Abstract
β(1-3)-Glucans, abundant in fungi, have the potential to activate the innate immune response against various pathogens. Although part of the action is exerted through the C-type lectin-like receptor Dectin-1, details of the interaction mechanism with respect to glucan chain-length remain unclear. In this study, we investigated a set of short β(1-3)-glucans with varying degree of polymerization (DP); 3, 6, 7, 16, and laminarin (average DP; 25), analyzing the relationship between the structure and interaction with the C-type lectin-like domain (CTLD) of Dectin-1. The interaction of short β(1-3)-glucans (DP6, DP16, and laminarin) with the CTLD of Dectin-1 was systematically analyzed by 1H-NMR titration as well as by saturation transfer difference (STD)-NMR. The domain interacted weakly with DP6, moderately with DP16 and strongly with laminarin, the latter plausibly forming oligomeric protein-laminarin complexes. To obtain structural insights of short β(1-3)-glucans, the exchange rates of hydroxy protons were analyzed by deuterium induced 13C-NMR isotope shifts. The hydroxy proton at C4 of laminarin has slower exchange with the solvent than those of DP7 and DP16, suggesting that laminarin has a secondary structure. Diffusion ordered spectroscopy revealed that none of the short β(1-3)-glucans including laminarin forms a double or triple helix in water. Insights into the interaction of the short β(1-3)-glucans with Dectin-1 CTLD provide a basis to understand the molecular mechanisms of β-glucan recognition and cellular activation by Dectin-1.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
β(1-3)-Glucan, a component of the fungal cell wall, is recognized by β(1-3)-glucan-binding proteins in host defense systems [1]. A diversity of binding modes of β(1-3)-glucan-binding proteins with their ligands has been revealed by X-ray crystallography and NMR approaches. For example, insect β(1-3)-glucan binding protein (βGRP/GNBP3) interacts with the β(1-3)-glucan laminarihexaose with pseudo-triple helical conformation [2], and with the laminarin forming oligomeric protein-glycan complexes [3]. On the other hand, the plant family 6 carbohydrate binding module interacts with the non-reducing terminus of the laminarihexaose [4]. Dectin-1 is a C-type lectin-like receptor that binds β-glucan and is expressed on immune cells such as macrophages, neutrophils, and dendritic cells [5, 6]. Knowledge of the Dectin-1 β-glucan-binding mode has been integrated from its protein structure [7] and ligand binding characteristics [8, 9]. While the putative binding residues, namely Trp221 and His223 are identified [10], the details of the carbohydrate binding site are unclear. The pattern recognition receptor Dectin-1 is characteristic from classical C-type lectins in terms of the metal-ion independent ligand binding. Upon binding with ligand β(1-3)-glucan, a tyrosine of the immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic tail receives phosphorylation, and the downstream of the signaling secrets certain inflammatory cytokines such as tumor-necrosis factor (TNF) and IL-12 [11]. It is interesting that the mono- and oligosaccharides of the β(1-3)-glucan do not inhibit receptor responses to the native β(1-3)-glucans which potentially form triple helix under a certain conditions [12, 13].
The triple helical fiber of β(1-3)-glucan polymers was initially revealed using X-ray diffraction [14, 15], and solid-state 13C-NMR spectroscopy [16]. At least partial triple-helix structure may exist in aqueous solution according to size exclusion chromatography with multi-angle laser-light scattering [17] and fluorescence resonance energy transfer [18]. Accumulating evidence suggests that degree of polymerization (DP) and β(1-3)-glucan structure (unstructured, helical, or triple-helical), are key recognition features in the activation of the innate immune response by mouse Dectin-1 [12, 13, 16, 19]. Recently, the triple helical structure of β-glucan has been exploited in the supramolecular chemistry field to give a hetero triple helical β-glycan DNA complex [20]. However, definitive evidence of structural transitions from unstructured β(1-3)-glucose(Glc)-oligomer/polymer to the helical β(1-3)-glucan, which should occur in a DP-dependent fashion, remains elusive [21]. Further, the point at which the chain length determines the change from simple secondary structural elements (single helix) to more complex structures (double and triple-helix) is unknown.
