Molecular toolkit for high content resolution of glycomes by expansion microscopy

项目摘要/摘要 碳水化合物链，又称糖链，是广泛的生物过程的基础，包括 抗凝、细胞生长和发育、细胞间通讯、免疫识别/反应以及 微生物致病机制。多糖在人类疾病中也占有重要地位，包括大多数人类疾病。 癌症。细胞表面多糖的空间排列现在被认为是生命中最关键的 细胞和多细胞行为，包括生长、细胞命运转变和组织组装。因此， 该提案的基本假设是，更好地理解多糖在两种健康中所起的作用 疾病可以通过一套全面的分子工具来实现，以成像高糖基 空间分辨率。近年来，出现了功能强大的光学成像新方法 在纳米尺度上。一种革命性的方法，称为扩张显微镜(EXM)，实现了超 通过在聚电解质基质中膨胀样品来进行详细的结构成像，从而可以 在传统的荧光显微镜上分辨。不幸的是，这些方法还没有被利用。 由于缺乏相容的成像试剂而导致糖生物学的进展 具有生物重要性的糖链表位(糖基)，为先进的超分辨率而定制 镜检方法包括Exm。为了应对这些挑战，这项提议寻求创建一种 一种称为糖共轭exm(GlycoExM)的技术，它利用多功能化学探针和 葡萄糖表位特异性亲和试剂，用于细胞多糖的简便检测和纳米分辨率 糖偶联物。(目标1)基于多功能的一套多功能化学探针 将开发用于代谢标记的非靶向GlycoExM成像的寡硫代乙酰胺(LOOTEA) 广泛使用的共聚焦显微镜上的葡聚糖，有效分辨率高达15 nm。(目标2)一种方法 将定制亲和试剂从非免疫库中分离出来将适用于多种选择 糖表位特异性抗体(糖体)。(目标3)糖体将针对目标GlycoExM进行优化 通过蛋白质工程和寡聚TEA偶联，导致试剂将提高分辨率 细胞和组织的Exm。将为表现最好的试剂制定详细的方案，并将包括 关于糖体的可再生生物合成、最佳试剂浓度和有效成像的信息 条件。除了开发该试剂的《操作手册》外，该项目还将使用寡聚乙酸乙酯 和糖体，以探讨不同正常人表面上真实的多糖签名的空间表达 和癌细胞系，例如，提供(1)免疫调节糖链的超分辨图谱 (2)推测的半乳糖凝集素晶格的第一个详细的分子结构。整体而言，建议的 GlycoExM平台有可能在糖糖范围内实现高内容成像，而低成本 用户友好性将确保它变得更广泛地为糖科学界和其他社区所接受。

Project Summary/Abstract Carbohydrate chains, known as glycans, are fundamental to a wide spectrum of biological processes, including anticoagulation, cell growth and development, cell-cell communication, immune recognition/response, and microbial pathogenesis. Glycans also feature prominently in human disease, including the majority of human cancers. The spatial arrangements of glycans on the cell surface are now recognized to inform life’s most critical cellular and multicellular behaviors, including growth, cell fate transitions, and tissue assembly. Thus, the underlying hypothesis of the proposal is that a better understanding of the role that glycans play in both health and disease could be achieved with a comprehensive set of molecular tools for imaging the glycome with high spatial resolution. Recent years have witnessed the emergence of powerful new approaches for optical imaging at nanometer lengths scales. One revolutionary approach, called expansion microscopy (ExM), achieves ultra- detailed structural imaging by swelling samples in a polyelectrolyte matrix so that nanoscale features can be resolved on conventional fluorescence microscopes. Unfortunately, these approaches have yet to be harnessed for advancement of glycobiology due to a lack of compatible imaging reagents that specifically recognize biologically important glycan epitopes (glycotopes) and that are customized for advanced super-resolution microscopy methods including ExM. To address these challenges, this proposal seeks to create an imaging technology called glycoconjugate ExM (GlycoExM) that leverages multifunctional chemical probes and glycotope-specific affinity reagents for facile detection and nanometer resolution of cellular glycans and glycoconjugates. (Aim 1) A suite of multifunctional chemical probes based on multifunctional oligothioetheramides (oligoTEAs) will be developed for untargeted GlycoExM imaging of metabolically labeled glycans on widely available confocal microscopes with up to 15 nm effective resolution. (Aim 2) A method for isolating custom affinity reagents from non-immune libraries will be adapted for multiplexable selections of glycotope-specific antibodies (glycobodies). (Aim 3) Glycobodies will be optimized for targeted GlycoExM through protein engineering and oligoTEA conjugation, leading to reagents that will enhance the resolution of ExM for cells and tissues. Detailed protocols will be developed for the top performing reagents and will include information on renewable biosynthesis of glycobodies, optimal reagent concentrations, and validated imaging conditions. In addition to the development of this reagent “operator’s manual,” the project will also use oligoTEAs and glycobodies to probe the spatial expression of authentic glycan signatures on the surfaces of different normal and cancer cell lines to, for example, provide (1) super-resolved maps of immunomodulatory glycans in the glycocalyx and (2) the first detailed molecular structure of the putative galectin lattice. Overall, the proposed GlycoExM platform has the potential to enable high-content imaging on a glycome-wide scale, while the low-cost and user-friendliness will ensure that it becomes broadly accessible to the glycoscience community and beyond.