β-Glucan is a homopolymer with a backbone of β-1,3-linked glucose residues. The solubility and biological activity of β-glucan can be influenced by the length of the backbone and the length/interval of the β-1,6 branches. Dectin-1 is crucial in innate immunity through its binding to exogenous β-glucans. However, there are few quantitative binding affinities available and there is no comprehensive comparative analysis of the binding of Dectin-1 to insoluble β-glucans. Here, we have developed a simple binding assay for the interaction between Dectin-1 lectin domain (Dectin-1 CTLD) and insoluble β-glucans. We utilized the paramylon particle as a model of insoluble β-glucans. Dectin-1 CTLD bound to paramylon (particle size 3.1 μm) was separated from unbound Dectin-1 CTLD by centrifugation using a membrane filter (pore size 0.2 μm). The protein in the filtrate was quantified by SDS-PAGE and densitometry. The amount decreased in proportion to the amount of paramylon in the mixture. A control experiment using the Dectin-1 CTLD inactive mutant W221A showed that the mutant passes through the filter without binding paramylon. These results are evidence of site-specific binding of Dectin-1 CTLD to paramylon and demonstrate that the separation of paramylon-bound/unbound Dectin-1 CTLD is achievable through centrifugation using a filter. The assay was extended to other insoluble β-glucans including curdlan. Additionally, it can be utilized in competitive inhibition experiments with soluble short-chain β-glucans such as laminarin. The assay system allows for quantitative comparison of the affinities between insoluble and soluble β-glucans and Dectin-1 CTLD, and should be useful because of its low-tech convenience.
Keywords: Binding assay; Dectin-1; Insoluble β-glucan; Paramylon.
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