cells also strongly decreased RANTES bound CCR5 and abolished the chemokine binding to SD-1 but did not modify its binding to SD-4 (Figure 9, lanes 2 versus 1, 6 versus 5 and 4 versus 3). This suggests that cholesterol and lipid rafts are involved in RANTES binding to CCR5 and SD-1 and indicates that rafts do not seem to play a role in RANTES binding to SD-4.
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Discussion |
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We have previously reported that RANTES binding to CCR5-positive primary macrophages is inhibited by anti-CCR5 mAb 2D7 and by heparitinase pretreatment of the cells (Mbemba et al., 2001) suggesting the role of both HSPGs and GPCRs. To further investigate the role of these molecules in RANTES binding, we compare here the binding characteristics of RANTES to HeLa cells expressing CCR5 or lacking specific RANTES GPCRs. Few studies have reported displacement binding assays to cells of iodine-labeled RANTES by unlabeled RANTES (Hoogewerf et al., 1997
). We show that RANTES specifically binds to HeLa cells expressing CCR5 with two classes of binding sites, characterized by 0.27 nM and 4.3 nM Kd, respectively. These Kd values are of the same order of magnitude as those previously reported for the binding of ß-chemokines to CCR5 (Samson et al., 1997
) and for that of RANTES to heparin, respectively (Martin et al., 2001
). Moreover, specific RANTES binding to CCR5- negative HeLa cells, lacking any RANTES GPCRs, was observed with a 56 nM Kd, which may be related with the chemokine binding to GAGs. The fact cannot be excluded that in all these experiments higher concentrations of the unlabeled chemokine may be required to further compete this binding. This suggests that other lower-affinity binding sites for RANTES on HeLa cells may occur. However, because RANTES aggregates at µM concentrations (Hoogewerf et al., 1997
) in this study, the cells were incubated in the presence of low nM concentrations of RANTES.
RANTES binding to the CCR5-negative cells was further investigated. Strong and significant inhibitions by exogeneous heparin as well as by RANTES heat denaturation were observed, which suggests the involvement of RANTES 3D structure and indicates that heparin may act as a competitive inhibitor.
The biological activity of chemokines has been shown to be influenced by their association with GAGs (Hoogewerf et al., 1997; Clasper et al., 1999
). The presence of GAGs attached to the cell surface has been reported to increase the binding affinity of RANTES to CCR1 and others GPCRs (Hoogewerf et al., 1997
). Enzymatic removal of GAGs from lymphocytes was reported to abrogate the ability of RANTES to elicit an intracellular Ca2+ signal (Burns et al., 1998
). However, in another work, no effect of cell surface GAGs was found on chemokine binding (Kuschert et al., 1999
). It was also demonstrated that removal of GAGs from the surface of a PM1 T cell line or from MDMs reduces the antiviral effect of RANTES (Amzazi et al., 1998
; Wagner et al., 1998
). Moreover, enzymatic removal of GAGs from the surface of CCR5-expressing Chinese hamster ovary cells by different glycosidases did not result in a reduction of the ability of RANTES to bind CCR5 or to induce a functional response (Martin et al., 2001
). Therefore studies on the role of GAGs on RANTES binding and function are conflicting.
In the present study, the staining of the plasma membrane of HeLa cells lacking CCR5 by fluorescently labeled biotinylated 1-RANTES was decreased when these cells were pretreated with a mixture of heparitinases I and III or with heparitinases I and III and chondroitinase ABC mixture. These data suggest that the binding of RANTES to these cells depends from GAGs.
By the use of coimmunoprecipitation experiments, we demonstrate that RANTES forms complexes with glycanated SD-1 molecules and mainly with homo- or hetero-oligomerized SD-4 molecules expressed on the plasma membrane of CCR5-negative HeLa cells, lacking any RANTES GPCRs. The fact that neither SD-2 nor betaglycan were associated to this complex indicates the specificity of the observed data. The fact that pretreatment of the CCR5-negative HeLa cells with glycosaminidases abolished RANTES binding to SD-1 and SD-4 further demonstrates, in accordance with the data already described, the involvement of GAGs in RANTES association to these PGs.
In addition, our data show that RANTES added to the plasma membrane of CCR5-positive HeLa cells forms complexes that comprise CCR5 and glycanated SD-1 and SD-4, both immunoreactive with anti-HS mAb 10 E4 but neither with SD-2 nor betaglycan. The glycanation of these SD-1 and SD-4 molecules was demonstrated by the fact that they were immunoreactive with anti-HS 10 E4 mAb and, after removal of their GAGs by glycosaminidase treatment, with 3G10 mAb. Moreover, removal of GAGs by glycosaminidases treatment of the complexes, RANTES/SD-1/SD-4/+/CCR5, immobilized on beads, reversed SD-1 and SD-4 bindings.
