Elucidating mechanisms underlying multivalency modulating lectin-glycan binding and assembly properties-implications for lectin function regulation

阐明多价调节凝集素-聚糖结合和组装特性的机制-对凝集素功能调节的影响

• 批准号: BB/Y005856/1
• 负责人: Yuan Guo
• 金额: $ 105.92万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FY005856%2F1
• 关键词: Elucidating; mechanisms; underlying; multivalency; modulating

Pathogen surfaces display high density carbohydrates to shield underneath elements from being recognized by antibodies for immune evasion. To recognize the unusually displayed carbohydrate, in immune systems, carbohydrate binding proteins (known as lectins) form multimeric structures where each lectin contains multiple carbohydrate recognition domains (CRDs). This also allows multiple contacts (multivalent binding) between them resulting in strong bindings similar to that observed with Velcro. Viruses and bacteria are much bigger than lectins, hence multiple lectins can bind to them, making the lectins cluster together. As many lectins are attached to immune cells, the cluster can be interpreted as signalling to initiate immune defence. However, some pathogens have developed strategies to exploit such strong binding to facilitate their infection. Currently, it is not clear whether they have changed the arrangement of carbohydrates to induce different lectin cluster patterns, and/or to make some CRDs unavailable for engagement which reduces binding strength and cluster stability, allowing more lectins to pack in to exclude other proteins from the area. These can be interpreted differently by immune cells.Here, we employ a dendritic cell (DC) surface lectin DC-SIGN to address these questions. DC-SIGN contains 4 CRDs and binds to mannose (a type of carbohydrate) on pathogens including bacteria, viruses (such as HIV, SARS-COV-2) and fungi. Binding leads to virus being engulfed, digested inside DC and results in small pieces further being used to instruct other immune cell to produce antibodies for pathogen elimination. Binding also stimulates DC to produce some proteins through DC-SIGN cross-talking to a TLR 4 protein as part of defence actions. However, how communication is achieved is unknown and it is unclear if they have to be closely associated. But viruses such as HIV and SARS-COV-2 exploit binding to enhance their infection. In the case of HIV, it somehow avoids being digested inside DC and escapes later to infect other cells. This makes DC-SIGN an excellent model lectin for this study. We will also include another tetrameric lectin named DC-SIGNR which is almost identical to DC-SIGN with only difference being that their 4 CRDs have different orientations. This makes them an excellent pair to study CRDs availability in multivalent binding strength and cluster formation.We will start with linking multiple pathogen glycans onto fluorescent quantum dot (QD) or rod (QR) surfaces with different densities to mimic possible displays on pathogen surfaces. We will develop a novel method to construct DC-SIGN or DC-SIGNR tetramers containing 4, 3 or 2 CRDs to investigate the effects of CRD engagement numbers on binding strength. To study cluster formation, we will exploit nanoparticles' high density to see their arrangement using electron microscopy to obtain information on lectin clusters: isolated particles mean lectins are assembled on the same particle and clustered particles are formed by proteins and particles cross-linking, these generate distinct cluster patterns. We will also label TLR 4 with a different shaped nanoparticle to see how it associates with DC-SIGN clusters. Proteins are invisible by electron microscope, by following nanoparticles we are able to gain their cluster information at nanometer level for the 1st time. Lectin binding can also be made to interfere with QD and QR's fluorescent property; we will follow the light signal by fluorescence microscopy to monitor the speed of signal change to gain information on cluster stability: the faster the change, the less stable the cluster. We will then use those QD/QR-glycans to stimulate DC to correlate observed cluster information with DC responses to explain how DC-SIGN instructs DC responses.Information obtained here will provide guidance to design treatments against infection and to suppress immune overreaction to treat diabetes, arthritis and allergy.

病原体表面显示出高密度的碳水化合物，以保护下面的元素不被抗体识别，从而逃避免疫。为了识别异常显示的碳水化合物，在免疫系统中，碳水化合物结合蛋白（称为凝集素）形成多聚体结构，其中每个凝集素包含多个碳水化合物识别结构域（CRD）。这也允许它们之间的多个接触（多价结合），导致类似于用Velcro观察到的强结合。病毒和细菌比凝集素大得多，因此多种凝集素可以与它们结合，使凝集素聚集在一起。由于许多凝集素附着在免疫细胞上，因此可以将该簇解释为启动免疫防御的信号。然而，一些病原体已经开发出利用这种强结合来促进其感染的策略。目前，尚不清楚它们是否改变了碳水化合物的排列以诱导不同的凝集素簇模式，和/或使一些CRD无法用于降低结合强度和簇稳定性的接合，从而允许更多的凝集素包装以将其他蛋白质排除在该区域之外。在这里，我们采用树突状细胞（DC）表面凝集素DC-SIGN来解决这些问题。DC-SIGN含有4个CRD，并与病原体（包括细菌，病毒（如HIV，SARS-COV-2）和真菌）上的甘露糖（一种碳水化合物）结合。结合导致病毒被吞噬，在DC内消化，并导致小片段进一步用于指导其他免疫细胞产生抗体以消除病原体。结合还刺激DC通过DC-SIGN与TLR 4蛋白质交叉对话产生一些蛋白质，作为防御作用的一部分。然而，如何实现通信是未知的，也不清楚它们是否必须密切相关。但是像HIV和SARS-COV-2这样的病毒利用结合来增强它们的感染。在HIV的情况下，它以某种方式避免在DC内被消化，并在稍后逃逸以感染其他细胞。这使得DC-SIGN成为这项研究的一个很好的模型凝集素。我们还将包括另一种名为DC-SIGNR的四聚体凝集素，其与DC-SIGN几乎相同，唯一的区别是它们的4个CRD具有不同的取向。这使得它们成为研究CRD在多价结合强度和簇形成方面的可用性的绝佳配对。我们将从将多个病原体聚糖连接到具有不同密度的荧光量子点（QD）或棒（QR）表面来模拟病原体表面上可能的展示开始。我们将开发一种新的方法来构建包含4个、3个或2个CRD的DC-SIGN或DC-SIGNR四聚体，以研究CRD接合数对结合强度的影响。为了研究簇的形成，我们将利用纳米颗粒的高密度，使用电子显微镜观察它们的排列，以获得凝集素簇的信息：孤立的颗粒意味着凝集素组装在同一颗粒上，而聚集的颗粒由蛋白质和颗粒交联形成，这些产生不同的簇模式。我们还将用不同形状的纳米颗粒标记TLR 4，以观察它如何与DC-SIGN簇结合。蛋白质在电子显微镜下是不可见的，通过跟踪纳米粒子，我们首次能够在纳米水平上获得蛋白质的团簇信息。也可以使凝集素结合干扰QD和QR的荧光性质;我们将通过荧光显微镜跟踪光信号以监测信号变化的速度，以获得关于簇稳定性的信息：变化越快，簇越不稳定。然后我们将使用这些QD/QR-聚糖刺激DC，将观察到的簇信息与DC反应相关联，以解释DC-SIGN如何指导DC反应。这里获得的信息将为设计抗感染治疗和抑制免疫过度反应以治疗糖尿病，关节炎和过敏提供指导。