Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M511461200 on December 21, 2005

J. Biol. Chem., Vol. 281, Issue 9, 5771-5779, March 3, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
281/9/5771    most recent
M511461200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palma, A. S.
Right arrow Articles by Chai, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palma, A. S.
Right arrow Articles by Chai, W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Ligands for the beta-Glucan Receptor, Dectin-1, Assigned Using "Designer" Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides*Formula

Angelina S. Palma{ddagger}§1, Ten Feizi{ddagger}2, Yibing Zhang{ddagger}, Mark S. Stoll{ddagger}, Alexander M. Lawson{ddagger}, Esther Díaz-Rodríguez{ddagger}, María Asunción Campanero-Rhodes{ddagger}, Júlia Costa§, Siamon Gordon, Gordon D. Brown||3, and Wengang Chai{ddagger}

From the {ddagger}Glycosciences Laboratory, Faculty of Medicine, Imperial College London, Northwick Park and St Mark's Campus, Watford Road, Harrow, Middlesex HA1 3UJ, United Kingdom, §Laboratório de Glicobiologia, Instituto de Tecnologia Química e Biológica, Apartado 127, 2781-901 Oeiras, Portugal, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom, ||Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Lower Ground Floor, Wernher and Beit Building South, Groote Schuur Campus Observatory, Cape Town 7925, South Africa

Received for publication, October 21, 2005 , and in revised form, December 1, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate beta-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in beta1,3- or both beta1,3- and beta1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate "designer" microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular beta-glucans and compete with zymosan binding and triggering of tumor necrosis factor-{alpha} secretion by a Dectin-1-expressing macrophage cell line.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dectin-1 is the main receptor on leukocytes that mediates innate immunity to fungal pathogens (1, 2). It is a germ line-encoded membrane-associated cell surface glycoprotein, first identified on a mouse dendritic cell line and cultured Langerhans cells (3) and on a murine macrophage cell line (4). It is now known to be also expressed at the surface of monocytes and neutrophils and at low levels on a subpopulation of T-cells (5). Studies of the tissue distribution of the murine Dectin-1 by immunohistochemistry have shown it to be expressed in the spleen, thymus, liver, lung, and gut and at low levels in the skin, but it is not detectable in the brain, heart, kidney, or eye (6). The human homologue of Dectin-1 has also been characterized (4, 7-9) and resembles the murine protein but differs in that the transcript is alternatively spliced, resulting in two major and several minor isoforms. The major isoforms appear to serve the same immune function as the murine receptor (10).

The deduced amino acid sequence of Dectin-1 is that of a type II transmembrane protein, with an extracellular lectin-like domain at the C terminus followed by a stalk region and a short cytoplasmic N-terminal domain with an immunoreceptor tyrosine-based activation (ITAM)4-like motif (3, 7-9). The lectin-like domain is of the C-type (Ca2+-dependent) family (11) but resembles those of natural killer cells in its lack of residues involved in the ligation of calcium that mediates carbohydrate binding in the classical lectins (3, 4, 8). The cell membrane-associated Dectin-1 nevertheless binds to zymosan, a particulate beta-glucan-rich extract derived from Saccharomyces cerevisiae, and the binding to zymosan is inhibited by soluble glucan polysaccharides, such as laminarin and a S. cerevisiae glucan solubilized by partial phosphorylation (referred to as glucan phosphate (4, 8)). The common feature of the glucan polysaccharides that inhibit Dectin-1 binding is the presence of beta1,3-linked or both beta1,3- and beta1,6-linked glucose. The beta-glucan binding has been shown to be mediated by the lectin-like domain (12) and is independent of calcium (4, 8). Intact yeasts, including S. cerevisiae and the fungal pathogens Candida albicans and Pneumocystis carinii, are also recognized by Dectin-1 in a glucan-dependent fashion (4, 13, 14).

beta-Glucans are homopolymers of D-glucose that have for long been used as anti-infective and anti-tumor drugs due to their ability to stimulate in vivo the phagocytic activity of leukocytes and to trigger the production of reactive oxygen species and of inflammatory mediators and cytokines, such as TNF-{alpha} (2, 15, 16). Dectin-1 is now thought to be the major receptor on leukocytes that mediates the biological effects of beta-glucans as immune cell activators (1, 17). These cellular responses to beta-glucans require the cytoplasmic tail and the ITAM-like domain of Dectin-1, and some of them involve collaboration with other signaling molecules, such as the Toll-like receptor, TLR-2, and the myeloid differentiation factor 88 known as Myd 88 (1, 18). Dectin-1 also mediates the internalization of intact yeasts and fungal pathogens (13, 19). This requires the tyrosine phosphorylation of the cytoplasmic ITAM-like motif, although downstream signaling cascades involved in the mechanism of uptake of the pathogens are different from that of the known phagocytic receptors (19). These findings implicate Dectin-1 as a pattern recognition receptor (20) for nonopsonized glucans on fungi that is involved in the control of these pathogens by the innate immune system.

The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not yet known whether Dectin-1 can interact with other types of carbohydrates. This is important to investigate, since it is known that, in addition to binding to glucans, Dectin-1 binds to a subset of T-lymphocytes and is involved in triggering the proliferation of these cells (3, 4). The T-cell binding is not inhibited by glucans (4, 8), and the ligand(s) on T-cells remain to be identified (4, 8).

Here we investigate the oligosaccharide ligands for Dectin-1 using the neoglycolipid (NGL)-based oligosaccharide microarray technology (21, 22), a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. Our approach is to first select ligand-positive and -negative beta-glucans, to partially fragment these and chemically link the oligosaccharide fragments to a lipid; the NGL probes thus generated are arrayed and used for Dectin-1 binding studies in conjunction with mass spectrometry (MS) and methylation analysis for chain length and sugar linkage assignments. Second, we use purified glucan fragments to examine their abilities to compete with the binding of zymosan to Dectin-1-expressing macrophages and the triggering of the secretion of the cytokine TNF-{alpha} by zymosan. Third, using glucan oligosaccharide ligands as positive controls, we evaluate Dectin-1 binding to microarrays that include almost 200 sequence-defined oligosaccharide probes, many of which are of the mammalian type.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The polysaccharides examined in this investigation, the predominant carbohydrate sequences reported in these, and their sources are given in Table 1. A mixture of oligosaccharide fragments of the polysaccharide curdlan, obtained by acid hydrolysis, was from Megazyme (Wicklow, Ireland). Laminari-hexaose and -heptaose, composed of beta1,3-linked glucose, were from Megazyme and Seikagaku America (Falmouth, MA), respectively.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Polysaccharides examined in this investigation and their reported properties

 
Recombinant Soluble Dectin-1—A soluble Dectin-1-human IgG Fc chimera (Fc-Dectin-1) was generated by cloning the extracellular domain of murine Dectin-1 into the pSecTag2C plasmid containing a mutated Fc portion of human IgG1 (23). This construct was transfected into HEL293T cells, and the protein was purified from the supernatant by protein A chromatography.

