Binding of the CC-chemokine RANTES to syndecan-1 and syndecan-4 expressed on HeLa cells

Hocine Slimani1,3, Nathalie Charnaux1,3, Elisabeth Mbemba3, Line Saffar3, Roger Vassy4, Claudio Vita5 and Liliane Gattegno2,3

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
It is believed that proteoglycans influence biological properties of chemokines. We show that the CC chemokine RANTES binds not only to high-affinity binding sites on CCR5-positive HeLa cells but also to low-affinity binding sites on HeLa cells expressing or lacking RANTES G protein–coupled receptors. Coimmunoprecipitation studies demonstrate that RANTES forms complexes with glycanated syndecan (SD)-1 and -4, in addition to CCR5 on the CCR5-positive HeLa cells. Moreover, confocal microscopy analysis shows the colocalization of RANTES with SD-1 and -4. Glycosaminoglycans removal from the cells by glycosaminidases treatment prevented RANTES binding to SD-1 and -4 and decreased RANTES binding to CCR5 on the CCR5-positive HeLa cells. Removal of glycosaminoglycans by glycosaminidases treatment of the complexes, RANTES/SD-1/SD-4/+/–CCR5, immobilized on beads, reversed SD-1 and -4 bindings. Therefore, RANTES bindings to SD-1 and -4 depend on glycosaminoglycans and facilitate RANTES interaction with CCR5. Extracting plasma membrane cholesterol abolished the coimmunoprecipitation of SD-1 with RANTES, suggesting that rafts are involved in RANTES association to SD-1. Confocal microscopy analysis as well as coimmunoprecipitation experiments show a RANTES-independent heteromeric complex on the CCR5-positive HeLa cells, SD-1, SD-4, and CCR5. This complex is likely a functional unit in which proteoglycans may modulate RANTES binding to CCR5.

Key words: CCR5 / HIV / RANTES / syndecans


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Chemokines mediate their biological activity through activation of G protein–coupled receptors (GPCRs) (D'Souza and Harden, 1996Go), but most chemokines, including regulated on activation normal T cell expressed and secreted (RANTES), also bind to glycosaminoglycans (GAGs) (Witt and Lander, 1994Go; Middleton et al., 1997Go; Kuschert et al., 1999Go; Mbemba et al., 2001Go). Virtually all GAGs exist in covalent linkage to a protein core as proteoglycans (PGs). RANTES exhibits selectivity in GAG binding with the highest affinity (nanomolar range) for heparin (Martin et al., 2001Go; Proudfoot et al., 2001Go). Chemokine receptors have been identified as human immunodeficiency virus (HIV) coreceptors, especially CCR5 for R5 HIV strains and CXCR4 for X4 HIV strains (Alkhatib et al., 1996Go; Bleul et al., 1996Go; Deng et al., 1996Go; Oberlin et al., 1996Go; Amara et al., 1997Go). We and others have reported that infection of human monocyte-derived macrophages (MDMs) and peripheral blood lymphocytes (PBLs) by HIV-1 R5 strains, but not PBL{Delta} by the X4 strain, is inhibited by ß-chemokines, such as RANTES or macrophase inflammatory protein (MIP)-1{alpha} (Amzazi et al., 1998Go; Rabehi et al., 1998Go), two physiological CCR5 ligands (Alkhatib et al., 1996Go). Opposite effects of RANTES on HIV-1 infection of macrophages have also been described (Gordon et al., 1999Go; Trkola et al., 1999Go; Chang et al., 2002Go).

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., 1991Go; Carre et al., 1995Go; Oravecz et al., 1997Go). It has recently been shown that a family of HSPGs, the syndecans (SDs), mediates HIV-1 attachment on human primary macrophages (Saphire et al., 2001Go). Cell surface HSPGs are anchored in the cell membrane either via a transmembrane domain (the SDs) or by glycosyl–phosphoinositol linkage (glypicans) (Saphire et al., 2001Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Immunofluorescence labeling of cells
Transfected CCR5 positive HeLa cells expressed CD4, CXCR4, and CCR5 (Table I). Nontransfected HeLa cells constitutively expressed CXCR4 (Chang et al., 2002Go) but neither CD4 nor CCR5 (Table I). Both cell lines expressed SD-1, -2, -4, and ß-glycan on their plasma membrane (Figure 1 and data not shown). Anti-SD-1 monoclonal antibodies (mAbs) DL-101 and BB-4 gave similar results (Figure 1). As expected (Saphire et al., 2001Go), CD4, CXCR4, CCR5, SD-1, -2, or -4 were not detected on human K562 leukemia cells. This indicates the specificity of the antibodies used here (Table I and data not shown).


