Chemical Cell Surface Engineering

描述(申请人提供)：多聚糖修饰真核细胞表面和分泌的蛋白质，在那里它们可能介导涉及附着病毒和细菌感染、免疫细胞激活、癌症转移和器官发育的各种分子识别事件。细胞表面多糖的许多生物学功能只能在活细胞的背景下进行研究。除了多糖的一级结构外，它的蛋白质支架的结构以及它在细胞表面的密度和分布都有助于生物活性。不幸的是，细胞表面的异质性，特别是关于糖共轭结构的异质性，阻碍了对糖功能的分子水平的研究。因此，受控地在活细胞上呈现结构定义的糖偶联物是加速糖生物学研究的重要能力。该项目的主要目标是开发用于工程细胞表面多糖结构的化学方法。在之前的授予期间，我们开发了一种使用二聚化策略的化学诱导剂来控制高尔基体中多糖生物合成酶活性的方法。我们设计了高尔基磺基转移酶和糖基转移酶，以供化学二聚体控制，并证明了细胞表面选择素配体和血型抗原的表达可以相应地调节。我们还开发了一种用于动物模型的化学二聚体。在接下来的授权期内，我们将重点关注三个具体目标。首先，我们将开发一种利用人工合成的、结构定义的多糖对细胞表面蛋白质进行定点化学修饰的方法。该方法使用甲甘氨酸生成酶(FGE)的6个残基共同序列，该序列驱动从遗传编码的半胱氨酸残基形成甲甘氨酸(FGly)。FGly的醛官能团提供了一个独特的反应部位，合成的氨基氧基多聚糖可以通过肟基连接到该部位。我们将利用“醛标签”，用量身定制的多糖修饰重组细胞表面蛋白。提出了在人工细胞表面粘蛋白的构建和P-选择素配体PSGL-1的研究中的应用。第二个目标涉及将醛标签应用于天然细胞表面粘蛋白MUC1的生物物理研究。第三个也是最终目标集中在第二项技术上，用于设计细胞表面的糖链。我们计划开发模拟粘蛋白糖蛋白的合成聚合物，并研究它们在插入活细胞膜时的行为。我们将研究它们的细胞表面动力学和膜取向。然后，合成的粘蛋白模拟物将被应用于符号识别和信号传递的研究。 与公共健康相关：复合糖装饰细胞表面，它们在正常生物过程中的细胞间相互作用中发挥作用，也在人类疾病中发挥作用。这项研究的目标是开发化学工具来研究复杂糖在细胞表面的功能。这些工具将提高我们对糖如何导致癌症和炎症等疾病的理解。

DESCRIPTION (provided by applicant): Glycans decorate eukaryotic cell surface and secreted proteins, where they are poised to mediate a variety of molecular recognition events involved in attachment viruses and bacterial infection, immune cell activation, cancer metastasis and organ development. Many biological functions of cell-surface glycans can only be studied in the context of living cells. In addition to a glycan's primary structure, the architecture of its protein scaffold, as well as its density and distribution on the cell surface can all contribute to biological activity. Unfortunately, the heterogeneous nature of cell surfaces, particularly with respect to glycoconjugate structures, has frustrated molecular-level studies of glycan function. The controlled presentation of structurally defined glycoconjugates on live cells is therefore an important capability for accelerating research in glycobiology. The broad objective of this project is to develop chemical approaches for engineering cell surface glycan structures. During the previous granting period, we developed a method for controlling the activity of glycan biosynthetic enzymes in the Golgi compartment using the chemical inducer of dimerization strategy. We engineered Golgi sulfotransferases and glycosyltransferases for control by chemical dimerizers and demonstrated that cell surface expression of selectin ligands and blood group antigens can be modulated accordingly. We also developed a chemical dimerizer for applications in animal models. The next granting period we will focus three specific Aims. First, we will develop a method for site- specific chemical modification of cell surface proteins with synthetic, structurally-defined glycans. The method employs the 6-residue consensus sequence for formylglycine generating enzyme (FGE), which drives the formation of formylglycine (FGly) from a genetically encoded cysteine residue. The aldehyde functionality of FGly provides a uniquely reactive site to which synthetic aminooxy glycans can be ligated via oxime formation. We will use the "aldehyde tag" to modify recombinant cell surface proteins with tailored glycans. Applications to the construction of artificial cell surface mucins and to studies of the P-selectin ligand PSGL-1 are proposed. The second Aim involves application of the aldehyde tag to biophysical studies of the natural cell-surface mucin MUC1. The third and final Aim focuses on a second technology for engineering cell surface glycans. We plan to develop synthetic polymers that mimic mucin glycoproteins and to study their behavior when inserted into live cell membranes. We will investigate their cell surface dynamics and membrane orientations. The synthetic mucin mimics will then be applied to studies of siglec recognition and signaling. PUBLIC HEALTH RELEVANCE: Complex sugars decorate the surfaces of cells, where they play a role in cell-cell interactions involved in normal biological processes and also in human disease. The goal of this research is to develop chemical tools for studying the functions of complex sugars on the cell surface. These tools will improve our understanding of how sugars contribute to diseases such as cancer and inflammation.