3 Laboratoire de Biologie Cellulaire, Biothérapies Bénéfices et Risques, UPRES 3410, and Hôpital Jean Verdier, 93, Bondy, France; 4 Laboratoire de Ciblage Fonctionnel des Tumeurs Solides, UPRES 2360, UFR-SMBH, Université Paris XIII, 74, rue Marcel Cachin, 93017, Bobigny, France
Received on July 17, 2003; revised on October 21, 2003; accepted on November 21, 2003
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Abstract |
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Key words: CXCR4 / HIV / SDF-1 / syndecans
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Introduction |
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Besides their importance in normal processes, such as leukocyte migration and organ development, SDF-1 and CXCR4 are involved in several pathological conditions. CXCR4 plays a central role in T cell accumulation in rheumatoid arthritis synovium (Nanki et al., 2000) and determines the metastatic destination of tumor cells (Murphy, 2001
). Moreover, SDF-1 can inhibit in vitro HIV entry (Bleul et al., 1996
; Deng et al., 1996
). Though CD4 is the HIV-1 primary receptor (Klatzmann et al., 1990
), other coreceptors were identifiedCCR5 for R5 HIV strains and CXCR4 for X4 HIV strains (Alkhatib et al., 1996
; Amara et al., 1997
; Bleul et al., 1996
; Deng et al., 1996
). Occupancy of CXCR4 by SDF-1 prevents the interaction of X4 HIV envelope glycoproteins (gp120) with CXCR4. Moreover, optimal inhibition of X4 HIV isolates by SDF-1 requires interaction with cell surface heparan sulfate (HS) (Valenzuela-Fernandez et al., 2001
). In addition, heparan-sulfated proteoglycans (HSPGs), the syndecans, were identified as HIV attachment receptors (Saphire et al., 2001
).
Most chemokines, including SDF-1, also bind to glycosaminoglycans (GAGs) (Kuschert et al., 1999; Mbemba et al., 2000
; Middleton et al., 1997
; Valenzuela-Fernandez et al., 2001
; Witt and Lander, 1994
). The concomitant binding of SDF-1 to CXCR4 and HSPGs was demonstrated (Valenzuela-Fernandez et al., 2001
). Four identified mammalian syndecans are transmembrane proteoglycans (PGs) whose core proteins range in size from 20 to 40 kDa (Bernfield et al., 1992
, 1999
; Carey, 1997
; Couchman et al., 2001
; Simons and Horowitz, 2001
; Woods and Couchman, 1998
; Zimmermann and David, 1999
). They are involved in specific binding of growth factors and growth factor receptors (Bernfield et al., 1992
, 1999
; Carey, 1997
; Couchman et al., 2001
; Simons and Horowitz, 2001
; Woods and Couchman, 1998
; Zimmermann and David, 1999
). Syndecan-2 expressed on human monocyte-derived macrophages (MDMs), selectively binds the macrophage-derived growth factors, fibroblast growth factor 2, vascular endothelial growth factor, and heparin-binding epidermal growth factor, but not the chemokines, MIP-1
, MIP-1ß, monocyte chemoattractant protein-1, interleukin-8, or RANTES (Clasper et al., 1999
).
In the present study, using SDF-1, the best characterized isoform of the chemokine, we identify the PGs involved in SDF-1 binding to human primary cells, permissive to HIV infection, such as lymphocytes and macrophages. We then confirm our investigation by the use of HeLa cell line. We also hypothesized that these PGs form a heteromeric complex with CXCR4 on the different cells tested here.
