Reengineering of Glycan Binding Specificity for Targeted Cellular Delivery.

重新设计用于靶向细胞递送的聚糖结合特异性。

• 批准号: EP/W022842/1
• 负责人: James Ross
• 金额: $ 55.66万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FW022842%2F1
• 关键词: Reengineering; Glycan; Binding; Specificity; Targeted

There are four major groups of biological molecules, nucleic acids (DNA and RNA), proteins, carbohydrates (sugars) and lipids (fats). The reengineering of these biological molecules is the focus of the field of synthetic biology; whereby proteins and nucleic acids are designed from scratch, or redesigned based on a natural blueprint, to perform functions not seen in nature. Proteins are the major workhorses of biological processes and over the last 20 years many advances have been made to reengineer proteins to perform new tasks. Proteins are responsible for many biological functions, for example: individually as enzymes; with each other to respond to environmental change; with nucleic acids to turn gene expression on or off; with sugars for communication between cells; or with fats to form transporters across biological membranes. Of these protein-based interactions, correspondingly little synthetic biology work has focused on the design of protein-sugar interactions, despite sugars being a major component of biological systems. Sugars are most commonly known as a foodstuff, such as sucrose (table sugar), or for maintaining structure, such as cellulose in trees. However, sugars are also present in great abundance on the outside of all our cells, forming a structure called the 'glycocalyx' (sugar-husk). The sugar molecules which form this husk are attached to the cell via proteins or lipids. Interaction with the glycocalyx allows for cellular adhesion and communication, controls cellular behaviour and defence, and forms a physical barrier against infection by microbes. There are many variations in the types of sugars which make up this glycocalyx and different cell types have a different sugary composition. In fact, the sugar composition of a cell can change during the phases of replication and when cells are diseased, such as in cancer. We call this sugar composition the 'glyco-code', and in this work, I will use this glyco-code to differentiate between cell types, including healthy from diseased.Often pathogens, microbial toxins and viruses, can use these sugars in the glycocalyx to trick the cell into absorbing them, including a group of toxins known as the AB5 toxin family, which includes cholera toxin from Vibrio cholera. This toxin attaches to a specific sugar which is found predominately in the glycocalyx of cells in the intestine. As a result, the toxin is absorbed, and the cell behaviour is changed, causing it to expel water into the intestine leading to the familiar cholera poisoning symptom, diarrhoea. These AB5 toxins are formed from two components the toxic A-subunit and the non-toxic B5-subunit. The cholera toxin B5-subunit (CTB), although non-toxic, is important as it binds to the sugars on the outside of the cell, and triggers absorption. This natural mechanism can be hijacked by redesigning CTB to bind to different sugar molecules; hence, reengineering this CTB molecule as a cell selective absorption tool would allow an attached cargo to be transported into the cell interior.In this fellowship I will use novel computational approaches combined with experimental selection and directed evolution to reengineer these B5-subunits to bind to a different sugar, which is only found on the surface of some cancer cells, including melanoma (skin cancer) and neuroblastoma (a childhood cancer of the nervous tissue). By reengineering CTB to bind to these sugars, it can be used to transport diagnostic and therapeutic molecules into specific cancer cells. The demonstration of this redesign process will allow future engineering of other protein-sugar interactions. Such as reengineering other B5-subunits against a range of glycolipids, to generate a selection of molecules which can specifically target a range of cells; such a synthetic biology toolkit will be of great importance in diagnostics and drug delivery.

有四个主要的生物分子，核酸（DNA和RNA），蛋白质，碳水化合物（糖）和脂质（脂肪）。这些生物分子的重新设计是合成生物学领域的重点。从头开始设计蛋白质和核酸，或根据自然蓝图进行重新设计，以执行自然界未见的功能。蛋白质是生物学过程的主要主持人，在过去的20年中，已经取得了许多进步来重新设计蛋白来执行新任务。蛋白质负责许多生物学功能，例如：单独作为酶；彼此响应环境变化；用核酸打开或关闭基因表达；用糖在细胞之间进行通信；或与脂肪一起形成跨生物膜的转运蛋白。在这些基于蛋白质的相互作用中，尽管糖是生物系统的主要组成部分，但几乎没有合成生物学的工作集中在蛋白质糖相互作用的设计上。糖最常被称为食品，例如蔗糖（餐糖）或维持结构，例如树木中的纤维素。但是，糖在我们所有细胞的外部也充满了大量，形成了一种称为“糖椰子”（Sugar-Husk）的结构。形成这种果壳的糖分子通过蛋白质或脂质附着在细胞上。与糖脂的相互作用允许细胞粘附和通信，控制细胞行为和防御，并形成微生物感染的物理屏障。构成该糖蛋白的糖类型的类型有很多变化，并且不同的细胞类型具有不同的含糖成分。实际上，在复制的阶段和细胞患病的阶段（例如在癌症中），细胞的糖组成可能会发生变化。 We call this sugar composition the 'glyco-code', and in this work, I will use this glyco-code to differentiate between cell types, including healthy from diseased.Often pathogens, microbial toxins and viruses, can use these sugars in the glycocalyx to trick the cell into absorbing them, including a group of toxins known as the AB5 toxin family, which includes cholera toxin from Vibrio cholera.这种毒素附着在特定的糖上，该糖主要在肠中细胞的糖囊中发现。结果，毒素被吸收并改变了细胞行为，导致将水排入肠道中，导致熟悉的霍乱中毒症状腹泻。这些AB5毒素由两个成分形成，有毒A-亚基和无毒的B5亚基。霍乱毒素B5亚基（CTB）虽然无毒，但很重要，因为它与细胞外部的糖结合并触发吸收。可以通过重新设计CTB与不同的糖分子结合来劫持这种自然机制。 hence, reengineering this CTB molecule as a cell selective absorption tool would allow an attached cargo to be transported into the cell interior.In this fellowship I will use novel computational approaches combined with experimental selection and directed evolution to reengineer these B5-subunits to bind to a different sugar, which is only found on the surface of some cancer cells, including melanoma (skin cancer) and neuroblastoma (a childhood cancer of the nervous 组织）。通过重新设计CTB与这些糖结合，可以将其用于将诊断和治疗分子转运到特定的癌细胞中。这种重新设计过程的演示将允许将来的其他蛋白质糖相互作用的工程。例如，将其他B5亚基重新设计针对一系列糖脂，以产生一系列可以特异性靶向一系列细胞的分子；这种合成生物学工具包将在诊断和药物输送中非常重要。

项目成果

The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity

• DOI: 10.3390/toxins16030133
• 发表时间: 2024-03-01
• 期刊: TOXINS
• 影响因子: 4.2
• 作者: Au，Cheuk W.;Manfield，Iain;Ross，James F.
• 通讯作者: Ross，James F.