The complexes RANTES/SD-1/SD-4 occuring on the plasma membrane of HeLa cells were further investigated by using confocal laser scanning microscopic analysis of fluorescently labeled molecules. By this approach, we observed that RANTES colocalizes with, respectively, SD-1 and SD-4 molecules on the plasma membrane of HeLa cells expressing or lacking CCR5.
Here again, GAG removal by glycosaminidases treatment of the CCR5-positive HeLa cells prevented RANTES binding to SD-1 and SD-4, as assessed by the results of the coimmunoprecipitation experiments. Moreover, this treatment also strongly decreased RANTES binding to CCR5. Therefore, GAG-dependent binding of RANTES to SD-1 and SD-4 may facilitate the subsequent interaction of this chemokine with CCR5 on the CCR5-positive HeLa cells tested here, characterized by low membrane expression of CCR5.
If the CCR5-positive HeLa cells were incubated in the presence of RANTES at physiological seric concentrations of 2.5 nM up to pathological 250 nM concentrations (similar to those observed in inflammatory diseases), no changes in bound CCR5 amount was detected. In contrast, after incubation of these cells with 2.5 nM RANTES, an increased amount of bound SD-1 and SD-4 molecules, immunoreactive with the anti-SD antibodies, was observed, as compared with the results observed with 250 nM RANTES. This suggests that SD-1 and SD-4 molecules, expressed by the CCR5-positive HeLa cells, may sequester RANTES in the presence of the chemokine at low nM concentration. However, whether RANTES is oligomerized after its binding to SD-1 and SD-4 and whether its aggregation at high (250 nM) concentrations decreases its binding to SD-1 and SD-4 are currently under investigation. Interestingly, we currently observe that RANTES forms a trimolecular complex with the ectodomain of SD-1 and SD-4, respectively, after their constitutive shedding from the HeLa cell surface; however, this chemokine does not induce an accelerated shedding of these PGs (Gattegno et al., unpublished data).
It was recently demonstrated that clustering induces redistribution of SD-4 core protein into raft membrane domains (Tkachenko and Simons, 2002). CCR5 molecules also associate with membrane rafts (Manes et al., 1999
; Nguyen and Taub, 2002
). In the present article, the pretreatment of the CCR5-positive HeLa cells with BCD, which is known to extract membrane cholesterol (Nguyen and Taub, 2002
), did not modify RANTES binding to SD-4; however, this pretreatment abolished the chemokine binding to SD-1 and strongly decreased RANTES bound CCR5. This suggests the involvement of cholesterol and lipid rafts in RANTES binding to most of the CCR5 molecules and also to SD-1 but not to SD-4. This also suggests that the complexes formed by RANTES and SD-1 may take place in different membrane compartements than those occuring between this chemokine and SD-4.
Otherwise, we did not detect any RANTES GPCRs or SD-1, SD-2, or SD-4 molecules on the plasma membrane of the human K562 leukemia cells and in their lysates. We also did not detect any RANTES binding to these lysates (data not shown). It has been previously reported that K562 cells express betaglycan and SD-3 (Saphire et al., 2001), so these data further rule out RANTES binding to betaglycan. However, whether RANTES also binds to PGs belonging to the glypican family has also to be investigated.
We have investigated whether SDs are coassociated with GPCRs in a RANTES-independent manner. We first observed by confocal microscopy analysis a partial spontaneous colocalization of CCR5 with SD-1 and SD-4, respectively, on the CCR5-positive HeLa cells. Moreover, we observed that in the absence of RANTES, CCR5 from the CCR5-positive HeLa cells coimmunoprecipated with glycanated SD-1 and SD-4 molecules, immunoreactive, respectively, with anti-SD-1 and anti-SD-4 mAbs as well as with anti-HS mAb 10E4. After GAG removal by glycosaminidases treatment, these PGs were immunoreactive with 3G10 mAb, which further demonstrates that they were glycanated. The fact that neither SD-2 nor betaglycan were associated to the heteromeric complex formed by CCR5, SD-1, and SD-4 indicates the specificity of the observed interactions.