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Table I. HeLa cell membrane expression (mean fluorescence intensity) of CXCR4, CCR5, and CD4 by FACScan analysis

 


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Fig. 1. Immunofluorescence analysis of PGs from HeLa cells. CCR5 positive HeLa cells were immunostained, as described in Materials and methods, with anti-SD-1 mAb, DL-101 (A) or B-B4 (I), goat anti-SD-2 Ab (B), anti-SD-4 mAb 5G9 (C), goat anti-ß glycan Ab (D), or with their isotypes, mouse IgG1 (E), mouse IgG2a (G), or goat IgG (F, H). Data are representative of three individual experiments. Bar: 5 µm.

 
Analysis of PGs of HeLa cells
Because BRIJ-97 does not modify the interactions of a ligand and its targets (Lapham et al., 1996Go; Mbemba et al., 1999Go, 2000Go), the living HeLa cells were lysed in the presence of this detergent in most experiments. In such conditions, lysates from HeLa cell lines contained proteins migrating as broad smears, immunoreactive with anti-SD-1 mAb DL-101, anti-SD-2 antibody, anti-SD-4 mAb 5G9 and anti-ß-glycan antibody, but not with the isotypes. Their apparent molecular masses ranged from 45 to 230 kDa for SD-1, 44 to 250 kDa for SD-4, 55 to 250 kDa for SD-2 and 40–100 kDa for ß-glycan (Figure 2A, lanes 1–4 and 5–7, and data not shown). These smears may be related with glycanation and also with homo- and hetero-oligomerization of the PGs.



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Fig. 2. Immunoblot analysis of PGs from HeLa cells. (A) Lysates from CCR5-positive HeLa cells (from 2 x 106 cells per lane) were prepared in the presence of BRIJ 97, run on SDS–PAGE 12%, electroblotted, and revealed with anti-SD-1 mAb DL-101 (lane 1), anti-SD-4 mAb 5G9 (lane 2), anti-SD-2 Ab (lane 3), anti ß-glycan (lane 4), or their isotypes, mouse IgG1 (lane 5), IgG2a (lane 6), or goat IgG (lane 7). Alternatively, CCR5-positive HeLa cells were incubated with anti-SD-1 mAb DL-101 (lanes 8, 10) or anti-SD-4 mAb 5G9 (lanes 9, 11) and lysed in the presence of BRIJ 97. Lysates were incubated with protein G beads. The anti-SD-1 mAb DL101- or anti-SD-4 mAb 5G9–interacting proteins (from 2 x 106 cells per lane) were then electroblotted and revealed, respectively, with anti-SD-1 DL-101 (lane 8), anti-SD-4 5G9 (lane 9), or their isotypes, IgG1 (lane 10) or IgG2a (lane 11). (B) HeLa cells were lysed in the presence of Triton X-100 and urea. PGs (from 2 x 106 cells per lane) were enriched by DEAE Sephacel anion exchange chromatography and then treated with heparitinases I and III and chondroitinase ABC mixture, electroblotted, and revealed with 3G10 mAb (lane 1) or the isotype, IgG2b (lane 2). Data are representative of three individual experiments.

 
No immunoreactivity with the anti-SD antibodies was observed in the electroblotted lysates from the K562 cells (data not shown). This argues, as reported (Saphire et al., 2001Go), for the lack of SD-1, -2, and -4 of these cells and the specificity of the anti-SD antibodies used here. To analyze some of the PGs expressed on their plasma membrane, HeLa cells expressing or lacking CCR5, were incubated with anti-SD mAbs and lysed in the presence of BRIJ-97. If the cells were incubated with anti-SD-1 mAb DL-101, the collected immunocomplexes contained 90-kDa and 60-kDa proteins and proteins migrating as a smear of apparent molecular masses of 50–70 kDa; all were immunoreactive with anti-SD 1 mAb DL-101. If the cells were pretreated with anti-SD-4 mAb 5G9, the immunocomplexes contained 48-kDa and 80-kDa proteins, beside a smear of apparent molecular masses of 48–80 kDa; all were immunoreactive with anti-SD-4 mAb 5G9. No immunoreactivity with the isotypes occurred (Figure 2A, lanes 8–11, and data not shown). No immunoreactivity with the tested antibodies was detected if the cells were incubated with the respective isotypes (data not shown).

