A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2

Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galβ1–3/4GlcNAcβ1–2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca2+ in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor. Blood dendritic cell antigen 2 (BDCA-2; also designated CLEC4C or CD303) is uniquely expressed on plasmacytoid dendritic cells. Stimulation of BDCA-2 with antibodies leads to an anti-inflammatory response in these cells, but the natural ligands for the receptor are not known. The C-type carbohydrate recognition domain in the extracellular portion of BDCA-2 contains a signature motif typical of C-type animal lectins that bind mannose, glucose, or GlcNAc, yet it has been reported that BDCA-2 binds selectively to galactose-terminated, biantennary N-linked glycans. A combination of glycan array analysis and binding competition studies with monosaccharides and natural and synthetic oligosaccharides have been used to define the binding epitope for BDCA-2 as the trisaccharide Galβ1–3/4GlcNAcβ1–2Man. X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca2+ in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site. As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins. Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor. Blood dendritic cell antigen 2 (BDCA-2) 3The abbreviations used are: BDCA-2blood dendritic cell antigen 2CRDcarbohydrate recognition domainDCIRdendritic cell immunoreceptor. is a defining feature of human plasmacytoid dendritic cells. It was originally identified as the target for one of several monoclonal antibodies that recognize antigens that are uniquely expressed on this unusual population of circulating leukocytes (1Dzionek A. Fuchs A. Schmidt P. Cremer S. Zysk M. Miltenyi S. Buck D.W. Schmitz J. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood.J. Immunol. 2000; 165: 6037-6046Crossref PubMed Scopus (1068) Google Scholar). Plasmacytoid dendritic cells have properties often associated with classical dendritic cells, such as the ability to internalize and present antigens, but they are circulating cells that develop through a distinct lineage (2Schraml B.U. Reis e Sousa C. Defining dendritic cells.Curr. Opin. Immunol. 2015; 32: 13-20Crossref PubMed Scopus (130) Google Scholar, 3Reizis B. Bunin A. Ghosh H.S. Lewis K.L. Sisirak V. Plasmacytoid dendritic cells: recent progress and open questions.Annu. Rev. Immunol. 2011; 29: 163-183Crossref PubMed Scopus (460) Google Scholar). They are believed to play an important role in initial stages of the adaptive immune response and have therefore been an important target in vaccine development. blood dendritic cell antigen 2 carbohydrate recognition domain dendritic cell immunoreceptor. Multiple receptors that mediate internalization of antigens in antigen-presenting cells are expressed on the surface of classical dendritic cells. Several of these endocytic receptors, such as DC-SIGN and the mannose receptor, have sugar binding activity and contain Ca2+-dependent carbohydrate recognition domains (C-type CRDs) (4Weis W.I. Taylor M.E. Drickamer K. The C-type lectin superfamily in the immune system.Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (888) Google Scholar, 5Guo Y. Feinberg H. Conroy E. Mitchell D.A. Alvarez R. Blixt O. Taylor M.E. Weis W.I. Drickamer K. Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR.Nat. Struct. Mol. Biol. 2004; 11: 591-598Crossref PubMed Scopus (484) Google Scholar). However, much less is known about C-type lectins on plasmacytoid dendritic cells. Sequence analysis reveals that BDCA-2 also contains a C-type CRD, despite the fact that it falls into a structurally distinct group of type 2 transmembrane receptors (6Dzionek A. Sohma Y. Nagafune J. Cella M. Colonna M. Facchetti F. Günther G. Johnston I. Lanzavecchia A. Nagasaka T. Okada T. Vermi W. Winkels G. Yamamoto T. Zysk M. Yamaguchi Y. Schmitz J. BDCA-2, a novel plasmacytoid dendritic cell-specific type II C-type lectin, mediates antigen capture and is a potent inhibitor of interferon alpha/beta induction.J. Exp. Med. 2001; 194: 1823-1834Crossref PubMed Scopus (624) Google Scholar, 7Plato A. Willment J.A. Brown G.D. C-Type lectin-like receptors of the dectin-1 cluster: ligands and signaling pathways.Int. Rev. Immunol. 