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• W2018840987 abstract "Dendritic cells are potent antigen-presenting cells that express several membrane lectins, including the mannose receptor and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). To identify highly specific ligands for these dendritic cell receptors, oligosaccharides were converted into glycosynthons (Os1) and were used to prepare oligolysine-based glycoclusters, Os-[Lys(Os)]n-Ala-Cys-NH2. Clusters containing two to six dimannosides as well as clusters containing four or five pentasaccharides (Lewisa or Lewisx) or hexasaccharides (Lewisb) were synthesized. The thiol group of the appended cysteine residue allows easy tagging by a fluorescent probe or convenient substitution with an antigen. Surface plasmon resonance was used to determine the affinity of the different glycoclusters for purified mannose receptor and DC-SIGN, whereas flow cytometry and confocal microscopy analysis allowed assessment of cell uptake of fluoresceinyl-labeled glycoclusters. Dimannoside clusters are recognized by the mannose receptor with an affinity constant close to 106 liter·mol–1 but have a very low affinity for DC-SIGN (less than 104 liter·mol–1). Conversely, Lewis clusters have a higher affinity toward DC-SIGN than toward the mannose receptor. Dimannoside clusters are efficiently taken up by human dendritic cells as well as by rat fibroblasts expressing the mannose receptor but not by HeLa cells or rat fibroblasts expressing DC-SIGN; DC-SIGN-expressing cells take up Lewis clusters. The results suggest that ligands containing dimannoside clusters can be used specifically to target the mannose receptor, whereas ligands containing Lewis clusters will be targeted to DC-SIGN. Dendritic cells are potent antigen-presenting cells that express several membrane lectins, including the mannose receptor and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). To identify highly specific ligands for these dendritic cell receptors, oligosaccharides were converted into glycosynthons (Os1) and were used to prepare oligolysine-based glycoclusters, Os-[Lys(Os)]n-Ala-Cys-NH2. Clusters containing two to six dimannosides as well as clusters containing four or five pentasaccharides (Lewisa or Lewisx) or hexasaccharides (Lewisb) were synthesized. The thiol group of the appended cysteine residue allows easy tagging by a fluorescent probe or convenient substitution with an antigen. Surface plasmon resonance was used to determine the affinity of the different glycoclusters for purified mannose receptor and DC-SIGN, whereas flow cytometry and confocal microscopy analysis allowed assessment of cell uptake of fluoresceinyl-labeled glycoclusters. Dimannoside clusters are recognized by the mannose receptor with an affinity constant close to 106 liter·mol–1 but have a very low affinity for DC-SIGN (less than 104 liter·mol–1). Conversely, Lewis clusters have a higher affinity toward DC-SIGN than toward the mannose receptor. Dimannoside clusters are efficiently taken up by human dendritic cells as well as by rat fibroblasts expressing the mannose receptor but not by HeLa cells or rat fibroblasts expressing DC-SIGN; DC-SIGN-expressing cells take up Lewis clusters. The results suggest that ligands containing dimannoside clusters can be used specifically to target the mannose receptor, whereas ligands containing Lewis clusters will be targeted to DC-SIGN. Dendritic cells are efficient antigen-presenting cells central to cellular immune responses (1Banchereau J. Steinman R.M. Nature. 