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 31–32-kDa, 34-kDa, 45-kDa, 60–62-kDa, and 90-kDa proteins, all immunoreactive with the anti-stub 3G10 mAb but not with the isotype. The 31–32-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 31–32-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, 2001Go). They are, however, higher than the predicted ones (Rosenberg et al., 1997Go). They may result, according to others (Oh et al., 1997Go; Zimmermann and David, 1999Go), from noncovalently linked sodium dodecyl sulfate–resistant homo- or hetero-oligomerization.

Binding of RANTES to HeLa cells expressing or lacking CCR5
Because RANTES aggregates at µM concentrations (Hoogewerf et al., 1997Go), 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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Fig. 3. Binding of RANTES to HeLa cells. Binding (A, C) and Scatchard (B, D) plots were obtained by incubating unlabeled RANTES at the indicated concentrations and 125I-RANTES at 7 pM with CCR5 + HeLa cells (A, B) or 125I-RANTES at 18 pM with CCR5-negative HeLa cells (C, D) for 2 h at 4°C. Results are the means ± SEM (bars) of three separate experiments performed in triplicate. (E) CCR5-negative HeLa cells, grown to confluence, were incubated for 2 h at 4°C with 125I-RANTES (at 18 pM) in the absence (control) or the presence of heparin (at 1, 0.1, 0.01 mg/ml) or dextran (at 1 mg/ml). In parallel, heat-inactivated RANTES was incubated with the cells. Bound cpm were measured. Results are mean percentages of controls of one individual experiment performed in triplicate and are representative of three different experiments. P of the coupled differences, as compared to control, were determined using the Student t-test: *<0.01, **<0.02, ***<0.05.

 
The maximum percent of 125I-RANTES binding (B/T) to the CCR5 negative HeLa cells was 20 ± 1%. The minimum percent of 125I-RANTES binding, determined in the presence of 10.4 nM unlabeled RANTES, was 14 ± 2%. Scatchard analysis of specific 125I-RANTES (18 pM) binding to these cells (Figure 3D) revealed only one class of binding sites with 1.5 x 106 ± 1.2 x 105 binding sites per cell and a 56.1 ± 7 nM Kd (Figure 3C). This Kd is closed to that reported for RANTES binding to heparin (Martin et al., 2001Go; Vives et al., 2002Go). Moreover, the binding of RANTES (from 7 pM up to 18 pM) to the CCR5-negative HeLa cells was significantly and strongly decreased by exogeneous heparin. This decrease was dose-dependent. Dextran, a polyssacharide that had the lowest inhibitory effect on RANTES binding to various primary cells or cell lines as compared to negatively charged polysaccharides (Mbemba et al., 2001Go), had only slight inhibitory effect. This binding was also strongly decreased by 125I-RANTES heat denaturation, whereas unlabeled heat-inactivated RANTES had no effect (Figure 3E and data not shown). This demonstrates the role of RANTES tridimensional structure and suggests that heparin behaves as a competitive inhibitor. Finally, fluorescently labeled biotinylated 1-RANTES decorated the plasma membrane of HeLa cells expressing or lacking CCR5, whereas no staining occurred in the absence of 1-RANTES (Figure 4).



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Fig. 4. Binding of fluorescently labeled biotinylated 1-RANTES to CCR5-negative HeLa cells. Adherent CCR5 negative HeLa cells were pretreated (B, C) or not (A, D) with heparitinases I and III (B) or heparitinases I, III, and chondroitinase ABC (C), as described in Materials and methods, and sequentially incubated at 4°C with biotinylated 1-RANTES and streptavidin–Alexa Fluor 488. As control, 1-RANTES was omitted (D). Data are representative of three individual experiments. Bar: 5 µm.