We here analyze a set of short β(1-3)-glucans having DPs of 3, 6, 7, and 16, and laminarin (average DP; 25) to define their structures and DP-dependent recognition by the Dectin-1 using various NMR techniques (Fig. 1). The glucan structures were characterized by dynamic 13C-NMR [22, 23] by observing deuterium induced 13C-isotope shifts (DIS). The technique provides H/D exchange rates of each sugar hydroxy group in a qualitative as well as a quantitative manner. Proton exchanges are sensitive to hydrogen-bond formation and complex structural elements and thus provide structural information. Diffusion ordered spectroscopy (DOSY) [24] was used to provide information on the oligomeric state of each glycan. DOSY directly determines the apparent diffusion coefficient of solutes [25, 26]. The DP dependent interaction of short β(1-3)-glucans to Dectin-1 CTLD was monitored by 1H-NMR titration. Epitope mapping was performed using 1D saturation transfer difference (STD)-NMR [27, 28], which is often used to measure various types of binding in solution, such as in protein-small molecular weight ligand screening [29].
Materials and methods
Materials
β(1-3)Glc3; DP3, β(1-3)Glc6,; DP6, β(1-3)Glc7; DP7 were purchased from Seikagaku Co. (Tokyo, Japan). β(1-3)Glc16-O-(CH2)6-NH2; DP16 was chemically synthesized according to the previously reported procedures [9, 13]. Laminarin from Laminaria digitata was purchased from Sigma-Aldrich, and the MALDI-TOF MS spectrum showed Gaussian distribution with the centered signal corresponding to DP = 25 (m/z = 4,091 [M+Na]+). The C-type lectin-like domain (CTLD) of mouse Dectin-1 (Gly113-Leu244; 16 kDa) was expressed by Escherichia coli, and the protein was refolded as described in a previous report [9]. Briefly, we constructed pET28a-Dectin-1 CTLD vector having N-terminal hexahistidine-tag and a thrombin cleavage site (Novagen). The plasmid was transformed into E. coli BL21(DE3)codonplus. Cells were cultured in LB medium and harvested after 6 h induction with isopropylthio-β-D-galactopyranoside at 37 °C, and then sonicated in PBS (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4) with BugBuster (Novagen). The resultant inclusion bodies were solubilized with 8 M urea and refolded by dilution into 200 mM Tris–HCl (pH 8.0), 0.4 M L-arginine, 5 mM reduced glutathione and 0.5 mM oxidized glutathione at 4 °C for 16 h. After refolding, the protein solution was purified with a Ni-Sepharose 6 Fast Flow column (GE Healthcare).
NMR experiments
NMR spectra were recorded with 500 MHz and 600 MHz spectrometers (Avance-500 and DRX-600, BrukerBioSpin) equipped with a triple resonance inverse cryogenic (cryo-TXI) probe and a triple resonance inverse probe (TXI), respectively. 1H chemical shifts indicated as parts per million (ppm) are calibrated based on an outer standard of the chemical shift of 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as a singlet at 0 ppm. 13C chemical shifts were calibrated using indirect reference based on the IUPAC-IUB recommended 13C/1H resonance ratio of 0.251449530 [30].
1H-NMR titration experiments were performed using mouse CTLD of Dectin-1 (10 μM, 500 μL) in PBS (8.1 mM Na2HPO4, 1.5 mM, KH2PO4, 137 mM NaCl and 2.7 mM KCl, pH 7.4) with 10 % D2O/90 % H2O, at a probe temperature of 25 °C. Ligand β(1-3)-glucans in the above mentioned buffer were gradually added into the protein solution. The residual water signals were suppressed with WATERGATE pulse sequence with a 3-9-19 pulse train.
1H-saturation transfer difference (STD)-NMR spectra were collected with a 500 MHz spectrometer, and the protein signal at 7.5 ppm (on-resonance) was saturated with Gaussian 25 Hz pulse train with 60 times. Reference spectra were obtained with irradiation at 40 ppm (off-resonance). The on-resonance and off-resonance spectra were collected in an interleaved manner, and accumulated in two different data sets. Water suppression was achieved using WATERGATE pulse sequence with a 3-9-19 pulse train, and the probe temperature set to 25 °C. To obtain good S/N ratios, 128 scans with 4 repetition loops were required, and the protein signals were partially suppressed using a T 1σ filter. Subtraction of saturation data from reference data was performed during free induction decay (FID) acquisition and repetition delay (total 3.1 s). With regard to the amplification factor (AF), relative signal intensities were calculated based on (I off−I on)/I off (where I on: intensity of on-resonance signals; I off: intensity of off-resonance signals) and the highest value of AF was assigned as 100 %. 1D STD-NMR spectra standardized with the AF of the highest H3 signal. Signal to noise (S/N) ratio was determined using the highest H5 signal and noise between 2 and 3 ppm.
13C-NMR spectra were collected with of β(1-3)-glucans in 10 mM sodium acetate buffer pH 6.0 composed of 50 % H2O/50 % D2O, unless otherwise stated [23]. DP7 and laminarin dissolved well in the buffer to a concentration of 20 mg/mL. DP16 was less soluble and provided 4 mg/mL.
