Structural biological studies on the sugar-binding mechanism of two sugar-binding domains having different physiological functions

为了阐明R型凝集素具有多种功能的原因，我们研究了R型凝集素家族的两个糖结合结构域的糖结合机制，一个是橄榄绿丝核菌木聚糖酶的木聚糖结合结构域（XBD），另一个是一种新的具有血凝活性的29-kDa凝集素（EW 29 Ch）的C-末端结构域。通过NMR滴定法，我们测定了两个糖结合结构域对以下糖的结合活性：XBD的乳糖、半乳糖、木糖、木二糖、木二糖、木四糖和木六糖;乳糖、蜜二糖、半乳糖、甲基-α-吡喃半乳糖苷和甲基-β-吡喃半乳糖苷。结果表明，XBD在α、β和γ 3个亚结构域中有3个结合位点，且3个结合位点对不同种类的糖具有不同的结合特异性，且3个结合位点对木四糖和木六糖均具有较高的结合活性。因此， ...更多信息 XBD可以结合不同的木聚糖链，同时在不溶性木聚糖的存在下有效的底物结合。NMR滴定实验表明，EW 29 Ch在α和γ两个亚结构域上有两个结合位点，且α结合位点的糖结合活性远高于γ结合位点。此外，α结合位点在糖中具有β-外消旋体偏好。这些结果表明，EW 29 Ch的两个糖结合位点中的每一个具有不同的糖结合模式。对乳糖与EW 29 Ch混合物的STD-NMR实验表明，乳糖的半乳糖残基主要与EW 29 Ch相互作用。EW 29 Ch与糖结合后的构象变化因糖的种类而异，这可能与其血凝活性有关。最后，我们认为R型凝集素的糖结合活性和糖结合位点的特异性与其生理功能密切相关。少

We investigated the sugar-binding mechanism of the two sugar-binding domains belonging to the R-type lectin family, one is xylan binding domain (XBD) in Stnaptamyees olivaasoviridis xylanase and another is the C-terminal domain of a novel 29-kDa lectin from earthworm (EW29Ch) having hemagglutinating activity, in order to clarify why the R-type lectins can have various functions. By the NMR titration method, we determined the binding activities of the two sugar-binding domains for the following sugars: lactose, galactose, xylose, xylobiose, xylobiose, xylotetraose, and xylohexaose for XBD; lactose, melibiose, galactose, methyl-α-galactopyranoside, and metbyl-β-galactopyranoskle. The results showed that XBD had three binding sites in the three subdomains α, β, and γ, and that each of the three binding sites had different sugarbinding specificities for kind of sugars and all of the binding sites had high binding activities for xylotetraose and xylohexaose. Thus, the three binding sites of … More XBD may bind different xylan chains at the same time as effective substrate binding in the presence of an insoluble xylan. The NMR titration experiments of EW29Ch with sugars showed that EW29Ch had two binding sites in the two subdomains, α and γ, and the sugar-binding activities of the α binding site are much higher than those of γ binding site. Further, the α binding site had a β-ranomer preference among the sugars. These results indicate that each of the two sugar-binding sites of EW29Ch has a distinct sugar-binding mode. STD-NMR experiments for the mixture of lactose with EW29Ch demonstrated that the galactose residue of the lactose mainly interacts with EW29Ch. Furthermore, the conformational changes of EW29Ch by binding with sugars differed among kind of sugars, which may relate with its hemagglutinating activity. Finally, we suggest that the sugar-binding activities and specificities of the sugar-binding sites in R-type lectins are closely related with their physiological functions. Less