 
Binding of glycanated SD-1 and -4 to the complexes formed by RANTES and GPCRs on the plasma membrane of HeLa cells expressing or lacking CCR5
Two protocols were carried out to collect the proteins interacting with RANTES on the plasma membranes of the living HeLa cells. After incubation with RANTES and subsequently with anti-RANTES antibodies, the cells were lysed in the presence of BRIJ-97 and the immunocomplexes were collected on beads. Alternatively, the cells were lysed just after their incubation with RANTES, and the RANTES-interacting proteins were collected on anti-RANTES-coated beads. Similar data were obtained in both experiments. The immunocomplexes collected from the CCR5-negative HeLa cells, contained proteins of 60 kDa, and proteins migrating as a smear of 50–70 kDa, all immunoreactive with the two anti-SD-1 mAbs, DL-101 and BB-4 (Figure 5, lane 1 and data not shown). They also contained proteins of 48–50 kDa and proteins migrating as a broad smear of 70–270 kDa, all immunoreactive with anti-SD-4 mAb 5G9 (Figure 5, lane 6). No immunoreactivity with anti-SD-2, anti-betaglycan antibodies, or with anti-CXCR4 mAb 12G5 was detected (Figure 5, lanes 5, 11, and 12). No immunoreactivity with anti-CCR5 mAb 2D7 was observed, as expected (Figure 5, lane 10). No immunoreactivity with all tested antibodies was detected when the cells were incubated in RANTES-free buffer (Figure 5, lanes 3 and 8). Therefore, RANTES binds selectively homo- or hetero-oligomerized, heavily glycanated SD-4 and also, but to a lower extent, glycanated SD-1, expressed on the CCR5-negative HeLa cells. The fact that neither SD-2 nor betaglycan were associated to the complex formed by RANTES, SD-1, and SD-4, suggests the selectivity of RANTES binding to the PGs.



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Fig. 5. Binding of SD-1 and SD-4 to RANTES at the plasma membrane of CCR5 negative HeLa cells. Nontransfected CCR5 negative HeLa cells were pretreated (lanes 2, 7) or not (lanes 1, 3–6, 8–12) with heparitinases I, III, and chondroitinase ABC and then incubated (lanes 1, 2, 4–7, 9–12) or not (lanes 3, 8) with RANTES and lysed in the presence of BRIJ 97. Lysates were incubated with anti-RANTES-coated beads. The RANTES-interacting proteins (from 2 x 106 cells per lane) were then electroblotted and revealed, respectively, with anti-SD-1 DL-101 (lanes 1–3), anti-SD-2 (lane 5), anti-SD-4 5G9 (lanes 6–8), anti-CCR5 2D7 (lane 10), anti-CXCR4 12G5 (lane 11), anti-ß-glycan (lane 12) antibodies or with their isotypes, IgG1 (lane 4) or IgG2a (lane 9). Data are representative of three individual experiments.

 
Using the same approach, we observed, as expected, that 46–48-kDa proteins from the immunocomplexes collected from the CCR5-positive HeLa cells were immunoreactive with anti-CCR5 mAb 2D7 but not with anti-CXCR4 mAb 12G5, anti-CXCR4 G19 Abs, or anti-CD4 mAb Q4120 (Figure 6, lanes 1 and 13, and data not shown). Sixty-kilodalton proteins collected from these immunocomplexes were immunoreactive with the two anti-SD-1 mAbs, DL-101 and BB-4, as well as with anti-HS mAb 10E4, whereas 50-kDa proteins were immunoreactive with anti-SD-4 5G9 and with anti-HS 10E4 mAbs (Figure 6, lanes 3, 5, and 7). Because mAb 10E4 reacts with an epitope that occurs in native HS chains and that is destroyed by N-desulfatation of the GAGs (David et al., 1992Go), the PGs bound by RANTES on the CCR5-positive HeLa cells have HS chains on them. Here again, the lack of immunoreactivity with anti-SD-2, anti-betaglycan antibodies, as well as the isotypes, indicates the specificity of the observed interactions (Figure 6, lanes 11, 12, 14–16). Furthermore, no immunoreactivity occurred with all tested antibodies when the CCR5-positive HeLa cells were incubated in RANTES-free buffer (Figure 7, lanes 4, 8, and 12).