1998; 392: 245-252Google Scholar). Internalized antigens are processed within the endosomal/lysosomal pathway for subsequent formation of MHC 1The abbreviations used are: MHC, major histocompatibility complex; Os, glycosynthons; BSA, bovine serum albumin; DABCO, 1,4- diazabicyclo[2.2.2]octane; ICAM-3, intercellular adhesion molecule 3; DC-SIGN, dendritic cell-specific ICAM-3-grabbing nonintegrin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; Glp, pyroglutamic acid; Flu, fluorescein-labeled; GM-CSF, granulocyte macrophage cell-stimulating factor; HPLC, high performance liquid chromatography; Rho, rhodamine Red-X™-labeled; RU, resonance units; CRDs, C-type carbohydrate recognition domains; Man, mannose; OBzl, O-benzyl ester. class II peptide complexes. Dendritic cell vaccination to elicit cellular responses rather than humoral responses requires peptide presentation with MHC I. Antigenic peptide loading onto class I MHC molecules in the endoplasmic reticulum requires the generation of peptides by the cytosolic proteasome and subsequent peptide transport into the endoplasmic reticulum. Dendritic cells express several endocytic receptors, including membrane lectins such as the mannose receptor and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) that may be used to increase antigen presentation (2Figdor C.G. van Kooyk Y. Adema G.J. Nat. Rev. Immunol. 2002; 2: 77-84Google Scholar). The mannose receptor, first characterized in macrophages (3Stahl P.D. Rodman J.S. Miller M.J. Schlesinger P.H. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 1399-1403Google Scholar), binds and takes up glycoproteins and glycoconjugates containing terminal mannose, GlcNAc, or fucose (4Shepherd V.L. Lee Y.C. Schlesinger P.H. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1019-1022Google Scholar). Calcium-dependent recognition of these sugars is mediated by C-type carbohydrate recognition domains (CRDs) in the extracellular region of the receptor (5Mullin N.P. Hitchen P.G. Taylor M.E. J. Biol. Chem. 1997; 272: 5668-5681Google Scholar). Glycoproteins internalized by the mannose receptor follow a well characterized endocytic path-way leading to lysosomes (6Stahl P.D. Wileman T.E. Diment S. Shepherd V.L. Biol. Cell. 1984; 51: 215-218Google Scholar). The cytoplasmic tail of the mannose receptor contains a tyrosine-based motif involved in the internalization process in association with clathrin-coated vesicles, and the mannose receptor traffics through early endosomes. Our experiments with fluorescein-labeled neoglycoproteins suggested that the mannose receptor is also expressed on monocyte-derived dendritic cells (7Avrameas A. McIlroy D. Hosmalin A. Autran B. Debré P. Monsigny M. Roche A.C. Midoux P. Eur. J. Immunol. 1996; 26: 394-400Google Scholar), and this finding was subsequently confirmed (8Engering A.J. Cella M. Fluitsma D.M. Hoefsmit E.C. Lanzavecchia A. Pieters J. Adv. Exp. Med. Biol. 1997; 417: 183-187Google Scholar). Recently, DC-SIGN, a new dendritic cell-specific membrane lectin was characterized (9Geijtenbeek T.B. Torensma R. van Vliet S.J. van Duijnhoven G.C. Adema G.J. van Kooyk Y. Figdor C.G. Cell. 2000; 100: 575-585Google Scholar). DC-SIGN binds the gp120 envelope glycoprotein on the surface of human immunodeficiency virus-1 (10Curtis B.M. Scharnowske S. Watson A.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8356-8360Google Scholar). Its extracellular domain is a tetramer stabilized by an α-helical stalk, whereas its C-type CRDs bind high mannose oligosaccharides (11Mitchell D.A. Fadden A.J. Drickamer K. J. Biol. Chem. 2001; 276: 28939-28945Google Scholar). The presence of an internalization motif in its cytoplasmic tail suggests that DC-SIGN acts as an endocytic receptor, and it has been shown that complexes of DC-SIGN with an anti DC-SIGN antibody are targeted to late endosomes/lysosomes (12Engering A. Geijtenbeek T.B. van Vliet S.J. Wijers M. van Liempt E. Demaurex N. Lanzavecchia A. Fransen J. Figdor C.G. Piguet V. van Kooyk Y. J. Immunol. 