Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance
Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance
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DOI：
10.1074/jbc.274.32.22604
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发表时间：
1999-08-06
影响因子：
4.8
通讯作者：
Buckley, JT
中科院分区：
文献类型：
--
作者：
MacKenzie, CR;Hirama, T;Buckley, JT
Aerolysin是一种与糖基磷脂酰肌醇（GPI）结合在宿主细胞表面结构上的糖基磷脂酰肌醇（GPI）锚固。使用表面等离子体共振研究了受体的性质和受体结合位点在毒素分子上的位置。与GPI锚定的蛋白THY-1，变体表面糖蛋白和接触蛋白结合的生气素与速率常数和亲和力相似。从THY-1中酶促去除N连接糖不会影响毒素的结合，表明这些糖与高亲和力与Aerololysin与Aerolylysin的高亲和力相互作用无关，而Aerolylysin是一种胆红素蛋白，并且两个裂片都表明是最佳结合所需的。大叶本身与THY-1的结合至少比整体毒素弱10倍，而小叶结合GPI锚定蛋白的蛋白质至少比完整的毒素更弱1000倍分析提供了进一步的证据，表明两个裂片都参与GPI锚固结合，在任何一个结构域中，某些单个氨基酸取代都会导致亲和力降低多达100倍。蛋白质两个叶片中具有单氨基酸取代的变体完全无法结合受体。膜蛋白糖蛋白已被大量糖基化但不是GPI锚定的，与固定的蛋白酶蛋白薄弱结合，表明除了GPI锚以外的细胞表面碳水化合物结构相互作用可能部分介导毒素与宿主细胞结合。
Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin, Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin, Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells.