Guo Yuan, Bruce Turnbull W, Zhou Dejian
School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, United Kingdom.
School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, West Yorkshire, United Kingdom.
Methods Enzymol. 2018;598:71-100. doi: 10.1016/bs.mie.2017.06.012. Epub 2017 Jul 18.
Cell surface carbohydrate-binding proteins (also known as lectins) recognize surface carbohydrates of viral, bacterial, and fungal pathogens to regulate host immune responses; however, such interactions are also often exploited by pathogens to enhance infection. Multivalent binding is typically involved to compensate for the intrinsically weak nature of protein-carbohydrate interactions. The spatial orientation of carbohydrate recognition domains (CRDs) in multimeric lectins plays a central role in governing their binding affinity and specificity. Such information is also key for designing potent, multivalent inhibitors, but is difficult to obtain in the absence of valid lectin crystal structures. In this chapter, we describe a method to allow controlled polyvalent display of carbohydrates on the surface of fluorescent quantum dots (QDs), which enables the use of Förster resonance energy transfer (FRET) efficiency between the QD and dye-labeled lectins to probe how well the displayed sugars match the presentation of the CRDs. A highly efficient ligand-exchange method has been developed to construct compact, polyvalent QD-mannose conjugates (QD-Man), which are essential for sensitive FRET readout and effective valency tuning. A rapid, sensitive, and ratiomeric FRET readout has been developed to quantify their binding affinity and specificity, which, in combination with the geometry of QD-Man, allow us to derive the spatial orientation of the CRDs. A good correlation between the specific binding of QD-Man to recombinant lectins and its effective inhibition of virus cell entry mediated by native cell surface lectins validates QD-FRET as a reliable technique to study multivalent receptor-ligand recognition mechanisms.
细胞表面碳水化合物结合蛋白（也称为凝集素）识别病毒、细菌和真菌病原体的表面碳水化合物，以调节宿主免疫反应；然而，病原体也常常利用这种相互作用来增强感染。通常涉及多价结合以弥补蛋白质 - 碳水化合物相互作用固有的弱性。多聚凝集素中碳水化合物识别结构域（CRD）的空间取向在决定其结合亲和力和特异性方面起着核心作用。此类信息对于设计有效的多价抑制剂也至关重要，但在缺乏有效的凝集素晶体结构的情况下很难获得。在本章中，我们描述了一种在荧光量子点（QD）表面实现碳水化合物可控多价展示的方法，该方法能够利用QD与染料标记的凝集素之间的Förster共振能量转移（FRET）效率来探测展示的糖与CRD的呈现匹配程度。已开发出一种高效的配体交换方法来构建紧密的多价QD - 甘露糖缀合物（QD - Man），这对于灵敏的FRET读出和有效的价态调节至关重要。已开发出一种快速、灵敏且具有比率性的FRET读出方法来量化它们的结合亲和力和特异性，结合QD - Man的几何结构，使我们能够推导CRD的空间取向。QD - Man与重组凝集素的特异性结合及其对天然细胞表面凝集素介导的病毒细胞进入的有效抑制之间的良好相关性，验证了QD - FRET作为研究多价受体 - 配体识别机制的可靠技术。
