β-Glucan-induced cooperative oligomerization of Dectin-1 C-type lectin-like domain

β-Glucan-induced cooperative oligomerization of Dectin-1 C-type lectin-like domain Abstract Dectin-1 is a C-type lectin-like pattern recognition receptor that recognizes β(1–3)-glucans present on non-self pathogens. It is of great importance in innate immunity to understand the mechanism whereby Dectin-1 senses β(1–3)-glucans and induces intracellular signaling. In this study, we characterize the ligand binding and ligand-induced oligomerization of murine Dectin-1 using its C-type lectin-like domain (CTLD). Interaction of CTLD with laminarin, a β-glucan ligand, induced a tetramer of CTLD, as evidenced by size exclusion chromatography and multi-angle light scattering. Component analysis suggested a stoichiometry of four CTLD molecules bound to four laminarin molecules. Dimers and trimers of CTLD were not detected suggesting cooperative oligomerization. In order to map the amino acid residues of CTLD involved in β-glucan binding and domain oligomerization, we performed site-directed mutagenesis on surface-exposed and most conserved amino acid residues. Among the mutants examined, W221A, H223A and Y228A abolished oligomer formation. Since these residues are spatially arranged to form a hydrophobic groove, it is likely that W221, H223 and Y228 are directly involved in β-glucan binding. Interestingly, mutation of the residues on the other side of the hydrophobic groove, including Y141, R145 and E243, also exhibited reduced oligomer formation, suggesting involvement in protein–protein interactions guided by laminarin. Ligand titration using intrinsic tryptophan fluorescence revealed that wild-type CTLD binds laminarin cooperatively with a Hill coefficient of ~3, while the oligomer-reducing mutations, inside and outside the putative binding site abolish or decrease cooperativity. We suggest that the ligand-induced cooperative oligomer formation of Dectin-1 is physiologically relevant in sensing exogenous β-glucan and triggering intracellular signaling. C-type lectin-like domain, Dectin-1, mutation, oligomerization, β-glucan Introduction The innate immune response is mediated by pattern recognition receptors (PRRs) which recognize highly conserved specific molecular structures called pathogen-associated molecular patterns (PAMPs) present on microbial cell surfaces (Robinson et al. 2006; Takeuchi and Akira 2010). Among PRRs, C-type lectin receptors (CLRs) recognize complex carbohydrate structures present on the cell surface of various fungal pathogens by means of their carbohydrate recognition domain (CRD) (Robinson et al. 2006; Hardison and Brown 2012). Many structurally homologous domains of CRD have been characterized and their function is not always restricted to just carbohydrate binding. These domains are called C-type lectin-like domains (CTLDs); receptors containing CTLDs are known as C-type lectin-like receptors (CTLRs) (Geijtenbeek et al. 2004). Over 100 different proteins encoded in the human genome contain CTLDs (Drickamer 1988, 1999; Zelensky and Gready 2005; Drickamer and Taylor 2015). Dectin-1 is a CTLR typified by having a single extracellular C-terminal CTLD, a short stalk region, a single transmembrane domain, and an N-terminal intracellular immunoreceptor tyrosine-based activation motif (ITAM) (Figure 1A). Two isoforms of murine Dectin-1 have been identified, a full-length A isoform and a stalk-less B isoform (Brown 2006; Willment et al. 2001). Human Dectin-1 shares 60% sequence identity and 71% sequence similarity with the mouse homolog (Hernanz-Falcon et al. 2001). Dectin-1 is found mainly on neutrophils, macrophages, and dendritic cells (Ariizumi et al. 2000; Brown and Gordon 2001; Sobanov et al. 2001; Taylor et al. 2002; Sancho and Reis e Sousa 2012). Fig. 1. View largeDownload slide Schematic diagram of the basic structure of Dectin-1. (A) Full length and stalk-less Dectin-1 showing N-linked glycosylation site, C-type lectin-like domain, stalk region, transmembrane domain, immunoreceptor tyrosine based activation motif (ITAM) and amino terminus. (B) Crystal structure of murine Dectin-1 CTLD showing its long loop region, α-helices and β-sheets. (N: N-terminus, C: C-terminus). (C) 3D crystal surface structure of mouse Dectin-1 CTLD showing the putative binding residues (W221 and H223, red), residues selected for mutagenesis around the putative binding site (cyan), and those spatially apart from the putative binding site (blue). Fig. 1. View largeDownload slide Schematic diagram of the basic structure of Dectin-1. (A) Full length and stalk-less Dectin-1 showing N-linked glycosylation site, C-type lectin-like domain, stalk region, transmembrane domain, immunoreceptor tyrosine based activation motif (ITAM) and amino terminus. (B) Crystal structure of murine Dectin-1 CTLD showing its long loop region, α-helices and β-sheets. (N: N-terminus, C: C-terminus). (C) 3D crystal surface structure of mouse Dectin-1 CTLD showing the putative binding residues (W221 and H223, red), residues selected for mutagenesis around the putative binding site (cyan), and those spatially apart from the putative binding site (blue). Dectin-1 signaling elicits a variety of cellular responses, but as yet, the mechanism of ligand-induced signaling has not been well studied. Typically, when a CRD, e.g., mannose binding protein, binds carbohydrate ligand, complexation is through coordination with a conserved bound Ca2+, as well as through hydrogen bonds with acid and amide side chains (Weis and Drickamer 1996). The most important functional role of the Ca2+ is monosaccharide binding, generally via a conserved galactose-type Gln-Pro-Asp (QPD) motif and a mannose-type Glu-Pro-Asn (EPN) motif (Childs et al. 1990; Drickamer 1992; Feinberg et al. 2001). In contrast, Dectin-1 does not contain QPD or EPN motifs and is involved in Ca2+-independent β(1–3)-glucan specific interaction (Brown et al. 2007). CTLDs are stabilized by three disulfide bonds between six highly conserved essential cysteine residues (Goodridge et al. 2009). As exemplified by the crystal structure of mouse Dectin-1 CTLD (Figure 1B), the CTLD fold has a double-loop structure with its N- and C-terminal β strands (β1, β5) coming close together to form an antiparallel β-sheet and a long loop region (LLR) that lies within the domain. Usually the LLR of CLRs contains residues responsible for Ca2+ binding, but these are absent in Dectin-1. When the Dectin-1 CTLD binds β-glucan, its ITAM motif is stimulated and tyrosine residues are phosphorylated causing intracellular signaling (Ariizumi et al. 2000; Engering et al. 2002). Such signaling can result in a variety of responses, including phagocytosis, oxidative burst, neutrophil degranulation, fungal killing, and the production of inflammatory lipid mediators, cytokines and chemokines that recruit and coordinate the activation of other immune cells (Goodridge et al. 2009). Through its recognition of β-glucans, Dectin-1 binds several fungal species such as Aspergillus, Candida, Coccidioides, Penicillium, Pneumocystis and Saccharomyces and plays an important role in fungal immunity (Plato et al. 2013). Structure–function relationships of Dectin-1 have identified critical amino acid residues and the likely binding site. Mouse Dectin-1 expressed on HEK293 cells normally binds the β(1–3)-d-glucan schizophyllan, but mutations at Trp221 and His223 in the β4 strand decreases its binding (Figure 1C) (Adachi et al. 2004). A Dectin-1 monoclonal antibody that inhibits the β-glucan interaction, failed to bind the Dectin-1 mutant W221A. An X-ray crystal structure of the Dectin-1 CTLD shows a shallow hydrophobic groove running between Trp221 and His223 (Figure 1C) (Brown et al. 2007). An NMR analysis of the interaction between Dectin-1 CTLD and short chain β(1–3)-glucans shows that CTLD weakly interacts with laminarihexaose (degree of polymerization, DP = 6), moderately with 16-mer (DP = 16) and strongly with laminarin from Laminaria digitata (<DP> = 25, DP ranging from 20 to 30) (Read et al. 1996; Hanashima et al. 2014). Fusion of a small protein tag GB1 (B1 domain of protein G, 6 kDa) on the N-terminus of recombinant murine Dectin-1 CTLD greatly enhances solubility and yield of the overexpressed protein (Dulal et al. 2016), and has now allowed characterization of ligand binding and ligand-induced oligomerization. Results Dectin-1 CTLD forms a uniformly-sized oligomer upon binding to laminarin Previously we performed preliminary NMR analysis of Dectin-1 CTLD in order to check the β-glucan binding property. We observed gross line broadening for the NMR signals originating from murine Dectin-1 CTLD upon addition of a β-glucan ligand, laminarin (Dulal et al. 2016). Consistent with this observation, dynamic light scattering and analytical ultracentrifugation analyses indicate that murine Dectin-1 CTLD forms higher-order complexes in the presence of laminarin (Brown et al. 2007). To gain more insights into the oligomer formation property of Dectin-1 CTLD, we performed size exclusion chromatography (Figure 2A). Without laminarin, Dectin-1 CTLD eluted at 26.7 min (13.4 mL) at the flow rate of 0.5 mL/min, consistent with it being a monomer (Dulal et al. 2016). Addition of 1 eq. laminarin to Dectin-1 CTLD produced two peaks at 22.2 min (11.1 mL) and 26.7 min (13.4 mL), suggesting partial oligomer formation. At 1:5 and 1:10 molar ratio the entire sample eluted at 22.2 min (11.1 mL), suggesting total conversion of all CTLD monomers into an oligomeric complex upon binding laminarin. Void volume of the size exclusion column was 8.0 mL using Blue Dextran 2000 (2000 kDa). Elution volume of standard proteins were 10.7 mL for immunoglobulin G (150 kDa), 11.5 mL conalbumin (75 kDa), 12.1 mL ovalbumin (44 kDa), 13.2 mL carbonic anhydrase (29 kDa), 14.3 mL ribonuclease A (13.7 kDa) and 16.0 mL aprotinin (6.5 kDa). Molecular masses (M) of Dectin-1 CTLD and CTLD–laminarin complex were analyzed using the elution volume of these standard proteins. A plot of log10M vs. elution volume of the standard proteins suggested a molecular mass of Dectin-1 CTLD of 24 kDa (almost equal to the calculated mass, 24.3 kDa) and Dectin-1 CTLD–laminarin oligomer complex as 102 kDa, which is about a fourfold increase over the molecular mass of monomeric Dectin-1 CTLD (Figure 2B). The elution volume for an imaginary dimeric or trimeric complex of CTLD was estimated to be 12.1 