Cells—The RAW264.7 cells transduced with full-length hemagglutinin-tagged Dectin-1 have been described previously (1). Chinese hamster ovary (CHO) cells expressing full-length Dectin-1 were generated by retroviral transduction, essentially as for the RAW264.7 cells. Cells were grown at 37 °C with 5% CO2 in Ham's F-12 medium with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B, 2 mM L-glutamine, and 0.6 mg/ml G418 (Gibco).

Depolymerization of Polysaccharides and Fractionation of the Oligosaccharide Fragments—Partial depolymerization of the neutral soluble glucan (NSG) isolated from S. cerevisiae was carried out by hydrolysis in 10 mM HCl. The reaction mixture was incubated at 100 °C for 2 h followed by neutralization with NaOH. Fragmentation of pustulan was performed by acetolysis with a solution of acetic anhydride/glacial acetic acid/concentrated H2SO4 (10:10:1 by volume) at 40 °C for 30 min. After stopping the reaction by neutralizing with anhydrous pyridine, the acetyl groups were removed by sodium methoxide (0.5 M in methanol at room temperature for 30 min). A further hydrolysis step with 0.1 M aqueous NaOH was introduced, for 30 min at ambient temperature, to ensure removal of residual acetyl groups detected by mass spectrometry. The product was neutralized with 10 mM HCl. The pustulan acetolysate and the NSG hydrolysate were desalted using a short Sephadex G10 column (1.6 x 30 cm) eluted with deionized water at a flow rate of 20 ml/h.

Fractionation of Oligosaccharides—The curdlan hydrolysate was pre-fractionated by ultrafiltration using a membrane (molecular weight cut-off 1000; Millipore, Watford, UK), and the retentate was investigated. The curdlan retentate, NSG hydrolysate, and pustulan acetolysate oligosaccharides were fractionated on a column of Bio-Gel P6 (1.6 x 90 cm) eluted with deionized water at a flow rate of 15 ml/h. The eluates were monitored on-line by refractive index and pooled according to their predominant glucose units, 7- to 13-mers, determined by matrix-assisted laser desorption/ionization (MALDI) MS, and lyophilized. Quantitation of oligosaccharides in the pooled fractions was carried out by a dot-orcinol assay using glucose as the standard, as described (24). Aliquots of each fraction (2 µg of hexose) were analyzed by high performance TLC (HPTLC) using a solvent system of n-propanol/water (8:3 by volume), developed twice, and stained with orcinol reagent.

Oligosaccharide Purification—NSG oligosaccharide fractions of interest, obtained by Bio-Gel P6 chromatography, were further separated into a, b, and c subfractions by preparative HPTLC using aluminum-backed plates, 10 x 10 cm (Merck), and the solvent system n-propanol/water (8:3 by volume), developed twice. Isolation of selected oligosaccharides was carried out by HPLC after conversion of oligosaccharide fractions into their aminopyridine derivatives as described (25). The derivatization was without reduction in order to recover the reducing oligosaccharides after chromatography (25). A Hypersil amino column, APS-2 (4.6 x 250 mm; Thermo Electron, Runcorn, UK), and a solvent system of a water/acetonitrile gradient were used for fractionation, at a flow rate of 0.5 ml/min, with detection at UV 247 nm. The collected oligosaccharide-2-aminopyridine derivatives were monitored by MALDI MS and converted back into the reducing forms by acid hydrolysis, 1 M AcOH at 70 °C, 16 h prior to conversion to NGLs or methylation analysis as described below.

Preparation and Purification of NGL Probes—Fluorescent NGLs were prepared as described (26) by chemical conjugation of oligosaccharides to the amino phospholipid N-aminoacetyl-N-(9-anthracenyl methyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (ADHP) with the following modifications. To the lyophilized oligosaccharide fraction (typically 100 nmol) were added 5 µl of water, 100 µl of a solution of ADHP (10 nmol/µl) in chloroform/methanol (1:3 by volume), and 20 µl of a fresh solution of the reducing agent tetrabutylammonium cyanoborohydride (20 µg/µl in methanol). The mixture was incubated at 75 °C for 72 h. For oligosaccharides higher than 9-mers, additional reagents (2.5 µl of water, 50 µl of ADHP solution, and 10 µl of reducing agent) were added after 24 h of incubation. The NGLs were visualized under UV light at 254 nm. The excess reagents in the reaction mixtures were removed (27, 28) either by preparative HPTLC using as solvent chloroform/methanol/water (105:100:28 by volume) or by silica cartridge (100 mg, Strata; Phenomenex, Macclesfield, UK). Purification of NGLs by HPLC was as described (26), except that 10 mM ammonium formate was used instead of sodium chloride, and the gradient was from 70 to 30% solvent C in D at ambient temperature over 35 min. NGLs were quantified by spectrophotometry in solution and by densitometry after arraying on HPTLC plates (27, 28). Molecular masses of the NGLs were determined by MALDI MS.

Methylation Analysis—Oligosaccharides were reduced with NaBD4 before they were methylated, hydrolyzed, reduced, and acetylated as described (29). Gas chromatography-MS analysis of the products, partially methylated alditol acetates, was performed on a ThermoQuest Trace system (Thermo Electron, Hemel Hempstead, UK), using a 15-m RTX-5 capillary column (Hewlett-Packard). The initial column temperature was 50 °C programmed to 100 °C at 25 °C/min, to 220 °C at 5 °C/min, and to 310 °C at 10 °C/min.

Mass Spectrometry—MALDI MS was carried out on a Tof Spec-2E instrument (Waters, Manchester, UK). Oligosaccharides and NGLs were dissolved in methanol and chloroform/methanol/water (25:25:8 by volume), respectively, at a concentration of 10-20 pmol/µl, and 0.5 µl was deposited on the sample target together with a matrix of 2-(4-hydroxyphenylazo)benzoic acid. Laser energy was 20% (coarse) and 60% (fine), and resolution was at 1000.

