Molecular toolkit for high content resolution of glycomes by expansion microscopy

项目总结/摘要 碳水化合物链，称为聚糖，是生物过程的基础，包括 抗凝、细胞生长和发育、细胞间通讯、免疫识别/应答，以及 微生物致病聚糖在人类疾病中也具有显著特征，包括大多数人类疾病。 癌的聚糖在细胞表面的空间排列被认为是生命中最重要的组成部分。 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，更好地了解聚糖在健康和免疫中的作用， 和疾病可以通过一套全面的分子工具来实现， 空间分辨率近年来，光学成像的新方法不断涌现 在纳米尺度上。一种革命性的方法，称为扩展显微镜（ExM），实现了超 详细的结构成像，通过膨胀的样品在一个纳米矩阵，使纳米尺度的功能， 在传统的荧光显微镜上分辨。不幸的是，这些方法尚未得到利用 由于缺乏特异性识别糖基化的相容性成像试剂， 生物学上重要的聚糖表位（糖表位），并为高级超分辨率定制 包括ExM的显微镜方法。为了应对这些挑战，该提案旨在创建一个成像 称为糖缀合物ExM（GlycoExM）的技术，其利用多功能化学探针， 用于细胞聚糖的简易检测和纳米分辨率的糖表位特异性亲和试剂， 糖缀合物(Aim 1）基于多功能化学探针的一套多功能化学探针 将开发低聚硫醚酰胺（oligoTEA）用于代谢标记的非靶向GlycoExM成像。 在广泛使用的共聚焦显微镜上观察聚糖，有效分辨率高达15 nm。(Aim 2）一种方法， 从非免疫文库中分离定制亲和试剂将适用于多重选择， 糖表位特异性抗体（糖体）。(Aim 3）将针对靶向GlycoExM优化糖体 通过蛋白质工程和oligoTEA缀合，导致试剂，将提高分辨率， ExM用于细胞和组织。将为性能最佳的试剂制定详细的方案，包括 关于糖体的可再生生物合成、最佳试剂浓度和验证成像的信息 条件除了开发这种试剂的“操作手册”，该项目还将使用oligoTEAs 和糖体，以探测不同正常细胞表面上真实聚糖特征的空间表达。 和癌细胞系，以例如提供（1）免疫调节聚糖的超分辨图谱， 糖萼和（2）推定的半乳糖凝集素晶格的第一个详细的分子结构。总体而言，拟议 GlycoExM平台具有在糖组范围内实现高内容成像的潜力，而低成本 和用户友好性，将确保它成为广泛访问的糖科学界和超越。

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.