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.

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The core fucose, a major modification of N‐glycans, is implicated in immune regulation, such as the attenuation of the antibody‐dependent cell‐mediated cytotoxicity of antibody drugs and the inhibition of anti‐tumor responses via the promotion of PD‐1 expression on T cells. Although the core fucose regulates many biological processes, no core fucose recognition molecule has been identified in mammals. Herein, we report that Dectin‐1, a known anti‐β‐glucan lectin, recognizes the core fucose on IgG antibodies. A combination of biophysical experiments further suggested that Dectin‐1 recognizes aromatic amino acids adjacent to the N‐terminal asparagine at the glycosylation site as well as the core fucose. Thus, Dectin‐1 appears to be the first lectin‐like molecule involved in the heterovalent and specific recognition of characteristic N‐glycans on antibodies.

The core fucose,amajor modification of N-glycans, is implicated in immune regulation, such as the attenuation of the antibody-dependent cell-mediated cytotoxicity of antibody drugs and the inhibition of anti-tumor responses via the promotion of PD-1 expression on Tc ells.A lthough the core fucose regulates many biological processes,n oc ore fucose recognition molecule has been identified in mammals.Herein, we report that Dectin-1, ak nowna nti-b-glucan lectin, recognizes the core fucose on IgG antibodies.Acombination of biophysical experiments further suggested that Dectin-1 recognizes aromatic amino acids adjacent to the N-terminal asparagine at the glycosylation site as well as the core fucose. Thus,D ectin-1 appears to be the first lectin-like molecule involved in the heterovalent and specific recognition of characteristic N-glycans on antibodies.