2013; 32: 134-156Crossref PubMed Scopus (133) Google Scholar). Although the cytoplasmic domain of the polypeptide is short and lacks obvious internalization or signaling motifs, BDCA-2 has a well defined link to signaling pathways as a result of its association with the common Fc receptor γ subunit (8Röck J. Schneider E. Grün J.R. Grützkau A. Küppers R. Schmitz J. Winkels G. CD303 (BDCA-2) signals in plasmacytoid dendritic cells via a BCR-like signalosome involving Syk, Slp65 and PLCγ2.Eur. J. Immunol. 2007; 37: 3564-3575Crossref PubMed Scopus (82) Google Scholar, 9Cao W. Zhang L. Rosen D.B. Bover L. Watanabe G. Bao M. Lanier L.L. Liu Y.J. BDCA2/FceR1g complex signals through a novel BCR-like pathway in human plasmcytoid dendritic cells.PLoS Biol. 2007; 5: e248Crossref PubMed Scopus (126) Google Scholar). Stimulation of BDCA-2 with antibodies initiates anti-inflammatory pathways through association with Syk kinase and adapter proteins such as Btk and BLNK, leading to stimulation of phospholipase C and a reduction in secretion of type 1 interferon (1Dzionek A. Fuchs A. Schmidt P. Cremer S. Zysk M. Miltenyi S. Buck D.W. Schmitz J. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood.J. Immunol. 2000; 165: 6037-6046Crossref PubMed Scopus (1068) Google Scholar, 10Jähn P.S. Zänker K.S. Schmitz J. Dzionek A. BDCA-2 signaling inhibits TLR-9-agonist-induced plasmacytoid dendritic cell activation and antigen presentation.Cell. Immunol. 2010; 265: 15-22Crossref PubMed Scopus (34) Google Scholar). Although the sequence of BDCA-2 suggests that it contains a C-type carbohydrate recognition domain, there is conflicting evidence about how it might bind such ligands. The primary sugar-binding site in a C-type CRD is usually centered on a conserved bound Ca2+ that makes coordination bonds to two adjacent hydroxyl groups in the pyranose ring of a monosaccharide residue (4Weis W.I. Taylor M.E. Drickamer K. The C-type lectin superfamily in the immune system.Immunol. Rev. 1998; 163: 19-34Crossref PubMed Scopus (888) Google Scholar). A group of conserved amino acid side chains simultaneously ligate to the Ca2+ and form hydrogen bonds with the sugar hydroxyl groups. Some of these amino acid residues are conserved in all sugar-binding C-type CRDs, whereas others vary with the type of sugar bound. C-type CRDs containing glutamic acid and asparagine residues in the sequence EPN at the primary Ca2+ site bind mannose, glucose, and N-acetylglucosamine through equatorial 3- and 4-OH groups, whereas in CRDs that bind sugars such as galactose and N-acetylgalactosamine, in which the 4-OH group is axial, the corresponding sequence is QPD (11Drickamer K. Engineering galactose-binding activity into a C-type mannose-binding protein.Nature. 1992; 360: 183-186Crossref PubMed Scopus (450) Google Scholar). In keeping with the presence of the EPN motif in BDCA-2, it binds to neoglycoproteins that carry mannose, GlcNAc, and glucose but not to those bearing galactose or N-acetylgalactosamine (12Lee R.T. Hsu T.L. Huang S.K. Hsieh S.L. Wong C.H. Lee Y.C. Survey of immune-related mannose/fucose-binding C-type lectin receptors reveals widely divergent sugar-binding specificities.Glycobiology. 