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Results |
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We then analyzed the PGs from HeLa cell lysates. If the PG-enriched fraction obtained from these cell lysates was prepared in the presence of Triton X-100 and urea and then treated with heparitinase I, III, and chondroitinase ABC mixture, proteins of 32 kDa and 5058 kDa immunoreactive with anti-syndecan-4 mAb 5G9 and with mAb 3G10 were observed (Figure 3C and data not shown). The 5058-kDa proteins may represent homo- or heterooligomerization of the protein core of syndecan-4. Moreover, proteins of 34 kDa, immunoreactive with anti-syndecan-2 antibody and with mAb 3G10 were observed, whereas proteins of about 60 kDa, immunoreactive with anti-CD44 mAb and with mAb 3G10, were also detected. Besides these proteins, proteins of 45 and 90 kDa, immunoreactive with anti-syndecan-1 mAb DL-101 and mAb 3G10 were observed (Figure 3C and data not shown). Because mAb 3G10 reacts with an epitope including a terminal unsaturated uronic acid residue that is unmasked after removal of HS by heparitinase treatment (David et al., 1992), these data demonstrate that these PGs are glycanated before the glycosaminidase treatment (Figure 3C). The apparent molecular masses of the protein cores of the PGs belonging to the syndecan family, observed here, are closed to the predicted ones (Bernfield et al., 1992
, 1999
; Couchman et al., 2001
; Woods and Couchman, 1998
; Zimmermann and David, 1999
). Moreover we previously observed that intact, glycanated CD44 from MDMs, PBLs, and HeLa cells are characterized by a 110-kDa apparent molecular mass (Slimani et al., forthcoming, and data not shown).
We then investigated the syndecan-4 molecules expressed on the plasma membranes of the different cells. If the PBL-5d, MDMs, or Hela cells were incubated with anti-syndecan-4 mAb 5G9 and lysed in the presence of Brij 97, the immunocomplexes collected on beads were characterized by the presence of proteins of >250 kDa for the PBL-5d and the HeLa cells and from 140 kDa to more than 250 kDa for the MDM. These proteins were all immunoreactive with anti-syndecan-4 mAb 5G9 but not with the isotype (Figure 3D and data not shown). If the same procedure was applied to PBL-1d, proteins immunoreactive with anti-syndecan-4 mAb 5G9 and migrating as a broad smear of 90250 kDa were revealed (data not shown). No immunoreactivity with 5G9 occurred when the cells were incubated as negative control with the isotype. Neither syndecan-1 nor ß-glycan were detected when the PBL-1d or the PBL-5d were incubated with anti-syndecan-1 or anti-ß-glycan antibodies and lysed before incubation of their lysates with beads (data not shown). Considered together, our data demonstrate that MDMs and HeLa cells express syndecan-1, syndecan-2, syndecan-4, ß-glycan, and CD44. They also demonstrate that PBLs express syndecan-4 and CD44 but neither syndecan-1, syndecan-2, nor ß-glycan and that changes in glycanation and/or homo- or heterooligomerization of syndecan-4 occurs during PBL culture.
SDF-1 binding to HeLa cells
We then investigated whether SDF-1 binds to different classes of specific binding sites expressed on HeLa cells. 125I-SDF-1 (50 pM) bound to HeLa cells (Figure 4) in a dose-dependent and saturable manner. Nonspecific binding, determined in the presence of unlabeled SDF-1
(up to 534 nM), was a mean of 14%. Scatchard analysis (Figure 4) of the specific binding of 125I-SDF-1
to HeLa cells revealed two classes of sites: high-affinity ones with a 13 nM Kd and 192,580 sites per cell and low-affinity ones with a 126 nM Kd and 3,828,700 sites per cell.
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If the immunocomplexes just described were collected on protein G beads and then treated with heparitinases I and III and chondroitinase ABC mixture, a shift in the apparent molecular masses of the syndecan-4 molecules, immunoreactive with anti-syndecan-4 5G9, to 32 kDa was observed. These 32-kDa proteins were also immunoreactive with 3G10 mAb (Figure 5, lane 4 versus lanes 13, 14, and data not shown). Therefore, the syndecan-4 molecules bound to the SDF-1/CXCR4 complex and had HS chains on them before their treatment with glycosaminidases. The fact that after GAG removal from the immobilized complex bound syndecan-4 were still detected suggests, however, that proteinprotein interactions between SDF-1 and its ligands (CXCR4 and/or syndecan-4) are involved in the formation of this complex (Figure 5).
