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Glycobiology Advance Access originally published online on January 19, 2006
Glycobiology 2006 16(5):422-430; doi:10.1093/glycob/cwj077
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose

Eamon P. McGreal2,6, Marcela Rosas6, Gordon D. Brown3,6, Susanne Zamze7, Simon Y.C. Wong4,7, Siamon Gordon6, Luisa Martinez-Pomares5,6 and Philip R. Taylor1,6

2 Present address: Department of Child Health, Wales College of Medicine, University of Cardiff, Heath Park, Cardiff CF14 4XN, UK
3 Present address: Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Lower Ground Floor, Wernher & Beit Building South, Groote Schuur Campus, Observatory, 7925, Cape Town, South Africa
4 Present address: School of Molecular Medical Sciences, University of Nottingham, Floor A, West Block, Queen’s Medical Centre, Nottingham NG7 2UH, UK
5 Present address: Inflammation and Immunity Theme, Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen, Scotland AB25 2ZD, UK
6 Sir William Dunn School of Pathology, Oxford University, South Parks Road, Oxford OX1 3RE, UK; and 7 The Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, UK

1 To whom correspondence should be addressed; e-mail: philip.taylor{at}path.ox.ac.uk

Received on November 16, 2005; revised on January 11, 2006; accepted on January 12, 2006


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Supplementary data
 Conflict of interest statement
 Acknowledgments
 References
 
We examined the carbohydrate-binding potential of the C-type lectin-like receptor Dectin-2 (Clecf4n). The carbohydrate-recognition domain (CRD) of Dectin-2 exhibited cation-dependent mannose/fucose-like lectin activity, with an IC50 for mannose of approximately 20 mM compared to an IC50 of 1.5 mM for the macrophage mannose receptor when assayed by similar methodology. The extracellular domain of Dectin-2 exhibited binding to live Candida albicans and the Saccharomyces-derived particle zymosan. This binding was completely abrogated by cation chelation and was competed by yeast mannans. We compared the lectin activity of Dectin-2 with that of two other C-type lectin receptors (mannose receptor and SIGNR1) known to bind fungal mannans. Both mannose receptor and SIGNR1 were able to bind bacterial capsular polysaccharides derived from Streptococcus pneumoniae, but interestingly they exhibited distinct binding profiles. The Dectin-2 CRD exhibited only weak interactions to some of these capsular polysaccharides, indicative of different structural or affinity requirements for binding, when compared with the other two lectins. Glycan array analysis of the carbohydrate recognition by Dectin-2 indicated specific recognition of high-mannose structures (Man9GlcNAc2). The differences in the specificity of these three mannose-specific lectins indicate that mannose recognition is mediated by distinct receptors, with unique specificity, that are expressed by discrete subpopulations of cells, and this further highlights the complex nature of carbohydrate recognition by immune cells.

Key words: lectin / macrophage / mannose


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Supplementary data
 Conflict of interest statement
 Acknowledgments
 References
 
C-Type lectins are a family of molecules that bind to carbohydrates in a calcium-dependent manner. The lectin activity is mediated by conserved carbohydrate-recognition domains (CRDs) (Drickamer, 1993Go). One of the best-characterized C-type lectins is the macrophage (MØ) mannose receptor (MR). The MØ MR has eight CRD-containing C-type lectin-like domains (CTLD) (Ezekowitz et al., 1990Go; Taylor et al., 1990Go) that contribute to receptor specificity, although five of these (CRD4–8) are considered adequate to reproduce similar affinity binding to the full-length molecule (Taylor et al., 1992Go). The crystal structure of CRD4 of the MØ MR has been determined, and it demonstrates conservation of structure and activity with the CRD of the well-characterized mannose-binding lectin (MBL) (Feinberg et al., 2000Go). The MØ MR was discovered because of its role in the clearance of endogenous glycoproteins (Wileman et al., 1986Go), and this activity has been recently verified in vivo by the generation of MR-deficient mice (Lee et al., 2002Go). It has also been speculated to have a broad role as a pattern-recognition receptor (PRR), recognizing mannose-like structures on the surface of many pathogens (Stahl and Ezekowitz, 1998Go).

Self, nonself discrimination on the basis of carbohydrate composition and spatial organization is an ancient mechanism of host defense. This is exemplified by the CRD of MBL, which shares many conserved features with CRD4 of MR. MBL binds hexoses such as mannose and N-acetylglucosamine with equatorial hydroxyl groups at positions C3 and C4 of the pyranose ring. This gives particularly high-affinity interactions with multivalent carbohydrates on a variety of pathogens including fungi and bacteria. In addition, the spatial arrangement of such carbohydrates provides extra specificity in ligand binding. In contrast to this, MBL has no detectable affinity for oligosaccharidess commonly found on mammalian glycoproteins.

