Reengineering of Glycan Binding Specificity for Targeted Cellular Delivery.

生物分子主要有四大类：核酸（DNA和RNA）、蛋白质、碳水化合物（糖）和脂质（脂肪）。这些生物分子的再工程是合成生物学领域的焦点;由此蛋白质和核酸从头开始设计，或基于天然蓝图重新设计，以执行自然界中看不到的功能。蛋白质是生物过程的主要工具，在过去的20年里，人们在重新设计蛋白质以执行新任务方面取得了许多进展。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;与核酸一起打开或关闭基因表达;与糖一起进行细胞之间的通信;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，相应地很少有合成生物学工作集中在蛋白质-糖相互作用的设计上，尽管糖是生物系统的主要组成部分。糖通常被称为食品，如蔗糖（蔗糖），或用于维持结构，如树木中的纤维素。然而，糖也大量存在于我们所有细胞的外部，形成一种称为“糖萼”（糖壳）的结构。形成这种外壳的糖分子通过蛋白质或脂质附着在细胞上。与糖萼的相互作用允许细胞粘附和通信，控制细胞行为和防御，并形成抵抗微生物感染的物理屏障。构成糖萼的糖的类型有许多变化，不同的细胞类型具有不同的糖组成。事实上，细胞的糖组成可以在复制阶段和细胞患病时（如癌症）发生变化。我们称这种糖成分为“糖密码”，在这项工作中，我将使用这种糖密码来区分细胞类型，包括健康和患病的细胞。通常病原体，微生物毒素和病毒可以利用糖萼中的这些糖来欺骗细胞吸收它们，包括一组称为AB 5毒素家族的毒素，其中包括来自霍乱弧菌的霍乱毒素。这种毒素附着在一种特殊的糖上，这种糖主要存在于肠细胞的糖萼中。结果，毒素被吸收，细胞行为改变，导致它将水排入肠道，导致常见的霍乱中毒症状，腹泻。这些AB 5毒素由两种组分形成：毒性A亚基和无毒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。这种天然机制可以通过重新设计CTB与不同的糖分子结合来劫持;因此，将这种CTB分子重新设计为细胞选择性吸收工具将允许将附着的货物运输到细胞内部。在本研究中，我将使用新的计算方法结合实验选择和定向进化来重新设计这些B5亚基以结合不同的糖，它只存在于一些癌细胞的表面，包括黑色素瘤（皮肤癌）和神经母细胞瘤（神经组织的儿童癌症）。通过重组CTB以结合这些糖，它可以用于将诊断和治疗分子运送到特定的癌细胞中。这种重新设计过程的演示将允许未来的其他蛋白质-糖相互作用的工程。例如针对一系列糖脂重新设计其他B5亚基，以产生可以特异性靶向一系列细胞的分子选择;这样的合成生物学工具包将在诊断和药物递送中具有重要意义。

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.