Interactions of Soluble Dectin-1 with Polysaccharides—Polysaccharides in 10 mM sodium phosphate buffer, 137 mM NaCl, pH 7.4 (PBS), were added to polystyrene wells (96-well Immulon 4 plates; Dynex Technologies Ltd., Worthing, UK) and allowed to dry at 37 °C for 16 h. Nonspecific binding sites were blocked with 1% (w/v) casein in Tris-buffered saline (TBS) (10 mM Tris buffer, pH 8.0, 150 mM NaCl, from Pierce), referred to as Pierce Blocker solution. Fc-Dectin-1 was used at 1 µg/ml, and the binding was detected using biotinylated goat anti-human IgG heavy and light (H + L) chains (anti-IgG) from Vector Laboratories (Peterborough, UK) (10 µg/ml), followed by streptavidin conjugated to horseradish peroxidase from Pierce (5 µg/ml) and colorimetric measurement (490 nm) using O-phenylenediamine hydrochloride (Sigma) as substrate. Variables investigated in initial experiments were (a) binding signals using Fc-Dectin-1 noncomplexed or precomplexed with the biotinylated anti-IgG, Dectin-1 (anti-IgG ratio 1:3 by weight) (30) and (b) calcium dependence using TBS in the presence of 2 mM CaCl2 or TBS in the presence of 10 mM EDTA for the blocking, incubation, and washing steps. For inhibition studies, NSG, used as the reference immobilized ligand (10 µg/ml in PBS), was added to wells and allowed to dry at 37 °C for 16 h. The Dectin-1 was used at the nonsaturating concentration of 0.5 µg/ml. The results were expressed as percentage of inhibition of binding as follows: percentage inhibition = ((OD no inhibitor - OD with inhibitor)/(OD no inhibitor - OD negative control)) x 100.

Flow Cytometric Studies of the Interactions of Cell Surface Expressed Dectin-1 with NSG—Binding of NSG by cell membrane-associated Dectin-1 was assayed by fluorescence-activated cell sorting using an antibody known to be directed at the polysaccharide binding site of Dectin-1 (17) (rat antibody to mouse Dectin-1, 2A11, from Serotec, Oxford, UK). Binding of this antibody was detected with R-phycoerythrin-labeled F(ab')2-goat anti-rat immunoglobulins (anti-rat-RPE), also from Serotec. The Dectin-1-transfected CHO cells were harvested in the presence of 2 mM EDTA and suspended at 2 x 106 cells/ml in flow cytometry buffer consisting of Ham's F-12 medium containing 2% fetal calf serum and 0.1% w/v NaN3. One hundred µl of cell suspension and 10 µl of 2A11 (1:5 working dilution) were added to Falcon 2054 tubes (BD Biosciences) and incubated for 30 min at 4 °C (all subsequent steps were carried out at 4 °C). The cells were washed three times with PBS containing 0.1% NaN3 and incubated for 30 min with 50 µl of goat anti-rat-RPE at a 1:5 working dilution. The cells were then washed twice and fixed with 1% (w/v) paraformaldehyde in PBS. Staining of the cells was detected using a FACSCalibur cell sorter (BD Biosciences). The inhibition of 2A11 binding using polysaccharides was performed essentially as described (31). The cells were first incubated for 30 min with 50 µl of NSG or dextran at 100 µg/ml, before the addition of 2A11. The percentage of inhibition of 2A11 binding in the presence of polysaccharides was determined as follows: percentage inhibition = ((mean fluorescence of cells without polysaccharides - mean fluorescence of cells with polysaccharides)/mean fluorescence of cells stained without polysaccharides) x 100.

Binding Assays of Soluble Dectin-1 to Arrays of Glucan-derived NGL Probes—Unless otherwise stated, NGLs (1 µl of 50 pmol/µl solutions of each, in chloroform/methanol/water (25:25:8 by volume)) were arrayed by jet spray as 2-mm bands onto nitrocellulose membranes or nitrocellulose-coated FASTTM glass slides (Schleicher & Schuell), and the binding of the soluble Dectin-1 was assayed essentially as described (21, 27). In brief, nonspecific binding sites on the slides were blocked for 1 h with Pierce Blocker solution; the binding of Fc-Dectin-1 (1 µg/ml in Pierce Blocker solution) was detected after 2 h of incubation, using biotinylated anti-IgG (10 µg/ml), followed by streptavidin-conjugated horseradish peroxidase (10 µg/ml) and color development with FAST-3, 3'-diaminobenzidine peroxidase substrate (Sigma).

Inhibition of Dectin-1-mediated Zymosan Binding and Triggering of TNF-{alpha} Secretion by Macrophages Using Ligand-positive NGL Probes—Zymosan binding and TNF-{alpha} production with the macrophage cell line RAW264.7 transduced with hemagglutinin-tagged Dectin-1, referred to as RAW-CTHA, was performed as described (1). Nontransduced cells, RAW-FB, were used as negative controls. For inhibition studies, NGLs derived from ligand-positive oligosaccharides, a pool of NSG F11-13, and ligand-negative NSG fraction 7 (F7) were incorporated into liposomes. The liposomes were prepared as described (32). In brief, 17 nmol of each NGL preparation and the carrier lipids egg lecithin (85 nmol) and cholesterol (51 nmol) in methanol were mixed and dried down at 37 °C; RPMI medium was added, and the mixtures were sonicated for 10 min in a sonic water bath. NGLs (3.75 nmol/ml final concentration) were added 30 min prior to the addition of zymosan.

Microarray Analysis of the Binding of Soluble Dectin-1 to Diverse Oligosaccharide Probes—Microarrays of 190 lipid-linked oligosaccharide probes, NGLs, and glycolipids (Ref. 22 and supplemental Table 1) were made available by courtesy of R. A. Childs of the Glycosciences Laboratory. The NGL probes derived from NSG F12 or curdlan F13 (containing 12-mers as major components) were included as positive controls, and their respective F7 and pustulan F13 were included as negative controls. Results shown are those with the curdlan F13 and F7 probes (IDs 159 and 158, respectively) and pustulan F13, ID 169 (supplemental Table 1). The majority of the 187 other oligosaccharide sequences in the microarrays were of the mammalian type, representative of N-glycans, glycolipids, and glycosaminoglycans, and the backbones and peripheral regions of O-glycans; others were microbial and plant-derived oligosaccharides. These had been printed in duplicate at ~2 and ~7 fmol/spot, using a noncontact arrayer (Piezorray; PerkinElmer Life Sciences), with Cy3 dye (Molecular Probes, Inc., Eugene, OR) included as a marker. The four arrayed spots for each oligosaccharide probe are referred to here as the "position" in the microarray for that probe. Fc-Dectin-1 binding was assayed as described above, except that blocking was with 1% (w/v) bovine serum albumin in Pierce Blocker solution (casein/BSA), Fc-Dectin-1 was used at 5 µg/ml in casein/BSA, and binding was detected using biotinylated anti-IgG (3 µg/ml in casein/BSA) followed by Alexa Fluor-647-labeled streptavidin (Molecular Probes) (1 µg/ml in casein/BSA). Slides were scanned using a ProScanArray (PerkinElmer Life Sciences), and Alexa Fluor-647-binding signals were quantified using ScanArray Express software (PerkinElmer Life Sciences).