Diffusion ordered spectroscopy (DOSY) experiments were carried out on the short β(1-3)-glucans (2.0 mg/mL, otherwise mentioned) in 99 % D2O. The pulse program was using stimulated echo sequence incorporating bipolar gradient pulses for diffusion (BPP-STE) [31]. The spectra were recorded in 16 scans for each gradient step. The linear rectangular shape gradient was logarithmically increased in 32 steps from 2 % to 95 % of the maximum gradient strength. The diffusion time (big delta) was set in 0.2 s, and the length of the square diffusion encoding gradient pulses (little delta) was set in 1 ms. The log10 D were determined at 27 °C, where D (m2/s) is the diffusion coefficient. The residual HOD signal was suppressed using WATERGATE pulse sequence with a 3-9-19 pulse train.
The 1H and 13C signal assignments of short β(1-3)-glucans were obtained by 1H-1H double quantum filtered correlation spectroscopy (DQF-COSY), 1H-13C heteronuclear single quantum coherence spectroscopy (HSQC) and 1H-13C HSQC-total correlation spectroscopy (TOCSY), measured at 25 °C. The results are summarized in Table S2 (DP16) and confirmed by references in previous reports [8, 9]. NMR data were processed with XWIN-NMR (ver.3.5) and Topspin 3.1, and the spectra were displayed using XWIN-PLOT and SPARKY (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco).
Results
1H-NMR spectra of CTLD of Dectin-1 in titration with short β(1-3)-glucans
NMR interaction analysis of short β(1-3)-glucans with mouse CTLD of Dectin-1 were performed by titration experiments (Fig. 1). Ligand was gradually added to a 10 μM Dectin-1 CTLD solution at a protein/ligand molar ratio of 1:1 to 1:10, in the case of DP6 and DP16, or 1:0.25 to 1:1, in the case of laminarin. The signal changes were monitored in the high field aliphatic region (−1.5 to −0.3 ppm) because it presents well separated protein signals. The full size 1H-NMR spectrum of the protein was shown in Fig. S1. The titration of DP6 induced quite limited changes to the 1H-signals of Dectin-1 CTLD (Fig. 2a), and DP16 caused slight but clear suppression of the protein signals (Fig. 2b). In contrast, laminarin induced significant signal broadening at a molar ratio of 1:1 (Fig. 2c), and further ligand additions to 5 equiv provided no further change (data not shown). The binding of DP6 is evidently quite weak, and DP16 moderately stronger.
Binding analysis of β(1-3)-glucans with Dectin-1 by STD-NMR
The signal perturbation patterns of the Dectin-1 in titration with β(1-3)-glucans were characteristic dependent on the ligand chain length, even though all ligand has identical Glcβ(1,3)Glc units. The interaction analysis was further performed by observing the ligand signal. To determine and compare the relative binding moiety, the interaction of Dectin-1 CTLD and β(1-3)-glucans are analyzed using STD-NMR. STD-NMR studies of the interaction of the CTLD of Dectin-1 with ligands DP6 [8] and DP16 [9] have been reported under different conditions especially in the temperature. In order to properly assess the DP-dependent differences, we here systematically compare the interactions of DP6, DP16 and laminarin under the same condition. Our results of the binding between the CTLD of Dectin-1 (10 μM) and short β(1-3)-glucans (in 25 or 10 molar excess) as measured using STD-NMR at 25 °C are shown in Fig. 3. The STD-NMR spectrum of DP6 with Dectin-1 CTLD (Fig. 3a) provides only the trace ligand signal under the condition. The findings indicated the saturation transfer occurring at 25 °C is nearly out of the detection range. In contrast, the STD-NMR spectra of DP16 and laminarin (Fig. 3b and c, respectively) showed significant saturation transfer from Dectin-1 CTLD to ligand. The S/N ratio was better in the case of laminarin, and the actual S/N ratio of the STD-NMR spectra are 9 (DP6), 15 (DP16), and 39 (laminarin), respectively. In addition, as we previously reported, the aminohexyl moiety of DP16 attached at the anomeric position shows very weak STD signals even at 5 °C [9]. Taken together with the 1H-NMR titration result, suggest that the binding of DP6 to the protein is weaker than DP16 and the dissociation quite fast. This is consistent with in vitro binding assays [13].