mL (24.2 min) and 11.6 mL (23.2 min), and importantly, no peak was observed with these elution volumes. The results suggest that Dectin-1 CTLD forms a tetramer upon binding laminarin and oligomerization appears to be strongly cooperative since intermediate dimer or trimer species were not formed. Fig. 2. View largeDownload slide (A) Elution pattern of GB1-Dectin-1 CTLD from a size exclusion chromatography column in the absence of ligand and at different equivalences of laminarin. (B) Estimation of the molecular mass (M) of laminarin bound Dectin-1 CTLD according to elution volume with reference to standard proteins. Correlation between log10M and elution volume is shown for immunoglobulin G (10.7 mL, 150 kDa), conalbumin (11.5 mL, 75 kDa), ovalbumin (12.1 mL, 44 kDa), carbonic anhydrase (13.2 mL, 29 kDa), ribonuclease A (14.3 mL, 13.7 kDa) and aprotinin (16 mL, 42.7 kDa) GB1-Dectin-1 CTLD (13.4 mL, 24 kDa) and laminarin-bound Dectin-1 CTLD (11.1 mL 102 kDa) with black spheres. The graph is plotted with a third-order fitting curve. Fig. 2. View largeDownload slide (A) Elution pattern of GB1-Dectin-1 CTLD from a size exclusion chromatography column in the absence of ligand and at different equivalences of laminarin. (B) Estimation of the molecular mass (M) of laminarin bound Dectin-1 CTLD according to elution volume with reference to standard proteins. Correlation between log10M and elution volume is shown for immunoglobulin G (10.7 mL, 150 kDa), conalbumin (11.5 mL, 75 kDa), ovalbumin (12.1 mL, 44 kDa), carbonic anhydrase (13.2 mL, 29 kDa), ribonuclease A (14.3 mL, 13.7 kDa) and aprotinin (16 mL, 42.7 kDa) GB1-Dectin-1 CTLD (13.4 mL, 24 kDa) and laminarin-bound Dectin-1 CTLD (11.1 mL 102 kDa) with black spheres. The graph is plotted with a third-order fitting curve. SEC-MALS analysis to estimate the stoichiometry of laminarin–CTLD complex For more rigorous analysis, we performed size exclusion chromatography coupled with multiple-angle light scattering (SEC-MALS). The details of the SEC-MALS experiments are described in the Materials and methods. The SEC-MALS analysis estimated the absolute molecular masses of CTLD, laminarin and CTLD–laminarin complex as 28.5 kDa, 6.2 kDa and 129 kDa, respectively (Figure 3). Since the calculated molecular sizes are basically uniform across the peaks, this analysis is valid and the CTLD–laminarin complex is rather homogeneous. These results are consistent with the finding that Dectin-1 CTLD forms a tetramer upon binding to laminarin. Furthermore, component analysis was conducted to estimate the molecular mass of the components (laminarin and Dectin-1 CTLD) in the complex. The rationale of the component analysis is using three observables, the intensity of light scattering signal, the refractive index signal and UV absorbance. The analysis is possible when each component has significantly different parameters, i.e., UV absorbance and differential refractive index increment (dn/dc). This case is especially suitable because laminarin has essentially no UV absorbance and each component showed different dn/dc values. Here, the molecular masses of Dectin-1 CTLD and laminarin within the complex were determined to be 103.5 kDa and 25.5 kDa. Considering the molecular mass of each component, the binding stoichiometry of CTLD–laminarin complex appears to be 4:4. Fig. 3. View largeDownload slide Characterization of Dectin-1 CTLD, laminarin and mixture of CTLD–laminarin complex by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The calculated molecular masses are indicated within each chart. In the CTLD–laminarin complex, component analysis was performed and the calculated molecular masses of the complex and each component are shown with different colors, the CTLD–laminarin complex in blue, CTLD in green and laminarin in red. Fig. 3. View largeDownload slide Characterization of Dectin-1 CTLD, laminarin and mixture of CTLD–laminarin complex by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The calculated molecular masses are indicated within each chart. In the CTLD–laminarin complex, component analysis was performed and the calculated molecular masses of the complex and each component are shown with different colors, the CTLD–laminarin complex in blue, CTLD in green and laminarin in red. Mapping ligand-binding residues The phenomenon of laminarin-induced oligomer formation motivated us to elucidate the ligand binding pattern of Dectin-1 at the amino acid residue level. A mutational study has indicated that Trp221 and His223 are critical for binding to β-glucan (Adachi et al. 2004). To confirm these findings and map more of the binding residues, we prepared a series of CTLD mutants selecting conserved and surface-exposed amino acid residues in and around the putative ligand binding site and replacing them with alanine (Adachi et al. 2004; Brown et al. 2007). Our first batch of 12 CTLD mutants comprised R174A, N176A, R207A, N208A, H217A, W221A, I222A, H223A, S225A, E226A, Y228A and Q230A (Figure 4). The folded status, stability and laminarin binding of wild-type CTLD and mutants were checked by a thermofluor assay in the absence and presence of laminarin. Wild-type Dectin-1 CTLD without ligand exhibited a melting temperature (Tm) of 52.0°C, which increased to 61.0°C in the presence of 10 eq. laminarin (Table I), confirming a previous thermal shift assay report (Brown et al. 2007). All the mutants in the absence of ligand, except for I222A, produced Tms from 51.0 to 57.5, suggesting proper folding and reasonable stability (Table I). Mutant I222A was not studied further. The Tms of W221A, H223A and Y228A were not significantly increased upon addition of laminarin (Table I), suggesting defective ligand binding and/or an inability to form oligomers. Interestingly, the TmS of the W221A mutant (57.5°C) was significantly higher than that of WT by 5.5°C. This implies that exposure of the hydrophobic indole side chain is thermodynamically unfavorable but compensated for by the involvement of this residue in energetically favorable ligand binding. Fig. 4. View largeDownload slide Sequence alignments of known Dectin-1 CTLD sequences of different mammalian species. Shadowed residues are the most conserved among the different species. Arrows indicate the residues mutated to alanine. Fig. 4. View largeDownload slide Sequence alignments of known Dectin-1 CTLD sequences of different mammalian species. Shadowed residues are the most conserved among the different species. Arrows indicate the residues mutated to alanine. Table I. Melting temperature (Tm) of WT and mutants with and without laminarin obtained by thermofluor assay Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 View Large Table I. Melting temperature (Tm) of WT and mutants with and without laminarin obtained by thermofluor assay Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 View Large To further evaluate the binding and oligomer formation properties of each mutant, the mutants were analyzed by size exclusion chromatography at protein–laminarin molar ratios of 1:0, 1:1, 1:5 and 1:10, and the elution profiles compared with those of wild type Dectin-1. In contrast to WT, mutants W221A, H223A and Y228A showed no change in retention time at all molar ratios of laminarin (Figure 5), consistent with these residues being directly involved in binding laminarin. Mutations N208A, H217A, S225A, E226A and Q230A weakly but significantly reduced oligomer formation (Table II), suggesting involvement in laminarin binding, but of less importance. Fig. 5. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Twelve mutants were prepared of possible binding residues selecting those in and around the putative ligand binding site, solvent exposed and most conserved among mammalian species. Fig. 5. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Twelve mutants were prepared of possible binding residues selecting those in and around the putative ligand binding site, solvent exposed and most conserved among mammalian species. Table II. Summary of mutagenesis results showing the effect of mutations on oligomer formation of Dectin-1 CTLD Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − aProtein was largely misfolded and the yield was too low to be analyzed. bThe average value calculated from two independent experiments. (+++: oligomer was formed similar to WT, ++: oligomer formation was weakly reduced, +: oligomer formation was significantly reduced, x: oligomer formation was not detected, –: not tested, NA; not applicable). View Large Table II. Summary of mutagenesis results showing the effect of mutations on oligomer formation of Dectin-1 CTLD Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − aProtein was largely misfolded and the yield was too low to be analyzed. bThe average value calculated from two independent experiments. (+++: oligomer was formed similar to WT, ++: oligomer formation was weakly reduced, +: oligomer formation was significantly reduced, x: oligomer formation was not detected, –: not tested, NA; not applicable). View Large Identification of residues involved in protein–protein interaction In order to try to identify the residues involved in protein–protein interactions, we prepared a second batch of 17 CTLD mutants selecting conserved residues over the entire protein surface, but away from the putative ligand binding site formed by W221, H223 and Y228. They comprised N122A, Y141A, K144A, R145A, Q149A, D158A, S160A, K161A, E164A, Q186A, E188A, F192A, D195A, Q205A, E213A, K242A and E243A. All mutants except K161A exhibited oligomer formation at 10 eq. laminarin, but some, namely N122A, Y141A, R145A, E164A, E213A, and E243A, only weakly at low ligand ratios compared to WT (Table II, Figure 6). We prepared double and triple mutants of these oligomer-impaired proteins and analyzed elution profiles in the absence and presence of laminarin. All the double mutants failed to form oligomers up to 1:5 protein–ligand molar ratio, and some conversion only occurred at 1:10 (Supplementary data, Figure S1). Triple mutants produced very similar results. Fig. 6. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Seventeen mutants were prepared with mutations to residues possibly involved in interprotein interactions by selecting residues outside of the putative ligand binding site, solvent exposed and most conserved among different mammalian species. Fig. 6. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Seventeen mutants were prepared with mutations to residues possibly involved in interprotein interactions by selecting residues outside of the putative ligand binding site, solvent exposed and most conserved among different mammalian species. Table II provides an overview of the results. We mapped the residues involved in oligomer formation on the crystal structure of mouse Dectin-1 CTLD, where color code and intensity reflect the degree of the mutational effect (Figure 7). The putative β-glucan binding site formed by W221, H223 and Y228 is highlighted in red. Surrounding residues, N208, H217, S225, E226 and Q230 contribute to binding but not as much. The residues outside of the putative ligand binding site, i.e., N122, Y141, R145, E164, E213 and E243 examined in the second batch of mutations, are distributed around the whole CTLD surface, indicative of extensive protein–protein interactions. Fig. 7. View largeDownload slide Mapping the residues on the crystal structure of mouse Dectin-1 CTLD whose mutation affected ligand-induced oligomer formation of CTLD (red: no oligomer formation upon mutation, bright cyan: oligomer formation significantly reduced upon mutation, faint cyan: oligomer formation weakly affected by mutation). Fig. 7. View largeDownload slide Mapping the residues on the crystal structure of mouse Dectin-1 CTLD whose mutation affected ligand-induced oligomer formation of CTLD (red: no oligomer formation upon mutation, bright cyan: oligomer formation significantly reduced upon mutation, faint cyan: oligomer formation weakly affected by mutation). Analysis of cooperative ligand binding by tryptophan fluorescence The absence of an intermediate dimeric or trimeric species of CTLD in the size exclusion chromatography analysis implies that oligomer formation is cooperative. In addition, mutation of many individual residues outside of the putative ligand binding site impeded oligomer formation, suggesting extensive protein–protein interaction on ligand binding. In order to analyze possible ligand cooperativity associated with the CTLD oligomerization, we employed a method to directly measure the ligand binding event namely intrinsic tryptophan fluorescence quenching due to the location of W221 in the putative ligand binding site. The residue is solvent-exposed in the ligand-free structure and expected to be buried in the bound form. As expected, the fluorescence intensity (F) was gradually quenched upon titration of CTLD with laminarin (Figure 8A). A plot of ΔF (F0–F) vs. total laminarin concentration [L] and fitting the data to a Langmuir-type equation yielded a sigmoidal curve for WT which is compared with a simple first-order curve setting the Hill coefficient (α) to 1 (Figure 8B). The root mean square deviation obtained from the fitted sigmoidal curve is found to be 78.5 as compared to 132.7 from the first-order curve, indicating that the sigmoidal fit is better than the first-order fit. Another WT binding experiment is shown in Supplementary data, Figure S2 and the average Hill coefficient (α) from the two independent experiments was calculated as 3.4. The value is compatible with four laminarin molecules binding to four CTLDs in a cooperative manner, i.e., the affinity for ligand increases with increasing oligomerization. Hill coefficients of H223A and Y228A are 0.9 and 1.8, respectively, suggesting the decreased ligand binding cooperativity noted above. Likewise, the Hill coefficients of N122A, Y141A, R145A, E164A, E213A and E243A are 1.4, 1.6, 1.1, 2.1, 1.5 and 1.0, respectively (Supplementary data, Figure S3, Table II), indicating that these residues are involved in cooperative ligand binding. The ligand affinity, which is here defied as the laminarin concentration needed to achieve a half-maximum binding of the Dectin-1 CTLD, is shown in Table II. The Kd of WT was calculated as 40 μM and the mutants roughly showed similar affinity. These data suggest that most mutants have the ability to bind laminarin but have partially defective protein–protein interactions, and thereby diminished ligand binding cooperativity. Fig. 8. View largeDownload slide Measurement of intrinsic tryptophan fluorescence of Dectin-1 CTLD. (A) ΔF was obtained by subtracting the fluorescence intensity at a particular ligand concentration (F) from initial fluorescence intensity (F0). (B) Plot of ΔF(F0– F) vs. [L] showing the ligand binding pattern of Dectin-1 to laminarin. Curve fitting was performed using Langmuir-type equation (solid line) and a simple first-order equation (dotted line) (see Materials and methods). Fig. 8. View largeDownload slide Measurement of intrinsic tryptophan fluorescence of Dectin-1 CTLD. (A) ΔF was obtained by subtracting the fluorescence intensity at a particular ligand concentration (F) from initial fluorescence intensity (F0). (B) Plot of ΔF(F0– F) vs. [L] showing the ligand binding pattern of Dectin-1 to laminarin. Curve fitting was performed using Langmuir-type equation (solid line) and a simple first-order equation (dotted line) (see Materials and methods). Discussion The recognition of carbohydrate by a lectin may involve nonpolar (hydrophobic) interactions as well as hydrogen bonds (Weis and Drickamer 1996), and Trp221 and His223 in a surface groove of murine Dectin-1 CTLD may be key residues for β-glucan binding (Adachi et al. 2004; Mochizuki et al. 2014) through mainly hydrophobic interactions (Brown et al. 2007). Our mutational analysis shows that in addition to W221 and H223, Y228 is also critical for laminarin binding, and all three are located in a triangular fashion in the shallow groove. Long chain β-glucans (e.g., curdlan) can form a triple helix, such a helix having a diameter of about 15 Å (Chuah et al. 1983). Rough estimation of the size of the Dectin-1·β-glucan complex suggests that the groove formed by W221, H223 and Y228 can accommodate a triple helical β-glucan. Interestingly, another β-glucan binding receptor βGRP/GNBP3 has Trp, His and Tyr in its β-glucan binding site (Kanagawa et al. 2011). Although the topology of Dectin-1 CTLD is different from that of βGRP N-terminal domain, these residues may interact with β-glucan in a similar way. Various approaches have indicated that Dectin-1 increases in molecular mass on binding laminarin by forming oligomers suggesting a role for oligomer formation in the recognition of β-glucan-containing pathogens and further intracellular signaling. Formation of such oligomers may contribute the aggregation of Dectin-1 receptor and its intracellular motif. Analytical ultracentrifugation of Dectin-1 CTLD and laminarin points to increases from 26 kDa (19 kDa Dectin-1 CTLD monomer + ~7 kDa laminarin) to 72–92 kDa (Brown et al. 2007). Dynamic light scattering recorded a fourfold increase, suggestive of a tetrameric complex (Brown et al. 2007). Our SEC and SEC-MALS data also favors a tetramer of CTLD with the CTLD and laminarin stoichiometry of 4:4 indicating the involvement of four CTLD and four laminarin in forming a complex. Evidenced from the sigmoidal curve in Langmuir-type equation and the Hill coefficient of ~3 suggests four laminarin bind cooperatively to four CTLD molecules (Figure 8B). Inhomogeneity of laminarin and variable chain length may contribute to some variations in the obtained values, and studies with homogeneous β-glucan of defined chain length may be needed to resolve this. The minimum chain length of β-glucan required for binding to Dectin-1 is 10-mer or 11-mer (Palma et al. 2006), which is long relative to the size of monomeric Dectin-1 CTLD and it is anticipated that there will be extensions beyond the ligand binding area containing W221, H223 and Y228 (Figure 9A). The binding possibilities preclude accurate model building at this stage and further structural studies are required. Fig. 9. View largeDownload slide Schematic diagram of (A) relative size of Dectin-1 CTLD and laminarin with 25 glucose residues. (B) Possible ligand-induced signaling by cooperative oligomerization of Dectin-1 CTLD. Fig. 9. View largeDownload slide Schematic diagram of (A) relative size of Dectin-1 CTLD and laminarin with 25 glucose residues. (B) Possible ligand-induced signaling by cooperative oligomerization of Dectin-1 CTLD. Mutation of individual amino acid residues outside of the ligand binding groove, namely N122A, Y141A, R145A, E164A, E213A and E243A, lowered Dectin-1 oligomer formation, even up to 1:5 molar ratio of Dectin-1 to laminarin. It points to their involvement in the protein–protein interactions of Dectin-1 CTLD in forming an oligomeric complex. The results were very similar with selected double mutants and triple mutants. These residues are distributed all around the surface outside the ligand binding site of Dectin-1 CTLD. Although it is difficult to distinguish between the direct ligand binding residues and protein–protein interacting residues, the distributing pattern of these residues and 4:4 stoichiometry of Dectin-1–laminarin complex suggests a role for these residues in intersubunit interaction. The property of Dectin-1 oligomerization and global participation of surface residues is suggestive of cooperative behavior of Dectin-1 CTLD monomers to form an oligomeric complex. Our intrinsic fluorescence analysis of Dectin-1–laminarin interaction bears this out. Laminarin binding to wild-type Dectin-1 CTLD exhibits the average Hill coefficient of 3.4, while binding to these mutants shows decreased Hill coefficient values, signifying impaired oligomerization. The mechanism how the cooperativity is achieved is unknown at this moment. One possibility is that the binding of laminarin to monomeric CTLD could promote binding of the 1:1 complex to three other monomeric empty CTLDs, which may on complex formation show increasing affinity for the ligand, and hence the cooperativity. In the complex, laminarin may form a triple-helical structure which is seen in longer β-glucan chains. 3D structural elucidation of laminarin–CTLD complex needs further analysis. Ligand-induced cooperative oligomerization of the single extracellular carbohydrate recognition domain of Dectin-1 likely underlies the clustering of these receptors to form high density regions of ITAM on the inner cell surface and facilitate binding of kinase and propagate intracellular signals. Structural details of these events are lacking but a tetramer of extracellular domains of Dectin-1 molecules on the surface may increase the chance of kinase binding and tyrosine phosphorylation activities, more than that achieved by the bridging of only two receptor monomers (Figure 9). Materials and methods Expression and purification of Dectin-1 CTLD of murine Dectin-1 (Gly113-Leu244; 15 kDa) was expressed as inclusion bodies in E. coli using pCold vector and refolded as described in the previous article (Dulal et al. 2016). In brief, we constructed pCold-Dectin-1 CTLD vector using an artificial codon optimized CTLD gene of mouse Dectin-1 with protein G B1 domain (GB1), an N-terminal hexahistidine tag and a tobacco etch virus (TEV) protease cleavage site (Novagen). The plasmid was transformed into E. coli BL21(DE3). The cells were cultured in LB medium, induced with IPTG (0.5 mM) at 15°C and harvested after 16 h, and then sonicated in PBS (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4) in presence of 0.3% (v/v) BugBuster (Novagen). Resultant inclusion bodies were solubilized in urea buffer (8 M Urea, 50 mM Tris-HCl, 50 mM NaCl, and pH 8.0) and refolded by 50-fold dilution in a refolding buffer (200 mM Tris-HCl, 0.4 M l-arginine, pH 8.0 with 5 mM reduced glutathione and 0.5 mM oxidized glutathione). The refolded protein was purified by Ni2+ Sepharose 6 Fast Flow column (GE Healthcare) and size exclusion chromatography (HiLoad 16/60 Superdex 75 prep grade column, GE Healthcare). Preparation of Dectin-1 mutants To prepare the mutants, forward and reverse mutation oligonucleotide primers composed of 30 nucleotide bases (Life Technologies Japan Ltd) were designed by replacing the desired code to be mutated with that for alanine. Forward primer was designed by selecting 30 nucleotide residues from −6 to +24 bases of mutating amino acid codon and the corresponding codon was replaced with alanine (GCC). Reverse primer was designed by selecting 30 nucleotide residues from +6 to −24 bases of mutating amino acid codon, replacing the corresponding codon with alanine (GCC) and making its reverse complement sequence. Expression plasmid vector (pCold) with wild-type Dectin-1 cDNA was replicated by high-fidelity PCR using primeSTAR HS DNA polymerase (TAKARA), 5x primeSTAR buffer (5xPS), dNTP mixture and mutation primers. The template plasmid in the resultant PCR mixture was incubated for 3 h at 37°C with Dpn-I (Roche) to digest wild-type cDNA. The mutated plasmids were transformed into DH5α competent cells and cultured overnight at 37°C on LB agar plate. Plasmids were purified using Wizard® Plus SV Minipreps DNA Purification System (Promega) and the DNA sequence checked by sequence analysis. The mutated plasmids were then transformed into BL21(DE3) and expression carried out. Size-exclusion chromatography For the purification of target protein, sample was concentrated to 5 mL, filtered and then subjected to size exclusion chromatography (HiLoad 16/60 Superdex 75 prep grade column, GE Healthcare). To calculate the molecular mass and ligand binding property, 1 mL of sample solution (10 μM) was applied to size exclusion chromatography (10/300 GL Superdex 75 column, GE Healthcare) at flow rate of 0.5 mL/min. Dectin-1 and laminarin solutions were prepared in 20 mM sodium phosphate buffer, pH 7.4 containing 150 mM NaCl. The size exclusion column was equilibrated with the same PBS mentioned above. Dectin-1 CTLD sample was injected with and without premixing of 1:1, 1:5 and 1:10 molar ratio of β-glucan ligand laminarin from L. digitata (Sigma-Aldrich). Likewise, several standard proteins i.e., immunoglobulin G (150 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (42.7 kDa) were analyzed with the same column and flow rate. Void volume was determined using Blue Dextran (2000 kDa). The elution volume of each sample was measured and the molecular mass of ligand-free Dectin-1 as well as laminarin-bound Dectin-1 was estimated with reference to the above mentioned standard protein samples. Size exclusion chromatography with multi-angle light scattering (SEC-MALS) The theoretical calculations for the determination of molecular mass by light scattering were described by Wyatt (Wyatt 1993). Our SEC-MALS experiments were performed at room temperature using an HPLC system (Shimadzu) coupled to and a Shimadzu UV detector SPD-20A (Shimazu), a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt Technology). For chromatographic separation, a 10/300 GL Superdex 75 size-exclusion column (GE Healthcare) was used at a flow rate of 0.5 mL/min equilibrated with 20 mM sodium phosphate buffer (pH 7.4) and 150 mM NaCl. For each run, a 100 μL of Dectin-1 CTLD (65 μM), laminarin (10 mM), or Dectin-1 CTLD–laminarin mixture (Dectin-1 CTLD 61 μM, laminarin 610 μM) was injected. Data analyses were performed using the software ASTRA 6.1 (Wyatt Technology). The differential refractive index increment (dn/dc) of Dectin-1 CTLD was assumed to be 0.185 mL/g (Zhao et al. 2011), and the dn/dc of laminarin was experimentally determined to be 0.1344 mL/g (Supplementary data, Figure S4). Component analysis of Dectin-1 CTLD–laminarin complex was performed using the ASTRA 6.1 software package to calculate the molecular mass of the entire complex as well as for each component of the complex (Hayashi et al. 1989; Kendrick et al. 2001). The theoretical background of the SEC-MALS analysis is shown below: (a) Determination of molecular weight of protein or ligand alone At the low sample concentrations typically attained in the column chromatography, the intensity of light-scattering signal (LS) is given as (LS)=KLSMc(dn/dc)2 where KLS is an instrument-specific constant, M is the molecular mass (Da), c is concentration in g/mL, dn/dc is the refractive index increment of the solute in mL/g. The refractive index signal (RI) is described as (RI)=KRIc(dn/dc) where KRI is an instrument-specific constant. Hence the molecular weight M can be determined from the two observables, (LS) and (RI), using the following relation: M=K′(LS)/(RI) where K′=KRI/[KLS(dn/dc)]. (b) Component analysis for the protein–ligand complex In addition to (LS) and (RI), UV absorbance (UV) is used for the component analysis. (UV)complex=KUV(cpεp+clεl) where KUV is a constant, ε is the extinction coefficient (mL/mg/cm) and the subscripts p, l and complex refer to the protein, ligand and protein–ligand complex, respectively. It can be assumed that the laminarin ligand has no UV absorbance at 280 nm, here the equation is simplified as (UV)complex=KUVcpεp (RI), (LS) and (dn/dc) for the protein–ligand complex are described as (RI)complex=KRIcp(dn/dc)p+KRIcl(dn/dc)l (LS)complex=KLSMcomplexccomplex(dn/dc)complex2 (dn/dc)complex=Mp/Mcomplex(dn/dc)p+Ml/Mcomplex(dn/dc)l Based on these equations, Mp, Ml and Mcomplex are determined. Fluorescence based thermal stability assay The melting temperatures of Dectin-1 CTLD and mutants were estimated by a thermal shift assay without and with 1:10 molar ratio of laminarin. Sample solution containing 20 μg protein (1 mg/mL) was mixed with 1000 times diluted SYPRO Orange solution (molecular probes) as a reporter dye in PBS (pH 7.4). The wavelengths for excitation and emission were 475–500 nm and 520–550 nm, respectively. The samples were heated in a real time PCR instrument (PikoReal 24, Thermo Scientific) from 293 to 368 K with increments of 0.5 K/s. Tryptophan fluorescence spectroscopy Intrinsic tryptophan fluorescence of Dectin-1 WT and mutants was measured using an f-4500 fluorescence spectrophotometer (Hitachi) at an excitation wavelength of 295 nm. Emission fluorescence intensity of the protein (100 μL, 10 μM, pH 6.5) solubilized in PBS (20 mM sodium phosphate, 50 mM NaCl, pH 6.5) was recorded without ligand and then by adding the laminarin solution prepared in the above mentioned PBS buffer in a protein/ligand ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15 and 1:20. The data were taken in duplicate. The maximum emission fluorescence intensities of the protein without ligand and laminarin-added samples of each protein ligand ratio were taken. These fluorescence values were adjusted for dilution effect caused by the addition of ligand solution by multiplying the fluorescence value with the ratio of total protein ligand solution volume to the volume of protein solution. ΔF values were calculated by subtracting the fluorescence value of each ligand ratio from the maximum fluorescence value of protein without ligand (F0). ΔF vs. total laminarin concentration [L] was plotted and binding analyzed using the following equation where ΔFmax is the maximum fluorescence decrease upon binding to laminarin, α, the Hill coefficient and Kd, the laminarin concentration needed to achieve a half-maximum binding of the protein at equilibrium. ΔF=ΔFmax[L]α/(Kdα+[L]α) Curve fitting was done using GraphPad Prism 7. The value of α indicates the nature of cooperative binding where α > 1, positive cooperative binding, α = 1, noncooperative binding, and α < 1, negative cooperative binding. To validate this curve fitting, a simple first-order curve fitting was additionally performed where the Hill coefficient (α) is set to 1 with the following equation: ΔF=ΔFmax[L]/(Kd+[L]) Supplementary data Supplementary data is available at Glycobiology online. Funding This work was supported in part by Grant-in-Aid for Scientific Research (C) [25460054 to Y.Y.] and Scientific Research (B) [16H04758 to Y.Y.] from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Acknowledgements We thank Prof. Naoyuki Taniguchi (Systems Glycobiology Group, RIKEN) for the opportunity to perform this study, Dr. Kurono Ken-ichiro (Shoko Scientific Co., Ltd.) for assistance with SEC-MALS experiment and Dr. Sushil K. Mishra for preparing coordinates of β-glucan chain. Conflict of interest statement None declared. 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β-Glucan-induced cooperative oligomerization of Dectin-1 C-type lectin-like domain

Glycobiology , Volume Advance Article (8) – May 11, 2018

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Abstract