Figure 1
View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 1.
Interactions of recombinant soluble and membrane-associated Dectin-1 with polysaccharides. The polysaccharides PGG (A), NSG, and mannan (B) were added and dried down in microwells at the indicated concentrations, and the binding of Fc-Dectin-1 (1 µg/ml) was assayed in the presence of 2 mM CaCl2 or 10 mM EDTA. Results are expressed as the means of duplicate wells with the range indicated by error bars. C, binding curve of Fc-Dectin-1 to immobilized NSG (applied to wells at 10 µg/ml); the arrow indicates the point on the binding curve (at 0.5 µg/ml) used as a positive control in the inhibition experiments. D, inhibition of the binding of Fc-Dectin-1 to NSG by three of the nine polysaccharides tested. Eight glucan polysaccharides and mannan were examined as inhibitors of the Fc-Decin-1 binding at the final concentrations indicated. E, flow cytometric analyses of Dectin-1-transfected CHO cells showing that in the presence of the polysaccharide NSG (but not dextran) there is inhibition of surface immunostaining with antibody, 2A11, directed at the glucan binding site of Dectin-1. Antibody binding was detected with R-phycoerythrin-labeled anti-rat immunoglobulins (anti-rat-RPE).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Interactions of Recombinant Soluble and Membrane-associated Dectin-1 with Polysaccharides—We performed binding experiments with Fc-Dectin-1 initially using three soluble S. cerevisiae polysaccharides immobilized in plastic microwells: polymeric glucan, poly-(1,6)-beta-D-glucopyranosyl-(1,3)-beta-D-glucopyranose (PGG), NSG, and mannan (Table 1). There was binding to PGG and NSG but not to mannan (Figs. 1, A and B). Saturation of binding was reached at 1 µg/ml PGG and 10 µg/ml NSG. The binding was cation-independent.

In order to exclude any effects of different degrees of immobilization of the polysaccharides on the plastic surface of microwells, we next investigated by inhibition of binding assays (Fig. 1, C and D) the interactions of the soluble Dectin-1 with the nine polysaccharides of fungal, bacterial, algal, and plant origins shown in Table 1. NSG was the immobilized reference compound. At the concentrations tested, three of the polysaccharides inhibited binding, with the following hierarchy: PGG > NSG > laminarin.

The NSG rather than the PGG was selected for further investigation and as a source of oligosaccharides to generate NGL probes, since the PGG was found to be relatively resistant to hydrolysis (not shown). The NSG is a biologically relevant molecule, since it is bound not only by the soluble Dectin-1 but also by the cell membrane-associated receptor, as shown by fluorescence-activated cell sorting (Fig. 1E). CHO cells transfected with the full-length Dectin-1 were used for surface fluorescent immunostaining with antibody 2A11, which recognizes the glucan-binding site of Dectin-1 (17). In the presence of NSG, there was ~70% inhibition of the surface immunostaining of the cells with antibody 2A11. The inhibition was specific, since dextran, a glucose polymer with predominantly {alpha}1,6-linkage not recognized by Dectin-1 (Fig. 1E), showed no inhibition.

Interactions of Recombinant Soluble Dectin-1 with NGL Probes Generated from Glucan Oligosaccharides—Mixtures of oligosaccharide fragments obtained after partial depolymerization of NSG were fractionated by gel filtration chromatography, and pooled F7 and F9-F13, containing mainly heptasaccharides and nona- to tridecasaccharides, respectively, as determined by MALDI MS (Table 2), were selected for Dectin-1 binding studies. The selection was based on our initial experiments (not shown), where laminari-heptaose gave no inhibition of Fc-Dectin-1 binding to NSG, and the NGL of laminari-hexaose was not bound by Fc-Dectin-1. This is in accord with earlier experiments using Dectin-1-transfected cells in which the binding to zymosan was not inhibited by laminari-heptaose (4, 8). NGL probes were prepared from oligosaccharide F7 and F9-F13 of NSG, arrayed on nitrocellulose membranes, and examined for binding by Fc-Dectin-1. Binding was detected to those derived from F10-F13, which contained 11-mer and higher oligomers (Fig. 2A and Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2
MALDI MS analysis of Bio-Gel P6 fractions of NSG fragments and derived NGLs and Dectin-1 binding to their NGLs arrayed by jet spray onto nitrocellulose

 
When the NSG oligosaccharide fractions were analyzed by HPTLC (Fig. 2B), it was noted that each comprises three groups with fast, intermediate, and slow migration, designated subfractions a, b, and c, respectively. Dectin-1 binding was detected predominantly to the NGL probes derived from a and c subfractions that had been isolated from F11 and F12, whereas there was little or no activity in the b subfractions (not shown). The ligand-positive NSG subfractions F11a and F11c contain predominantly 1,3-linked glucose with small amounts of 1,6- and/or 1,3,6-linked glucose revealed by methylation analyses (Table 5). In contrast, the major feature of subfraction F11b is glucose with 1,4-linkage with low levels of 1,3-, 1,4-, 1,6-, and 1,4,6-linkages. Incomplete separation during TLC may be responsible for small proportions of some components in adjoining subfractions.


View this table:
[in this window]
[in a new window]
  		TABLE 5
Linkage determination by gas chromatography-MS methylation analysis

The percentages shown are based on total internally linked residues.

 
Since both of the Dectin-1-binding NSG subfractions, 11a and 11c, contain mainly 1,3-linked glucose, we evaluated binding to NGL probes prepared from oligosaccharide fragments of curdlan, a polysaccharide that contains only beta1,3-linked glucose (32) (Table 5). As with the NGLs derived from NSG, binding was detected to those derived from curdlan fractions (Fig. 2B) that contained 11-mer and higher oligomers (Fig. 2A and Table 3). To closely compare the binding intensities of 11-mers of NSG and curdlan and to substantiate the specificity toward beta1,3-linked glucose, we used NGL probes derived from 11-mers of NSG F11a and curdlan F11 purified by HPLC. Both of these showed almost exclusively 1,3-linked glucose by methylation analysis (not shown). The two 11-mer NGL probes were further purified by HPLC and spotted at 1, 5, 10, and 20 pmol. The binding signals to the two sets were virtually identical (Fig. 2C).