In conclusion, the present data strongly suggest that glycanated SD-1 and SD-4 but not SD-2 nor betaglycan serve as ligands for both RANTES and CCR5. The GAG dependence of RANTES binding to these PGs does not exclude that additional proteinprotein interactions may take place. Our data suggest that the heteromeric complex SD-1/SD-4/CCR5 is likely a functional unit that may modulate RANTES binding to CCR5-positive cells. Whether these PGs also serve as signaling receptors involved in specific RANTES functions remains to be investigated. The role of these interactions in the pathophysiology of RANTES and in HIV infection deserves further study.
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Materials and methods |
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FACScan analysis of the cells
Aliquots of 5 x 105 scraped HeLa cells were incubated for 30 min at 4°C in 100 µl phosphate buffered saline (PBS), supplemented with 0.05% bovine serum albumin (BSA, Sigma-Aldrich, Saint Quentin Fallavier, France) and with 12.5 µg anti-CD4 mAb (murine IgG1; clone Q4120; Sigma-Aldrich), anti-CD14 mAb (murine IgG2b; Becton Dickinson, Pont de Claix, France), anti-CCR5 mAb 2D7 or anti-CXCR4 mAb 12G5 (mouse IgG2a; both from Pharmingen, Pont de Claix, France), anti-CCR5 mAb 182 (mouse IgG2b, clone 45531), anti-CCR1 mAb (mouse IgG2b; clone 55504.111; both from R&D systems, Abigdon, U.K), or the isotypes (Pharmingen). After washing, cells were incubated for 30 min at 4°C with fluorescein isothiocyanate(FITC) labeled goat anti-mouse Ig antibodies (1/20; Pharmingen), fixed in 1% paraformaldehyde (PFA) (Sigma-Aldrich) in PBS (PBS-PFA) and analyzed on a FACScan (Becton Dickinson). In some experiments, to extract the cholesterol from the plasma membrane, the CCR5 + HeLa cells were preincubated for 1 h at 37°C in 20 mM BCD (Sigma-Aldrich) as described (Nguyen and Taub, 2002).
Immunofluorescence staining and microscope analysis of the cells
Adherent HeLa cells, cultured on glass coverslips, were incubated for 1 h at room temperature with anti-SD-1 mAb (10 µg/ml; mouse IgG-1; clone DL-101, specific for an epitope corresponding to the ectodomain of human SD-1, Santa Cruz Biotechnology, Santa Cruz, CA), anti-SD-4 mAb (10 µg/ml; mouse IgG2a; clone 5G9, specific for an epitope corresponding to the ectodomain of human SD-4; Santa Cruz Biotechnology), or their isotypes (Pharmingen). Bound antibodies were revealed by a subsequent incubation of the cells for 30 min at room temperature in the darkness with a Cy-3-conjugated donkey anti-mouse antibody (1:400; Jackson Immunoresearch, Baltimore, MD). Cells were then fixed in PBS-PFA and mounted in fluorescent mounting medium (Dako, Glostrop, Denmark). Alternatively, HeLa cells were fixed in PBS-PFA and incubated for 1 h at room temperature with anti-SD-1 mAb (10 µg/ml; mouse IgG1; clone B-B4, Serotec, Oxford, U.K.), anti-ß-glycan Ab (10 µg/ml; goat IgG, R&D Systems) or with their isotypes (mouse IgG1 or goat IgG, Jackson Immunoresearch). Bound antibodies were revealed by Cy3-conjugated donkey anti-mouse antibody or Alexa Fluor 488labeled donkey anti-goat antibody (Molecular Probes, Eugene, OR). In parallel, HeLa cells were fixed with methanol, air-dried, rehydrated with PBS, and incubated for 1 h at room temperature with anti-SD-2 goat Ab (10 µg/ml; goat IgG, specific for an epitope corresponding to the C-terminal domain of human SD-2; Santa Cruz Biotechnology) or its isotype. Bound antibodies were revealed with Alexa Fluor 488labeled donkey anti-goat secondary antibody and observed using an Olympus fluorescence microscope.