2002; 168: 2118-2126Google Scholar). Therefore, in dendritic cells that express both the mannose receptor and DC-SIGN, the trafficking of an internalized glycosylated ligand will depend on its relative ability to bind each of these lectins. In search of synthetic ligands suitable for use in human therapy, we selected chemically defined compounds that should have low molecular weight and be recognized by either the mannose receptor or DC-SIGN with high affinity. Accordingly, we developed sugar clusters using disaccharides (13Bédouet L. Bousser M.T. Frison N. Boccaccio C. Abastado J.P. Marceau P. Mayer R. Monsigny M. Roche A.C. Biosci. Rep. 2001; 21: 839-855Google Scholar, 14Frison N. Marceau P. Roche A.C. Monsigny M. Mayer R. Biochem. J. 2002; 368: 111-119Google Scholar). These small multivalent synthetic glycopeptides were made by coupling glycosynthons (diglycosylpyroglutamyl-β-alanine derivatives (15Quétard C. Bourgerie S. Normand-Sdiqui N. Mayer R. Strecker G. Midoux P. Roche A.C. Monsigny M. Bioconjug. Chem. 1998; 9: 268-276Google Scholar)) onto a peptide containing up to five lysine residues. Such glycoclusters have previously been shown to bind various lectins with a high specificity. Glycoclusters containing lactose are taken up by HepG2 cells (13Bédouet L. Bousser M.T. Frison N. Boccaccio C. Abastado J.P. Marceau P. Mayer R. Monsigny M. Roche A.C. Biosci. Rep. 2001; 21: 839-855Google Scholar), a human hepatoma cell line expressing the galactose-specific lectin (16Schwartz A.L. Fridovich S.E. Knowles B.B. Lodish H.F. J. Biol. Chem. 1981; 256: 8878-8881Google Scholar) but not by dendritic cells, whereas those containing dimannosides are taken up avidly by dendritic cells but not by HepG2 cells (14Frison N. Marceau P. Roche A.C. Monsigny M. Mayer R. Biochem. J. 2002; 368: 111-119Google Scholar). This study describes the use of a series of glycoclusters made of di- or oligosaccharides containing either mannose or fucose to identify the best ligands to allow selective recognition by either the mannose receptor or DC-SIGN. Surface plasmon resonance was used to determine glycocluster affinity for the purified lectins immobilized on sensor chips. Flow cytometry was used to examine glycocluster endocytosis by cells expressing either the mannose receptor or DC-SIGN. Materials—Lactose was purchased from Janssen Chimica (Beerse, Belgium); oligomannoses (α2 mannobiose, α3 mannobiose, and α6 mannobiose) were from Dextra Laboratories (Reading, UK). Lewisa, Lewisx, and Lewisb oligosaccharides were kindly donated by Gérard Strecker (F-Lille). Flu-neoglycoproteins were prepared by coupling bovine serum albumin (BSA) with phenylisothiocyanate monosaccharides and disaccharides and labeled with fluorescein isothiocyanate (17Roche A.C. Barzilay M. Midoux P. Junqua S. Sharon N. Monsigny M. J. Cell. Biochem. 1983; 22: 131-140Google Scholar, 18Monsigny M. Roche A.C. Midoux P. Biol. Cell. 1984; 51: 187-196Google Scholar). The number of sugar residues bound per BSA, determined by the resorcinol sulfuric acid micromethod (19Monsigny M. Petit C. Roche A.C. Anal. Biochem. 1988; 175: 525-530Google Scholar), under conditions of optimal affinity and specificity, was found to be 25 ± 3. In addition, to assess the clustering effect, Man-BSA containing about 17 mannose residues was also used. Chromatography—Conjugates were purified by gel filtration on a column (2 × 45 cm) of either Trisacryl GF05 (Biosepra, Villeneuve-la-Garenne, France), stabilized and eluted with distilled water containing 5% n-butanol, or BioGel (P2, P4, or P6 from Bio-Rad, Oxfordshire, England), stabilized and eluted with 50 mm acetic acid. The flow rate was 0.35 ml/min. High performance liquid chromatography (HPLC) was performed on a Waters 2690 apparatus (Milford, MA) linked to a Waters 2996 photodiode array detector. For glycosynthon analysis, a 5-μm LC-NH2 (4.6 × 250 mm) column was stabilized and eluted with acetonitrile/water (84/16, v/v) at 35 °C. The flow rate was 1 ml/min. For glycocluster analysis, a Vydac 5-μm C4 (4.6 × 250 mm) column (Hesperia, CA) was stabilized at 35 °C in the starting eluent: 2% of solvent B (acetonitrile 95%/water 5%/trifluoroacetic acid 0.1%) in solvent A (water 95%/acetonitrile 5%/trifluoroacetic acid 0.1%). Upon injection, the compounds were eluted using this mixture for 5 min and then a linear gradient of 2–15% solvent B in solvent A over 15 min. The flow rate was 1 ml/min. 1H NMR Spectroscopy—For 1H NMR analysis, compounds were dis-solved at room temperature in deuterated water, once with D2O containing 99.9% D, and, after freeze-drying, with D2O containing 99.96% D (Sigma). 1H NMR spectroscopy was performed at 300 K with a Varian Unity 500 MHz NMR spectrometer. Synthesis and Characterization of Oligosaccharyl-Glp-βAla-OH— The first step was to synthesize the glycosynthon oligosaccharyl-Glp-βAla-OH (15Quétard C. Bourgerie S. Normand-Sdiqui N. Mayer R. Strecker G. Midoux P. Roche A.C. Monsigny M. Bioconjug. Chem. 1998; 9: 268-276Google Scholar) in a one-pot, two-step reaction, by coupling an oligosaccharide with a protected dipeptide H-Glu-βAla-OBzl in the presence of imidazole followed by intramolecular acylation to stabilize the glycosylamine formed. After purification on Trisacryl column, the glycosynthon was deprotected by catalytic hydrogenation. The glycosynthons synthesized from lactose and mannobioses, Manα2Man, Manα3Man, and Manα6Man, are fully described in Frison et al. (14Frison N. Marceau P. Roche A.C. Monsigny M. Mayer R. Biochem. J. 2002; 368: 111-119Google Scholar). The same procedures were followed for the synthesis of glycosynthons from Lewisa/Lewisx oligosaccharides and Lewisb oligosaccharide. Lewisa and Lewisx oligosaccharides (Galβ3(Fucα4)GlcNAcβ3Galβ4Glc and Galβ4(Fucα3)GlcNAcβ3Galβ4Glc) (Fig. 1) were isolated as a mixture from biological fluids that included human milk. However, because they behave almost identically in most chromatographic systems, it is difficult to obtain a pure preparation of each. Remarkably, we found that they were easily separated by HPLC as glycosynthons (Galβ3(Fucα4)G-lcNAcβ3Galβ4Glc-Glp-βAla-OBzl and Galβ4(Fucα3)GlcNAcβ3Galβ4G-lc-Glp-βAla-OBzl) after substitution of the terminal reducing sugar by a short peptide (pyroglutamyl-β-alanyl-O-benzyl ester) (20Frison N. Meunier L. Marceau P. Quétard C. Roche A.C. Mayer R. Monsigny M. Biochimie (Paris). 2003; 85: 47-51Google Scholar). Consequently, we were able to prepare and use the pure Lewisa and Lewisx glycosynthons. Synthesis and Characterization of Oligosaccharyl-Glp-βAla-[Lys(oligosaccharyl-Glp-βAla-)]n-Ala-Cys(Flu)-NH2—As described previously (14Frison N. Marceau P. Roche A.C. Monsigny M. Mayer R. Biochem. J. 2002; 368: 111-119Google Scholar), the purified glycosynthons were linked through an amide bond to α- and ϵ-amino groups of the lysine residues of a peptide core Lysn-Ala-Cys(SPy)NH2 (with n = 1, 2, 3, 4 or 5)(Fig. 2). The resulting glycoclusters, oligosaccharyl-Glp-βAla-[Lys(oligosaccharyl-Glp-βAla-)]n-Ala-Cys(SPy)-NH2, were purified on a BioGel column and analyzed by HPLC and by 1H NMR to control the substitution. The cysteine residue was used to label the glycocluster: the thiol group was made free in the presence of Tris-(2-catboxyethyl)phosphine and then substituted by reaction with iodoacetamidofluorescein under nitrogen in a 50 mm sodium phosphate buffer (at pH 7.2, to avoid the formation of dimers), leading to oligosaccharyl-Glp-βAla-[Lys(oligosaccharyl-Glp-βAla-)]n-Ala-Cys(Flu)-NH2. All molecules were characterized by HPLC and 1H NMR. Production of Cells Expressing DC-SIGN or the Mannose Receptor— HeLa cells transiently expressing DC-SIGN were produced by transfection of full-length cDNA (21Soilleux E.J. Barten R. Trowsdale J. J. Immunol. 