View this table:
[in this window]
[in a new window]
  		TABLE 3
MALDI MS analysis of Bio-Gel P6 fractions of curdlan fragments and derived NGLs and Dectin-1 binding to their NGLs arrayed by jet spray onto nitrocellulose

 
To compare the relative potencies of glucose oligomers with exclusively beta1,3- and beta1,6-linkage, we prepared NGL probes from pustulan, reported to contain predominantly glucose with beta1,6-linkage (33) and confirmed by methylation analysis (Table 5). We evaluated Fc-Dectin-1 binding to NGL probes of pustulan F7 and F9-F13 (Fig. 2B and Table 4); binding was not detectable (Fig. 2A and Table 4). We conclude that in the range of chain lengths investigated, beta1,3-linked rather than beta1,6-linked glucose sequences are recognized by Dectin-1.


View this table:
[in this window]
[in a new window]
  		TABLE 4
MALDI-MS analysis of Bio-Gel P6 fractions of pustulan fragments and derived NGLs and Dectin-1 binding to their NGLs arrayed by jet spray onto nitrocellulose

 
Inhibition of Dectin-1-mediated Zymosan Binding and Triggering of TNF-{alpha} Secretion by Macrophages Using Ligand-positive NGL Probes—To investigate biological activities associated with the glucan ligands, we examined the ability of these oligosaccharides to inhibit Dectin-1 function in RAW-CTHA macrophages, which express high levels of Dectin-1 (1). For these experiments, the NGLs of the ligand-positive NSG F11-13, containing mainly the 11- to 13-mers, and the ligand-negative F7 as negative controls were rendered multivalent by incorporation into liposomes. The ability of the ligand-positive but not the ligand-negative NGLs to partially inhibit the binding of zymosan to RAW-CTHA macrophages (Fig. 3A) and inhibit the zymosan-induced TNF-{alpha} secretion by these cells (Fig. 3B) demonstrates that these oligosaccharides can act as functional antagonists of the receptor in situ. These properties of the ligand-positive glucan oligosaccharides are shared with macromolecular beta-glucans, which also inhibit partially the zymosan binding and the zymosan-induced TNF-{alpha} secretion by these cells (1, 34).


Figure 2
View larger version (114K):
[in this window]
[in a new window]
  		FIGURE 2.
Analyses of the glucan oligosaccharides (fractions F7-F13) from NSG, curdlan, and pustulan and of interactions of NGL probes derived from them with recombinant soluble Dectin-1. A, arrays of NGL probes derived from oligosaccharide fractions F7 and F9-F13 of NSG, curdlan, and pustulan, 50 pmol/spot on nitrocellulose membranes, were revealed under UV light and examined for Fc-Dectin-1 binding. B, the glucan oligosaccharide fractions from NSG, curdlan, and pustulan were analyzed by HPTLC using a solvent system of n-propanol/water, 8:3 (by volume), developed twice. The arrow indicates positions of application. The carbohydrates were revealed by staining with orcinol reagent. C, binding of Fc-Dectin-1 to purified 11-mer probes from NSG and curdlan. These were arrayed at 1, 5, 10, and 20 pmol/spot, on a silica gel plate for fluorescent detection and on nitrocellulose-coated glass slides for binding.

 
Microarray Analysis of Soluble Dectin-1 with Diverse Oligosaccharide Probes—As it is known that, in addition to interactions with glucans, Dectin-1 binds to a subset of T-lymphocytes (3, 4), we used Fc-Dectin-1 to probe in a microarray format (22) a panel of 190 oligosaccharide sequences that include diverse mammalian types. The positive control shown here is the curdlan F13 probe (ID 159) spotted at positions 53 and 115 in the microarray layout, and the negative controls are curdlan F7 (ID 158) at positions 54 and 116 and pustulan F13 (ID 169) at position 52 (supplemental Table 1 and Fig. 4). The 187 other oligosaccharide probes were selected from our (the Glycosciences Laboratory) unique carbohydrate library. They include numerous mammalian-type carbohydrate sequences: N-glycans (neutral and acidic, high mannose, and complex types), blood group types (A, B, H, Lewisa, Lewisb, Lewisx, and Lewisy) on linear or branched backbones and their sialylated and sulfated analogs, gangliosides, glycosaminoglycans, homo-oligomers of sialic acid, and fragments of other polysaccharides, ranging in size from 2 to 20 monosaccharides. They amount to 112 neutral, 46 sialylated, 18 sulfated, four both sialylated and sulfated, and seven miscellaneous acidic oligosaccharide sequences. Fc-Dectin-1 showed binding only to the NSG F12 and curdlan F13 probes and not to their F7 and pustulan F13 probes or to any of the other oligosaccharide probes printed on the microarrays. Results using the curdlan probes are shown in Fig. 4.


Figure 3
View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 3.
Inhibition of Dectin-1-mediated zymosan binding and triggering of TNF-{alpha} secretion by macrophages using ligand-positive NGL probes displayed on liposomes. A, the NGL probes derived from the ligand-positive NSG F11-13 and the ligand-negative NSG F7 were incorporated into liposomes and examined in A as inhibitors of zymosan recognition by RAW264.7 cells transduced to express high levels of Dectin-1 (RAW-CTHA), and in B they were examined as inhibitors of TNF-{alpha} production by these cells in response to zymosan. Control cells (RAW-FB) are shown for comparison. In B, the carbohydrates themselves in the absence of zymosan were also tested. The final concentration of the NGLs was 3.75 nmol/ml.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Polysaccharides are heterogeneous and may contain contaminants and substituents that are difficult to detect. The present experiments with the purified glucan oligosaccharides show that Dectin-1 binds to the glucan sequences, rather than other components, although it lacks the calcium ligating elements that are characteristic of the classical C-type lectins. Thus, it is established that the 1,3-linked NGL probes of 11-mers and higher oligosaccharides of NSG and curdlan are bound by Dectin-1, whereas the 1,6-linked analogues of pustulan show no binding.

In earlier studies of Dectin-1 interactions with glucans, it was observed that pustulan could inhibit the binding of zymosan by Dectin-1-expressing cells (4, 8). The inhibitory activity of pustulan was 10-50 times less than that of laminarin. Small amounts of 1,3,6-linked glucose are detectable in the polysaccharide (Table 5) and could account for this activity. In the acetolysis procedure used in the present experiments, fragmentation of the 1,6-linked sequences is favored, whereas 1,3-linked sequences would be relatively resistant (35) and would tend not to feature among the oligosaccharide fragments generated. This is corroborated by the results of methylation analysis in which 1,3- or 1,3,6-linked sequences were not detected in the pustulan F11 oligosaccharide fraction (Table 5). Thus, it has been possible to exclude Dectin-1 binding to 1,6-linked glucan sequence oligosaccharides investigated. It will be interesting to investigate whether Dectin-1 recognizes branched glucan sequences containing both 1,3- and 1,6-linkages. Studies of such heterogeneous oligosaccharides are subjects for future investigation.

