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
复制标题
DOI：
10.1093/glycob/cwy039
复制
发表时间：
2018-08-01
期刊：
影响因子：
4.3
通讯作者：
Yamaguchi, Yoshiki
中科院分区：
文献类型：
--
作者：
Dulal, Hari P.;Adachi, Yoshiyuki;Yamaguchi, Yoshiki
Dectin-1是一种c型凝集素样模式识别受体，可识别存在于非自身病原体上的β(1-3)-葡聚糖。Dectin-1感知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具有重要意义。在这项研究中，我们利用其c型凝集素样结构域（CTLD）表征了小鼠Dectin-1的配体结合和配体诱导的寡聚化。CTLD与β -葡聚糖配体laminarin的相互作用诱导了CTLD的四聚体，通过尺寸排斥色谱和多角度光散射证实了这一点。成分分析表明四个CTLD分子与四个层状蛋白分子结合的化学计量。未检测到CTLD的二聚体和三聚体，提示其存在协同寡聚现象。为了绘制CTLD中参与β -葡聚糖结合和结构域寡聚化的氨基酸残基，我们对表面暴露的和最保守的氨基酸残基进行了定点诱变。在所检测的突变体中，W221A、H223A和Y228A消除了低聚物的形成。由于这些残基在空间上排列形成疏水槽，因此W221、H223和Y228可能直接参与了β -葡聚糖的结合。有趣的是，疏水沟槽另一侧的残基（包括Y141、R145和E243）的突变也表现出低聚物形成的减少，这表明参与了层粘连蛋白引导下的蛋白质-蛋白质相互作用。利用固有色氨酸荧光进行配体滴定，发现野生型CTLD与层粘连蛋白的希尔系数接近于3，而推测结合位点内外的低聚物还原突变会破坏或降低层粘连蛋白的协同性。我们认为配体诱导的Dectin-1的协同寡聚物形成在生理上与感知外源性β -葡聚糖和触发细胞内信号传导有关。
Dectin-1 is a C-type lectin-like pattern recognition receptor that recognizes beta(1-3)-glucans present on non-self pathogens. It is of great importance in innate immunity to understand the mechanism whereby Dectin-1 senses beta(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 beta-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 beta-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 beta-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 similar to 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 beta-glucan and triggering intracellular signaling.