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Fig. 6. Binding of SD-1 and SD-4 to the complex formed by RANTES and CCR5 at the plasma membrane of CCR5-positive HeLa cells. CCR5-positive HeLa cells were pretreated (lanes 2, 4, 6, 8, 10) or not (lanes 1, 3, 5, 7, 9, 11–17) with heparitinase I, heparitinase III, and chondroitinase ABC, incubated with RANTES, and lysed in the presence of BRIJ 97. The RANTES-interacting proteins (from 2 x 106 cells per lane) were collected on anti-RANTES–coated beads, electroblotted, and revealed with anti-CCR5 2D7 (lanes 1, 2), anti-SD-4 5G9 (lanes 3, 4), anti-SD-1 DL-101 (lanes 5, 6), anti-HS 10E4 (lanes 7, 8), anti-SD-2 (lane 11), anti-ß-glycan (lane 12), anti-CXCR4 12G5 (lane 13) antibodies, or their respective isotypes, IgG1, IgM, IgG2a, or IgG2b (lanes 14–17). In parallel, the complexes formed by RANTES and its targets expressed on the intact (lane 9) or the glycosaminidases-treated living cells (lane 10) were eluted from the beads and then treated with heparitinases I and III and chondroitinase ABC mixture, electroblotted, and revealed with 3G10 mAb (lanes 9, 10) or its isotype (lane 17). The arrows represent the bands that were also immunoreactive with anti-SD-4 5G9 or anti-SD-1 DL-101 mAbs. Data are representative of three individual experiments.

 


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Fig. 7. Effect of RANTES concentration on the complex formed by RANTES and CCR5 at the plasma membrane of CCR5-positive HeLa cells. CCR5-positive HeLa cells were pretreated with different concentrations of RANTES and lysed. The RANTES-interacting proteins (from 2 x 106 cells per lane) were collected on anti-RANTES-coated beads, electroblotted, and revealed with anti-CCR5 2D7 (lanes 1–4), anti-SD-4 5G9 (lanes 5–8), anti-SD-1 DL-101 (lanes 9–12). Data are representative of three individual experiments.

 
If the complexes RANTES/SD-1/SD-4/±CCR5 were eluted from the beads and then treated with heparitinase I, heparitinase III, and chondroitinase ABC mixture, 45-kDa and 31–32-kDa proteins, all immunoreactive with mAb 3G10 but not with its isotype, were observed (Figure 6, lanes 9 and 17, and data not shown). The 31–32-kDa proteins were also immunoreactive with anti-SD-4 mAb 5G9, whereas the 45-kDa proteins were immunoreactive with anti-SD-1 mAb DL-101 (Figure 6, lane 9). Therefore, the SD-1 and -4 molecules bound by RANTES had HS chains on them before GAG removal. These results demonstrate that RANTES binds, beside CCR5 on the CCR5-positive HeLa cells, glycanated SD-1 and -4 but neither SD-2 nor betaglycan. They suggest that differences in glycanation and/or homo- or hetero-oligomerization of the SD-4 molecules bound by RANTES occur, according to the presence or absence of CCR5 on the HeLa cells.

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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Fig. 8. Colocalization of fluorescently labeled biotinylated 1-RANTES with SD-1 or SD-4, at the plasma membrane of HeLa cells. HeLa cells expressing (A, C) or lacking (B, D) CCR5 were double-stained for fluorescently labeled biotinylated 1-RANTES (green) and either anti-SD-1 DL-101 (red) (C, D) or anti-SD-4 5G9 (red) (A, B). Colocalization of biotinylated 1-RANTES with either SD-1 or SD-4 is demonstrated by the yellow (red-green) colocalization, suggesting their clustering. Data are representative of three individual experiments. Bar: 5 µm.

 
GAG dependence of RANTES binding to the HeLa cells
GAG removal from the CCR5-negative HeLa cells by treatment with heparitinases I and III or with heparitinases I and III and chondroitinase ABC mixtures decreased the staining of these cells by biotinylated 1-RANTES, but heat-inactivated enzymes had no effect (Figure 4). Therefore, GAGs are involved in RANTES binding to CCR5 negative HeLa cells. Moreover, GAGs removal from the living HeLa cells, expressing or lacking CCR5, by their pretreatment with heparitinases I and III and chondroitinase ABC mixture abolished the coimmunoprecipitation of SD-1 and -4 with RANTES, as assessed by the lack of immunoreactivity of the collected immunocomplexes with anti-SD-1, anti-SD-4, and 3G10 mAbs (Figure 5, lanes 2 and 7; data not shown; and Figure 6, lanes 4, 6, 8, and 10). Therefore, RANTES assembly with SD-1 and -4 on living HeLa cells, is GAG-dependent. In addition, if the CCR5-positive HeLa cells were pretreated with the glycosaminidases mixture, the immunoreactivity of the RANTES-interacting material with anti-CCR5 2D7 mAb was also strongly decreased (Figure 6, lane 2). This indicates that GAG-dependent RANTES binding to glycanated SD-1 and -4 facilitates RANTES interaction with CCR5. Finally, if the complexes formed by RANTES and its targets expressed on HeLa cells were immobilized on beads and treated with the glycosaminidases mixture, no RANTES-bound SD-1 and -4 were detected, as assessed by the lack of immunoreactivity with anti-SD-1, anti-SD-4, and 3G10 mAbs (data not shown). Therefore, GAG removal from the complexes, RANTES/SD-4/SD-1/±CCR5, has eliminated the two PGs. This further demonstrates the GAG dependence of RANTES binding to SD-1 and SD-4.