2000; 165: 2937-2942Google Scholar), tagged with an AU1 epitope tag introduced in the extracellular 3′ region of the plasmid. HeLa human carcinoma cells (Eurobio, Les Ulis, France) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Paisley, Scotland) supplemented with 10% heat-inactivated fetal bovine serum (Clontech), 2 mm l-alanylglutamine (Invitrogen), 10 units/ml penicillin, and 10 μg/ml streptomycin (Invitrogen) (complete medium). HeLa cells or rat fibroblasts were seeded 1 day before transfection at 1.5 × 105 cells per well in a 24-well culture plate or 106 cells in a 6-cm diameter Petri dish. ExGen 500 (Euromedex, Souffelweyersheim, France), a linear polyethyleneimine (22 kDa), was used to transfect the cells (22Boussif O. Lezoualc'h F. Zanta M.A. Mergny M.D. Scherman D. Demeneix B. Behr J.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7297-7301Google Scholar). 10 μl of ExGen diluted in 40 μl of 150 mm NaCl was added dropwise under vortex to plasmid DNA (1 μg from a 1 mg/ml DNA solution, diluted in 50 μlof150 mm NaCl). This mixture was kept for 10 min at room temperature to allow complex formation, then 100 μl of serum-free DMEM medium was added, and this final mixture was poured into each well (1.5 cm in diameter). Following incubation for 3 h at 37 °C in a CO2 humidified atmosphere, the medium was discarded, complete medium containing serum was added, and cells were further incubated for 48 h at 37 °C. In some experiments a large number of cells was transfected in Petri dishes, and, after a 24-h incubation, cells were released by trypsin-EDTA treatment; 2 × 105 cells were either put in 24-well plates for flow cytometry analysis or on glass coverslips for confocal microscopy analysis and incubated for a further 24 h before uptake experiments. As a control of transfected cells, we used cDNA (pMR60Δ378Myc-His) (23Carrière V. Piller V. Legrand A. Monsigny M. Roche A.C. Glycobiology. 1999; 9: 995-1002Google Scholar) encoding the myc-tagged truncated intracellular mannose-specific lectin MR60/ERGIC-53 (24Arar C. Carpentier V. Le Caer J.P. Monsigny M. Legrand A. Roche A.C. J. Biol. Chem. 1995; 270: 3551-3553Google Scholar). Production of stably transfected rat fibroblast cell lines expressing the human mannose receptor using retroviral infection has been described previously (25Taylor M.E. Conary J.T. Lennartz M.R. Stahl P.D. Drickamer K. J. Biol. Chem. 1990; 265: 12156-12162Google Scholar). The cell line used in this study was produced using the same method. Mannose receptor expression in this line is approximately twice as high as in the transfected cell line described originally, as assessed by pulse labeling and immunoprecipitation with an anti-mannose receptor antibody and uptake of 125I-labeled mannose-BSA. 2C. E. Napper and M. E. Taylor, unpublished observations. Dendritophages—These were kindly provided by J. P. Abastado (Immuno Designed Molecules, Paris). Briefly, peripheral blood mononuclear cells were obtained from apheresis from healthy volunteers (on average, 1010 peripheral blood mononuclear cells), washed three times in phosphate-buffered saline (PBS), and cultured for 7 days in nonadherent hydrophobic bags (Sdedim, Aubagne, France) in serum-free AIM-V medium (VacCell medium, IDM Life Technologies, France), which was supplemented with 500 units/ml GM-CSF and 50 ng/ml IL-13, at a density of 5 × 106 cells/ml. Fresh IL-13 was added on day 4 of culture. On day 7, dendritophages were isolated by elutriation (26Goxe B. Latour N. Chokri M. Abastado J.P. Salcedo M. Immunol. Invest. 