Additional C-type lectins with specificity for mannose, which have more recently been characterized, are DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) and DC-SIGNR (DC-SIGN-related; L-SIGN, Liver-SIGN) (Geijtenbeek TB, Torensma R et al., 2000Go; Bashirova et al., 2001Go). These molecules may function in DC migration and DC/T-cell synapse formation through interaction with ICAM-2 and ICAM-3, respectively (Geijtenbeek TB, Krooshoop DJ et al., 2000Go ; Geijtenbeek TB, Torensma R et al., 2000Go), as well as playing roles in pattern recognition by binding carbohydrates on the surface of viruses and other microbes in a manner similar to other C-type lectin receptors (Figdor et al., 2002Go). Determination of the crystal structure of DC-SIGN/DC-SIGNR has revealed high-affinity binding to an internal feature of high-mannose oligosaccharides (Feinberg et al., 2001Go). This is in contrast to MBL and the MR, which have preferred specificity for terminal mannose residues. This, taken with evidence that DC-SIGN forms tetramers at the cell surface (Mitchell et al., 2001Go), provides a mechanism for lectins to exhibit different specificity, whilst all being loosely classified as ‘mannose specific’. Recent experiments with model oligosaccharide ligands demonstrated distinct ligand preferences for both MR and DC-SIGN (Frison et al., 2003Go). The MØ-expressed murine homolog of DC-SIGNR (SIGNR1) appears to function analogously to its human counterpart (Geijtenbeek et al., 2002Go; Kang et al., 2003Go) but has distinct activities compared to other murine DC-SIGN homologs (Koppel et al., 2004Go; Takahara et al., 2004Go).

Dendritic cell-associated lectin-2 (Dectin-2/Clecf4n) was discovered in an experimental murine model of acute myeloid leukemia as a receptor over-expressed in the spleen (Fernandes et al., 1999Go). We recently generated monoclonals against Dectin-2 and have found that its tissue distribution is tightly restricted to myeloid cells of the MØ and DC lineages (Taylor et al., 2005Go). Dectin-2 surface expression on these cells was also found to be low but exhibited enhanced expression on inflammatory monocytes during the acute phase of an inflammatory reaction (Taylor et al., 2005Go).

Dectin-2 is encoded in the natural killer complex of C-type lectin-like genes and shares features with other classical C-type lectins in that it has conserved motifs for the recognition of mannose in a Ca2+-dependent manner (Fernandes et al., 1999Go). Despite this, reports of a lectin activity of Dectin-2 have been contradictory (Fernandes et al., 1999Go; Ariizumi et al., 2000Go). Recent experiments with a soluble recombinant form of Dectin-2 have suggested the presence of a ligand on CD4+CD25+ T cells, and in vivo administration of this protein in mice impaired the development and maintenance of ultraviolet (UV)-induced tolerance (Aragane et al., 2003Go). It has been speculated that prevention of the interaction between endogenous Dectin-2 and T cells causes this defect, although the nature of this ligand is as yet uncharacterized.

In this study, we have addressed the role of Dectin-2 as a C-type lectin. We demonstrate that the CRD of Dectin-2 functions as a C-type lectin with specificity for glycoconjugates bearing high-mannose sugars. We show that the CRD of Dectin-2 can bind live Candida albicans and zymosan. We also compare the lectin activity of the Dectin-2 CRD toward complex bacterial polysaccharides with that of two other mannose-specific lectins, MR and SIGNR1. Together, the data presented here indicate that Dectin-2 could play a role in the recognition of complex polysaccharides and shows that mannose-binding receptors on immune cells not only have distinct expression profiles but also distinct specificities.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Supplementary data
 Conflict of interest statement
 Acknowledgments
 References
 
Generation of soluble recombinant Dectin-2
To assess the lectin activity of Dectin-2, we generated a soluble form of Dectin-2 (Dectin-2-Fc) to test the lectin activity of the CRD. This strategy has been previously used to analyze the lectin activity of the C-type lectin domains of MR, Endo180, and DC-SIGN family members (Linehan et al., 2001Go; East et al., 2002Go; Appelmelk et al., 2003Go). The fusion protein is shown schematically in Figure 1A. Dectin-2-Fc was purified by affinity chromatography on protein-A sepharose and its purity assessed by SDS-PAGE and Coomassie blue staining (Figure 1B).