In the same conditions, we did not detect any SDF-1 ligands from the K562 cells (data not shown). It has been previously reported that K562 cells express ß-glycan and syndecan-3 (Saphire et al., 2001), so these data further rule out SDF-1 binding to ß-glycan and indicate that syndecan-3 is not involved in this binding.
Moreover, fluorescently labeled biotinylated SDF-1 colocalizes with syndecan-4: the yellow (mixed red-green) staining also strongly suggests an association between SDF-1 and syndecan-4. Controls, without labeled SDF-1
or with the isotype, were not stained (data not shown).
These data demonstrate that glycanated syndecan-4 is associated to the complexes formed by SDF-1 and CXCR4 on PBLs, MDMs, and HeLa cells.
Involvement of GAGs in the complex formed by SDF-1, CXCR4, and syndecan-4
To analyze the role of GAGs on SDF-1 binding, MDMs or HeLa cells were treated with the glycosaminidases mixture. To preserve cell viability, lower concentrations of glycosaminidases were used to treat the cells, as compared to those used to treat the immunocomplexes collected on the protein G beads. Pretreatment of the HeLa cells (Figure 6, lane 6 versus 4) or MDMs (Figure 7, lane 5 versus 4) with the glycosaminidases mixture decreased the amount and the apparent molecular masses of the syndecan-4 molecules bound to the SDF-1/CXCR4 complex, as compared with the results observed if the cells were untreated. This indicates that GAG-dependent interactions are involved in the formation of the complex, SDF-1/CXCR4/syndecan-4 and presumably in the binding of SDF-1 to syndecan-4. Moreover, such glycosaminidases pretreatment of MDMs also decreased the amount of CXCR4 bound to SDF-1 (Figure 7, lane 2 versus 1). This indicates that SDF-1 binding to syndecan-4 may facilitate on these cells, the chemokine binding to CXCR4. By contrast, glycosaminidases pretreatment of the HeLa cells did not change the amount of CXCR4 bound by SDF-1 (Figure 6, lane 3 versus 1).
Syndecan-4 is coassociated with CXCR4
To analyze the possible codistribution of CXCR4 and syndecan-4, HeLa cells were selected. Using confocal microscopy, we observed an high degree of colocalization between CXCR4 and syndecan-4 in the absence of SDF-1 (Figure 8A). These data suggest a molecular interaction between both molecules. This was further confirmed by electron microscopy analysis: clear colocalization of syndecan-4 and CXCR4 was found by transmission electron microscopy using secondary antibodies linked to 6-nm and 15-nm beads, respectively (Figure 8B). To further demonstrate the occurrence of a coassociation between CXCR4 and syndecan-4, coimmunoprecipitation experiments were performed. PBL-1d, PBL-5d, MDMs, or HeLa cells were incubated with anti-CXCR4 12G5 mAb. After cell lysis, immunocomplexes were collected on protein G beads. Western blot analysis of these complexes revealed, as expected, 48-kDa proteins from HeLa cells (Figure 9, lane 1), PBL-1d, and PBL-5d (Figure 10A, lanes 1, 2) and 75-kDa proteins from MDMs (Figure 10B, lane 1), all immunoreactive with anti-CXCR4 12G5 and G19 antibodies (Figures 9 and 10 and data not shown). No immunoreactivity with anti-CCR5 2D7 or 3A9 mAbs nor with anti-CCR1 mAb was observed (Figures 9 and 10 and data not shown). In addition, the immunocomplexes contained immunoreactive proteins with anti-syndecan-4 mAb 5G9 but neither with anti-syndecan-1, anti-syndecan-2, nor anti-ß-glycan antibodies nor with the isotypes (Figures 9 and 10). No immunoreactivity with the tested antibodies was observed when the cells were preincubated in buffer supplemented with the isotype (data not shown). Therefore, syndecan-4 and CXCR4 form a SDF-1-independent heteromeric complex on PBLs, MDMs, and HeLa cells.
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The apparent molecular mass of 50 kDa, observed besides the 32-kDa one, may be related with homo- or heterooligomerization of the protein core of some syndecan-4 molecules.