The minimum chain length of gluco-oligosaccharides required for Dectin-1 binding is unusually long for a lectin of this family. Binding is detected only to 11-mer or longer oligomers of glucose when these are examined as NGL probes. Taking into account that the core monosaccharide adjoining the lipid is reduced (ring-opened), it is likely that among the glucan oligosaccharides we have investigated, the minimum chain length bearing the recognition motif for Dectin-1 is a decasaccharide. Other lectins of this family recognize mono- to tetrasaccharides. With E-selectin, for example, which recognizes the tri- and tetrasaccharide capping oligosaccharides, sulfo- and sialyl-Lex and -Lea, an additional monosaccharide on the backbone, adjoining the lipid of the NGL, allows adequate presentation for binding (36). The requirement of Dectin-1 for decasaccharides or longer oligosaccharides of glucans is reminiscent of the properties of antibodies specific for another homo-oligomer, {alpha}2-8-linked polysialic acid. NMR studies together with potential energy calculations led to the postulate that the unusual oligosaccharide length dependence for recognition by these antibodies arises from the existence of a conformational epitope, a high order local helix (37).


Figure 4
View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 4.
Microarrays of lipid-linked oligosaccharide probes showing Dectin-1 binding exclusively to the NGL probe derived from the 1,3-linked glucose oligomers F13 of curdlan. A, Oligosaccharide probes were printed in duplicate on nitrocellulose-coated glass slides at ~2 and ~7 fmol/spot (four spots per "position") with Cy3 dye included as a marker (green emission). Binding was detected with Alexa Fluor 647-labeled streptavidin (red emission); the respective images for arrayed and bound components were then merged, after adjustment of the background, so that the spots bound by Dectin-1 appear yellow. The image is that of a subset of 256 arrayed spots of 64 oligosaccharide probes, at positions 115-178 (supplemental Table 1). B, numerical scores are shown of the binding signals (means of duplicate spots) at 2 (blue) and 7 fmol/spot (red) with error bars for the 64 probes at positions 115-178. The inset shows numerical scores of the binding signals at 7 fmol/spot for all of the 190 oligosaccharide probes arrayed (some at more than one position, as shown in supplemental Table 1). Probes at positions 53 and 115 correspond to the curdlan F13 probe (ID 159) used as the positive control.

 
Oligosaccharide microarray technologies have been emerging only recently. In most studies, emphasis has been on chemically synthesized oligosaccharide probes (38-41). The results from the present study using diverse known oligosaccharide sequences of mammalian types, naturally occurring and chemically synthesized, indicate that these are not recognized by Dectin-1. Among them are oligosaccharide probes recognized by other mammalian carbohydrate-binding proteins (21, 31, 42). Here, the ability to generate tailor-made oligosaccharide probes from targeted sources, fungal and bacterial polysaccharides, in conjunction with MS and methylation analysis has been crucial for assigning oligosaccharide ligands for Dectin-1. Thus, it is established that Dectin-1 binds specifically to exogenous oligosaccharides, and these are unrelated to the known endogenous, mammalian type sequences included in our microarrays. The nature of the endogenous ligand expressed on T-lymphocyte subsets to which Dectin-1 binds is an open question and will remain under review as new oligosaccharide probes become available. However, the glucan ligands that we have identified are functional antagonists of the receptor's response to fungal particles.

This microarray approach and its further development for designs of microarrays of glycomes of mammalian tissues, also of microorganisms and plants, promises to be a powerful means of discovering novel carbohydrate ligands for lectin-type proteins and novel antigenic determinants for pathological antibodies such as those directed to allergens. Designer microarrays from T-lymphocytes for Dectin-1 binding studies would be an appropriate sequel to the present studies.


   FOOTNOTES
 
This article is dedicated to the late Gordon Ross, who was a great facilitator of this work.

* This work was supported by United Kingdom Medical Research Council Program Grant G9601454 (to A. M. L. and T. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1. Back

1 Supported by a Ph.D. fellowship from Fundação para a Ciência e Tecnologia, Portugal. Back

3 A Wellcome Senior Fellow in Biomedical Science in South Africa. Back

2 To whom correspondence should be addressed. Tel.: 44-20-8869-3460/3461; Fax: 44-20-8869-3455; E-mail: t.feizi{at}imperial.ac.uk.