2011; 21: 512-520Crossref PubMed Scopus (114) Google Scholar). Therefore, it is surprising that glycan array analysis indicates that BDCA-2 binds selectively to galactose-terminated biantennary glycans (13Riboldi E. Daniele R. Parola C. Inforzato A. Arnold P.L. Bosisio D. Fremont D.H. Bastone A. Colonna M. Sozzani S. Human C-type lectin domain family 4, member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides.J. Biol. Chem. 2011; 286: 35329-35333Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In addition, a published structure of BDCA-2 reveals a domain-swapped structure that lacks many elements of the typical Ca2+-dependent binding site (14Nagae M. Ikeda A. Kitago Y. Matsumoto N. Yamamoto K. Yamaguchi Y. Crystal structures of carbohydrate recognition domain of blood dendritic cell antigen-2 (BDCA2) reveal a common domain-swapped dimer.Proteins. 2014; 82: 1512-1518Crossref PubMed Scopus (10) Google Scholar). To clarify the apparent contradictions between the known mechanism of sugar binding to C-type CRDs and the apparent properties of BDCA-2, the ligand binding properties of the CRD have been investigated further. Binding, structural, and mutational analysis of the receptor suggest that it has a primary binding site for mannose, but that selectivity for galactose-terminated glycans is achieved through a secondary binding site. The results reconcile the existing data and help to define potential biological ligands for BDCA-2. Methyl glycosides were purchased from Sigma and Carbosynth. Egg yolk glycopeptide was purified according to the published protocol (15Zou Y. Wu Z. Chen L. Liu X. Gu G. Xue M. Wang P.G. Chen M. An efficient approach for large-scale production of sialylglycopeptides from egg yolks.J. Carbohydr. Chem. 2012; 31: 436-446Crossref Scopus (17) Google Scholar), except that Sephacryl S-75 and S-peptide columns (1.5 × 300 mm; GE Life Sciences) were used for gel filtration. Biantennary oligosaccharides were prepared by digestion of the egg yolk glycopeptide (40 μmol) with peptide N-glycosidase (7,500 units; New England Biolabs) at 37 °C for 72 h in 100 mm Tris-Cl, pH 7.8, and was purified by chromatography on an S-peptide column run in 1% acetic acid followed by passage over an Oasis C18 column (Waters) pre-equilibrated with 5 ml of methanol and 5 ml of water. Partial desialylation was achieved by incubation in 1 ml of 40 mm HCl for 6 h at 37 °C followed by neutralization with 0.4 ml of 1 m NaHCO3. The oligosaccharide was purified by two passes over the S-peptide column run in 2 mm Tris. Separation of charged forms was performed by chromatography on a 1-ml column of QAE-Sephadex (A25 resin from GE Life Sciences) equilibrated in 2 mm Tris (16Varki A. Analysis of oligosaccharide negative charge by anion-exchange chromatography.Curr. Prot. Mol. Biol. 2001; 17: 20.21-20.27Google Scholar). Fully desialylated oligosaccharide was eluted with 6 × 1 ml of 2 mm Tris, monosialylated oligosaccharide was eluted with 6 × 1 ml of 2 mm Tris containing 20 mm NaCl, and the remaining disialylated oligosaccharide was eluted with 6 × 1 ml of 2 mm Tris containing 70 mm NaCl. Eluted pools were lyophilized and desalted on the S-peptide column run in 1% acetic acid. The separated oligosaccharides were quantified by anthrone assay and characterized by NMR and mass spectrometry. The disaccharide GlcNAcβ1–2Man, purchased from Dextra, was extended by incubation of 25 μmol in 2 ml of 50 mm Tris-Cl, pH 7.4, containing 20 mm MgCl2 with 35 μmol of UDP-galactose (Sigma), 100 milliunits of galactosyltransferase from bovine milk (Sigma), and 20 milliunits of alkaline phosphatase (Sigma) for 4 h at 37 °C. The reaction was lyophilized, dissolved in 4 ml of chromatography solvent A (n-butanol:acetic acid:H2O, 3/1/1), and fractionated on a 5-ml column of silica gel. The trisaccharide product eluted between 18 and 30 ml, as determined by chromatography of aliquots on high performance thin layer silica plates that were run in solvent A and stained with orcinol. The product was further purified by gel filtration on a 15 × 300-mm S-peptide column run in water. The final product was quantified by anthrone assay and characterized by NMR and mass spectrometry (supplemental Figs. S1 and S2). Mannose-Sepharose was prepared by the divinyl sulfone coupling method (17Fornstedt N. Porath J. Characterization studies on a new lectin found in seed of Vicia ervilia.FEBS Lett. 1975; 57: 187-191Crossref PubMed Scopus (212) Google Scholar). Glycopeptide (25 mg), partially desialylated by heating at 70 °C for 30 min in 50 mm HCl and purified by gel filtration on a 15-ml Sephadex G-25 column run in 1% acetic acid, was coupled to Affi-Gel 10 (10 ml) in 6 ml of 0.1 m sodium HEPES buffer, pH 7.5, for 3 h at 4 °C. A cDNA for BDCA-2 was amplified from a human testis cDNA library (Clontech/Takara) by PCR (Advantage 2 polymerase mix; Takara), cloned into the pCR2.1-TOPO vector (Invitrogen), and sequenced using an Applied Biosystems 310 genetic analyzer. The portion of the cDNA encoding the CRD was reamplified with forward primers designed to provide an initiation sequence in the pT5T expression vector (18Eisenberg S.P. Evans R.J. Arend W.P. Verderber E. Brewer M.T. Hannum C.H. Thompson R.C. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist.Nature. 1990; 343: 341-346Crossref PubMed Scopus (925) Google Scholar). In addition to the natural termination codon, a version with nucleotides encoding the biotinylation sequence Gly-Leu-Asn-Asp-Ile-Phe-Glu-Ala-Gln-Lys-Ile-Glu-Trp-His-Glu (19Schatz P.J. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli.Biotechnology. 1993; 11: 1138-1143Crossref PubMed Scopus (510) Google Scholar) at the 3′ end was created using an alternative reverse primer. Mutagenesis was performed by two-step PCR (20Cormack B. Directed mutagenesis using the polymerase chain reaction.Curr. Protoc. Neurosci. 2001; 4: 11PubMed Google Scholar) using the original cDNA clone as template. Expression was performed in Escherichia coli strain BL21(DE3), which was co-transformed with pBirA plasmid encoding biotin ligase (19Schatz P.J. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli.Biotechnology. 1993; 11: 1138-1143Crossref PubMed Scopus (510) Google Scholar) for expression of biotin-tagged proteins. Expression of the wild type CRD from human BDCA-2 following published protocols for other C-type CRDs resulted in inclusion bodies that were isolated as described (21Jégouzo S.A. Harding E.C. Acton O. Rex M.J. Fadden A.J. Taylor M.E. Drickamer K. Defining the conformation of human mincle that interacts with mycobacterial trehalose dimycolate.Glycobiology. 2014; 24: 1291-1300Crossref PubMed Scopus (38) Google Scholar). Inclusion bodies from 2 liters of bacterial culture were dissolved in 30 ml of 6 m guanidine HCl containing 100 mm Tris-Cl (pH 7.8) and incubated in the presence of 0.01% (v/v) 2-mercaptoethanol for 30 min at 4 °C. Following centrifugation for 30 min at 100,000 × g in a Beckman Ti70.1 rotor, the supernatant was diluted dropwise into 120 ml of 0.5 m NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2 at 4 °C, followed by dialysis against 2 changes of 2 liters of the same buffer. Insoluble material was removed by centrifugation for 30 min at 50,000 × g in a Beckman JA20 rotor, and the supernatant was applied to a 5-ml column of glycopeptide-agarose. After rinsing with 12 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2, the bound protein was eluted with 10 × 1-ml aliquots of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 2.5 mm EDTA. Fractions containing the CRD were identified by analyzing aliquots on SDS-polyacrylamide gels, with protein detected by staining with Coomassie Blue. Mutant forms of the CRD were expressed in the same way, but following initial dialysis against the renaturation buffer, the proteins from 4–6 liters of culture were further dialyzed against two changes of 2 liters of H2O and lyophilized. The lyophilized proteins were taken up in 6 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2 and centrifuged at 100,000 × g in a Beckman TLA100.4 rotor for 30 min at 4 °C. The supernatant was applied to a 10-ml column of mannose-Sepharose, which was washed five times with 2-ml aliquots