After GAG removal from the immobilized immunocomplexes, bound syndecan-4 was still detected. This suggests the occurrence of proteinprotein interactions between this PG and CXCR4 (Figure 10). In addition, high amounts of syndecan-4 were coimmunoprecipitated with CXCR4 if the cells were pretreated with glycosaminidases (data not shown). This indicates that GAG-dependent interactions may not be involved in syndecan-4 association with CXCR4.
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Discussion |
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The best characterized cell surface HSPGs fall into three groupsthe syndecan, glypican, and ß-glycan family proteins. Most cells express at least one syndecan family member. Syndecan-3 is found predominantly in the central nervous system, whereas syndecan-4 is more ubiquitous. Glypicans and ß-glycan are widely expressed (Bernfield et al., 1999). However, Saphire et al. (2001)
have recently observed very low levels of syndecan-3 and glypican on MDMs and low levels of HS on PBLs. In the present work, we observed that MDMs and HeLa cells express high levels of syndecan-1, syndecan-2, syndecan-4, ß-glycan, and CD44, and we found no evidence of MDM expression of glypicans, which are GPI-anchored PGs, sensitive to phospholipase C treatment. In addition, we detected only the presence of low levels of syndecan-4 and high levels of CD44 on PBLs from human healthy blood donors, but not syndecan-1, syndecan-2, or ß-glycan. Such syndecan-4 expression on PBLs has been recently reported (Kaneider et al., 2002
). The apparent molecular masses of syndecan-4 very much depended on the conditions used: they were significantly higher if the cell lysates were prepared in the presence of Brij 97, a detergent that does not disrupt the intermolecular associations (Mbemba et al., 1999
), as compared to the results observed if these lysates were prepared in the presence of Triton X-100 and urea.
We then investigated whether SDF-1 directly binds syndecan-4. We observed that biotinylated SDF-1 binds to electroblotted PGs from PBLs, which were characterized as syndecan-4 according to their immunoreactivity with anti-syndecan-4 mAb 5G9. Glycosaminidase pretreatment of these PGs abolished SDF-1 binding to syndecan-4. Therefore SDF-1 directly binds syndecan-4, and GAG-dependent interactions are involved.
The fact that SDF-1 bound to two classes of sites (high and low affinity) on HeLa cells further suggests that SDF-1 binds not only to CXCR4 but also to others ligands or receptors. Interestingly, Amara et al. (1999) have observed, in agreement with our data, CXCR4-independent as well as CXCR4-dependent binding of SDF-1 to HeLa cells.
To characterize SDF-1 ligands or receptors on PBLs, MDMs, and HeLa cell line, coimmunoprecipitation experiments were performed. These experiments show that SDF-1 forms complexes on these cells, which make up, beside CXCR4, homo- or heterooligomerized syndecan-4 but not syndecan-1, syndecan-2, ß-glycan, or CD44. The fact that syndecan-4 from these complexes migrated as smears indicates the glycanation of these PGs. This glycanation was further demonstrated by the immunoreactivity of these syndecan-4 molecules with 3G10 mAb after removal of their GAGs by glycosaminidase treatment. However, the fact that after GAGs removal from the immobilized complex (SDF-1/syndecan-4/CXCR4) bound syndecan-4 was still detected suggests the occurence of proteinprotein interactions between syndecan-4 and its ligands (CXCR4 and/or SDF-1).
SDF-1 association to syndecan-4 was visualized in parallel on the plasma membrane of HeLa cells by confocal microscopy analysis, which showed that biotinylated SDF-1 colocalizes with syndecan-4.