Figure 1
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  		Fig. 1. Production of a soluble recombinant form of Dectin-2. (A) Schematic representation of Dectin-2 and Dectin-2-Fc. The extracellular domain of Dectin-2 was cloned between the Ig{kappa} signal peptide and the mutant hIgG1 Fc domain. (B) GelCode Blue protein stain of 2 µg protein-A purified Dectin-2-Fc resolved under non-reducing (NR) and reducing (R) conditions by 7% SDS-PAGE.

 

Carbohydrate specificity of Dectin-2
As Dectin-2 was predicted to recognize mannose the lectin activity of Dectin-2-Fc was tested in a FACS assay using zymosan particles as a model poly-mannose ligand. Dectin-2-Fc bound to zymosan in a manner which was completely abrogated by 5mM EDTA and inhibited by mannose in a dose-dependent manner (Figure 2A), indicating that the CRD of Dectin-2 possesses specific Ca2+-dependent lectin activity.


Figure 2
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  		Fig. 2. Definition of the lectin specificity of Dectin-2. (A) FACS analysis of Dectin-2-Fc binding zymosan particles. Binding of Dectin-2-Fc to zymosan was prevented by EDTA and competed with increasing concentrations of mannose. Data shown are from one experiment, which is representative of 3 individual experiments. (B) Binding of Dectin-2-Fc to zymosan particles was assessed by Flow cytometry and competitively inhibited with increasing concentrations of the indicated monosaccharides. For comparison, results obtained with MRCRD4–7-Fc, competed with mannose, are included (IC50 of ~1.5 mM; dotted line). The IC50 for the inhibition of Dectin-2-Fc by mannose was approximately 21 mM compared to 140 mM for galactose. Similar results were obtained from two individual experiments. CRW117A.-Fc (see Materials and Methods) was used a negative control Fc chimeric protein. (C) Binding of Dectin-2-Fc to live C. albicans. Mannan specifically inhibited the binding of Dectin-2-Fc to the surface of the Candida. Similar results were obtained from two individual experiments. Data are expressed as geometric mean fluorescent intensity (geoMFI).

 

To determine the sugar specificity of the CRD of Dectin-2, we used different monosaccharides in competition assays in a similar way to previous studies of the specificity of MR and other lectins (Taylor et al., 1990Go; Holmskov et al., 1993Go; Mitchell et al., 2001Go; Zamze et al., 2002Go). The monosaccharides were titrated in competition assays to block the interaction of Dectin-2-Fc with zymosan particles (Figure 2B). Mannose and fucose were the best inhibitors of the binding of Dectin-2-Fc (the IC50 of mannose was approximately 21 mM compared to an IC50 of approximately 140 mM for galactose). Dimeric Fc fusion chimeras derived from CRD4–7 of the MØ MR have an IC50 of approximately 1.5 mM for mannose in this and similar assays (dotted line in Figure 2B; Zamze et al., 2002Go), which indicates that Dectin-2, when compared to the MØ MR, has a significantly lower affinity for mannose/fucose.

To test the possibility that Dectin-2 may be involved in the recognition of exogenous pathogen-derived carbohydrates, Dectin-2-Fc was also tested for its ability to recognize live C. albicans (Figure 2C). Dectin-2-Fc bound to live C. albicans and this was completely blocked in the absence of Ca2+ and competed by yeast mannan but not galactan (Figure 2C).

Recognition of carbohydrates on pathogens by Dectin-2
We extended our study of the recognition of pathogen-derived carbohydrates by the Dectin-2 CRD by studying the binding of Dectin-2 to several pathogens which have been shown to expose mannose-like structures and be recognized by lectins with ‘mannose-like’ specificity. As previously shown, the CRD of Dectin-2 bound well to C. albicans and, as expected from the recognition of zymosan, Saccharomyces cerevisiae, and this binding was inhibitable by chelation of Ca2+ and competition with mannose (Figure 3). Binding was also observed to Mycobacterium tuberculosis, Paracoccoides brasiliensis, Histoplasma capsulatum and also the capsule-deficient Cryptococcus neoformans, with different levels of binding evident to the different microorganisms (Figure 3).


Figure 3
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  		Fig. 3. Recognition of fungi by the CRD of Dectin-2. The expression of potential Dectin-2 ligands was investigated on pathogens known to contain mannose structures by flow-cytometry. Dectin-2-Fc binding is indicated by bold-line histograms and binding of a control chimeric protein (CRW117A-Fc) is indicated by red filled histograms. Binding, where observed, was competed by mannose (green histograms) and Ca2+-chelation with EDTA (blue histograms). All FACS profiles are representative of at least two experiments.