Confocal microscopy
To visualize a colocalization of RANTES and, respectively, SD-1 or SD-4, HeLa cells grown on glass coverslip were incubated with either anti-SD-1 mAb DL-101, anti-SD-4 mAb 5G9 or their isotypes and revealed as described. Cells were then incubated for 1 h at 4°C in the presence or the absence of 1-biotinylated RANTES (biotinylated on the first amino acid of RANTES; Vita et al., 2002). Bound 1-biotinylated RANTES was revealed with a streptavidinAlexa Fluor 488 complex (1:100, Molecular Probes). Cells were fixed with PFA, mounted, and observed using a Zeiss microscope (Axiovert 135 M) in conjunction with a confocal laser scanning unit (Zeiss LSM 410). In parallel, nontransfected HeLa cells were pretreated for 1 h at 37°C with a mixture of intact or heat-treated (5 min at 100°C) heparitinases I (E.C. 4.2.2.7) and III (E.C. 4.2.2.8), or heparitinases and chondroitinase ABC (E.C. 4.2.2.4) (all at 0.02 U/ml; Sigma-Aldrich) (Linhardt et al., 1990
; Yamada et al., 1994
; Sugahara et al., 1994
).
To determine whether SD-1 and CCR5 are colocalized, HeLa cells were fixed with PFA 1% for 10 min, incubated with anti-SD-1 (clone B-B4, 10 µg/ml) for 1 h at room temperature, and revealed by an Alexa Fluor 594 goat anti-mouse IgG1 secondary antibody (1/100). Cells were then incubated for 1 h at room temperature with anti-CCR5 mAb 182, revealed by an Alexa Fluor 488 goat anti mouse IgG2b Ab (1/100; Molecular Probes).
For SD-4 and CCR5 colocalization experiments, HeLa cells were incubated for 1 h at room temperature with anti-SD-4 mAb 5G9 (10 µg/ml). Bound antibodies were revealed by an Alexa Fluor 568 goat anti-mouse IgG2a secondary antibody (1/100). After PFA fixation, the cells were then incubated with anti-CCR5 mAb 182; bound antibodies were revealed by an Alexa Fluor 488 goat anti-mouse IgG2b Ab (1/100; Molecular Probes). Cells were mounted and observed using a Zeiss microscope (Axiovert 135 M) in conjonction with a confocal laser scanning unit (Zeiss LSM 410).
RANTES binding to HeLa cells
125I-RANTES (81 TBq/mmol) used in displacement binding assays was from Perkin Elmer Life Sciences (Boston, MA). Aliquots of adherent HeLa cells, grown to confluence (all at 5 x 105), were cultured in 24-well plates (Falcon, Strasbourg, France). After 48 h incubation in serum-free medium and three washings with ice-cold binding buffer (PBS/0.1% BSA), cells were incubated for 2 h at 4°C in 0.3 ml binding buffer containing 125I-RANTES (from 7.5 pM to 18 pM; Perkin Elmer) in the presence or absence of unlabeled RANTES (up to 10.4 nM). Incubation was terminated by removing the medium and washing the cells. After cell lysis in 5% NaOH, bound radioactivity was measured using a -counter (LKB 1261 Multigamma). Data were analyzed by fitting to a logistic curve or according to Scatchard. Results are means ± SEM of three independent assays, each performed in triplicate.
In some experiments, 125I-RANTES was incubated with the cells in the presence of exogeneous heparin (at 101000 µg/ml) or dextran (at 1000 µg/ml). In parallel, heat-treated 125I-RANTES (10 min at 100°C, in the presence of 0.3% ß-mercaptoethanol) was incubated with the cells.
Preparation of PGs from HeLa cell lysates
HeLa cells grown to confluence were scraped, washed on ice with HEPES saline buffer (30 mM HEPES, 150 mM NaCl, pH 7.4), and extracted by sonication in the presence of lysis buffer (10 mmol/L Tris, 8 M urea, 0.1% [w/v] Triton X-100, 1 mM Na2SO4, 1 mM phenylmethylsulfonyl fluoride, 1 mM N-ethylmaleimide, pH 8; Sigma-Aldrich). Lysates were submitted to anion exchange chromatography on DEAE Sephacel beads (Sigma-Aldrich) previously equilibrated with lysis buffer. After washing the beads with Tris-buffered saline/ethylenediamine tetra-acetic acid (EDTA) buffer (10 mM Tris, 150 mM NaCl, 0.5 mM EDTA, pH 7.4), bound PGs were eluted with buffer (100 mM HEPES, 1 M NaCl, 10 mM CaCl2, 20 mM NaOAc, 0.2 mg/ml ß-casein, 0.5 % [w/v] CHAPS, pH 6.5, all from Sigma-Aldrich). Eluates were diluted with doubledistilled H2O to reduce the NaCl concentration to 200 mM. Aliquots of the PG fraction were then incubated for 6 h at 37°C with a mixture of heparitinase I (0.2 U/ml), heparitinase III (1.2 U/ml), and chondroitinase ABC (0.4 U/ml). Alternatively, the PGs were incubated as described with protein-G-Sepharose beads (Pharmacia, Paris), precoated (Mbemba et al., 1999, 2000
, 2002
) either with anti-SD-1mAb DL-101 or BB4, or anti-SD-4 mAb 5G9.