The binding of DP16 and laminarin were analyzed using the relative (%) amplification factor (AF) (Fig. 4). The H3 positions were adjusted to a %AF of 100 %, and the relative AFs of H2, H4/5, H6a, and H6b determined. In both results, the H3 position showed highest relative AFs, and position H4, H5, and H6 showed moderate AFs. The effective saturation transfer to H3 position compared with the other positions. It should be noted that not all H3 protons may be involved in binding because we averaged the AFs of H3 from different glucose residues. Considering the major conformation of β(1-3) linkage (Fig. S8), the orientation of H3 in residue i displays a different spatial orientation that in i + 1 and i-1 residues. Nonetheless, it can be concluded that at least some of the H3 protons are included in the interaction. Interestingly, the relative AF of the H2 position increases from DP16 to laminarin.
Dynamic NMR of deuterium induced 13C-NMR isotope shifts (DIS)
To investigate correlations between glycan structure and interaction with Dectin-1 CTLD, an insight of the solution structure of short β(1-3)-glucans was analyzed using DIS.
Glycans are richly hydroxylated and the hydroxy groups are used to interact with binding partners through hydrogen bonds. Normally sugar hydroxy protons exchange rapidly with water protons, which make measurements by NMR difficult. However, we have developed a dynamic 13C-NMR technique employing DIS [32]. which provides an indirect observation of an exchangeable hydroxy proton through a neighboring 13C-signal (Fig. 5) [22, 23]. It is possible because the 13C-NMR line shape is highly dependent on the H/D to water exchange rate, and has been used as a good indicator of low-barrier hydrogen bonds [33–35]. The doublet of each isotopomer, namely 13C-OH and 13C-OD, are observed under a slow H/D exchanging environment with up to 0.15 ppm difference. In contrast, a fast exchanging environment makes each isotopomers indistinguishable and a singlet signal appears at the averaged chemical shift.
To analyze the structure of short β(1-3)-glucans and focusing on the proton exchanges of the hydroxy groups, we performed 13C-NMR on DP7, synthetic DP16, and laminarin (average DP; 25) [36] in 10 mM sodium acetate buffer (pH 6.0) composed of 50 % H2O/50 % D2O (Fig. 6a and b). The signal splitting of laminarin based on DIS was confirmed by measuring 13C-NMR in either 90 % D2O or 90 % H2O solutions (Fig. S5). 13C-NMR at 5 °C provided no significant difference in signal pattern at the C2 position in any of the three β(1-3)-glucans. The signals at 76.4 ppm gave sets of 13C-OH/D isotopomer doublets with 0.06 ppm difference (Fig. 6a). The DIS doublets all coalesced on raising the probe temperature from 5 °C to 10 °C. Thus, there were no significant differences in the 13C-OH/D exchange rate at the C2 hydroxy group among DP7, DP16, and laminarin. Although minor signals that overlapped at 76.4 ppm gradually shifted with changes in temperature, the major C2 signals of all three β(1-3)-glucans showed almost no chemical shift changes with temperature. Evidently, the micro environments surrounding C2 are insensitive to temperature. On the other hand, in the experiments performed at 5 °C, the C4 signals at 71.1 ppm gave broad coalesced signals for DP7 and DP16 and a set of 13C-OH/D isotopomer doublet signals 0.07 ppm apart for laminarin (Fig. 7b). The DIS doublet of laminarin coalesces at 15 to 20 °C. Evidently, the exchanges of 13C-OH/D at C4 of laminarin are significantly slower than those of DP7 and DP16. This is likely due to the C4-OH of laminarin forming a hydrogen bond to either O3, O5, or O6 of the nascent Glc residue. The chemical shifts of all C4 signals exhibited slight but significant down field shifts with elevation of temperature.
Characterization of oligomeric state by diffusion ordered spectroscopy (DOSY)
The 13C-NMR-based DIS experiments suggest that laminarin is structured as a helix under the experimental conditions. We then analyzed the oligomeric state of the β(1-3)-glucans using DOSY experiments. Long β(1-3)-glucans are known to form higher order structures of double- and triple-helices [14–18]. DOSY has been utilized to analyze higher order complexes of low molecular weight compounds, including biological molecules, in solution [24], through gradual z-gradient changes. DOSY spectra of DP3, 6, 16 and laminarin were collected at 27 °C (Fig. 7). The corresponding self-diffusion coefficient D (m2/s) of DP3, DP6, DP16, and laminarin were 3.9 × 10−10 (the common logarithm; −9.41), 3.2 × 10−10 (−9.50), 2.1 × 10−10 (−9.68), and 1.4 × 10−10 (−9.84), respectively (Fig. 7a–d). The average molecular weight of the glycans were estimated using the scaling relationship,
where K and α are scaling parameters [37, 38]. This relationship has been shown to be valid only at low concentrations (up to 1 wt.%). Based on the Eq. (1), a double-logarithmic plot of log10 D values against log10 M w was linear and a line could be fitted with the following values (Fig. 7e).
