Reengineering of Glycan Binding Specificity for Targeted Cellular Delivery.

生物分子有四大类：核酸(DNA和RNA)、蛋白质、碳水化合物(糖)和脂类(脂肪)。这些生物分子的再工程是合成生物学领域的焦点；借此蛋白质和核酸被从头开始设计，或根据自然蓝图重新设计，以执行自然界中未曾见过的功能。蛋白质是生物过程的主要主力，在过去的20年里，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；相互作用以对环境变化作出反应；与核酸一起开启或关闭基因表达；与糖一起在细胞间交流；或与脂肪一起形成跨越生物膜的转运体。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，但相应地，很少有合成生物学工作专注于蛋白质-糖相互作用的设计。糖通常被认为是一种食物，如蔗糖(食用糖)，或用于维持结构，如树木中的纤维素。然而，糖类也大量存在于我们所有细胞的外部，形成了一种被称为“糖皮”的结构。形成这种外壳的糖分子通过蛋白质或脂类附着在细胞上。与糖萼的相互作用允许细胞黏附和交流，控制细胞行为和防御，并形成防止微生物感染的物理屏障。组成这个糖萼的糖类有许多变化，不同的细胞类型有不同的糖组成。事实上，细胞的糖组成在复制阶段和细胞患病时都会发生变化，比如癌症。我们将这种糖的组成称为‘糖码’，在这项工作中，我将使用这种糖码来区分细胞类型，包括健康和疾病。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，包括一组被称为AB5毒素家族的毒素，其中包括霍乱弧菌的霍乱毒素。这种毒素附着在一种特定的糖上，这种糖主要存在于肠道细胞的糖萼中。结果，毒素被吸收，细胞行为改变，导致它将水分排入肠道，导致常见的霍乱中毒症状-腹泻。这些AB5毒素由两种成分组成，有毒的A亚单位和无毒的B5亚单位。霍乱毒素B5亚单位(CTB)虽然无毒，但它很重要，因为它与细胞外部的糖结合，并引发吸收。这种自然的机制可以通过重新设计CTB来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附着的货物被运送到细胞内部。在这个团契中，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亚基以结合到不同的糖上，这种糖只在一些癌细胞的表面发现，包括黑色素瘤(皮肤癌)和神经母细胞瘤(一种儿童神经组织癌)。通过重组CTB与这些糖结合，它可以用来将诊断和治疗分子输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对其他蛋白质-糖相互作用进行工程设计。例如针对一系列糖脂对其他B5亚基进行重组，以产生一系列特定靶向一系列细胞的分子；这样的合成生物学工具包在诊断和药物输送方面将非常重要。

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.