Abstract Dectin-1 is a C-type lectin-like pattern recognition receptor that recognizes β(1–3)-glucans present on non-self pathogens. It is of great importance in innate immunity to understand the mechanism whereby Dectin-1 senses β(1–3)-glucans and induces intracellular signaling. In this study, we characterize the ligand binding and ligand-induced oligomerization of murine Dectin-1 using its C-type lectin-like domain (CTLD). Interaction of CTLD with laminarin, a β-glucan ligand, induced a tetramer of CTLD, as evidenced by size exclusion chromatography and multi-angle light scattering. Component analysis suggested a stoichiometry of four CTLD molecules bound to four laminarin molecules. Dimers and trimers of CTLD were not detected suggesting cooperative oligomerization. In order to map the amino acid residues of CTLD involved in β-glucan binding and domain oligomerization, we performed site-directed mutagenesis on surface-exposed and most conserved amino acid residues. Among the mutants examined, W221A, H223A and Y228A abolished oligomer formation. Since these residues are spatially arranged to form a hydrophobic groove, it is likely that W221, H223 and Y228 are directly involved in β-glucan binding. Interestingly, mutation of the residues on the other side of the hydrophobic groove, including Y141, R145 and E243, also exhibited reduced oligomer formation, suggesting involvement in protein–protein interactions guided by laminarin. Ligand titration using intrinsic tryptophan fluorescence revealed that wild-type CTLD binds laminarin cooperatively with a Hill coefficient of ~3, while the oligomer-reducing mutations, inside and outside the putative binding site abolish or decrease cooperativity. We suggest that the ligand-induced cooperative oligomer formation of Dectin-1 is physiologically relevant in sensing exogenous β-glucan and triggering intracellular signaling. C-type lectin-like domain, Dectin-1, mutation, oligomerization, β-glucan Introduction The innate immune response is mediated by pattern recognition receptors (PRRs) which recognize highly conserved specific molecular structures called pathogen-associated molecular patterns (PAMPs) present on microbial cell surfaces (Robinson et al. 2006; Takeuchi and Akira 2010). Among PRRs, C-type lectin receptors (CLRs) recognize complex carbohydrate structures present on the cell surface of various fungal pathogens by means of their carbohydrate recognition domain (CRD) (Robinson et al. 2006; Hardison and Brown 2012). Many structurally homologous domains of CRD have been characterized and their function is not always restricted to just carbohydrate binding. These domains are called C-type lectin-like domains (CTLDs); receptors containing CTLDs are known as C-type lectin-like receptors (CTLRs) (Geijtenbeek et al. 2004). Over 100 different proteins encoded in the human genome contain CTLDs (Drickamer 1988, 1999; Zelensky and Gready 2005; Drickamer and Taylor 2015). Dectin-1 is a CTLR typified by having a single extracellular C-terminal CTLD, a short stalk region, a single transmembrane domain, and an N-terminal intracellular immunoreceptor tyrosine-based activation motif (ITAM) (Figure 1A). Two isoforms of murine Dectin-1 have been identified, a full-length A isoform and a stalk-less B isoform (Brown 2006; Willment et al. 2001). Human Dectin-1 shares 60% sequence identity and 71% sequence similarity with the mouse homolog (Hernanz-Falcon et al. 2001). Dectin-1 is found mainly on neutrophils, macrophages, and dendritic cells (Ariizumi et al. 2000; Brown and Gordon 2001; Sobanov et al. 2001; Taylor et al. 2002; Sancho and Reis e Sousa 2012). Fig. 1. View largeDownload slide Schematic diagram of the basic structure of Dectin-1. (A) Full length and stalk-less Dectin-1 showing N-linked glycosylation site, C-type lectin-like domain, stalk region, transmembrane domain, immunoreceptor tyrosine based activation motif (ITAM) and amino terminus. (B) Crystal structure of murine Dectin-1 CTLD showing its long loop region, α-helices and β-sheets. (N: N-terminus, C: C-terminus). (C) 3D crystal surface structure of mouse Dectin-1 CTLD showing the putative binding residues (W221 and H223, red), residues selected for mutagenesis around the putative binding site (cyan), and those spatially apart from the putative binding site (blue). Fig. 1. View largeDownload slide Schematic diagram of the basic structure of Dectin-1. (A) Full length and stalk-less Dectin-1 showing N-linked glycosylation site, C-type lectin-like domain, stalk region, transmembrane domain, immunoreceptor tyrosine based activation motif (ITAM) and amino terminus. (B) Crystal structure of murine Dectin-1 CTLD showing its long loop region, α-helices and β-sheets. (N: N-terminus, C: C-terminus). (C) 3D crystal surface structure of mouse Dectin-1 CTLD showing the putative binding residues (W221 and H223, red), residues selected for mutagenesis around the putative binding site (cyan), and those spatially apart from the putative binding site (blue). Dectin-1 signaling elicits a variety of cellular responses, but as yet, the mechanism of ligand-induced signaling has not been well studied. Typically, when a CRD, e.g., mannose binding protein, binds carbohydrate ligand, complexation is through coordination with a conserved bound Ca2+, as well as through hydrogen bonds with acid and amide side chains (Weis and Drickamer 1996). The most important functional role of the Ca2+ is monosaccharide binding, generally via a conserved galactose-type Gln-Pro-Asp (QPD) motif and a mannose-type Glu-Pro-Asn (EPN) motif (Childs et al. 1990; Drickamer 1992; Feinberg et al. 2001). In contrast, Dectin-1 does not contain QPD or EPN motifs and is involved in Ca2+-independent β(1–3)-glucan specific interaction (Brown et al. 2007). CTLDs are stabilized by three disulfide bonds between six highly conserved essential cysteine residues (Goodridge et al. 2009). As exemplified by the crystal structure of mouse Dectin-1 CTLD (Figure 1B), the CTLD fold has a double-loop structure with its N- and C-terminal β strands (β1, β5) coming close together to form an antiparallel β-sheet and a long loop region (LLR) that lies within the domain. Usually the LLR of CLRs contains residues responsible for Ca2+ binding, but these are absent in Dectin-1. When the Dectin-1 CTLD binds β-glucan, its ITAM motif is stimulated and tyrosine residues are phosphorylated causing intracellular signaling (Ariizumi et al. 2000; Engering et al. 2002). Such signaling can result in a variety of responses, including phagocytosis, oxidative burst, neutrophil degranulation, fungal killing, and the production of inflammatory lipid mediators, cytokines and chemokines that recruit and coordinate the activation of other immune cells (Goodridge et al. 2009). Through its recognition of β-glucans, Dectin-1 binds several fungal species such as Aspergillus, Candida, Coccidioides, Penicillium, Pneumocystis and Saccharomyces and plays an important role in fungal immunity (Plato et al. 2013). Structure–function relationships of Dectin-1 have identified critical amino acid residues and the likely binding site. Mouse Dectin-1 expressed on HEK293 cells normally binds the β(1–3)-d-glucan schizophyllan, but mutations at Trp221 and His223 in the β4 strand decreases its binding (Figure 1C) (Adachi et al. 2004). A Dectin-1 monoclonal antibody that inhibits the β-glucan interaction, failed to bind the Dectin-1 mutant W221A. An X-ray crystal structure of the Dectin-1 CTLD shows a shallow hydrophobic groove running between Trp221 and His223 (Figure 1C) (Brown et al. 2007). An NMR analysis of the interaction between Dectin-1 CTLD and short chain β(1–3)-glucans shows that CTLD weakly interacts with laminarihexaose (degree of polymerization, DP = 6), moderately with 16-mer (DP = 16) and strongly with laminarin from Laminaria digitata (<DP> = 25, DP ranging from 20 to 30) (Read et al. 1996; Hanashima et al. 2014). Fusion of a small protein tag GB1 (B1 domain of protein G, 6 kDa) on the N-terminus of recombinant murine Dectin-1 CTLD greatly enhances solubility and yield of the overexpressed protein (Dulal et al. 2016), and has now allowed characterization of ligand binding and ligand-induced oligomerization. Results Dectin-1 CTLD forms a uniformly-sized oligomer upon binding to laminarin Previously we performed preliminary NMR analysis of Dectin-1 CTLD in order to check the β-glucan binding property. We observed gross line broadening for the NMR signals originating from murine Dectin-1 CTLD upon addition of a β-glucan ligand, laminarin (Dulal et al. 2016). Consistent with this observation, dynamic light scattering and analytical ultracentrifugation analyses indicate that murine Dectin-1 CTLD forms higher-order complexes in the presence of laminarin (Brown et al. 2007). To gain more insights into the oligomer formation property of Dectin-1 CTLD, we performed size exclusion chromatography (Figure 2A). Without laminarin, Dectin-1 CTLD eluted at 26.7 min (13.4 mL) at the flow rate of 0.5 mL/min, consistent with it being a monomer (Dulal et al. 2016). Addition of 1 eq. laminarin to Dectin-1 CTLD produced two peaks at 22.2 min (11.1 mL) and 26.7 min (13.4 mL), suggesting partial oligomer formation. At 1:5 and 1:10 molar ratio the entire sample eluted at 22.2 min (11.1 mL), suggesting total conversion of all CTLD monomers into an oligomeric complex upon binding laminarin. Void volume of the size exclusion column was 8.0 mL using Blue Dextran 2000 (2000 kDa). Elution volume of standard proteins were 10.7 mL for immunoglobulin G (150 kDa), 11.5 mL conalbumin (75 kDa), 12.1 mL ovalbumin (44 kDa), 13.2 mL carbonic anhydrase (29 kDa), 14.3 mL ribonuclease A (13.7 kDa) and 16.0 mL aprotinin (6.5 kDa). Molecular masses (M) of Dectin-1 CTLD and CTLD–laminarin complex were analyzed using the elution volume of these standard proteins. A plot of log10M vs. elution volume of the standard proteins suggested a molecular mass of Dectin-1 CTLD of 24 kDa (almost equal to the calculated mass, 24.3 kDa) and Dectin-1 CTLD–laminarin oligomer complex as 102 kDa, which is about a fourfold increase over the molecular mass of monomeric Dectin-1 CTLD (Figure 2B). The elution volume for an imaginary dimeric or trimeric complex of CTLD was estimated to be 12.1 mL (24.2 min) and 11.6 mL (23.2 min), and importantly, no peak was observed with these elution volumes. The results suggest that Dectin-1 CTLD forms a tetramer upon binding laminarin and oligomerization appears to be strongly cooperative since intermediate dimer or trimer species were not formed. Fig. 2. View largeDownload slide (A) Elution pattern of GB1-Dectin-1 CTLD from a size exclusion chromatography column in the absence of ligand and at different equivalences of laminarin. (B) Estimation of the molecular mass (M) of laminarin bound Dectin-1 CTLD according to elution volume with reference to standard proteins. Correlation