The monomeric form of the laminarin was confirmed at various concentrations and temperatures (Fig. S6 and S7). The increase of the temperature from 5 to 35 °C, the diffusion coefficients were also increased (Fig. S6), which was mainly due to the change of the solvent viscosity, and significant change of their hydrodynamic radius was not observed. The concentration dependent change of the diffusion coefficient of laminarin was collected (Fig. S7), and the infinite diffusion coefficient (D 0) of laminarin is 1.4 × 10−10 (m2/s) based on the linear fitting. The drastic change of the concentration dependent self-diffusion coefficient was not observed under the experimental conditions. We also performed the DOSY experiment of Dectin-1/laminarin complexes however the estimation of diffusion constant was unsuccessful due to the formation of larger oligmeric complexes and hence inducing the broadening of 1H signals (data not shown).
Discussion
In 1H-NMR titration experiment, observing Dectin-1 signal, the significant line broadening effect induced by laminarin seems more than just simple tight binding and may reflect a large change in the hydrodynamic properties of the complex (Fig. 2). It is interesting because the significant change is induced by the nine (in average) β(1-3)-glucose extensions of laminarin. Another possibility is that Dectin-1 strongly binds to the occasional β(1-6)-branch which found in the interval of 0 to 2 on a β(1-3)-Glc backbone of laminarin. However, the previous result using synthetic glucans suggested that a β(1-6)-Glc branch does not show significant enhancement of the binding [9]. The additional numbers of β(1-3)-glucose residue lead laminarin’s tighter binding and slower dissociation rate compared with DP16. Perhaps, the formation of an oligomeric protein-glucan complex occurs. The consideration is consistent with a previous report on laminarin-Dectin-1 complex, which was analyzed by dynamic light scattering and sedimentation coefficient by analytical centrifugation [7, 39]. Then DP16 may only have a weak propensity for inducing such an oligomeric state. Interestingly, laminarin induces a macro oligomeric state of a moth β(1-3)-glucan recognition protein [3], which is likely to be similar to our observation in Dectin-1 CTLD.
In STD-NMR, the relative AF of the H3, in both DP16 and laminarin, are significantly higher (~20 %) than the other positions. The effective saturation transfer to H3 position suggests that Dectin-1 CTLD discriminates β(1-3)-glucans from other connections through direct interaction around the glycoside bond. The H3 atom is mapped in the tightly packed triple helical β(1-3)-glucan structure model with blue ball (Fig. 8). It is apparent that the H3 atoms locate not the surface but the inside of the triple helix. The tight triple-helical model is unlikely in the interaction between short β(1-3)-glucan and Dectin-1 because H3 atoms is incapable to access the protein binding site. Therefore, in the case of short β(1-3)-glucans including laminarin, monomeric form or partially relaxed triple helix interact with Dectin-1 CTLD. The proportional relation in the logarithm of the molecular weight (M w) and diffusion coefficient (D) suggests that the short β(1-3)-glucans are not triple helical but monomeric (Fig. 7e).
Interestingly, the relative AF of the H2 position increases the longer the chain length (59 % in DP16 and 78 % in laminarin), and this may correlate with decreased flexibility due to greater secondary structure formation the longer the chain, expected from DIS experiment shown in Fig. 6. The β(1-3)-glucan structure possibly plays a role in interaction with Dectin-1 CTLD.
We observed correlations between DP and the proton exchanges of C4-OH (those of laminarin were slower than in DP7 and DP16), but not C2-OH (no differences between the three) in dynamic 13C-NMR study (Fig. 6). Since the β(1-3)-glucans have hydroxy groups at C2, C4, and C6 positions, the torsion of the glycoside bond is partially restricted by their inter-residual hydrogen bonds especially between O4-O5’ and O2-O2’ [40]. Furthermore, the φ (H1'-C1'-O-C3) and ψ (C1'-O-C3-H3) angles of laminaribiose determined by X-ray crystallography and NMR are (28°, −38°), (43°, −52°), and (40°, −9°) and their distance in hydrogen bonds of O4-O5’ are 2.8 Å in (Table S1) [41–43]. The dihedral angle of Glcβ(1-3)Glc in Protein Data Bank show that majority of the φ and ψ angles are 20° < φ < 60° and −60° < ψ < 40° (Fig. S8). The relatively narrow range of φ is due to the exo-anomeric effect. Considering to the O4-O5’ hydrogen bonds in our data, the averaged ψ angle of laminarin is restricted into −60° < ψ < −10°. It suggests that the O4-O5’ hydrogen bond partially restricts the alteration of the torsion as well as of the distribution in DP dependent manner, which is expected to induce the helical conformation. Combined with the DOSY results, laminarin uniquely tends to form a partial single helical conformation stabilized by hydrogen bond of C4-OH.