 

Recognition of complex microbial polysaccharides by Dectin-2; comparison with SIGNR1 and MR
Others and we have recently reported that the MØ MR and SIGNR1 play a role in the recognition of a range of capsular polysaccharides (CPS) derived from different capsular serotypes of S. pneumoniae (Zamze et al., 2002Go; Kang et al., 2004Go). To compare the specificity of the lectin activity of Dectin-2 to the MØ MR and SIGNR1, we used S. pneumoniae CPS to block the interaction of Dectin-2-Fc with yeast mannan immobilized on an ELISA plate as a measure of the ability of Dectin-2 to interact with those polysaccharides. In these assays EDTA effectively blocked the interaction and it was competed with mannan (Figure 4). The majority of S. pneumoniae CPS tested exhibited no significant inhibitory activity. Although three CPS did demonstrate mild inhibitory activity, this was low in most instances despite the high concentration of inhibitory CPS used (250 µg/ml) (Figure 4) and thus represents at best a weak interaction. Other ELISAs analyzing the direct interaction of Dectin-2-Fc with immobilized CPS did not yield positive results although a weak interaction with type 3 CPS (SP3) was noted (data not shown). In contrast to this, the interaction of MRCRD4–7-Fc with mannan in parallel experiments was significantly inhibited by the same CPS preparations, with a profile in agreement with our previously reported data (Zamze et al., 2002Go). The ability of SP4 to inhibit MRCRD4–7-Fc binding was unexpected based on our previous study; however, this may reflect the high concentration of CPS used here as well as the different assay system and again most likely reflects a weak interaction not previously detected. Interestingly, LPS derived from capsular serotype 55 of Klebsiella pneumoniae (K55 LPS) (LPS serotype 03), which has previously been shown to be a good ligand for MR and is known to contain a poly-mannose side chain, significantly blocked Dectin-2 binding (Figure 4).


Figure 4
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  		Fig. 4. Comparative interactions between Dectin-2-Fc, MRCRD4–7-Fc and capsular polysaccharidesCapsular polysaccharides from S. pneumoniae (250 µg/ml) were used to inhibit the interaction of Fc-chimeras with yeast mannan immobilized on wells of a 96 well ELISA plate. Data presented here are from two separate experiments. Percent inhibition values were calculated compared to binding observed in control wells in the absence of a specific inhibitor. Data represent two separate experiments ± SD.

 

We recently identified SIGNR1 as a MØ fungal pattern-recognition receptor, and others have shown interactions of S. pneumoniae CPS with SIGNR1 (Kang et al., 2004Go) and a non-redundant role for SIGNR1 in host defense against S. pneumoniae (Lanoue et al., 2004Go). Consequently we compared its ability to bind to S. pneumoniae CPS and K55 LPS, determined by their ability to compete the interaction of SIGNR1 transduced cells with FITC-labeled zymosan as previously described (Taylor PR, Brown GD et al., 2004Go) (Figure 5A and B), to the binding of the CRDs of Dectin-2 and the MØ MR (the results are summarized comparatively in Table I). We observed significant binding of SIGNR1 to many of the polysaccharides, but interestingly the binding profile exhibited notable differences in binding specificity to that of the MØ MR (Zamze et al., 2002Go). SIGNR1 but not the MØ MR bound to SP1 and SP18C (Zamze et al., 2002Go) (Table I), whereas the MR but not SIGNR1 bound to SP2 and SP3 (Zamze et al., 2002Go) (Table I).


Figure 5
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  		Fig. 5. Recognition of bacterial capsular polysaccharides by SIGNR1.The ability of an additional ‘mannose-binding’ lectin, SIGNR1, to recognize CPS was examined by competing the binding of FITC-labeled zymosan from cell surface expressed receptor. (A) Representative FACS profiles demonstrating the ability of 10 µg of mannan and SP9V CPS to inhibit the binding of FITC-labeled zymosan to NIH3T3 cells expressing virally transduced SIGNR1. Plots indicate the two populations of cells analyzed, those binding FITC-zymosan (upper right quadrant) and those not (upper left quadrant). Excess zymosan particles are evident in the lower right quadrant. The inability of CWPS to inhibit zymosan binding is evident by comparison of cell density in the upper right quadrant of the first panel and the fourth panel, which are experiments without an inhibitor and with CWPS respectively. (B) Background binding data obtained from parallel experiments using wild type cells was subtracted and % inhibition values were calculated as described in the materials and methods. Data represent 2 (for K55 LPS) or 3 experiments ± SD.