of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2 and eluted with three 2-ml aliquots and eight 1-ml aliquots of 50 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 2.5 mm EDTA. Gel filtration was performed on a 1 × 30-cm Superdex 200 column (GE Healthcare) eluted with 10 mm Tris-Cl (pH 7.8), 100 mm NaCl, and 2.5 mm EDTA at a flow rate of 0.5 ml/min, with absorbance monitored at 280 nm. Gel electrophoresis was performed on SDS-polyacrylamide gels containing 17.5% (w/v) acrylamide. Biotinylated protein was incubated overnight with Alexa 488-labeled streptavidin (Invitrogen) at a ratio of ∼2 mol of CRD to 1 mol of streptavidin subunit. The mixture was applied to a 1-ml column of mannose-Sepharose, which was washed with loading buffer, and the complex was eluted with 0.5-ml aliquots of elution buffer. The protein was tested against version 5.1 of the glycan array of the Consortium for Functional Glycomics using the standard protocol. Competition binding assays were performed as previously described for mincle (22Feinberg H. Jégouzo S.A. Rowntree T.J. Guan Y. Brash M.A. Taylor M.E. Weis W.I. Drickamer K. Mechanism for recognition of an unusual mycobacterial glycolipid by the macrophage receptor mincle.J. Biol. Chem. 2013; 288: 28457-28465Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). 125I-Man-BSA and 125I-IgG reporter ligands were prepared by radioiodination (23Greenwood F.C. Hunter W.M. Glover J.S. The preparation of 131I-labelled human growth hormone of high specific radioactivity.Biochem. J. 1963; 89: 114-123Crossref PubMed Scopus (6739) Google Scholar) of Man31-BSA (E-Y Laboratories) and human IgG (Sigma). Crystals of human BDCA-2 complexed with α-methyl mannoside were grown by hanging drop vapor diffusion at 22 °C using a mixture of 0.13:0.13 μl of protein:reservoir solution in the drop, with the protein solution comprising 5 mg/ml CRD from BDCA-2, 5 mm CaCl2, 10 mm Tris-Cl, pH 8.0, 25 mm NaCl, and 50 mm α-methyl mannoside. The reservoir solution contained 0.2 m MgCl2 and 20% polyethylene glycol 3.35 K. Crystals were dipped in a freezing solution containing 30% polyethylene glycol 3.35 K, 0.2 m MgCl2, 5 mm CaCl2, 10 mm Tris, pH 8.0, 25 mm NaCl, and 50 mm α-methyl mannoside, before being frozen in liquid nitrogen for data collection. Diffraction data were measured at 100 K on Beamline 23-ID-D at the Advanced Photon Source of Argonne National Laboratory. Crystals of human BDCA-2 complexed with Galβ1-4GlcNAcβ1–2Man were grown using a mixture of 0.2:0.1 μl of protein:reservoir solution, at 22 °C, from a protein solution comprising 6.2 mg/ml BDCA-2, 5 mm CaCl2, 10 mm Tris-Cl, pH 8.0, 25 mm NaCl, and 20 mm Galβ1–4GlcNAcβ1-2Man. The reservoir solution contained 0.2 NH4Cl and 20% polyethylene glycol 3.35 K. Crystals were dipped in oil (Lancaster PFO-XR75) before being frozen in liquid nitrogen for data collection. Diffraction data were measured at 100 K on Beamline 12-2 at Stanford Synchrotron Radiation Laboratory. All diffraction data were integrated with XDS (24Kabsch W. XDS.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 125-132Crossref PubMed Scopus (11318) Google Scholar) and scaled with AIMLESS (25Evans P.R. Murshudov G.N. How good are my data and what is the resolution?.Acta Crystallogr. D Biol. Crystallogr. 2013; 69: 1204-1214Crossref PubMed Scopus (2724) Google Scholar). The statistics are summarized in Table 1.TABLE 1Crystallographic data statisticsDataBDCA-2 with α-methyl mannosideBDCA-2 with Galβ1–4GlcNAcβ1–2ManSymmetryP22121P22121Wavelength (Å)1.033190.97950Unit cell lengths (Å)a = 37.73, b = 69.58, c = 117.51a = 37.56, b = 67.64, c = 118.14Resolution Å (last shell)28.88–1.65 (1.69–1.65)37.56–2.90 (3.07–2.90)RsymaRsym = 100 × ΣhΣi (|Ii(h) − |)/ΣhΣiIi(h), where Ii(h) = observed intensity, and = mean intensity obtained from multiple measurements.5.4 (54.6)14.6 (47.2)Mn(I) half-set correlation CC(1/2)0.999 (0.749)0.985 (0.823)Mean (I/σ(I))17.5 (2.5)8.7 (4.3)% complete99.8 (96.5)99.2 (97.4)Number of unique reflections381387094Average multiplicity6.4 (4.9)5.3 (5.2)a Rsym = 100 × ΣhΣi (|Ii(h) − |)/ΣhΣiIi(h), where Ii(h) = observed intensity, and = mean intensity obtained from multiple measurements. Open table in a new tab The high resolution structure of BDCA-2 complexed with α-methyl mannoside was solved by molecular replacement, using the program Phaser (26McCoy A.J. Grosse-Kunstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. Phaser crystallographic software.J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14528) Google Scholar). The model used for molecular replacement was prepared from the coordinates for the CRD of cow mincle (Protein Data Bank entry 4ZRV), with three Ca2+ molecules and no carbohydrate or water molecules. The molecular replacement solution confirmed that the space group was P22121, with two monomers in the asymmetric unit. The lower resolution structure of BDCA-2 complexed with Galβ1-4GlcNAcβ1–2Man was solved using the partially refined high resolution structure of BDCA-2 complexed with α-methyl mannoside. Difference Fourier maps clearly showed Galβ1-4GlcNAcβ1–2Man in both monomers. Model building and refinement were performed with Coot (27Emsley P. Cowtan K. Coot: model-building tools for molecular graphics.Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23344) Google Scholar) and PHENIX (28Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. PHENIX: building new software for automated crystallographic structure determination.Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3640) Google Scholar). Refinement included individual positional and isotropic temperature factor refinement and occupancy refinement for residues with alternate conformations. Refinement statistics are shown in Table 2.TABLE 2Crystallographic refinement statisticsDataBDCA-2 with α-methyl mannosideBDCA-2 with Galβ1–4GlcNAcβ1–2ManNumber of reflections used for refinement380837062Reflections marked for Rfree1,905354RfreeaR and Rfree = 100 × Σh|Fo(h) − Fc(h)|/ΣhFo(h), where Fo(h) = observed structure factor amplitude, and Fc(h) = calculated structure factor amplitude for the working and test sets, respectively.21.426.3RcrystaR and Rfree = 100 × Σh|Fo(h) − Fc(h)|/ΣhFo(h), where Fo(h) = observed structure factor amplitude, and Fc(h) = calculated structure factor amplitude for the working and test sets, respectively.17.317.8B factors (Å2)Average26.032.1Protein24.732.1Ca2+21.622.0Sugars26.531.8Waters34.0Bond length root mean square deviation (Å)0.0110.013Angle root mean square deviation (°)1.031.18Ramachandran plot: (% in each region)bAs defined in Coot. (preferred/allowed/outliers)93.5/5.4/1.189.3/8.3/2.4a R and Rfree = 100 × Σh|Fo(h) − Fc(h)|/ΣhFo(h), where Fo(h) = observed structure factor amplitude, and Fc(h) = calculated structure factor amplitude for the working and test sets, respectively.b As defined in Coot. Open table in a new tab Comparison of both the overall structural organization of BDCA-2 and the sequence of the carbohydrate recognition domain place it in a subgroup of the family of type II transmembrane receptors that includes the macrophage-inducible C-type lectin (mincle), the macrophage C-type lectin, dendritic cell C-type lectin (dectin-2), and the dendritic cell immunoreceptor (DCIR) (Fig. 1, A and B) (7Plato A. Willment J.A. Brown G.D. C-Type lectin-like receptors of the dectin-1 cluster: ligands and signaling pathways.Int. Rev. Immunol. 