Our DOSY results, the values of slope (−0.47) and intercept (−8.1) from Eq. (2) are similar to those in a report analyzing monomeric α(1-4)/α(1-6)-glucan pullulans, namely −0.49 and −8.1, respectively [44]. The similarity suggests that the hydrodynamic radii of short β(1-3)-glucans correspond closely to those of pullulans of the same chain length. In water, pullulan has been shown to behave as a random coil [45]. Thus the laminarin’s helix seems to be flexible.
The binding mode of long β(1-3)-glucans including curdlan and schizophyllan is possibly different from the case of short β(1-3)-glucans. While their solution structure is unclear, the φ and ψ angles of the solid curdlan are different from the laminaribiose (Table S1). They potentially form triple-helix and actually the affinity to Dectin-1 is higher than the short β(1-3)-glucans [12, 13]. To make this point clear, an artificial triple helix model consisting of short β(1-3)-glucans will be a good probe.
In this study we have analyzed the structure of several short β(1-3)-glucans with varying degrees of polymerization and studied their interaction with the C-type lectin-like domain of Dectin-1 using NMR. 1H-NMR titrations and STD-NMR revealed that DP6 interacts weakly with the C-type lectin-like domain of Dectin-1, DP16 rather stronger, and laminarin very significantly, possibly even inducing oligomeric protein-glycan complexes. The increasing ligand-protein interaction with increasing chain length may correlate with increasing secondary structure formation. H/D exchanges of the proton of hydroxy groups were analyzed by deuterium induced 13C-NMR isotope shifts and indicated that the hydroxy group at C4 of laminarin uniquely had a slow exchange rate. DOSY experiments revealed that the short β(1-3)-glucans are monomeric in solution, forming neither double nor triple helical structures. Insights into the molecular mechanism of how short β(1-3)-glucans interact with Dectin-1 will contribute to the development novel immuno-activating medicines against unidentified pathogens.
References
Goodridge, H.S., Wolf, A.J., Underhill, D.M.: β-glucan recognition by the innate immune system. Immunol. Rev. 230, 38–50 (2009)
Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., Yamaguchi, Y.: Structural insights into recognition of triple-helical β-glucans by an insect fungal receptor. J. Biol. Chem. 286, 29158–29165 (2011)
Dai, H., Hiromasa, Y., Takahashi, D., Vandervelde, D., Fabrick, J.A., Kanost, M.R., Krishnamoorthi, R.: An initial event in the insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein. Biochemistry 52, 161–170 (2013)
van Bueren, A.L., Morland, C., Gilbert, H.J., Boraston, A.B.: Family 6 carbohydrate binding modules recognize the non-reducing end of β-1,3-linked glucans by presenting a unique ligand binding surface. J. Biol. Chem. 280, 530–537 (2005)
Brown, G.D., Gordon, S.: Immune recognition. A new receptor for β-glucans. Nature 413, 36–37 (2001)
Palma, A.S., Feizi, T., Zhang, Y., Stoll, M.S., Lawson, A.M., Díaz-Rodríguez, E., Campanero-Rhodes, M.A., Costa, J., Gordon, S., Brown, G.D., Chai, W.: Ligands for the β-glucan receptor, Dectin-1, assigned using “designer” microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides. J. Biol. Chem. 281, 5771–5779 (2006)
Brown, J., O’Callaghan, C.A., Marshall, A.S., Gilbert, R.J., Siebold, C., Gordon, S., Brown, G.D., Jones, E.Y.: Structure of the fungal β-glucan-binding immune receptor dectin-1: implications for function. Protein Sci. 16, 1042–1052 (2007)
Sylla, B., Guégan, J.P., Wieruszeski, J.M., Nugier-Chauvin, C., Legentil, L., Daniellou, R., Ferrières, V.: Probing β-(1→3)-D-glucans interactions with recombinant human receptors using high-resolution NMR studies. Carbohydr. Res. 346, 1490–1494 (2011)
Tanaka, H., Kawai, T., Adachi, Y., Hanashima, S., Yamaguchi, Y., Ohno, N., Takahashi, T.: Synthesis of β(1,3) oligoglucans exhibiting a Dectin-1 binding affinity and their biological evaluation. Bioorg. Med. Chem. 20, 3898–3914 (2012)