2000; 29: 319-336Google Scholar). The purity of dendritophages ranged from 75 to 95%, and their viability was in all cases greater than 95%. Dendritophages were plated at 4 × 105 per well in 24-well culture plates and used 3 days after elutriation. Flow Cytometry Analysis of Endocytosis—Plated cells were incubated for 2 h at 37 °C in complete culture medium in the presence of the fluorescein-labeled conjugates (1.33 nm to 1.3 μm (0.1–100 μg/ml) Fluneoglycoprotein, 1–10 μm glycoclusters). After washing in PBS, cells were either recovered by trypsin-EDTA treatment for direct flow cytometry analysis or further incubated with a specific antibody to visualize the membrane lectin (see below, immunofluorescence). Cell suspensions were incubated for a further 30 min at 4 °C in the presence or in the absence of 50 μm monensin (18Monsigny M. Roche A.C. Midoux P. Biol. Cell. 1984; 51: 187-196Google Scholar, 27Midoux P. Roche A.C. Monsigny M. Cytometry. 1987; 8: 327-334Google Scholar), and the cell-associated fluorescence was determined by flow cytometry. To look for specific uptake of fluoresceinated conjugates, cells were preincubated for 30 min in the presence of fluorescein-free Man-BSA (1 mg/ml) as previously described (7Avrameas A. McIlroy D. Hosmalin A. Autran B. Debré P. Monsigny M. Roche A.C. Midoux P. Eur. J. Immunol. 1996; 26: 394-400Google Scholar), followed by incubation at 37 °C for 2 h in the presence of both 1 mg/ml fluorescein-free Man-BSA and a fluorescein-labeled conjugate. Cell-associated fluorescence was assessed using a BD-LSR flow cytometer, and the data were analyzed with Cell Quest software (BD Biosciences). In the case of DC-SIGN, the fluorescein fluorescence intensity reported is that of cells expressing DC-SIGN as evidenced by their labeling with an anti-AU1 antibody. Immunofluorescence—DC-SIGN-expressing cells were visualized using mouse monoclonal anti-AU1 epitope tag (RDI, Flanders). Cell surface DC-SIGN was labeled either by incubating living cells or cells fixed with 2% (w/v) paraformaldehyde with anti-AU1 (1/300, 45 min at room temperature) followed by incubation with R-phycoerythrin conjugated to a goat anti-mouse immunoglobulin-specific polyclonal antibody (1/ 100) (BD Biosciences/Pharmingen, labeled anti-mouse immunoglobulin) for flow cytometry analysis or rhodamine Red™-X conjugated to an AffiniPure F(ab′)2 fragment goat anti-mouse IgG(H+L) antibody (1/100) for confocal microscopy analysis. The myc-tagged truncated MR60 was detected using anti-myc antibodies (Invitrogen, Leek, The Netherlands) following fixation and permeabilization of cells with saponin (0.1% saponin in PBS-glycine, 20 min at room temperature) (23Carrière V. Piller V. Legrand A. Monsigny M. Roche A.C. Glycobiology. 1999; 9: 995-1002Google Scholar). Confocal Microscopy Analysis—Cells were fixed at 37 °C in PBS containing 2% paraformaldehyde for 20 min. Coverslips were mounted on slides in a PBS/glycerol mixture (1:1 v/v) containing 1% 1,4- diazabicyclo[2.2.2]octane (DABCO) as an anti-fading agent (28Johnson G.D. Davidson R.S. McNamee K.C. Russell G. Goodwin D. Holborow E.J. J. Immunol. Methods. 