 

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  		Table I. A summary of the comparative binding of S pneumoniae CPS by mannose-specific C-type lectins

 

Glycan array analysis of the specificity of the CRD of Dectin-2
Our data indicated that Dectin-2 clearly recognizes mannose type ligands, although the precise nature of these ligands is different to those recognized by SIGNR1 and MØ MR. To more accurately determine the ligand specificity of the CRD of Dectin-2, it was screened by ELISA plate assay against 109 distinct synthetic carbohydrates (a detailed list of which is in Supplemental Table I). The plate assay indicated that Dectin-2 was very specific for high-mannose structures with greatest recognition of Man9GlcNAc2>Man8GlcNAc2 and also some recognition of Man7GlcNAc2 (Figure 6).


Figure 6
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  		Fig. 6. Glycan Array analysis of Dectin-2 CRD specificity. Graph showing the recognition of carbohydrates by Dectin-2-Fc in ELISA-based glycan array. A detailed numbered list of the carbohydrates can be found in Supplementary Data Table I. The CRD of Dectin-2 exhibited specific recognition of the indicated high-mannose structures, which are also shown schematically according to the conventions endorsed by the consortium for functional glycomics (http://www.functionalglycomics.org).

 


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Supplementary data
 Conflict of interest statement
 Acknowledgments
 References
 
C-type lectins expressed by cells of the myeloid lineage represent an important recognition system for carbohydrates found on a range of pathogens including viruses, bacteria and fungi as well as endogenously expressed glycoproteins. Consequently, C-type lectins play important roles in innate immune responses, homeostasis of serum protein levels and the regulation of cell–cell interactions by binding endogenous adhesion molecules. The CRDs of many C-type lectins have been defined as mannose-specific on the basis of binding to simple defined sugars and the ability of mannose and mannose-like monosaccharides to inhibit the interaction of the CRD with complex oligosaccharides. In addition to a well-conserved CTLD and the ability to bind Ca2+, the primary indicator of mannose-type specificity in the CRDs of MBL, MR and the DC-SIGN family is the presence of a consensus triplet of amino acids with the sequence EPN (Drickamer, 1992Go). The presence of such a motif within the CRD of Dectin-2 prompted our investigation of its ligand specificity. In this study, we have shown that the CRD of Dectin-2, a recently discovered and poorly understood member of the type-II CTLD family of proteins, has cation-dependent mannose/fucose-like lectin activity which is different to that of the MØ MR and SIGNR1.

A soluble recombinant Dectin-2-Fc chimera consisting of the extracellular domain of Dectin-2 fused to the Fc portion of human IgG1 was generated as a probe to test the ligand specificity of this molecule. Dectin-2-Fc bound to zymosan and competition assays in which varying concentrations of mannose were used as inhibitors revealed that, in comparison to MRCRD4–7-Fc, Dectin-2-Fc had a 15-fold lower affinity for mannose (IC50 of 1.5mM for MRCRD4–7-Fc compared with 21mM for Dectin-2-Fc). Fucose was also identified as an effective inhibitor of Dectin-2 binding with an IC50 comparable to that of mannose. Consistent with its ability to bind to mannose, Dectin-2-Fc also bound to live C. albicans and other pathogens, suggestive of a role in innate pathogen recognition similar to other myeloid-expressed C-type lectin receptors.

The much lower affinity of the CRD of Dectin-2 for these ligands in comparison to CRDs 4–7 of MR is not readily explained, although it is probable that beyond calcium coordinating residues, the EPN motif and disulfide bonds, other less well defined residues within the domain play a critical role in tailoring the relative affinity of different CRDs (Quesenberry and Drickamer, 1992Go). In addition, oligomerization of CRDs has recently been described for cell-derived type II C-type lectins which confers a 6–7-fold enhanced affinity of DC-SIGN and DC-SIGNR for a high-mannose glycoprotein (Man30-BSA) (Mitchell et al., 2001Go). The avidity of cell surface Dectin-2 for its ligand might therefore be expected to differ from that seen with Dectin-2-Fc if such oligomerization occurs. However, RAW 264.7 cells over-expressing HA-tagged Dectin-2 at the cell surface did not show enhanced binding of zymosan particles or C. albicans when compared to wild type cells (data not shown). These data therefore indicate that whilst the CRD of Dectin-2 is capable of recognizing polysaccharides on the surface of fungi, it does not play a significant role in the tethering of these particles to the cell surface, an observation in agreement with the relatively poor affinity for mannose-type ligands observed in this study.

There are many mannose-specific lectins expressed by myeloid cells, and members of the DC-SIGN family and MR have already been shown to bind yeast and fungi (Marodi et al., 1991Go; Cambi et al., 2003Go). The functional differences between these receptors will therefore depend not only on their relative affinities for ligand but also the post-ligation events such as intracellular trafficking and signal transduction. Accordingly, identification of the co-receptor required for Dectin-2 surface expression (Taylor et al., 2005Go) will be a crucial next step in understanding the full implications of the interactions described in this study.