Whether GAG-dependent interactions are involved in the formation of SDF-1/CXCR4/syndecan-4 complexes on intact cells was then investigated. Glycosaminidase pretreatment of MDMs or HeLa cells decreased the amount of syndecan-4 bound by SDF-1 which suggests the role of GAGs in these complexes formations and presumably in the binding of SDF-1 to syndecan-4. Also, this pretreatment decreased the binding of SDF-1 to CXCR4, expressed by MDMs. This last effect was not observed in the case of HeLa cells. This suggests the involvement of GAGs in the association of SDF-1 to syndecan-4 and that such association facilitates the subsequent interaction of SDF-1 with CXCR4 on MDMs but not on HeLa cells. The fact that no SDF-1 ligands were detected on K562 cells rules out the possibility that SDF-1 also binds syndecan-3. Otherwise, glypicans were absent or weakly expressed on MDMs and HeLa cells, as assessed by the lack of effect of phospholipase C on HS expression of these cells. Therefore, whether glypicans may also be involved in SDF-1 binding to other cell lines or primary cells needs further investigation.
We then tested whether syndecan-4 is associated to CXCR4, in the absence of SDF-1. A spontaneous colocalization between syndecan-4 and CXCR4 was observed on the plasma membrane of HeLa cells, using confocal microscopy as well as electron microscopy. Moreover, syndecan-4 from PBLs, MDMs, and HeLa cells coimmunoprecipitated with CXCR4 in the absence of SDF-1, whereas neither CCR5, CCR1, syndecan-1, syndecan-2, nor ß-glycan were present. These syndecan-4 molecules were glycanated, as assessed by their immunoreactivity with 3G10 mAb, after removal of their GAGs by glycosaminidases treatment. The glycosaminidase-treated syndecan-4 had an apparent molecular mass of 50 kDa, besides that of 32 kDa, also observed in the complex SDF-1/CXCR4/syndecan-4. This suggests that a homo- or heterooligomerization of the protein core of some CXCR4 bound syndecan-4 molecules occurs in the absence of SDF-1 and that SDF-1 may disrupt such homo- or heterooligomerization. In these experiments, the presence of bound syndecan-4, after GAG removal from the immobilized heteromeric complex, suggests the occurrence of proteinprotein interactions between the PG and CXCR4. The fact that if the cells were pretreated with glycosaminidases, high amounts of syndecan-4 still coimmunoprecipitated with CXCR4 suggests that GAG-dependent interactions may not be necessary for the association of syndecan-4 with CXCR4.
Nevertheless, the fact that a direct GAG-dependent binding of SDF-1 to electroblotted syndecan-4 was observed rules out that the interaction of SDF-1 and syndecan-4 only results from CXCR4 association with syndecan-4.
In conclusion, our data suggest that the binding of SDF-1 to its specific GPCR CXCR4 is associated with a GAG-dependent binding of the chemokine to syndecan-4 expressed on PBLs, MDMs, and HeLa cells and that an SDF-1-independent heteromeric complex formed by CXCR4 and syndecan-4, involving proteinprotein interactions, occurs on these cells. They also suggest that the association of SDF-1 to syndecan-4 may facilitate on primary macrophages the binding of SDF-1 to CXCR4, which seems not to be the case for the HeLa cell line. Therefore, the syndecan-4/CXCR4 complex is likely a functional unit in which syndecan-4 may modulate the binding of SDF-1 to CXCR4 on some primary cells. Interestingly, we currently observe that HIV-1LAI gp120 also binds to syndecan-4/CXCR4 expressed by CD4+ CXCR4+ cells, which may further explain at least in part the inhibitory effect of SDF-1 on HIV infection mediated by X4 HIV isolates (Gattegno, unpublished data). Moreover, although PGs may also play important roles in the regulation of the immune response (Van der Voort et al., 2000), data on the expression and function of PGs in lymphocytes are scarce. The role of the interactions of syndecan-4 with SDF-1 and CXCR4 in the pathophysiology of SDF-1, such as inflammation, neoplasic diseases, and in HIV infection, deserves further study. For these purposes, whether syndecan-4 modulate the activity of SDF-1 is currently investigated.
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Materials and methods |
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Transfected CD4+ CXCR4+ CCR5+ HeLa cells (a gift from A. Benjouad, CERVI, Hôpital Pitié-Salpétrière, Paris), were cultured in Dulbecco's modified Eagle medium (Invitrogen, Paris) supplemented with 10% fetal calf serum (Biowhittaker, Paris). K562 cells were cultured in RPMI (Roswell Park Memorial Institute) 1640 (Invitrogen) supplemented with 10% fetal calf serum.