1982; 55: 231-242Google Scholar). Cell fluorescence was analyzed with a confocal imaging system MRC-1024 (Bio-Rad, Ivry sur Seine, France) equipped with a Nikon Optiphot epifluorescence microscope (Nikon, Tokyo, Japan) and a ×60 Planapo objective (numerical aperture 1.4). The krypton/argon laser was tuned to produce both 488-nm (fluorescein excitation) and 568-nm (rhodamine excitation) wavelengths. Images were recorded with a Kalman filter average of 10–15 images and treated with Adobe Photoshop software. Analysis of Glycocluster Binding to Soluble Mannose Receptor and DC-SIGN Fragments by Surface Plasmon Resonance—A soluble fragment of the human mannose receptor consisting of the eight C-type CRDs (MMR1–8) was produced in Chinese hamster ovary cells as described previously (29Simpson D.Z. Hitchen P.G. Elmhirst E.L. Taylor M.E. Biochem. J. 1999; 343: 403-411Google Scholar, 30Napper C.E. Dyson M.H. Taylor M.E. J. Biol. Chem. 2001; 276: 14759-14766Google Scholar). This fragment of the receptor contains all of the domains necessary for Ca2+-dependent binding of Man/GlcNAc/Fuc-terminated glycoconjugates and binds such ligands as well as the whole receptor does (29Simpson D.Z. Hitchen P.G. Elmhirst E.L. Taylor M.E. Biochem. J. 1999; 343: 403-411Google Scholar, 31Taylor M.E. Drickamer K. J. Biol. Chem. 1993; 268: 399-404Google Scholar). MMR1–8 was isolated from the medium by affinity chromatography on mannose-substituted Sepharose. A soluble fragment of DC-SIGN consisting of the whole extracellular region was produced by bacterial expression as described previously (11Mitchell D.A. Fadden A.J. Drickamer K. J. Biol. Chem. 2001; 276: 28939-28945Google Scholar). Protein was purified by affinity chromatography and ion exchange chromatography. To compare the affinities of different glycoclusters for the mannose receptor and DC-SIGN, we chose to immobilize the lectins, because previous studies (32Shinohara Y. Kim F. Shimizu M. Goto M. Tosu M. Hasegawa Y. Eur. J. Biochem. 1994; 223: 189-194Google Scholar) have shown that, when a lectin is immobilized, the affinity constant calculated is in good agreement with values obtained using classic methods in solution. Soluble MMR1–8 and DC-SIGN proteins were linked to the sensor chip CM5, starting with lectin concentrations of 10 μg/ml in 10 mm sodium acetate at pH 4.5. Efficient fixation was indicated by a large increase in the refractive index: ΔRU = 5000 for MMR1–8 and ΔRU = 2000 for DC-SIGN. The neoglycoproteins (Man-BSA or Fuc-BSA) in the absence or in the presence of inhibitors were injected in the running buffer (pH 7.4, 10 mm HEPES, 1 mm CaCl2, 1 mm MgCl2, 150 mm NaCl, 17 mm NaN3, 0.5% P20 (BIAcore surfactant)) at a flow rate of 20 μl/min to avoid mass transfer limitations. Regeneration of the chip was achieved by adding 0.3 m α-methylmannopyranoside to the running buffer for all flow cells. Inhibition constants were derived from experiments using a solution of glycocluster (0.25–25 μm concentration range), containing 0.125 μm Man-BSA or Fuc-BSA neoglycoprotein. These neoglycoprotein concentrations were those that gave the half-maximal value of resonance units (RUmax/2). The procedure to determine RUmax as well as the equations used to analyze row data are fully described in Ref. 33Duverger E. Frison N. Roche A.C. Monsigny M. Biochimie (Paris). 2003; 85: 167-179Google Scholar. The number of resonance units (RUs) was obtained from the sensorgrams; RUmax and the affinity constant K were deduced from the linear transformation (1/RU) versus (1/g)α, (1/g)α=(Kα×RUmax)×(1/RU)+(RUmax)−1(Eq. 1) where g is the concentration of the neoglycoprotein expressed as mol·liter–1. The factor α stands for the Sips coefficient (34Sips R. J. Chem. Phys. 1948; 16: 490-495Google Scholar), which takes into account the avidity effect as well as the heterogeneity of the neoglycoprotein (Man-BSA and Fuc-BSA containing between 20 and 30 sugar units) and/or of the surface density of the lectin immobilized on the chip. Data were fitted into a straight line corresponding to the equation, (RUmax×RU−1−1)1/α=(K×g)−1+Ka×(K×g)−1×i(Eq. 2) where i is the concentration of the glycocluster inhibitor expressed as mol·liter–1, and Ka is the association constant of the glycocluster tested. Neoglycoprotein Uptake by Cells Expressing Either the Mannose Receptor or DC-SIGN—Our previous studies have demonstrated the presence of mannose-specific membrane lectins on the surface of human dendritic cells, but the molecules responsible for this binding were not characterized (7Avrameas A. McIlroy D. Hosmalin A. Autran B. Debré P. Monsigny M. Roche A.C. Midoux P. Eur. J. Immunol. 