Many of the studies describing pathogen-derived ligands for mannose-specific lectins have focused on their capacity to bind carbohydrate ligands with high-mannose structures. In a recent study by our group, the interaction of MR with complex CPS from S. pneumoniae revealed a much more broad ligand specificity than might have been expected (Zamze et al., 2002Go). These polysaccharides have none of the structural features associated with known MR specificity such as multiple terminal mannose or even glucose residues. It was speculated that MR might interact with internal residues within the polysaccharides as S. pneumoniae CPS are known to have a flexible ribbon-like structure which could facilitate such events. As MR, SIGNR1 and Dectin-2 all have ‘mannose-specificity’ we compared all three receptors for their ability to bind CPS from S. pneumoniae. The structures of these polysaccharides have been described before (Zamze et al., 2002Go). Despite the apparent similarities in ligand preference of these receptors, they displayed a distinct binding profile when complex bacterial polysaccharides were used in binding assays. There was no obvious structural correlation between the ligands recognized by these lectins.

When Dectin-2-Fc was compared directly with MRCRD4–7-Fc in solid phase assays which assessed the ability of CPS to inhibit the interaction with immobilized mannan, a striking disparity between both receptors was observed. The binding activity of both molecules could be abrogated with EDTA and competed with soluble mannan indicating that the binding was a result of specific lectin activity. CPS that have previously been characterized as MR ligands effectively blocked the interaction of MRCRD4–7-Fc with mannan, but had little effect on Dectin-2-Fc binding. Notable differences were also observed when MR and SIGNR1 were compared. In contrast to the MR, SIGNR1 did not appear to have any affinity for CPS from serotypes 2 and 3, but unlike MR, SIGNR1 did bind CPS from serotypes 1 and 18C. The affinity of SIGNR1 for different CPS varied but the level of inhibition of zymosan binding achieved was comparable to that achieved with an equal amount (10 µg/well) of mannan. Notably, all three lectins recognized the mannosylated K55 LPS. This comparison highlights the differential specificity of CRDs from lectins with similar simple ligand specificity toward complex polysaccharides indicating that the spectrum of pathogens/carbohydrates recognized by the family of ‘mannose-specific’ lectins may be quite different.

When assayed against multiple carbohydrate structures by glycan array, Dectin-2 exhibited very specific recognition of high-mannose structures (Man9GlcNAc2 > Man8GlcNAc2 > Man7GlcNAc2). Similar lectin activity is exhibited by DC-SIGN, although this lectin exhibits broader specificity, for example recognizing ligands containing Lewis x (Koppel et al., 2005Go). High mannose is expressed on endogenous glycoproteins, lysosomal enzymes and many proteins in the Golgi and endoplasmic reticulum. Recognition of endogenous carbohydrates could be suggestive of a role in the clearance of self (as with MR; Lee et al., 2002Go), cell:cell interactions (as with DC-SIGN; Koppel et al., 2005Go), or possibly even in the recognition of abnormal self. It is not clear whether the lectin activity is required for the recognition of the putative ligand on CD4+CD25+ T cells.

Collectively these data indicate that the lectin domain of Dectin-2 is a low affinity mannose-binding lectin with specificity for high-mannose structures. These interactions may play an important role in the detection of pathogenic material by the host and may also be involved in the recognition of endogenous glycoproteins. Identification of the co-receptor required for Dectin-2 surface expression may help to elucidate the specific role of Dectin-2 in response to ligand recognition.


   Materials and methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Supplementary data
 Conflict of interest statement
 Acknowledgments
 References
 