Immunofluorescence staining and FACScan analysis of the cells
Aliquots of 5 x 105 PBL-1d, PBL-5d, scraped MDMs, or HeLa cells were incubated for 30 min on ice in 100 µl phosphate buffered saline (PBS), supplemented with 0.05% bovine serum albumin (BSA; Sigma-Aldrich, Saint Quentin Fallavier, France) and with 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-CD3 mAb (mouse IgG1; Pharmingen), anti-syndecan-1 mAb (mouse IgG-1; clone DL-101; specific for the ectodomain of syndecan-1 of human origin), anti-syndecan-4 mAb (clone 5G9; mouse IgG2a; specific for the ectodomain of syndecan-4 of human origin; both from Santa Cruz Biotechnology, Santa Cruz, CA), anti-ß-glycan Abs (goat IgG, R&D systems), or their respective isotypes (mouse IgG1, IgG2b, IgG2a or goat IgG; Jackson Immunoresearch Laboratories, Baltimore, MD, or Pharmingen), anti-CD44 mAb (Serotec, Oxford, U.K.). Cells were then labeled with 1/20 fluorescein isothiocyanatelabeled-goat anti-mouse- (Pharmingen) or mouse anti-goat- (Santa Cruz Biotechnology) Ig antibodies. PBLs were also incubated with 1 µg Phyc-labeled anti-CD19 (IgG1; clone HD37; Dako, Paris). Cells were fixed in 1% paraformaldehyde (PFA) (Sigma-Aldrich) in PBS and analyzed on a FACScan (Becton Dickinson).
Alternatively, adherent MDMs or HeLa cells were cultured in 24-well flat-bottom plates (at about 5 x 105 cells per well) in 1 ml culture medium. After three washes with PBS, cells were incubated for 30 min at 4°C in 300 µl PBS-BSA supplemented either with anti-syndecan-1 mAb DL-101, anti-syndecan-4 mAb 5G9, anti-ß-glycan Abs (all at 10 µg/ml) or their isotypes (Pharmingen). In parallel, aliquots of these cells (5 x 105 cells per well) were permeabilized in the presence of 300 µl RPMI supplemented with 2% fetal calf serum and 0.3% saponin (Sigma-Aldrich) and then incubated in this medium for 30 min at 4°C with anti-syndecan-2 goat antibody (10 µg/ml; goat IgG, specific for an epitope corresponding to the C-terminal domain of human syndecan-2; Santa Cruz Biotechnology) or its isotype. In parallel, adherent MDMs or HeLa cells were incubated for 1 h at 37°C with 100 mU of phospholipase C (E.C. 3.1.4.10; 250 mU/ml; Sigma-Aldrich) in 300 µl PBS, washed three times with PBS, and incubated for 90 min at 4°C with 2.5 µg anti-HS mAb 10E4 (Seigakaku, Japan) or the isotype (mouse IgM; Pharmingen). After washing, cells were incubated for 30 min at 4°C in 300 µl PBS-BSA supplemented with fluorescein isothiocyanatelabeled goat anti-mouse or mouse anti-goat Ig antibodies (1/20; Pharmingen), fixed in 1% PFA in PBS, scraped, and analyzed by flow cytometry.