1996; 26: 394-400Google Scholar). Using flow cytometry analysis and a panel of fluorescein-labeled neoglycoproteins bearing 25 sugar units (on average), we showed that Fuc-BSA and Man-BSA are the neoglycoproteins most efficiently internalized and Lac-BSA one of the least efficiently internalized (7Avrameas A. McIlroy D. Hosmalin A. Autran B. Debré P. Monsigny M. Roche A.C. Midoux P. Eur. J. Immunol. 1996; 26: 394-400Google Scholar). Thus, we decided to study the uptake of such neoglycoproteins by cells expressing either the mannose receptor or DC-SIGN to get more precise insights than can be obtained using immature dendritic cells that are known to express both lectins. Rat fibroblast-derived cell lines stably transfected with the mannose receptor have previously been shown to mediate efficient endocytosis of 125I-labeled mannose-BSA (25Taylor M.E. Conary J.T. Lennartz M.R. Stahl P.D. Drickamer K. J. Biol. Chem. 1990; 265: 12156-12162Google Scholar, 35Taylor M.E. Bezouska K. Drickamer K. J. Biol. Chem. 1992; 267: 1719-1726Google Scholar). Consistent with these results, rat fibroblasts expressing the mannose receptor efficiently took up fluorescein-labeled mannose-BSA bearing on average 25 mannose residues (Flu,Man25-BSA) (Fig. 3). Fucose-BSA (Flu,Fuc25-BSA) was also taken up by the mannose receptor-expressing cells, but the cells did not endocytose lactose-BSA (Flu,Lac25-BSA). The ligands of the mannose receptor are known to be internalized into an acidic compartment (see Ref. 36Stahl P. Schlesinger P.H. Sigardson E. Rodman J.S. Lee Y.C. Cell. 1980; 19: 207-215Google Scholar for a review). This finding was confirmed by the 10- to 20-fold fluorescence intensity increase seen following monensin postincubation of cells expressing the mannose receptor that had previously been incubated at 37 °C for 2 h with Flu,Man25-BSA or Flu,Fuc25-BSA. Monensin is known to neutralize intracellular acidic compartments allowing the recovery of fluorescein fluorescence that is quenched in an acidic environment (37Maxfield F.R. J. Cell Biol. 1982; 95: 676-681Google Scholar). Thus, the increase in fluorescence intensity seen upon monensin treatment indicated that Flu, Man25-BSA and Flu,Fuc25-BSA internalized by the mannose receptor are routed to an acidic compartment. We have previously shown, using different types of cells, that neoglycoproteins bearing about 25 sugars are specifically recognized by endogenous lectins (18Monsigny M. Roche A.C. Midoux P. Biol. Cell. 1984; 51: 187-196Google Scholar). Higher substitution increases nonspecific binding, and lower substitution leads to inefficient neoglycoprotein binding and consequently to poor uptake by membrane lectins because of the loss of avidity (38Monsigny M. Mayer R. Roche A.C. Carbohydr. Lett. 2000; 4: 35-42Google Scholar). Consistent with these results, in the case of cells expressing the mannose receptor, the cell fluorescence measured upon incubation at 37 °C in the presence of Flu-neoglycoprotein and a postincubation at 4 °C with monensin was higher with Flu, Man25-BSA (1.5-fold) than with Flu,Man17-BSA (data not shown). Neoglycoproteins were not internalized by rat fibroblasts transfected with an empty vector (data not shown). Peripheral blood mononuclear cells differentiate" @default.
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• W2018840987 title "Oligolysine-based Oligosaccharide Clusters" @default.
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