Generation of soluble recombinant Dectin-2
A mutated Fc domain with its own stop codon, derived from human IgG1, but containing four mutations which remove Fc receptor binding and complement activation (a kind gift from Christine Ambrose) (Canfield and Morrison, 1991Go; Lund et al., 1991Go; Tao et al., 1993Go; Taylor PR, Zamze S et al., 2004Go) was transferred into the pSecTag2(C) vector (Invitrogen, UK), which contains the Ig{kappa} signal peptide. The extracellular domain of mouse Dectin-2 (amino acids 50–209) was amplified by PCR (primers: 5'-AGG TGG TAC CGA AGA AGA CTA TAT GAA CTT CAC-3' and 5'-AGG GAA TTC TAG GTA AAT CTT CTT CAT TTC-3') and introduced in-frame into the KpnI and EcoRI restriction sites between the signal peptide and Fc generating a vector encoding a Dectin-2-Fc chimeric protein (henceforth referred to as Dectin-2-Fc) under the control of the CMV promoter. Dectin-2-Fc was introduced into the HEK293T cell line by transient transfection with Gene-Juice reagent (Novagen, UK), which was used in accordance with the manufacturers’ guidelines. After transfection, the HEK293T were selected in Zeocin (Invitrogen) at 0.2mg/ml and cloned to establish stable transfected cell lines. The cells were cultured in media containing 4% IgG-depleted bovine serum instead of 10% fetal bovine serum and the transfected cells were left to condition this medium for 7 days. After this time, Dectin-2-Fc was isolated by affinity purification on Protein A-sepharose (Amersham Biosciences) and elution with 0.1 M glycine, pH 2.9. After neutralization with 0.1 volumes of 1M Tris, pH 9.5, Dectin-2-Fc was dialyzed against PBS. The purity of individual batches of Dectin-2-Fc was assessed by GelCode blue (Perbio, Sweeden) staining of protein resolved under both reducing and non-reducing conditions by 7% SDS-PAGE.

FACS assay for the binding of Dectin-2-Fc to zymosan and fungi
Dectin-2-Fc was added at 2–4 µg/ml to 2 x 106 zymosan particles or live C. albicans in a total volume 100 µl of binding buffer (HBSS with Ca2+ and Mg2+ or tris-buffered saline (TBS), 1M NaCl, 10mM Tris-HCl, 10mM CaCl2 or 5mM EDTA, 0.05% Tween-20, pH 8.0) for 1 hour at room temperature. The particles were washed twice with binding buffer and phycoerythrin labeled anti-human Fc (Jackson Laboratories) was added at a dilution of 1 in 100 in the appropriate binding buffer for 30 minutes at room temperature. The particles were then washed a further 3 times with binding buffer before bound protein was fixed with 1% formaldehyde in binding buffer. Bound Dectin-2-Fc was quantified by flow cytometry. For monosaccharide inhibition assays, Dectin-2-Fc was preincubated with the indicated concentration of monosaccharide prior to the addition of zymosan. MRCRD4–7-Fc (Linehan et al., 2001Go), a chimeric protein containing the CRD4–7 of mouse MR, was used as an affinity comparison, and 5mM EDTA-treatment, which abrogated the binding of both proteins, was used as a negative control. Candida neoformans, P. brasiliensis, and M. tuberculosis were supplied by KJ Kwong Chug (Molecular Microbiology NIH), G. Pereira (State University of Rio de Janeiro) and GS Besra (School of Biosciences, University of Birmingham) respectively.

Capsular polysaccharides, LPS, and carbohydrates
Highly pure CPS from S. pneumoniae were provided either by Dr Chris Jones (National Institute for Biological Standards and Controls, Hertfordshire, UK) or purchased from ATCC. These polysaccharides contain less than 1% by weight protein and nucleic acid contaminants. Common cell wall polysaccharide of S. pneumoniae (CWPS) was obtained from the Statens Serum Institute (Copenhagen, Denmark). LPS from K. pneumoniae, strain K55:03 was isolated and purified as previously described (Zamze et al., 2002Go). All monosaccharides used in inhibition assays and S. cerevisiae-derived mannan were purchased from Sigma (Dorset, UK). Galactan was purchased from Megazyme (Co. Wicklow, Ireland).

Carbohydrate-binding ELISA
For ELISA-based lectin assays to determine the ligand specificity of Dectin-2 CRD, all washes and incubations were carried out in 10 mM Tris-HCl, pH 7.5, 10 mM Ca2+, 0.154 M NaCl, and 0.05% (w/v) Tween 20. Carbohydrate ligands were coated onto the wells of Maxisorb ELISA plates (Nunc, Roskilde, Denmark) by incubation in 0.154 M NaCl overnight at 37 °C (50 µl/well) in a sealed humid box. After coating, plates were washed three times. Dectin-2-Fc and MRCRD4–7-Fc (Linehan et al., 2001Go) were incubated in the wells of coated plates at 2 µg/ml (50 µl/well) for 2 h at room temperature and then the plates were washed three times. In all assays using Fc-chimeras, a negative control protein was used, which consisted of a mutated, non-functional cysteine-rich domain from the MR (CRW117A) fused with the same Fc domain of human IgG1 that was used for the construction of Dectin-2-Fc (Taylor PR, Zamze S et al., 2004Go). The fusion proteins were detected by incubation with anti-human IgG Fc-specific, alkaline phosphatase conjugate, species-absorbed (Jackson Laboratories). Plates were washed three times and developed with p-nitrophenyl phosphate substrate (Sigma). Absorbance was measured at 405 nm. Readings were measured against a blank of uncoated wells incubated with the appropriate Fc-chimera. All assays were carried out in duplicate or triplicate. Yeast mannan-coated wells were used as a positive control in assays measuring MRCRD4–7-Fc binding.