Immunofluorescence staining and microscopic analysis of the cells
Alternatively, adherent MDMs or HeLa cells, grown on glass coverslips, were incubated for 1 h at room temperature with anti-syndecan-1 mAb DL-101 (10 µg/ml), anti-syndecan-4 mAb 5G9 (10 µg/ml), or the isotypes, IgG1 or IgG2a. Cells were then labeled with Cy-3-conjugated donkey anti-mouse antibody (1:400; Jackson Immunoresearch), fixed with PFA, and mounted in fluorescent mounting medium (Dako, Glostrop, Denmark). In parallel, adherent HeLa cells were fixed with PFA and incubated for 1 h at room temperature with anti-syndecan-1 mAb (10 µg/ml, mouse IgG1, MCA682, clone B-B4, Serotec, Oxford, U.K.), anti-ß-glycan Ab (10 µg/ml) or the isotypes, mouse IgG1. In some experiments, adherent MDMs or HeLa cells were fixed with methanol, air-dried, rehydrated with PBS, and incubated for 1 h at room temperature with anti-syndecan-2 goat antibodies (10 µg/ml; goat IgG, specific for the C-terminal domain of syndecan-2 of human origin; Santa Cruz Biotechnology) or the isotype (Jackson Immunoresearch). Cells were then labeled with Alexa-fluor 488 donkey anti-goat antibody (Molecular Probes, Eugene, OR) and observed using an Olympus fluorescence microscope.
To determine whether SDF-1 colocalizes with syndecan-4, HeLa cells were incubated with anti-syndecan-4 mAb, revealed by Cy-3 donkey anti-mouse antibodies, and then incubated for 1 h at 4°C with 1-biotinylated SDF-1 (gift from F. Baleux, Laboratoire de chimie organique, Institut Pasteur, Paris), labeled for 30 min at 4°C with a streptavidin-Alexa Fluor 488 complex (1:100, Molecular Probes), and fixed with PFA. As controls, cells were incubated with the isotypes or biotinylated SDF-1 was omitted.
For syndecan-4 and CXCR4 colocalization experiments, HeLa cells were first incubated with anti-syndecan-4 mAb 5G9 and revealed by a Cy3 donkey anti-mouse antibody. Thereafter, cells were incubated with biotinylated anti-CXCR4 mAb 12G5 (1/25) and revealed by Alexa Fluor 488-streptavidin complex.
Immunoelectron microscopy on HeLa cells
The HeLa cells were grown until 80% confluence in multiwell chambers. After washes with PBS, HeLa cells were first incubated for 1 h at 4°C with anti-syndecan-4 mAb 5G9 (at 20 µg/ml), which was followed by an incubation for 30 min at 4°C with a donkey-anti-mouse IgG linked to 6 nm colloidal gold particles (Aurion, AA Wageningen, The Netherlands). The cells were then incubated for 1 h at 4°C with goat anti-CXCR4 antibody (clone G19, Santa Cruz Biotechnology) (at 20 µg/ml), which was followed by an incubation with a donkey-anti-goat IgG linked to 15 nm colloidal gold particles (Aurion). Cells were postfixed with 2.5% glutaraldehyde, dehydrated in graded ethanol series, and embedded in epoxy resin. Ultrathin sections (100 nm) were performed and observed in transmission electron microscopy (CM-10, Philips) at high magnification (27,500x).
Binding of 125I-SDF-1 to HeLa cells
For displacement binding assays, 125I-SDF-1 (specific activity 81 TBq/mmol) was from Perkin Elmer Life Sciences (Boston, MA).
Confluent HeLa cells were detached with PBS-5 mM ethylenediamine tetra-acetic acid (EDTA), washed and incubated for 1 h at 20°C in 0.3 ml PBS supplemented with 0.1% BSA (PBS-BSA) containing 50 pM 125I-SDF-1. Incubations were stopped by three washings. Binding of 125I-SDF-1
(50 pM) to the cells was determined in the presence or absence of unlabeled SDF-1
(up to 534 nM). Bound radioactivity was measured using a
-counter (LKB 1261 Multigamma). Values for the displacement of binding of 125I-SDF-1
to the cells (5 x 105 cells) by cold SDF-1
(a gift from F. Baleux, Institut Pasteur, Paris) were analyzed by fitting to a logistic curve or according to Scatchard. Results are means of three independent assays, each performed in triplicate.