between log10M and elution volume is shown for immunoglobulin G (10.7 mL, 150 kDa), conalbumin (11.5 mL, 75 kDa), ovalbumin (12.1 mL, 44 kDa), carbonic anhydrase (13.2 mL, 29 kDa), ribonuclease A (14.3 mL, 13.7 kDa) and aprotinin (16 mL, 42.7 kDa) GB1-Dectin-1 CTLD (13.4 mL, 24 kDa) and laminarin-bound Dectin-1 CTLD (11.1 mL 102 kDa) with black spheres. The graph is plotted with a third-order fitting curve. Fig. 2. View largeDownload slide (A) Elution pattern of GB1-Dectin-1 CTLD from a size exclusion chromatography column in the absence of ligand and at different equivalences of laminarin. (B) Estimation of the molecular mass (M) of laminarin bound Dectin-1 CTLD according to elution volume with reference to standard proteins. Correlation between log10M and elution volume is shown for immunoglobulin G (10.7 mL, 150 kDa), conalbumin (11.5 mL, 75 kDa), ovalbumin (12.1 mL, 44 kDa), carbonic anhydrase (13.2 mL, 29 kDa), ribonuclease A (14.3 mL, 13.7 kDa) and aprotinin (16 mL, 42.7 kDa) GB1-Dectin-1 CTLD (13.4 mL, 24 kDa) and laminarin-bound Dectin-1 CTLD (11.1 mL 102 kDa) with black spheres. The graph is plotted with a third-order fitting curve. SEC-MALS analysis to estimate the stoichiometry of laminarin–CTLD complex For more rigorous analysis, we performed size exclusion chromatography coupled with multiple-angle light scattering (SEC-MALS). The details of the SEC-MALS experiments are described in the Materials and methods. The SEC-MALS analysis estimated the absolute molecular masses of CTLD, laminarin and CTLD–laminarin complex as 28.5 kDa, 6.2 kDa and 129 kDa, respectively (Figure 3). Since the calculated molecular sizes are basically uniform across the peaks, this analysis is valid and the CTLD–laminarin complex is rather homogeneous. These results are consistent with the finding that Dectin-1 CTLD forms a tetramer upon binding to laminarin. Furthermore, component analysis was conducted to estimate the molecular mass of the components (laminarin and Dectin-1 CTLD) in the complex. The rationale of the component analysis is using three observables, the intensity of light scattering signal, the refractive index signal and UV absorbance. The analysis is possible when each component has significantly different parameters, i.e., UV absorbance and differential refractive index increment (dn/dc). This case is especially suitable because laminarin has essentially no UV absorbance and each component showed different dn/dc values. Here, the molecular masses of Dectin-1 CTLD and laminarin within the complex were determined to be 103.5 kDa and 25.5 kDa. Considering the molecular mass of each component, the binding stoichiometry of CTLD–laminarin complex appears to be 4:4. Fig. 3. View largeDownload slide Characterization of Dectin-1 CTLD, laminarin and mixture of CTLD–laminarin complex by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The calculated molecular masses are indicated within each chart. In the CTLD–laminarin complex, component analysis was performed and the calculated molecular masses of the complex and each component are shown with different colors, the CTLD–laminarin complex in blue, CTLD in green and laminarin in red. Fig. 3. View largeDownload slide Characterization of Dectin-1 CTLD, laminarin and mixture of CTLD–laminarin complex by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The calculated molecular masses are indicated within each chart. In the CTLD–laminarin complex, component analysis was performed and the calculated molecular masses of the complex and each component are shown with different colors, the CTLD–laminarin complex in blue, CTLD in green and laminarin in red. Mapping ligand-binding residues The phenomenon of laminarin-induced oligomer formation motivated us to elucidate the ligand binding pattern of Dectin-1 at the amino acid residue level. A mutational study has indicated that Trp221 and His223 are critical for binding to β-glucan (Adachi et al. 2004). To confirm these findings and map more of the binding residues, we prepared a series of CTLD mutants selecting conserved and surface-exposed amino acid residues in and around the putative ligand binding site and replacing them with alanine (Adachi et al. 2004; Brown et al. 2007). Our first batch of 12 CTLD mutants comprised R174A, N176A, R207A, N208A, H217A, W221A, I222A, H223A, S225A, E226A, Y228A and Q230A (Figure 4). The folded status, stability and laminarin binding of wild-type CTLD and mutants were checked by a thermofluor assay in the absence and presence of laminarin. Wild-type Dectin-1 CTLD without ligand exhibited a melting temperature (Tm) of 52.0°C, which increased to 61.0°C in the presence of 10 eq. laminarin (Table I), confirming a previous thermal shift assay report (Brown et al. 2007). All the mutants in the absence of ligand, except for I222A, produced Tms from 51.0 to 57.5, suggesting proper folding and reasonable stability (Table I). Mutant I222A was not studied further. The Tms of W221A, H223A and Y228A were not significantly increased upon addition of laminarin (Table I), suggesting defective ligand binding and/or an inability to form oligomers. Interestingly, the TmS of the W221A mutant (57.5°C) was significantly higher than that of WT by 5.5°C. This implies that exposure of the hydrophobic indole side chain is thermodynamically unfavorable but compensated for by the involvement of this residue in energetically favorable ligand binding. Fig. 4. View largeDownload slide Sequence alignments of known Dectin-1 CTLD sequences of different mammalian species. Shadowed residues are the most conserved among the different species. Arrows indicate the residues mutated to alanine. Fig. 4. View largeDownload slide Sequence alignments of known Dectin-1 CTLD sequences of different mammalian species. Shadowed residues are the most conserved among the different species. Arrows indicate the residues mutated to alanine. Table I. Melting temperature (Tm) of WT and mutants with and without laminarin obtained by thermofluor assay Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 View Large Table I. Melting temperature (Tm) of WT and mutants with and without laminarin obtained by thermofluor assay Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 Item WT N176A R207A H217A W221A H223A Y228A Q230A Tm (−) ligand 52.0 52.0 53.0 55.0 57.5 52.0 51.0 53.0 Tm (+) 1:10 laminarin 61.0 58.0 57.5 60.0 57.5 54.0 54.0 59.5 View Large To further evaluate the binding and oligomer formation properties of each mutant, the mutants were analyzed by size exclusion chromatography at protein–laminarin molar ratios of 1:0, 1:1, 1:5 and 1:10, and the elution profiles compared with those of wild type Dectin-1. In contrast to WT, mutants W221A, H223A and Y228A showed no change in retention time at all molar ratios of laminarin (Figure 5), consistent with these residues being directly involved in binding laminarin. Mutations N208A, H217A, S225A, E226A and Q230A weakly but significantly reduced oligomer formation (Table II), suggesting involvement in laminarin binding, but of less importance. Fig. 5. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Twelve mutants were prepared of possible binding residues selecting those in and around the putative ligand binding site, solvent exposed and most conserved among mammalian species. Fig. 5. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Twelve mutants were prepared of possible binding residues selecting those in and around the putative ligand binding site, solvent exposed and most conserved among mammalian species. Table II. Summary of mutagenesis results showing the effect of mutations on oligomer formation of Dectin-1 CTLD Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − aProtein was largely misfolded and the yield was too low to be analyzed. bThe average value calculated from two independent experiments. (+++: oligomer was formed similar to WT, ++: oligomer formation was weakly reduced, +: oligomer formation was significantly reduced, x: oligomer formation was not detected, –: not tested, NA; not applicable). View Large Table II. Summary of mutagenesis results showing the effect of mutations on oligomer formation of Dectin-1 CTLD Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − Mutation △Tm [°C] (thermal shift assay) Oligomer formation (size exclusion chromatography) Hill coefficient (tryptophan fluorescence) Kd [μM] (tryptophan fluorescence) WT 9.0 +++ 3.4b 40b First-batch mutation R174A − +++ − − N176A 6.0 +++ − − R207A 4.5 +++ − − H217A 5.0 ++ − − W221A 0 − NA NA I222Aa NA NA NA NA H223A 2.0 − 0.9 39 S225A − ++ − − E226A − ++ − − Y228A 3.0 − 1.8 13 Q230A 6.5 ++ − − Second-batch mutation Y141A − + 1.6 85 K144A − ++ − − R145A − + 1.1 84 Q149A − ++ − − D158A − ++ − − S160A − +++ − − K161Aa NA NA NA NA E164A − + 2.1 37 Q186A − ++ − − E188A − ++ − − F192A − ++ − − D195A − ++ − − Q205A − ++ − − N208A − ++ − − E213A − + 1.5 31 K242A − ++ − − E243A − + 1.0 19 Double and triple mutation N122A and Y141A − + − − Y141A and R145A − + − − Y141A and E213A − + − − R145A and E213A − + − − N122A, Y141A and E164A − + − − Y141A, R145A and E164A − + − − Y141A, E164A and E213A − + − − R145 A, E164A and E213A − + − − aProtein was largely misfolded and the yield was too low to be analyzed. bThe average value calculated from two independent experiments. (+++: oligomer was formed similar to WT, ++: oligomer formation was weakly reduced, +: oligomer formation was significantly reduced, x: oligomer formation was not detected, –: not tested, NA; not applicable). View Large Identification of residues involved in protein–protein interaction In order to try to identify the residues involved in protein–protein interactions, we prepared a second batch of 17 CTLD mutants selecting conserved residues over the entire protein surface, but away from the putative ligand binding site formed by W221, H223 and Y228. They comprised N122A, Y141A, K144A, R145A, Q149A, D158A, S160A, K161A, E164A, Q186A, E188A, F192A, D195A, Q205A, E213A, K242A and E243A. All mutants except K161A exhibited oligomer formation at 10 eq. laminarin, but some, namely N122A, Y141A, R145A, E164A, E213A, and E243A, only weakly at low ligand ratios compared to WT (Table II, Figure 6). We prepared double and triple mutants of these oligomer-impaired proteins and analyzed elution profiles in the absence and presence of laminarin. All the double mutants failed to form oligomers up to 1:5 protein–ligand molar ratio, and some conversion only occurred at 1:10 (Supplementary data, Figure S1). Triple mutants produced very similar results. Fig. 6. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Seventeen mutants were prepared with mutations to residues possibly involved in interprotein interactions by selecting residues outside of the putative ligand binding site, solvent exposed and most conserved among different mammalian species. Fig. 6. View largeDownload slide Characterization of Dectin-1 CTLD (WT and mutants) upon binding to laminarin by size exclusion chromatography. Seventeen mutants were prepared with mutations to residues possibly involved in interprotein interactions by selecting residues outside of the putative ligand binding site, solvent exposed and most conserved among different mammalian species. Table II provides an overview of the results. We mapped the residues involved in oligomer formation on the crystal structure of mouse Dectin-1 CTLD, where color code and intensity reflect the degree of the mutational effect (Figure 7). The putative β-glucan binding site formed by W221, H223 and Y228 is highlighted in red. Surrounding residues, N208, H217, S225, E226 and Q230 contribute to binding but not as much. The residues outside of the putative ligand binding site, i.e., N122, Y141, R145, E164, E213 and E243 examined in the second batch of mutations, are distributed around the whole CTLD surface, indicative of extensive protein–protein interactions. Fig. 7. View largeDownload slide Mapping the residues on the crystal structure of mouse Dectin-1 CTLD whose mutation affected ligand-induced oligomer formation of CTLD (red: no oligomer formation upon mutation, bright cyan: oligomer formation significantly reduced upon mutation, faint cyan: oligomer formation weakly affected by mutation). Fig. 7. View largeDownload slide Mapping the residues on the crystal structure of mouse Dectin-1 CTLD whose mutation affected ligand-induced oligomer formation of CTLD (red: no oligomer formation upon mutation, bright cyan: oligomer formation significantly reduced upon mutation, faint cyan: oligomer formation weakly affected by mutation). Analysis of cooperative ligand binding by tryptophan fluorescence The absence of an intermediate dimeric or trimeric species of CTLD in the size exclusion chromatography analysis implies that oligomer formation is cooperative. In addition, mutation of many individual residues outside of the putative ligand binding site impeded oligomer formation, suggesting extensive protein–protein interaction on ligand binding. In order to analyze possible ligand cooperativity associated with the CTLD oligomerization, we employed a method to directly measure the ligand binding event namely intrinsic tryptophan fluorescence quenching due to the location of W221 in the putative ligand binding site. The residue is solvent-exposed in the ligand-free structure and expected to be buried in the bound form. As expected, the fluorescence intensity (F) was gradually quenched upon titration of CTLD with laminarin (Figure 8A). A plot of ΔF (F0–F) vs. total laminarin concentration [L] and fitting the data to a Langmuir-type equation yielded a sigmoidal curve for WT which is compared with a simple first-order curve setting the Hill coefficient (α) to 1 (Figure 8B). The root mean square deviation obtained from the fitted sigmoidal curve is found to be 78.5 as compared to 132.7 from the first-order curve, indicating that the sigmoidal fit is better than the first-order fit. Another WT binding experiment is shown in Supplementary data, Figure S2 and the average Hill coefficient (α) from the two independent experiments was calculated as 3.4. The value is compatible with four laminarin molecules binding to four CTLDs in a cooperative manner, i.e., the affinity for ligand increases with increasing oligomerization. Hill coefficients of H223A and Y228A are 0.9 and 1.8, respectively, suggesting the decreased ligand binding cooperativity noted above. Likewise, the Hill coefficients of N122A, Y141A, R145A, E164A, E213A and E243A are 1.4, 1.6, 1.1, 2.1, 1.5 and 1.0, respectively (Supplementary data, Figure S3, Table II), indicating that these residues are involved in cooperative ligand binding. The ligand affinity, which is here defied as the laminarin concentration needed to achieve a half-maximum binding of the Dectin-1 CTLD, is shown in Table II. The Kd of WT was calculated as 40 μM and the mutants roughly showed similar affinity. These data suggest that most mutants have the ability to bind laminarin but have partially defective protein–protein interactions, and thereby diminished ligand binding cooperativity. Fig. 8. View largeDownload slide Measurement of intrinsic tryptophan fluorescence of Dectin-1 CTLD. (A) ΔF was obtained by subtracting the fluorescence intensity at a particular ligand concentration (F) from initial fluorescence intensity (F0). (B) Plot of ΔF(F0– F) vs. [L] showing the ligand binding pattern of Dectin-1 to laminarin. Curve fitting was performed using Langmuir-type equation (solid line) and a simple first-order equation (dotted line) (see Materials and methods). Fig. 8. View largeDownload slide Measurement of intrinsic tryptophan fluorescence of Dectin-1 CTLD. (A) ΔF was obtained by subtracting the fluorescence intensity at a particular ligand concentration (F) from initial fluorescence intensity (F0). (B) Plot of ΔF(F0– F) vs. [L] showing the ligand binding pattern of Dectin-1 to laminarin. Curve fitting was performed using Langmuir-type equation (solid line) and a simple first-order equation (dotted line) (see Materials and methods). Discussion The recognition of carbohydrate by a lectin may involve nonpolar (hydrophobic) interactions as well as hydrogen bonds (Weis and Drickamer 1996), and Trp221 and His223 in a surface groove of murine Dectin-1 CTLD may be key residues for β-glucan binding (Adachi et al. 2004; Mochizuki et al. 2014) through mainly hydrophobic interactions (Brown et al. 2007). Our mutational analysis shows that in addition to W221 and H223, Y228 is also critical for laminarin binding, and all three are located in a triangular fashion in the shallow groove. Long chain β-glucans (e.g., curdlan) can form a triple helix, such a helix having a diameter of about 15 Å (Chuah et al. 1983). Rough estimation of the size of the Dectin-1·β-glucan complex suggests that the groove formed by W221, H223 and Y228 can accommodate a triple helical β-glucan. Interestingly, another β-glucan binding receptor βGRP/GNBP3 has Trp, His and Tyr in its β-glucan binding site (Kanagawa et al. 2011). Although the topology of Dectin-1 CTLD is different from that of βGRP N-terminal domain, these residues may interact with β-glucan in a similar way. Various approaches have indicated that Dectin-1 increases in molecular mass on binding laminarin by forming oligomers suggesting a role for oligomer formation in the recognition of β-glucan-containing pathogens and further intracellular signaling. Formation of such oligomers may contribute the aggregation of Dectin-1 receptor and its intracellular motif. Analytical ultracentrifugation of Dectin-1 CTLD and laminarin points to increases from 26 kDa (19 kDa Dectin-1 CTLD monomer + ~7 kDa laminarin) to 72–92 kDa (Brown et al. 2007). Dynamic light scattering recorded a fourfold increase, suggestive of a tetrameric complex (Brown et al. 2007). Our SEC and SEC-MALS data also favors a tetramer of CTLD with the CTLD and laminarin stoichiometry of 4:4 indicating the involvement of four CTLD and four laminarin in forming a complex. Evidenced from the sigmoidal curve in Langmuir-type equation and the Hill coefficient of ~3 suggests four laminarin bind cooperatively to four CTLD molecules (Figure 8B). Inhomogeneity of laminarin and variable chain length may contribute to some variations in the obtained values, and studies with homogeneous β-glucan of defined chain length may be needed to resolve this. The minimum chain length of β-glucan required for binding to Dectin-1 is 10-mer or 11-mer (Palma et al. 2006), which is long relative to the size of monomeric Dectin-1 CTLD and it is anticipated that there will be extensions beyond the ligand binding area containing W221, H223 and Y228 (Figure 9A). The binding possibilities preclude accurate model building at this stage and further structural studies are required. Fig. 9. View largeDownload slide Schematic diagram of (A) relative size of Dectin-1 CTLD and laminarin with 25 glucose residues. (B) Possible ligand-induced signaling by cooperative oligomerization of Dectin-1 CTLD. Fig. 9. View largeDownload slide Schematic diagram of (A) relative size of Dectin-1 CTLD and laminarin with 25 glucose residues. (B) Possible ligand-induced signaling by cooperative oligomerization of Dectin-1 CTLD. Mutation of individual amino acid residues outside of the ligand binding groove, namely N122A, Y141A, R145A, E164A, E213A and E243A, lowered Dectin-1 oligomer formation, even up to 1:5 molar ratio of Dectin-1 to laminarin. It points to their involvement in the protein–protein interactions of Dectin-1 CTLD in forming an oligomeric complex. The results were very similar with selected double mutants and triple mutants. These residues are distributed all around the surface outside the ligand binding site of Dectin-1 CTLD. Although it is difficult to distinguish between the direct ligand binding residues and protein–protein interacting residues, the distributing pattern of these residues and 4:4 stoichiometry of Dectin-1–laminarin complex suggests a role for these residues in intersubunit interaction. The property of Dectin-1 oligomerization and global participation of surface residues is suggestive of cooperative behavior of Dectin-1 CTLD monomers to form an oligomeric complex. Our intrinsic fluorescence analysis of Dectin-1–laminarin interaction bears this out. Laminarin binding to wild-type Dectin-1 CTLD exhibits the average Hill coefficient of 3.4, while binding to these mutants shows decreased Hill coefficient values, signifying impaired oligomerization. The mechanism how the cooperativity is achieved is unknown at this moment. One possibility is that the binding of laminarin to monomeric CTLD could promote binding of the 1:1 complex to three other monomeric empty CTLDs, which may on complex formation show increasing affinity for the ligand, and hence the cooperativity. In the complex, laminarin may form a triple-helical structure which is seen in longer β-glucan chains. 