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 1–3). 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 9–11 and 5–7). 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, 2002Go), strongly decreased, as expected (Nguyen and Taub, 2002Go), 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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Fig. 9. Effect of BCD on the complex formed by RANTES and CCR5 at the plasma membrane of CCR5-positive HeLa cells. CCR5-positive HeLa cells were pretreated (lanes 2, 4, 6) or not with BCD (lanes 1, 3, 5) and incubated with RANTES. The RANTES-interacting proteins (from 2 x 106 cells per lane) were collected on anti-RANTES–coated beads, electroblotted, and revealed with anti-CCR5 2D7 (lanes 1, 2), anti-SD-4 5G9 (lanes 3, 4), anti-SD-1 DL-101 (lanes 5, 6). Data are representative of three individual experiments.

 
Coassociation of SDs and GPCRs
We next investigated whether CCR5 forms an heteromeric complex with PGs in the absence of RANTES. Using confocal microscopy, we observed that CCR5 is spontaneously colocalized with SD-1 and -4 respectively, on the plasma membrane of CCR5-positive HeLa cell. This suggests that these molecules are closely localized in a ligand-independent manner (Figure 10). If the CCR5-positive HeLa cells were incubated with anti-CCR5 mAb 2D7, the immunocomplexes collected on beads contained 48-kDa proteins that were immunoreactive with anti-CCR5 mAb 2D7 (Figure 11, lane 2). In addition, proteins migrating respectively as broad bands of 60 kDa and 50 kDa were coimmunoprecipitated with CCR5 and were immunoreactive with anti-SD-1 DL-101 and anti-SD-4 5G9, respectively (Figure 11, lanes 4 and 6). These proteins were also immunoreactive with anti-HS 10E4 mAb, which indicates that they have HS on them (Figure 11, lane 7).



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Fig. 10. Colocalization of CCR5 with SD-1 and SD-4. CCR5-positive HeLa were double-stained for anti-CCR5 mAb 182 (green) (A, D) and either anti-SD-4 mAb 5G9 (red) (B) or anti-SD-1 mAb B-B4 (red) (E) as described in Materials and methods. The merged images (C, F) show the yellow colocalization of both molecules. Data are representative of three individual experiments. Bar: 5 µm.

 


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Fig. 11. Coassociation of SD-1 and SD-4 with CCR5 at the plasma membrane of CCR5-positive HeLa cells. CCR5-positive HeLa cells were incubated either with anti-CCR5 2D7 mAb (lanes 2, 4, 6, 8–14) or its isotype, IgG2a (lanes 1, 3, 5) and lysed. Lysates were incubated with protein G beads. Collected immunocomplexes (from 2 x 106 cells per lane) were electroblotted and revealed with anti-CCR5 2D7 (lanes 1, 2), anti-SD-1 DL-101 (lanes 3, 4), anti-SD-4 5G9 (lanes 5, 6), anti-HS 10E4 (lane 7), anti-SD-2 (lane 10), anti-ß-glycan (lane 11) antibodies or their isotypes, IgG2a (lane 12), IgG1 (lane 13), and IgM (lane 14). In parallel, the heteromeric complexes were eluted from the beads and then treated with heparitinases I and III and chondroitinase ABC mixture, electroblotted, and revealed with 3G10 mAb (lane 8) or the isotype, IgG2b (lane 9). The arrows represent the bands that were also immunoreactive with anti-SD-4 5G9 or anti-SD-1 DL-101 mAbs. Data shown are representative of three individual experiments.

 
If the complex SD-1/SD-4/CCR5 was eluted from the beads and then treated with heparitinases I and III and chondroitinase ABC mixture, two proteins of, respectively, 45 kDa and 31 kDa, both immunoreactive with 3G10 mAb, were observed (Figure 11, lane 8). The 45-kDa proteins were also immunoreactive with anti-SD-1 mAb DL-101, whereas the 31-kDa proteins were immunoreactive with anti-SD-4 5G9 mAb (Figure 11, lane 8). No immunoreactivity with anti-betaglycan antibody, anti-SD-2 antibody, or all the isotypes was observed (Figure 11, lanes 9–14). No immunoreactivity with all tested antibodies was observed when the cells were preincubated in buffer supplemented with the isotype (Figure 11, lanes 1, 3, and 5 and data not shown). Therefore, glycanated SD-1 and SD-4, but neither SD-2 nor betaglycan form an heteromeric complex with CCR5 on the CCR5-positive HeLa cells.


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
SDs are HSPGs involved in specific binding of growth factors and growth factor receptors. We investigate here whether RANTES uses HSPGs, beside its GPCRs, to bind to HeLa cells and whether these HSPGs are associated with CCR5 on CCR5-positive HeLa cells. We first confirmed by specific immunostaining HeLa cells membrane expression of SD-1, SD-2, SD-4, and betaglycan. Moreover, western blot analysis of these cells lysates also revealed the presence of these PGs.

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., 2001Go) 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., 1997Go). 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., 1997Go) and for that of RANTES to heparin, respectively (Martin et al., 2001Go). 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., 1997Go) 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., 1997Go; Clasper et al., 1999Go). 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., 1997Go). Enzymatic removal of GAGs from lymphocytes was reported to abrogate the ability of RANTES to elicit an intracellular Ca2+ signal (Burns et al., 1998Go). However, in another work, no effect of cell surface GAGs was found on chemokine binding (Kuschert et al., 1999Go). 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., 1998Go; Wagner et al., 1998Go). 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., 2001Go). 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, 2002Go). CCR5 molecules also associate with membrane rafts (Manes et al., 1999Go; Nguyen and Taub, 2002Go). In the present article, the pretreatment of the CCR5-positive HeLa cells with BCD, which is known to extract membrane cholesterol (Nguyen and Taub, 2002Go), 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., 2001Go), 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 protein–protein 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.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Cells and cell culture
Nontransfected HeLa cells, which constitutively express CXCR4 but not RANTES GPCRs, CCR1, CCR5, CCR3, CCR4, nor the D6 receptor and the Duffy antigen (Chang et al., 2002Go), and transfected HeLa cells, which express CD4, CXCR4, and CCR5 (a gift from A. Benjouad, CERVI, Hôpital Pitié-Salpétrière, Paris), were cultured in Dulbecco's modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS) (Biowhittaker, Paris). K562 cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS.

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 1–2.5 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, 2002Go).

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 488–labeled 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 488–labeled 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., 2002Go). Bound 1-biotinylated RANTES was revealed with a streptavidin–Alexa 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., 1990Go; Yamada et al., 1994Go; Sugahara et al., 1994Go).

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 {gamma}-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 10–1000 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 double–distilled 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., 1999Go, 2000Go, 2002Go) 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 (2–5 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 1–2.5 µg in 300–500 µl PBS). In some experiments, cells were incubated in parallel with various RANTES concentrations (2.5–250 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 Tris–HCl, 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 G–Sepharose 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, 1999Go). 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 sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) 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% SDS–PAGE. 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 1/1000–1/5000). After three washes, strips were incubated with horseradish peroxidase–labeled anti-mouse or anti-goat IgG (at 1/5000–1/20,000). 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).


    Acknowledgements
 
This work was supported by the Agence Nationale de Recherche sur le SIDA and the Direction de la Recherche et des Enseignements Doctoraux (Ministère de l'Enseignement Supérieur et de la Recherche), Université Paris XIII. We thank P. Leclerc for help with confocal microscopy (Université Paris XI). We are grateful to Jenny Vaysse for her suggestions.


    Footnotes
 
1 These authors contributed equally to this work. Back

2 To whom correspondence should be addressed; e-mail: liliane.gattegno{at}jvr.ap-hop-paris.fr Back


    Abbreviations
 
BCD, hydroxypropyl-ß-cyclodextrin; BSA, bovine serum albumin; EDTA, ethylenediamine tetra-acetic acid; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GAGs, glycosaminoglycans; GPCRs, G protein–coupled receptors; mAb, monoclonal antibody; MDM, monocyte-derived macrophage; MIP, macrophage inflammatory protein; PBL, peripheral blood lymphocyte; PBS, phosphate buffered saline; PFA, paraformaldehyde; PGs, proteoglycans; RANTES, regulated on activation normal T cell expressed and secreted; SD, syndecan; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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