4 The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation; ADHP, N-aminoacetyl-N-(9-anthracenyl methyl)-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine; BSA, bovine serum albumin; CHO, Chinese hamster ovary; HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; NGL, neoglycolipid; NSG, neutral soluble glucan; PBS, phosphate-buffered saline; PGG, poly-(1,6)-beta-D-glucopyranosyl-(1,3)-beta-D-glucopyranose; RPE, R-phycoerythrin-labeled anti-rat immunoglobulins; TBS, Tris-buffered saline; TNF, tumor necrosis factor; F7-F13, fraction 7-13, respectively. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Biothera for providing NSG and PGG and Robert A. Childs for invaluable advice and for providing the microarrays. Also acknowledged is the help of Chunhao Yu and Colin Herbert in oligosaccharide and NGL preparations and Penelope Bedford in the flow cytometric analyses.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Brown, G. D., Herre, J., Williams, D. L., Willment, J. A., Marshall, A. S., and Gordon, S. (2003) J. Exp. Med. 197, 1119-1124[Abstract/Free Full Text]
  2. Herre, J., Willment, J. A., Gordon, S., and Brown, G. D. (2004) Crit. Rev. Immunol. 24, 193-203[CrossRef][Medline] [Order article via Infotrieve]
  3. Ariizumi, K., Shen, G. L., Shikano, S., Xu, S., Ritter, R., III, Kumamoto, T., Edelbaum, D., Morita, A., Bergstresser, P. R., and Takashima, A. (2000) J. Biol. Chem. 275, 20157-20167[Abstract/Free Full Text]
  4. Brown, G. D., and Gordon, S. (2001) Nature 413, 36-37[Medline] [Order article via Infotrieve]
  5. Taylor, P. R., Brown, G. D., Reid, D. M., Willment, J. A., Martinez-Pomares, L., Gordon, S., and Wong, S. Y. (2002) J. Immunol. 169, 3876-3882[Abstract/Free Full Text]
  6. Reid, D. M., Montoya, M., Taylor, P. R., Borrow, P., Gordon, S., Brown, G. D., and Wong, S. Y. (2004) J. Leukocyte Biol. 76, 86-94[Abstract/Free Full Text]
  7. Sobanov, Y., Bernreiter, A., Derdak, S., Mechtcheriakova, D., Schweighofer, B., Duchler, M., Kalthoff, F., and Hofer, E. (2001) Eur. J. Immunol. 31, 3493-3503[CrossRef][Medline] [Order article via Infotrieve]
  8. Willment, J. A., Gordon, S., and Brown, G. D. (2001) J. Biol. Chem. 276, 43818-43823[Abstract/Free Full Text]
  9. Grunebach, F., Weck, M. M., Reichert, J., and Brossart, P. (2002) Exp. Hematol. 30, 1309-1315[CrossRef][Medline] [Order article via Infotrieve]
  10. Willment, J. A., Marshall, A. S., Reid, D. M., Williams, D. L., Wong, S. Y., Gordon, S., and Brown, G. D. (2005) Eur. J. Immunol. 35, 1539-1547[CrossRef][Medline] [Order article via Infotrieve]
  11. Weis, W. I., Taylor, M. E., and Drickamer, K. (1998) Immunol. Rev. 163, 19-34[CrossRef][Medline] [Order article via Infotrieve]
  12. Adachi, Y., Ishii, T., Ikeda, Y., Hoshino, A., Tamura, H., Aketagawa, J., Tanaka, S., and Ohno, N. (2004) Infect. Immun. 72, 4159-4171[Abstract/Free Full Text]
  13. Steele, C., Marrero, L., Swain, S., Harmsen, A. G., Zheng, M., Brown, G. D., Gordon, S., Shellito, J. E., and Kolls, J. K. (2003) J. Exp. Med. 198, 1677-1688[Abstract/Free Full Text]
  14. Gantner, B. N., Simmons, R. M., and Underhill, D. M. (2005) EMBO J. 24, 1277-1286[CrossRef][Medline] [Order article via Infotrieve]
  15. Bohn, J. A., and BeMiller, N. (1995) Carbohydr. Polymers 28, 3-14
  16. Brown, G. D., and Gordon, S. (2003) Immunity 19, 311-315[CrossRef][Medline] [Order article via Infotrieve]
  17. Brown, G. D., Taylor, P. R., Reid, D. M., Willment, J. A., Williams, D. L., Martinez-Pomares, L., Wong, S. Y., and Gordon, S. (2002) J. Exp. Med. 196, 407-412[Abstract/Free Full Text]
  18. Gantner, B. N., Simmons, R. M., Canavera, S. J., Akira, S., and Underhill, D. M. (2003) J. Exp. Med. 197, 1107-1117[Abstract/Free Full Text]
  19. Herre, J., Marshall, A. S., J., Caron, E., Edwards, A. D., Williams, D. L., Schweighoffer, E., Tybulewicz, V., Reis e Sousa, C., Gordon, S., and Brown, G. D. (2004) Blood 104, 4038-4045[Abstract/Free Full Text]
  20. Gordon, S. (2002) Cell 111, 927-930[CrossRef][Medline] [Order article via Infotrieve]
  21. Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai, W. (2002) Nat. Biotechnol. 20, 1011-1017[CrossRef][Medline] [Order article via Infotrieve]
  22. Feizi, T., and Chai, W. (2004) Nat. Rev. Mol. Cell Biol. 5, 582-588[CrossRef][Medline] [Order article via Infotrieve]
  23. Ettinger, R., Browning, J. L., Michie, S. A., van, E. W., and McDevitt, H. O. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13102-13107[Abstract/Free Full Text]
  24. Chai, W., Feizi, T., Yuen, C.-T., and Lawson, A. M. (1997) Glycobiology 7, 861-872[Abstract/Free Full Text]
  25. Her, G. R., Santikarn, S., Reinhold, V. N., and Williams, J. C. (1987) J. Carbohydr. Chem. 6, 129-139
  26. Stoll, M. S., Feizi, T., Loveless, R. W., Chai, W., Lawson, A. M., and Yuen, C. T. (2000) Eur. J. Biochem. 267, 1795-1804[Medline] [Order article via Infotrieve]
  27. Chai, W., Stoll, M. S., Galustian, C., Lawson, A. M., and Feizi, T. (2003) Methods Enzymol. 362, 160-195[CrossRef][Medline] [Order article via Infotrieve]
  28. Stoll, M. S., and Feizi, T. (1997) Preparation of Neoglycolipids for Structure and Function Assignments of Oligosaccharides: A Laboratory Guide to Glycoconjugate Analysis BioMethods, Vol. 9, pp. 329-348, Birkhauser Verlag AG, Basel, Switzerland
  29. Chai, W. G., Hounsell, E. F., Cashmore, G. C., Rosankiewicz, J. R., Bauer, C. J., Feeney, J., Feizi, T., and Lawson, A. M. (1992) Eur. J. Biochem. 203, 257-268[Medline] [Order article via Infotrieve]
  30. Galustian, C., Childs, R. A., Yuen, C.-T., Hasegawa, A., Kiso, M., Lubineau, A., Shaw, G., and Feizi, T. (1997) Biochemistry 36, 5260-5266[CrossRef][Medline] [Order article via Infotrieve]
  31. Galustian, C., Park, C. G., Chai, W., Kiso, M., Bruening, S. A., Kang, Y. S., Steinman, R. M., and Feizi, T. (2004) Int. Immunol. 16, 853-866[Abstract/Free Full Text]
  32. Feizi, T., Childs, R. A., Watanabe, K., and Hakomori, S. I. (1979) J. Exp. Med. 149, 975-980[Abstract/Free Full Text]
  33. de la Cruz, C. J., Pintor-Toro, J. A., Benitez, T., and Llobell, A. (1995) J. Bacteriol. 177, 1864-1871[Abstract/Free Full Text]
  34. Rogers, N. C., Slack, E. C., Edwards, A. D., Nolte, M. A., Schulz, O., Schweighoffer, E., Williams, D. L., Gordon, S., Tybulewicz, V. L., Brown, G. D., and Reis e Sousa, C. (2005) Immunity 22, 507-517[CrossRef][Medline] [Order article via Infotrieve]
  35. Lindberg, B., Lonngren, J., and Svensson, S. (1975) Adv. Carbohydr. Chem. Biochem. 31, 185-240
  36. Yuen, C.-T., Lawson, A. M., Chai, W., Larkin, M., Stoll, M. S., Stuart, A. C., Sullivan, F. X., Ahern, T. J., and Feizi, T. (1992) Biochemistry 31, 9126-9131[CrossRef][Medline] [Order article via Infotrieve]
  37. Brisson, J. R., Baumann, H., Imberty, A., Perez, S., and Jennings, H. J. (1992) Biochemistry 31, 4996-5004[CrossRef][Medline] [Order article via Infotrieve]
  38. Feizi, T., Fazio, F., Chai, W., and Wong, C. H. (2003) Curr. Opin. Struct. Biol. 13, 637-645[CrossRef][Medline] [Order article via Infotrieve]
  39. Ratner, D. M., Adams, E. W., Disney, M. D., and Seeberger, P. H. (2004) Chembiochem 5, 1375-1383[CrossRef][Medline] [Order article via Infotrieve]
  40. Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M. E., Alvarez, R., Bryan, M. C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D. J., Skehel, J. J., Van, D., I, Burton, D. R., Wilson, I. A., Cummings, R., Bovin, N., Wong, C. H., and Paulson, J. C. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 17033-17038[Abstract/Free Full Text]
  41. Shin, I., Park, S., and Lee, M. R. (2005) Chem. Eur. J. 11, 2894-2901[CrossRef]
  42. Reddy, S. T., Chai, W., Childs, R. A., Page, J. D., Feizi, T., and Dahms, N. M. (2004) J. Biol. Chem. 279, 38658-38667[Abstract/Free Full Text]
  43. Jamas, S., Easson, D. D. J., Ostroff, G. R., and Onderdonk, A. B. (1991) Am. Chem. Soc. Symp. Ser. 469, 44-51
  44. Hong, F., Hansen, R. D., Yan, J., Allendorf, D. J., Baran, J. T., Ostroff, G. R., and Ross, G. D. (2003) Cancer Res. 63, 9023-9031[Abstract/Free Full Text]
  45. Black, W. A. P., Cornhill, W. J., Dewar, E. J., and Woodward, F. N. (1951) J. Appl. Chem. 501-517
  46. Chanda, N. B., Hirst, E. L., and Manners, D. J. (1957) J. Chem. Soc. 1951-1958
  47. Mccleary, B. V., and Matheson, N. K. (1987) Adv. Carbohdydr. Chem. Biochem. 44, 147-276
  48. de Belder, A. N. (1993) in Industrial Gums (Whistler, R. L., and BeMiller, J., eds) pp. 399-426, Academic Press, Inc., New York
  49. Haworth, W. N., Hirst, E. L., and Isherwood, F. A. (1937) J. Chem. Soc. 748-790

