Comparison of Binding Platforms Yields Insights into Receptor Binding Differences between Shiga Toxins 1 and 2
Comparison of Binding Platforms Yields Insights into Receptor Binding Differences between Shiga Toxins 1 and 2
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DOI：
10.1021/bi902084y
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发表时间：
2010-03-02
期刊：
影响因子：
2.9
通讯作者：
Weiss, Alison A.
中科院分区：
文献类型：
--
作者：
Flagler, Michael J.;Mahajan, Sujit S.;Weiss, Alison A.
蛋白质-聚糖相互作用通常非常弱，而当蛋白质表达多个聚糖结合位点时，就会实现狂热的结合。志贺毒素（Stx）利用聚糖受体进入细胞。 Stx 具有五个相同的结合亚基，每个亚基具有三个不同的聚糖结合位点。先前的研究检查了与表达Ilk三糖的双触角聚糖的结合模拟固定化油链霉亲和素，导致每个链霉亲和素表面显示四种三糖。 Stx1优选其天然受体三糖神经酰胺(Gb3)的Ilk三糖，而更有效且临床相关的变体Stx2优选末端半乳糖被N-乙酰半乳糖胺(NHAc-Pk)取代的Ilk三糖。在本研究中，使用 ELISA 和表面等离子共振 (SPR) 两个实验平台检查了 Stxs 与 Ilk 类似物的结合。 ELISA 比 SPR 更敏感。 ELISA 的灵敏度是由于链霉亲和素密度高，表明亲和力结合可能需要接合或超过四种三糖。在两个实验平台中都保持了 Pk 类似物的选择性。聚糖偏好被映射到结合位点 2，因为单个氨基酸（Stx1 B 亚基的天冬酰胺 32/Stx2 B 亚基的丝氨酸 31）的相互突变逆转了结合偏好，然而，天然 Stx1 与装载有 50:50 Pk-NHAc-Pk 混合物的板很好地结合，而 Stx2 结合效率较低，这表明 Stx1 结合位点之一可能 只参与 Pk，而另一个则可以耐受 Pk 或 NHAc-Pk。不同体外结合平台上不同的聚糖结构和密度揭示了 Stx1 和 Stx2 之间受体结合特性的重要差异。
Protein-glycan interactions Dire typically very weak, and avid binding is achieved when proteins express multiple glycan binding sites. Shiga toxin (Stx) uses glycan receptors to enter cells. Stx has five identical binding subunits, each with three nonidentical glycan binding sites. Previous studies examined binding to biantennary glycans expressing Ilk trisaccharide mimics immobilized oil streptavidin, resulting in display of four trisaccharides per streptavidin face. Stx1 preferred the Ilk trisaccharide of its native receptor, globotriaosylceramide (Gb3), while the more potent and clinically relevant variant, Stx2, preferred the Ilk trisaccharide with the terminal galactose replaced with N-acetylgalactosamine (NHAc-Pk). In the present study, binding of Stxs to Ilk analogues was examined Using two experimental platforms, ELISA and surface plasmon resonance (SPR). ELISA was more sensitive than SPR. Sensitivity in the ELISA Was due to high streptavidin density, suggesting that avid binding may require engagement or more than four trisaccharides. Selectivity for the Pk analogues was maintained in both experimental platforms. Glycan preference was mapped to binding site 2, since reciprocal mutation of a single amino acid (asparagine 32 of Stx1 B-subunit/serine 31 of Stx2 B-subunit) reversed binding preference, However, native Stx1 bound well to plates loaded with a 50:50 mixture of Pk-NHAc-Pk, while Stx2 bound less efficiently, suggesting that one of the Stx1 binding sites may only engage Pk, while another may tolerate either Pk or NHAc-Pk. Varying glycan structure and density across different in vitro binding platforms revealed important differences in receptor binding properties between Stx1 and Stx2.