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.

中文翻译：

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β-葡聚糖诱导的Dectin-1 C型凝集素样结构域的协同寡聚

Dectin-1是一种C型凝集素样模式识别受体，可识别非自身病原体上存在的β（1-3）-葡聚糖。理解Dectin-1感应β（1-3）-葡聚糖并诱导细胞内信号传导的机制在先天免疫中非常重要。在这项研究中，我们表征了使用其C型凝集素样结构域（CTLD）的鼠Dectin-1的配体结合和配体诱导的寡聚。如尺寸排阻色谱法和多角度光散射所证明的，CTLD与层粘连蛋白（β-葡聚糖配体）的相互作用诱导了CTLD的四聚体。成分分析表明与四个层板蛋白分子结合的四个CTLD分子的化学计量。未检测到CTLD的二聚体和三聚体，表明存在协同寡聚。为了绘制参与β-葡聚糖结合和结构域寡聚化的CTLD的氨基酸残基，我们对暴露于表面和最保守的氨基酸残基进行了定点诱变。在检查的突变体中，W221A，H223A和Y228A消除了寡聚物的形成。由于这些残基在空间上排列以形成疏水性凹槽，因此W221，H223和Y228可能直接参与β-葡聚糖的结合。有趣的是，疏水槽另一侧的残基（包括Y141，R145和E243）的突变也表现出低聚物形成的减少，表明参与了层粘连蛋白引导的蛋白质间相互作用。使用固有色氨酸荧光进行配体滴定表明，野生型CTLD以约3的Hill系数协同结合层粘连蛋白，而减少低聚物的突变，推定的结合位点内部和外部消除或降低了协同作用。我们建议，Dectin-1的配体诱导的协同寡聚体形成在感知外源β-葡聚糖和触发细胞内信号传导方面具有生理学意义。