Adachi, Y., Ishii, T., Ikeda, Y., Hoshino, A., Tamura, H., Aketagawa, J., Tanaka, S., Ohno, N.: Characterization of β-glucan recognition site on C-type lectin, dectin 1. Infect. Immun. 72, 4159–4171 (2004)
Brown, G.D.: Dectin-1: a signalling non-TLR pattern-recognition receptor. Nat. Rev. Immunol. 6, 33–43 (2006)
Adams, E.L., Rice, P.J., Graves, B., Ensley, H.E., Yu, H., Brown, G.D., Gordon, S., Monteiro, M.A., Papp-Szabo, E., Lowman, D.W., Power, T.D., Wempe, M.F., Williams, D.L.: Differential high-affinity interaction of dectin-1 with natural or synthetic glucans is dependent upon primary structure and is influenced by polymer chain length and side-chain branching. J. Pharmacol. Exp. Ther. 325, 115–123 (2008)
Tanaka, H., Kawai, T., Adachi, Y., Ohno, N., Takahashi, T.: β(1,3) Branched heptadeca- and linear hexadeca-saccharides possessing an aminoalkyl group as a strong ligand to dectin-1. Chem. Commun. (Camb.) 46, 8249–8251 (2010)
Bluhm, T.L., Sarko, A.: The triple helical structure of lentinan, a linear β-(1→3)-D-glucan. Can. J. Chem. 55, 293–299 (1977)
Chuah, C.T., Sarko, A., Deslandes, Y., Marchessault, R.H.: Packing analysis of carbohydrates and polysaccharides.14. Triple-helical crystalline-structure of curdlan and paramylon hydrates. Macromolecules 16, 1375–1382 (1983)
Yoshioka, Y., Uehara, N., Saito, H.: Conformation-dependent change in antitumor activity of linear and branched (1 → 3)-β-D-glucans on the basis of conformational elucidation by carbon-13 nuclear magnetic resonance spectroscopy. Chem. Pharm. Bull. 40, 1221–1226 (1992)
Kulicke, W.M., Lettau, A.I., Thielking, H.: Correlation between immunological activity, molar mass, and molecular structure of different (1→3)-β-D-glucans. Carbohydr. Res. 297, 135–143 (1997)
Young, S.H., Dong, W.J., Jacobs, R.R.: Observation of a partially opened triple-helix conformation in 1–>3-β-glucan by fluorescence resonance energy transfer spectroscopy. J. Biol. Chem. 275, 11874–11879 (2000)
Goodridge, H.S., Reyes, C.N., Becker, C.A., Katsumoto, T.R., Ma, J., Wolf, A.J., Bose, N., Chan, A.S., Magee, A.S., Danielson, M.E., Weiss, A., Vasilakos, J.P., Underhill, D.M.: Activation of the innate immune receptor Dectin-1 upon formation of a ‘phagocytic synapse’. Nature 472, 471–475 (2011)
Sakurai, K., Uezu, K., Numata, M., Hasegawa, T., Li, C., Kaneko, K., Shinkai, S.: β-1,3-glucan polysaccharides as novel one-dimensional hosts for DNA/RNA, conjugated polymers and nanoparticles. Chem. Commun. (Camb), 4383–4398 (2005)
Sletmoen, M., Stokke, B.T.: Higher order structure of (1,3)-β-D-glucans and its influence on their biological activities and complexation abilities. Biopolymers 89, 310–321 (2008)
Uchida, K., Markley, J.L., Kainosho, M.: Carbon-13 NMR method for the detection of correlated hydrogen exchange at adjacent backbone peptide amides and its application to hydrogen exchange in five antiparallel β strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI). Biochemistry 44, 11811–11820 (2005)
Hanashima, S., Kato, K., Yamaguchi, Y.: 13C-NMR quantification of proton exchange at LewisX hydroxyl groups in water. Chem. Commun. (Camb.) 47, 10800–10802 (2011)
Johnson, C.S.: Diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications. Prog. NMR Spect. 34, 203–256 (1999)
Díaz, M.D., Berger, S.: Studies of the complexation of sugars by diffusion-ordered NMR spectroscopy. Carbohydr. Res. 329, 1–5 (2000)
Groves, P., Rasmussen, M.O., Molero, M.D., Samain, E., Cañada, F.J., Driguez, H., Jiménez-Barbero, J.: Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis. Glycobiology 14, 451–456 (2004)
Mayer, M., Meyer, B.: Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J. Am. Chem. Soc. 123, 6108–6117 (2001)
Mayer, M., Meyer, B.: Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew. Chem. Int. Ed. Engl. 38, 1784–1788 (1999)
Wang, Y.S., Liu, D., Wyss, D.F.: Competition STD NMR for the detection of high-affinity ligands and NMR-based screening. Magn. Reson. Chem. 42, 485–489 (2004)
Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J., Oldfield, E., Markley, J.L., Sykes, B.D.: 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR 6, 135–140 (1995)
Gibbs, S.J., Johnson, C.S.: A PFG NMR experiment for accurate diffusion and flow studies in the presence of eddy currents. J. Magn. Reson. 93, 395–402 (1991)
Pfeffer, P.E., Valentine, K.M., Parrish, F.W.: Deuterium-induced differential isotope shift 13C NMR.1. resonance reassignments of mono- and disaccharides. J. Am. Chem. Soc. 101, 1265–1274 (1979)
Dziembowska, T., Hansen, P.E., Rozwadowski, Z.: Studies based on deuterium isotope effect on 13C chemical shifts. Prog. NMR Spect. 45, 1–29 (2004)
Smirnov, S.N., Golubev, N.S., Denisov, G.S., Benedict, H., SchahMohammedi, P., Limbach, H.H.: Hydrogen/deuterium isotope effects on the NMR chemical shifts and geometries of intermolecular low-barrier hydrogen-bonded complexes. J. Am. Chem. Soc. 118, 4094–4101 (1996)
Marshall, T.W.: Isotope shifts in the NMR spectra of H2, HD and D2 due to zero-point vibration. Mol. Phys. 4, 61–63 (1961)
Read, S.M., Currie, G., Bacic, A.: Analysis of the structural heterogeneity of laminarin by electrospray-ionisation-mass spectrometry. Carbohydr. Res. 281, 187–201 (1996)
Chen, A., Wu, D.H., Johnson, C.S.: Determination of molecular-weight distributions for polymers by diffusion-ordered NMR. J. Am. Chem. Soc. 117, 7965–7970 (1995)
Suárez, E.R., Syvitski, R., Kralovec, J.A., Noseda, M.D., Barrow, C.J., Ewart, H.S., Lumsden, M.D., Grindley, T.B.: Immunostimulatory polysaccharides from Chlorella pyrenoidosa. A new galactofuranan. measurement of molecular weight and molecular weight dispersion by DOSY NMR. Biomacromolecules 7, 2368–2376 (2006)
Friedlaender, M.H., Cook, W.H., Martin, W.G.: Molecular weight and hydrodynamic properties of laminarin. Biochim. Biophys. Acta 14, 136–144 (1954)
Sundaralingam, M.: Some aspects of stereochemistry and hydrogen bonding of carbohydrate related to polysaccharide conformations. Biopolymers 6, 189–213 (1968)
Takeda, H., Yasuoka, N., Kasai, N.: Crystal and molecular-structure of a 3:2 mixture of laminarabiose and O-α-D-glucopyranosyl-(1 → 3)-β-D-glucopyranose. Carbohydr. Res. 53, 137–152 (1977)
Noguchi, K., Okuyama, K., Kitamura, S., Takeo, K.: Crystal structure of methyl 3-O-β-D-glucopyranosyl-β-D-glucopyranoside (methyl β-D-laminarabioside) monohydrate. Carbohydr. Res. 237, 33–43 (1992)
Olsson, U., Serianni, A.S., Stenutz, R.: Conformational analysis of β-glycosidic linkages in 13C-labeled glucobiosides using inter-residue scalar coupling constants. J. Phys. Chem. B 112, 4447–4453 (2008)
Viel, S., Capitani, D., Mannina, L., Segre, A.: Diffusion-ordered NMR spectroscopy: A versatile tool for the molecular weight determination of uncharged polysaccharides. Biomacromolecules 4, 1843–1847 (2003)
Kato, T., Katsuki, T., Takahashi, A.: Static and dynamic solution properties of pullulan in a dilute-solution. Macromolecules 17, 1726–1730 (1984)
Acknowledgments
We thank Dr. Yukishige Ito (RIKEN/ERATO) and Prof. Osamu Kanie (ERATO/Tokai University) for providing us with opportunities to use the 500 MHz NMR equipped with a cryoprobe. We thank Dr. Mayumi Kanagawa for discussion and kind help for preparation of the figures. We thank Ms. Kana Matsumoto-Morita for sample preparation and Ms. Noriko Tanaka for secretarial assistance. This work was supported in part by Grant-in-Aid for Young Scientists (B) (No. 24710257) from Japan Society of the Promotion of Science (JSPS) and by SUNBOR GRANT from Suntory Foundation of Life Sciences (S.H.).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 679 KB)
Rights and permissions
About this article
Cite this article
Hanashima, S., Ikeda, A., Tanaka, H. et al. NMR study of short β(1-3)-glucans provides insights into the structure and interaction with Dectin-1. Glycoconj J 31, 199–207 (2014). https://doi.org/10.1007/s10719-013-9510-x
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10719-013-9510-x