Probing Multivalent DC-SIGN/R-Glycan Interactions Using Polyvalent Multifunctional Glycan-Quantum Dot

所有活细胞和许多病毒都被特定的糖所包裹，使它们能够与携带特定糖结合蛋白（凝集素）的伴侣相互作用。虽然每种凝集素-糖相互作用通常是弱的且生物学上无活性的，但通过用特定糖的阵列涂覆它们的表面，病毒可以与多个细胞表面凝集素相互作用以加强相互作用，使它们能够进入细胞，最终导致感染。尽管有新的抗病毒和疫苗治疗方法，病毒感染引起的疾病仍然很高。例如，2015年，约有3700万和1.5亿人感染艾滋病毒和HCV，每年分别造成约110万和50万的全球死亡。幸运的是，具有特定糖涂层的病毒模拟物可以阻止这种相互作用，从而防止感染。抑制效力关键取决于结合配偶体之间个体相互作用的间距和方向的匹配。因此，了解凝集素的多个糖结合位点（CRD）是如何排列的，对于设计有效的病毒抑制剂至关重要。然而，在研究中的进展受到了阻碍，目前的方法无法揭示关键的结构信息（如结合位点的方向，间距和灵活性）的重要细胞表面凝集素。例如，尽管在全球范围内进行了20年的广泛研究，但两种至关重要的凝集素DC-SIGN和DC-SIGNR的结构仍然未知。它们都含有四个CRD，并与HIV和埃博拉病毒表面的多种糖结合，以增强病毒感染。然而，为什么它们对多种糖和病毒具有不同的结合偏好仍然知之甚少，我们将通过开发被称为量子点（QD）的糖包被的微小荧光颗粒作为病毒模拟物来解决当前方法的能力差距，并研究它们与DC-SIGN/R的相互作用与溶液中的单个凝集素和细胞表面的多个凝集素。我们计划通过充分利用QD的独特性质来实现这一目标：用于结合测量的强荧光;用于可视化结合诱导的颗粒排列以揭示结合位点方向的电子显微镜中的高对比度;用于用多种糖装饰以增强结合强度的固体核心，以及用于调整糖数量和糖间距离以探测凝集素的CRD排列。我们组建了一个在量子点、糖合成、电子显微镜和凝集素生物化学方面拥有丰富专业知识的团队，他们将共同努力应对这一重大挑战，每个成员都为该项目贡献了重要的专业知识。我们将首先制备一系列具有不同糖数量和结构、糖间距离和灵活性的糖衣量子点。然后，我们将通过荧光测量它们与溶液中单个DC-SIGN/R分子的相互作用，以了解分子相互作用的强度和速度，以及每个QD-糖-凝集素伴侣的结合偏好。我们将通过电子显微镜测量与不同凝集素结合后的颗粒排列，并监测每次相互作用后颗粒大小的变化。我们将联合收割机结合这些结果，找出DC-SIGN/R CRD是如何排列和定向的，以及它们的结合位点间隔多远。我们还将研究为什么DC-SIGN/R CRD以这种特殊的方式排列，蛋白质的哪些部分控制这种排列。我们将进一步测试糖衣量子点阻断埃博拉病毒感染靶细胞的能力，并找出单个量子点-糖-DC-SIGN/R结合强度与其病毒阻断效率之间的联系。这项研究是非常及时和重要的，因为它将开发一种新的方法来揭示DC-SIGN/R病毒相互作用的关键结构机制，解决这一重要研究领域目前面临的未满足的技术挑战。这也将有助于揭示配体结合强度和病毒抑制效力之间的联系，从而指导新的抗病毒策略的开发。

All living cells and many viruses are coated with specific sugars, allowing them to interact with partners bearing specific sugar binding proteins (lectins). While each lectin-sugar interaction is often weak and biologically inactive, by coating their surfaces with arrays of specific sugars, viruses can interact with multiple cell surface lectins to strengthen the interaction, allowing them to gain cell entry which ultimately leads to infection. Despite new anti-viral and vaccine treatments, disease caused by virus infection remains high. For example, ~37 and 150 million people are living with HIV and HCV infections in 2015, causing annual global deaths of ~1.1 and 0.5 million, respectively. Fortunately, virus mimics with specific sugar coatings can block such interactions, thereby preventing infection. The inhibition potency depends critically on matching the spacing and orientation of individual interactions between the binding partners. Hence understanding how a lectin's multiple sugar binding sites (CRDs) are arranged is vital to design effective virus inhibitors. However, the advances in research have been hampered by the inability of current methods to reveal key structural information (e.g. binding site orientation, spacing and flexibility) of important cell surface lectins. For example, despite 20 years of extensive research worldwide, the structure of two critically important lectins, DC-SIGN and DC-SIGNR, remain unknown. They both contain four CRDs and bind to multiple sugars on the HIV and Ebola surface to enhance virus infection. However, why they have different binding preferences to multiple sugars and virus remain poorly understood.We will address the capability gap of current methods by developing sugar coated tiny fluorescent particles called quantum dots (QDs) as virus mimics and study their interactions with DC-SIGN/R with single lectins in solution and multiple lectins on cell surface. We plan to achieve this goal by fully exploiting QD's unique properties: strong fluorescence for binding measurement; high contrast in electron microscopy for visualising binding induced particle arrangement to reveal binding site orientation; solid core for decorating with multiple sugars to enhance binding strength, and for adjusting sugar number and inter-sugar distance to probe lectin's CRD arrangement. We have assembled a team with extensive expertise in QD, sugar synthesis, electron microscopy and lectin biochemistry who will work together to address this significant challenge, each member contributing an essential expertise to this project.We will first prepare a series of sugar-coated QDs with varying number and structure of sugars, inter-sugar distance and flexibility. We will then measure their interactions by fluorescence with individual DC-SIGN/R molecules in solution to find out how strong and how fast the molecules interact, what binding preference is for each QD-sugar-lectin partner. We will measure the particle arrangement after binding to different lectins by electron microscopy, and monitor their size changes upon each interaction. We will combine these results to find out how DC-SIGN/R CRDs are arranged and oriented, and how far apart their binding sites are spaced. We will also study why DC-SIGN/R CRDs are arranged in this particular way, which parts of the protein control such arrangement. We will further test the ability of the sugar-coated QDs to block Ebola virus infection of target cells and find out the link between individual QD-sugar-DC-SIGN/R binding strength and its virus blocking efficiency. This study is extremely timely and important because it will develop a novel method to reveal key structural mechanisms of DC-SIGN/R-virus interactions, addressing an unmet technical challenge currently facing this important research area. It will also help to reveal the link between ligand binding strength and virus inhibition potency, and so guide the development new anti-viral strategies.