Analysis of PGs from PBLs or HeLa cells
PBL-5d or scraped HeLa cells were washed on ice with HEPES saline buffer (30 mM HEPES, 150 mM NaCl, pH 7.4) and incubated for 30 min with lysis buffer (10 mmol/L Tris, 8 M urea, 0.1% [w/v] Triton X-100, 1 mM Na2SO4, 1 mM phenylmethylsulfonyl fluoride, pH 8, all from Sigma-Aldrich). Lysates were incubated for 12 h at 4°C with DEAE Sephacel beads (Sigma-Aldrich). The beads were then washed with Tris-buffered saline/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 BSA, 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 these eluates were then treated with heparitinases I (HS lyase; E.C. 4.2.2.8; 1 U/ml) and III (heparin lyase; E.C. 4.2.2.7; 15 U/ml) and chondroitinase ABC (chondroitin ABC lyase; E.C. 4.2.2.4; 5 U/ml) (all from Sigma-Aldrich) mixture. Intact and glycosaminidase-treated PGs were electroblotted and characterized as will be described. Alternatively, to test whether SDF-1 directly binds PGs, strips were exposed for 1 h at room temperature to biotinylated SDF-1
(0.02 µg/ml).
Coimmunoprecipitation of CXCR4 and syndecans with SDF-1
To characterize SDF-1 targets, PBL-1d, PBL-5d, K562 cells, scraped MDMs, or HeLa cells (2 x 107) were incubated for 2 h at 4°C in 500 µl PBS supplemented with SDF-1 (2 µg; synthetic, a gift from F. Baleux). As negative control, cells were also incubated with SDF-1
-free buffer. In parallel, the cells were incubated with anti-CXCR4 mAb 12G5 or its isotype, IgG2a (all at 22.5 µg in 300500 µl PBS; 0.51 µM). In some experiments, cells were pretreated for 2 h at 37°C with a mixture of heparitinase I (0.1 U/ml) and III (0.5 U/ml) and chondroitinase ABC (0.2 U/ml). To further characterize the syndecan-4 molecules, cells were also incubated with anti-syndecan-4 mAb 5G9 or its isotype.
Previous studies have shown that the detergent Brij 97 does not modify the interactions between a ligand and its targets (Mbemba et al., 1999). Therefore, the cells were lysed by incubation for 30 min at 4°C in 500 µl lysis 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). Lysates were cleared by centrifugation. Immunocomplexes were collected in the presence of 10 mM dithiothreitol (Sigma-Aldrich), by incubation for 18 h at 4°C with 100 µl protein GSepharose beads (Pharmacia, Paris), precoated (Mbemba et al., 1999
, 2000
, 2002
) or not by anti-SDF-1
mAb (goat IgG; R&D) or its isotype (R&D) (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 heparitinases I (1 U/ml) and III (15 U/ml) and chondroitinase ABC (5 U/ml) mixture. To release bound components, beads were boiled for 10 min with 120 µl 2x sample buffer for sodium dodecyl sulfatepolycrylamide gel electrophoresis and centrifuged (400 x g; 5 min at 15°C). Cell lysates, eluates, or eluted proteins were submitted to sodium dodecyl sulfatepolycrylamide gel electrophoresis (12%) under nonreducing conditions and transferred onto immobilon strips (Mbemba et al., 1999
, 2000
, 2002
). Complexes were revealed by incubation for 1 h at room temperature with anti-CXCR4 12G5, anti-CXCR4 G19 (goat IgG; Santa Cruz Biotechnology; specific [Brelot et al., 1997
] for the first extracellular domain of CXCR4), anti-CCR5 2D7, anti-CCR5 3A9 (Pharmingen), anti-syndecan-1 DL-101, anti-syndecan-2, anti-syndecan-4 5G9, anti-ß-glycan, anti-CD4 Q4120, anti-HS 10E4 or 3G10 antibodies (both from Seigakaku), anti-CD44 mAb, or their isotypes (all at 1/10001/5000). After washing, strips were incubated with horseradish peroxidaslabeled anti-mouse or anti-goat IgG (at 1/50001/20,000) and revealed by enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, U.K., or Supersignal West Dura Extended, Pierce, Rockford, IL).
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Acknowledgements |
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Footnotes |
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1 These authors contributed equally to this work.
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References |
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