2013; 32: 134-156Crossref PubMed Scopus (133) Google Scholar). Comparison of the sequences of the CRDs of mincle and BDCA-2 (Fig. 1C) reveals conservation of structural framework residues including disulfide bonds and the features associated with the characteristic sugar-binding site in C-type CRDs, including five amino acids that form the conserved Ca2+-binding site. Following expression of the CRD from BDCA-2 in E. coli as inclusion bodies, several protocols for renaturation were investigated, and attempts were made to purify the CRD on high density mannose-Sepharose resin, by analogy to protocols used for mincle and other C-type lectins. When renatured protein was applied to an extended affinity column in a small sample volume, retardation of a band corresponding to the CRD from BDCA-2 was observed (Fig. 2A). Although the protein did not stick tightly to the column and washed through in the presence of Ca2+, the results provide evidence that BDCA-2 has at least weak mannose binding activity. Based on the previous finding that a galactose-terminated biantennary glycan is a ligand for BDCA-2 (13Riboldi E. Daniele R. Parola C. Inforzato A. Arnold P.L. Bosisio D. Fremont D.H. Bastone A. Colonna M. Sozzani S. Human C-type lectin domain family 4, member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides.J. Biol. Chem. 2011; 286: 35329-35333Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), an alternative affinity resin was created by immobilizing a desialylated preparation of glycopeptide bearing a biantennary glycan, isolated from chicken egg yolks. With this oligosaccharide resin, the expressed CRD of BDCA-2 bound tightly in the presence of Ca2+ and could be eluted with EDTA (Fig. 2B). This approach provided a rapid method for isolation of natively folded protein and confirmed that BDCA-2 binds to the desialylated oligosaccharide in a Ca2+-dependent manner. In addition to the untagged CRD, a version in which a biotinylation tag was attached to the C terminus was also prepared by the same procedures. The biotin tag attached to the C terminus of the CRD was used to generate tetrameric complexes of CRD with fluorescently labeled streptavidin for screening of the most recent version of the glycan array at the Consortium for Functional Glycomics, which has been expanded to 610 glycans (29Blixt 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 Die I. Burton D.R. Wilson I.A. Cummings R. Bovin N. Wong C.-H. Paulson J.C. Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 17033-17038Crossref PubMed Scopus (967) Google Scholar). Screening of the expanded array confirmed binding to biantennary glycans with terminal galactose residues, as previously observed with versions of the array containing 264 and 320 glycans (13Riboldi E. Daniele R. Parola C. Inforzato A. Arnold P.L. Bosisio D. Fremont D.H. Bastone A. Colonna M. Sozzani S. Human C-type lectin domain family 4, member C (CLEC4C/BDCA-2/CD303) is a receptor for asialo-galactosyl-oligosaccharides.J. Biol. Chem. 2011; 286: 35329-35333Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and revealed a number of additional ligands. However, there were still a limited number of strong signals (Fig. 3). The 13 highest signals were obtained for glycans bearing terminal Galβ1–4GlcNAcβ1–2Man or Galβ1–3GlcNAcβ1–2Man epitopes. All but two of the glycans containing such epitopes appear in the top 28 signals. However, the signals are consistently lower when the epitope is displayed on the 1–3 branch of bi- or triantennary glycans. Glycans bearing just a trisaccharide epitope bind relatively weakly, but these trisaccharides are displayed on a relatively short two-carbon linker that may restrict access to the ligand-binding site. These results suggest that the primary ligands for BDCA-2 are a very specific subset of galactose-terminated glycans. Three of the remaining glycan ligands that lack terminal galactose residues contain terminal GlcNAcβ1–2Man epitopes on two branches, whereas seven other glycans that contain one or more copies of this terminal epitope, without galactose, rank lower on the array (glycans ranked 48, 49, 65, 100, 110, 305, and 361), suggesting that this epitope binds more weakly. The only glycan that does not fit into one of these patterns is the 15th ranked signal. Previous versions of the array had no tri- or tetra-antennary glycans, which is