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

为了阐明R型凝集素具有不同功能的原因，我们研究了R型凝集素家族的两个糖结合域的糖结合机制，一个是StnapTamyees olivaasoviridis木聚糖酶中的木聚糖结合域(XBD)，另一个是具有血凝活性的新的29 kDa蚯蚓凝集素(EW29CH)的C末端结构域。通过核磁共振滴定的方法，我们测定了乳糖、半乳糖、木糖、木二糖、木四糖和木六糖；乳糖、蜜二糖、半乳糖、甲基-α-半乳糖苷和甲基-β-半乳吡喃糖苷的结合活性。结果表明，XBD在α，β、γ3个亚区均有3个结合位点，每个结合位点对多种糖类具有不同的糖结合特性，且对木糖四糖和木六糖均有较高的结合活性。因此，…的三个结合位点在不溶于水的木聚糖存在的情况下，更多的XBD可能与不同的木聚糖链同时结合，同时与有效的底物结合。EW29CH与糖的核磁共振滴定实验表明，EW29CH在α和γ两个亚区上有两个结合部位，且α结合部位的糖结合活性远高于γ结合部位。此外，α结合位点在糖中具有β-单体偏好。这些结果表明，EW29CH的两个糖结合位点都有不同的糖结合模式。乳糖与EW29CH混合物的STD-核磁共振实验表明，乳糖的半乳糖残基主要与EW29CH相互作用。此外，EW29CH与糖结合的构象变化因糖的种类不同而不同，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部位的特异性与其生理功能密切相关。较少

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