For soluble inhibition assays to determine potential interactions of the CRD of Dectin-2 with S. pneumoniae CPS, the binding of F(ab')2 crosslinked Dectin-2-Fc and MRCRD4-7-Fc (2 µg/ml Fc-chimera final) to mannan (25 µg/ml) coated ELISA plates was carried out in the presence of competitive inhibitors in a high salt buffer, to ensure specificity, consisting of 10 mM Tris-HCl, pH 7.5, 10 mM Ca2+ or 10mM EDTA, 1M NaCl, and 0.05% (w/v) Tween-20. The crosslinked fusion proteins were preincubated with different concentrations of inhibitor, for 30 min followed by incubation for 2 h in the wells of coated ELISA plates prior to detection as described above. All steps were performed at room temperature.

Flow cytometric assay for non-opsonic recognition of zymosan by SIGNR1
Zymosan binding assays were performed as previously described (Taylor PR, Brown GD et al., 2004Go). In brief, subconfluent NIH3T3 cells transduced with SIGNR1 (Taylor PR, Brown GD et al., 2004Go) were lifted by scraping in PBS/5mM EDTA and were washed twice with 0.5% BSA, 2 mM NaN3 in HBSS with Ca2+ and Mg2+. 1 x 105 NIH3T3 cells were placed in 96-well V-bottom bacterial plastic plates. Inhibitors (carbohydrates or EDTA) were added (final concentrations stated where appropriate) and incubated on ice for 20 minutes prior to the addition of FITC-labeled zymosan (Molecular Probes) at a particle:cell ratio of 25:1. The cells and particles were brought into contact by centrifugation at 350 x g for 5 minutes and left for 1 hour on ice before resuspension and fixation in 1% formaldehyde. Zymosan binding was evaluated by flow-cytometry. Results from binding assays were expressed as a binding index (% of cells interacting with particles x by the geometric mean fluorescent intensity (gMFI) of those cells), and the capacity of the polysaccharides to bind to SIGNR1 was expressed as % inhibition of binding when background binding to control wild type cells had been subtracted.

Glycan array analysis
Dectin-2-Fc chimera was screened on the streptavidin/biotin array (V3) in binding buffer (20mM Tris-HCl pH 7.4, 150mM NaCl, 2mM CaCl2, 2mM MgCl2, 0.05% Tween 20, 1% BSA) as previously described (Bochner et al., 2005Go). A stock solution of Dectin-2-Fc (30 µg/ml) was added to each well and incubated at room temperature for 1 hour. This was followed by incubation with goat anti-human IgG-Alexa488 (5 µg/ml). The plates were washed and read in 25 µl of wash buffer on a Victor-2 1420 Multi-label Counter (PerkinElmer Life Sciences) at excitation 485/emission 535. A list of the carbohydrates screened in the array is found in Supplementary Data Table I. This ELISA assay is considered semi-quantitative because of the nature of the streptavidin/biotin-based carbohydrate coating.


   Supplementary data
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Supplementary data
Conflict of interest statement
 Acknowledgments
 References
 
Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/).


   Conflict of interest statement
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Supplementary data
 Conflict of interest statement
Acknowledgments
 References
 
None declared.


   Acknowledgments
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Supplementary data
 Conflict of interest statement
 Acknowledgments
References
 
This work was supported by grants from The Wellcome Trust, the Jenner Institute for Vaccine Research and the Medical Research Council. P.R.T. is a Wellcome Trust Research Career Development Fellow (Grant No. 70579).

The glycan-array analysis was conducted by the Protein–Carbohydrate Interaction Core of The Consortium for Functional Glycomics funded by the National Institute of General Medical Sciences grant GM62116. We also thank (Richard Alvarez, Director Core H, and Angela Lee, glycan assistant) of the Core for helpful advice and assistance.


   Abbreviations
 
CPS, capsular polysaccharide; CRD, carbohydrate-recognition domain; CWPS, common cell wall polysaccharide; DC-SIGN, dendritic cell-specific ICAM-3 grabbing non-integrin; DC-SIGNR, DC-SIGN-related; Dectin-2, dendritic cell associated lectin-2; MBL, mannose-binding lectin; MØ, macrophage; MR, mannose receptor; SIGNR1, SIGN-related-1


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Supplementary data
 Conflict of interest statement
 Acknowledgments
 References
 
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