3D structural elucidation of laminarin–CTLD complex needs further analysis. Ligand-induced cooperative oligomerization of the single extracellular carbohydrate recognition domain of Dectin-1 likely underlies the clustering of these receptors to form high density regions of ITAM on the inner cell surface and facilitate binding of kinase and propagate intracellular signals. Structural details of these events are lacking but a tetramer of extracellular domains of Dectin-1 molecules on the surface may increase the chance of kinase binding and tyrosine phosphorylation activities, more than that achieved by the bridging of only two receptor monomers (Figure 9). Materials and methods Expression and purification of Dectin-1 CTLD of murine Dectin-1 (Gly113-Leu244; 15 kDa) was expressed as inclusion bodies in E. coli using pCold vector and refolded as described in the previous article (Dulal et al. 2016). In brief, we constructed pCold-Dectin-1 CTLD vector using an artificial codon optimized CTLD gene of mouse Dectin-1 with protein G B1 domain (GB1), an N-terminal hexahistidine tag and a tobacco etch virus (TEV) protease cleavage site (Novagen). The plasmid was transformed into E. coli BL21(DE3). The cells were cultured in LB medium, induced with IPTG (0.5 mM) at 15°C and harvested after 16 h, and then sonicated in PBS (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4) in presence of 0.3% (v/v) BugBuster (Novagen). Resultant inclusion bodies were solubilized in urea buffer (8 M Urea, 50 mM Tris-HCl, 50 mM NaCl, and pH 8.0) and refolded by 50-fold dilution in a refolding buffer (200 mM Tris-HCl, 0.4 M l-arginine, pH 8.0 with 5 mM reduced glutathione and 0.5 mM oxidized glutathione). The refolded protein was purified by Ni2+ Sepharose 6 Fast Flow column (GE Healthcare) and size exclusion chromatography (HiLoad 16/60 Superdex 75 prep grade column, GE Healthcare). Preparation of Dectin-1 mutants To prepare the mutants, forward and reverse mutation oligonucleotide primers composed of 30 nucleotide bases (Life Technologies Japan Ltd) were designed by replacing the desired code to be mutated with that for alanine. Forward primer was designed by selecting 30 nucleotide residues from −6 to +24 bases of mutating amino acid codon and the corresponding codon was replaced with alanine (GCC). Reverse primer was designed by selecting 30 nucleotide residues from +6 to −24 bases of mutating amino acid codon, replacing the corresponding codon with alanine (GCC) and making its reverse complement sequence. Expression plasmid vector (pCold) with wild-type Dectin-1 cDNA was replicated by high-fidelity PCR using primeSTAR HS DNA polymerase (TAKARA), 5x primeSTAR buffer (5xPS), dNTP mixture and mutation primers. The template plasmid in the resultant PCR mixture was incubated for 3 h at 37°C with Dpn-I (Roche) to digest wild-type cDNA. The mutated plasmids were transformed into DH5α competent cells and cultured overnight at 37°C on LB agar plate. Plasmids were purified using Wizard® Plus SV Minipreps DNA Purification System (Promega) and the DNA sequence checked by sequence analysis. The mutated plasmids were then transformed into BL21(DE3) and expression carried out. Size-exclusion chromatography For the purification of target protein, sample was concentrated to 5 mL, filtered and then subjected to size exclusion chromatography (HiLoad 16/60 Superdex 75 prep grade column, GE Healthcare). To calculate the molecular mass and ligand binding property, 1 mL of sample solution (10 μM) was applied to size exclusion chromatography (10/300 GL Superdex 75 column, GE Healthcare) at flow rate of 0.5 mL/min. Dectin-1 and laminarin solutions were prepared in 20 mM sodium phosphate buffer, pH 7.4 containing 150 mM NaCl. The size exclusion column was equilibrated with the same PBS mentioned above. Dectin-1 CTLD sample was injected with and without premixing of 1:1, 1:5 and 1:10 molar ratio of β-glucan ligand laminarin from L. digitata (Sigma-Aldrich). Likewise, several standard proteins i.e., immunoglobulin G (150 kDa), conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (42.7 kDa) were analyzed with the same column and flow rate. Void volume was determined using Blue Dextran (2000 kDa). The elution volume of each sample was measured and the molecular mass of ligand-free Dectin-1 as well as laminarin-bound Dectin-1 was estimated with reference to the above mentioned standard protein samples. Size exclusion chromatography with multi-angle light scattering (SEC-MALS) The theoretical calculations for the determination of molecular mass by light scattering were described by Wyatt (Wyatt 1993). Our SEC-MALS experiments were performed at room temperature using an HPLC system (Shimadzu) coupled to and a Shimadzu UV detector SPD-20A (Shimazu), a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt Technology). For chromatographic separation, a 10/300 GL Superdex 75 size-exclusion column (GE Healthcare) was used at a flow rate of 0.5 mL/min equilibrated with 20 mM sodium phosphate buffer (pH 7.4) and 150 mM NaCl. For each run, a 100 μL of Dectin-1 CTLD (65 μM), laminarin (10 mM), or Dectin-1 CTLD–laminarin mixture (Dectin-1 CTLD 61 μM, laminarin 610 μM) was injected. Data analyses were performed using the software ASTRA 6.1 (Wyatt Technology). The differential refractive index increment (dn/dc) of Dectin-1 CTLD was assumed to be 0.185 mL/g (Zhao et al. 2011), and the dn/dc of laminarin was experimentally determined to be 0.1344 mL/g (Supplementary data, Figure S4). Component analysis of Dectin-1 CTLD–laminarin complex was performed using the ASTRA 6.1 software package to calculate the molecular mass of the entire complex as well as for each component of the complex (Hayashi et al. 1989; Kendrick et al. 2001). The theoretical background of the SEC-MALS analysis is shown below: (a) Determination of molecular weight of protein or ligand alone At the low sample concentrations typically attained in the column chromatography, the intensity of light-scattering signal (LS) is given as (LS)=KLSMc(dn/dc)2 where KLS is an instrument-specific constant, M is the molecular mass (Da), c is concentration in g/mL, dn/dc is the refractive index increment of the solute in mL/g. The refractive index signal (RI) is described as (RI)=KRIc(dn/dc) where KRI is an instrument-specific constant. Hence the molecular weight M can be determined from the two observables, (LS) and (RI), using the following relation: M=K′(LS)/(RI) where K′=KRI/[KLS(dn/dc)]. (b) Component analysis for the protein–ligand complex In addition to (LS) and (RI), UV absorbance (UV) is used for the component analysis. (UV)complex=KUV(cpεp+clεl) where KUV is a constant, ε is the extinction coefficient (mL/mg/cm) and the subscripts p, l and complex refer to the protein, ligand and protein–ligand complex, respectively. It can be assumed that the laminarin ligand has no UV absorbance at 280 nm, here the equation is simplified as (UV)complex=KUVcpεp (RI), (LS) and (dn/dc) for the protein–ligand complex are described as (RI)complex=KRIcp(dn/dc)p+KRIcl(dn/dc)l (LS)complex=KLSMcomplexccomplex(dn/dc)complex2 (dn/dc)complex=Mp/Mcomplex(dn/dc)p+Ml/Mcomplex(dn/dc)l Based on these equations, Mp, Ml and Mcomplex are determined. Fluorescence based thermal stability assay The melting temperatures of Dectin-1 CTLD and mutants were estimated by a thermal shift assay without and with 1:10 molar ratio of laminarin. Sample solution containing 20 μg protein (1 mg/mL) was mixed with 1000 times diluted SYPRO Orange solution (molecular probes) as a reporter dye in PBS (pH 7.4). The wavelengths for excitation and emission were 475–500 nm and 520–550 nm, respectively. The samples were heated in a real time PCR instrument (PikoReal 24, Thermo Scientific) from 293 to 368 K with increments of 0.5 K/s. Tryptophan fluorescence spectroscopy Intrinsic tryptophan fluorescence of Dectin-1 WT and mutants was measured using an f-4500 fluorescence spectrophotometer (Hitachi) at an excitation wavelength of 295 nm. Emission fluorescence intensity of the protein (100 μL, 10 μM, pH 6.5) solubilized in PBS (20 mM sodium phosphate, 50 mM NaCl, pH 6.5) was recorded without ligand and then by adding the laminarin solution prepared in the above mentioned PBS buffer in a protein/ligand ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15 and 1:20. The data were taken in duplicate. The maximum emission fluorescence intensities of the protein without ligand and laminarin-added samples of each protein ligand ratio were taken. These fluorescence values were adjusted for dilution effect caused by the addition of ligand solution by multiplying the fluorescence value with the ratio of total protein ligand solution volume to the volume of protein solution. ΔF values were calculated by subtracting the fluorescence value of each ligand ratio from the maximum fluorescence value of protein without ligand (F0). ΔF vs. total laminarin concentration [L] was plotted and binding analyzed using the following equation where ΔFmax is the maximum fluorescence decrease upon binding to laminarin, α, the Hill coefficient and Kd, the laminarin concentration needed to achieve a half-maximum binding of the protein at equilibrium. ΔF=ΔFmax[L]α/(Kdα+[L]α) Curve fitting was done using GraphPad Prism 7. The value of α indicates the nature of cooperative binding where α > 1, positive cooperative binding, α = 1, noncooperative binding, and α < 1, negative cooperative binding. To validate this curve fitting, a simple first-order curve fitting was additionally performed where the Hill coefficient (α) is set to 1 with the following equation: ΔF=ΔFmax[L]/(Kd+[L]) Supplementary data Supplementary data is available at Glycobiology online. Funding This work was supported in part by Grant-in-Aid for Scientific Research (C) [25460054 to Y.Y.] and Scientific Research (B) [16H04758 to Y.Y.] from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. Acknowledgements We thank Prof. Naoyuki Taniguchi (Systems Glycobiology Group, RIKEN) for the opportunity to perform this study, Dr. Kurono Ken-ichiro (Shoko Scientific Co., Ltd.) for assistance with SEC-MALS experiment and Dr. Sushil K. Mishra for preparing coordinates of β-glucan chain. Conflict of interest statement None declared. 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GlycobiologyOxford University Press

Published: May 11, 2018

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