RANTES binding to ligands or receptors and association of GPCRs and PGs
To collect either RANTES ligands or CCR5 ligands, scraped HeLa cells (25 x 107) were incubated in parallel for 2 h at 4°C with or without RANTES, anti-CCR5 mAb 2D7, or its isotype, mouse IgG2a (all at 12.5 µg in 300500 µl PBS). In some experiments, cells were incubated in parallel with various RANTES concentrations (2.5250 nM). Alternatively, the CCR5-positive HeLa cells were preincubated for 1 h at 37°C with 20 mM BCD or for 2 h at 37°C with 0.02 U/ml heparitinases I and III and chondroitinase ABC mixture. In other experiments, cells were subsequently incubated with RANTES (2 µg) and, after washing, with anti-RANTES mAb (2 µg; mouse IgG1; clone 21445.1; R&D Systems) for 2 h at 4°C. In parallel, to immunoprecipitate the SD-1 or SD-4 molecules expressed on the plasma membranes, cells were incubated for 2 h at 4°C either with anti-SD-1 mAb DL-101 or anti-SD-4 mAb 5G9 or their respective isotypes. Cells were then lysed at 4°C in 500 µl buffer (150 mM NaCl, 20 mM TrisHCl, pH 8.2, supplemented with 1% Brij 97 and 10 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 25 mM phenanthrolin, 20 µg/ml aprotinin; Sigma-Aldrich). Lysates were cleared by centrifugation at 1000 x g for 30 min at 4°C. Immunocomplexes were collected in the presence of 10 mM dithiothreitol (Sigma-Aldrich, by incubation for 18 h at 4°C with 100 µl of Protein GSepharose beads, precoated or not with anti-RANTES mAb or its isotype (each at 2.5 µg). Weak reducing conditions during the collection of the immunocomplexes were used to eliminate cross-reactivity with nonspecific proteins (Jordan and Devi, 1999). In some experiments, the complexes immobilized on the beads were treated with heparitinase I (0.2 U/ml), heparatinase III (1.2 U/ml), and chondroitinase ABC (0.4 U/ml) mixture for 6 h at 37°C.
To release bound ligands, beads were boiled for 10 min with 120 µl 2x sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) sample buffer and centrifuged (400 x g; 5 min; 15°C). Alternatively, bound ligands were eluted by 1 M NaCl supplemented with 3% (w/v) CHAPS. After elution, medium was diluted with 10 mM CaCl2, 100 mM HEPES, 20 mM NaOAc, 0.2 mg/ml beta casein, pH 6.5, to reduce the NaCl concentration to 200 mM. In some experiments, eluted proteins were treated with heparitinase I (0. 2 U/ml), heparatinase III (1.2 U/ml), and chondroitinase ABC mixture (0.4 U/ml) for 6 h at 37°C.
Cell lysates or eluted proteins were resolved on nonreducing 12% SDSPAGE. The proteins were transferred to Immobilon strips or to polyvinylidene difluoride Hybond-P membrane (Amersham), blocked by incubation for 18 h at 37°C with PBS or Tris-buffered saline supplemented with 5% BSA (w/v). Excess BSA was washed out with PBS or Tris-buffered saline supplemented with 0.5% BSA and 0.2% Tween 20 (v/v) (Sigma-Aldrich). Strips were then incubated for 1 h at room temperature with anti-CCR5 2D7, anti-SD-1 DL-101 or BB-4, anti-SD-2, anti-SD-4 5G9, anti-betaglycan, anti-HS 10E4 (mouse IgM; Seikagaku, Tokyo), anti-CD4 Q4120, anti-CXCR4 12G5, 3G10 mAb (mouse IgG2b; Seikagaku) antibodies or their isotypes (all at 1/10001/5000). After three washes, strips were incubated with horseradish peroxidaselabeled anti-mouse or anti-goat IgG (at 1/50001/20,000). Strips were then washed and revealed by enhanced chemilumiscence reagent (Amersham Pharmacia Biotech, UK, Supersignal West Dura Extended, or Supersignal Femto Maximum Sensitivity, both from Pierce).
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Acknowledgements |
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Footnotes |
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2 To whom correspondence should be addressed; e-mail: liliane.gattegno{at}jvr.ap-hop-paris.fr
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Abbreviations |
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References |
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