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 GlycobiologyHome page
S T. Hollmig, K. Ariizumi, and P. D Cruz Jr
Recognition of non-self-polysaccharides by C-type lectin receptors dectin-1 and dectin-2
Glycobiology, June 1, 2009; 19(6): 568 - 575.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
G. J. Jones, J. C. D. Wiseman, K. J. Marr, S. Wei, J. Y. Djeu, and C. H. Mody
In contrast to anti-tumor activity, YT cell and primary NK cell cytotoxicity for Cryptococcus neoformans bypasses LFA-1
Int. Immunol., April 1, 2009; 21(4): 423 - 432.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
T. Schallus, C. Jaeckh, K. Feher, A. S. Palma, Y. Liu, J. C. Simpson, M. Mackeen, G. Stier, T. J. Gibson, T. Feizi, et al.
Malectin: A Novel Carbohydrate-binding Protein of the Endoplasmic Reticulum and a Candidate Player in the Early Steps of Protein N-Glycosylation
Mol. Biol. Cell, August 1, 2008; 19(8): 3404 - 3414.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
G D. Sprott, C. J Dicaire, J.-P. Cote, and D. M Whitfield
Adjuvant potential of archaeal synthetic glycolipid mimetics critically depends on the glyco head group structure
Glycobiology, July 1, 2008; 18(7): 559 - 565.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. M. Weck, S. Appel, D. Werth, C. Sinzger, A. Bringmann, F. Grunebach, and P. Brossart
hDectin-1 is involved in uptake and cross-presentation of cellular antigens
Blood, April 15, 2008; 111(8): 4264 - 4272.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
E. L. Adams, P. J. Rice, B. Graves, H. E. Ensley, H. Yu, G. D. Brown, S. Gordon, M. A. Monteiro, E. Papp-Szabo, D. W. Lowman, et al.
Differential High-Affinity Interaction of Dectin-1 with Natural or Synthetic Glucans Is Dependent upon Primary Structure and Is Influenced by Polymer Chain Length and Side-Chain Branching
J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 115 - 123.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
M. A. Campanero-Rhodes, A. Smith, W. Chai, S. Sonnino, L. Mauri, R. A. Childs, Y. Zhang, H. Ewers, A. Helenius, A. Imberty, et al.
N-Glycolyl GM1 Ganglioside as a Receptor for Simian Virus 40
J. Virol., December 1, 2007; 81(23): 12846 - 12858.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
R. Nisini, A. Torosantucci, G. Romagnoli, P. Chiani, S. Donati, M. C. Gagliardi, R. Teloni, V. Sargentini, S. Mariotti, E. Iorio, et al.
{beta}-Glucan of Candida albicans cell wall causes the subversion of human monocyte differentiation into dendritic cells
J. Leukoc. Biol., November 1, 2007; 82(5): 1136 - 1142.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. G. Rothfuchs, A. Bafica, C. G. Feng, J. G. Egen, D. L. Williams, G. D. Brown, and A. Sher
Dectin-1 Interaction with Mycobacterium tuberculosis Leads to Enhanced IL-12p40 Production by Splenic Dendritic Cells
J. Immunol., September 15, 2007; 179(6): 3463 - 3471.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
K. M. Dennehy and G. D. Brown
The role of the {beta}-glucan receptor Dectin-1 in control of fungal infection
J. Leukoc. Biol., August 1, 2007; 82(2): 253 - 258.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
C. A. Rappleye, L. G. Eissenberg, and W. E. Goldman
Histoplasma capsulatum {alpha}-(1,3)-glucan blocks innate immune recognition by the beta-glucan receptor
PNAS, January 23, 2007; 104(4): 1366 - 1370.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow An addition or correction has been published
Right arrow All Versions of this Article:
281/9/5771    most recent
M511461200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Palma, A. S.
Right arrow Articles by Chai, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Palma, A. S.
Right arrow Articles by Chai, W.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement