Mechanistic insight into tumor-associated MUC1 glycopeptides binding to macrophage galactose-type lectin

项目总结 糖类识别是生物系统中最复杂的识别过程之一。这个 低聚糖对高密度信息编码的特殊能力是细胞发挥中介作用的关键 细胞-细胞识别的许多重要方面。被称为凝集素的内源性糖链结合蛋白， 越来越多地被认为是基于碳水化合物编码的生物信息的“解码器” 它们区分密切相关的糖链结构的能力。这种固有的复杂性构成了 在分子水平上理解这些修饰的功能意义的主要障碍。帮助 为了解决这一知识差距，拟议的研究旨在表征TN/STN之间的相互作用 肿瘤相关MUC1的糖链与巨噬细胞半乳糖特异性凝集素(MGL)结合。到目前为止，它的意义 肿瘤相关O-糖链类型和位置的多样性对免疫反应的调节作用 MGL目前仍不清楚。因此，单糖化和多糖化的MUC1多肽模型和MUC1衍生的 糖肽定位扫描组合文库显示类天然的异质性和变异性 将制备TN/STN O-葡聚糖表位，以检测表位的异质性、糖苷簇效应以及 邻近多聚糖与凝集素结合的空间位阻效应(目标1)。单独的糖肽与 增强的结合MGL的能力将在多价配体展示中合成，以模拟生物多价 系统(目标2)。中央支架(线性或分支)的效果，呈现了多个副本 凝集素结合特性的识别元件将通过等温滴定量热法进行评估 国际贸易中心(ITC)和原子力光谱学(AFM)(目标3)。配体-受体相互作用的热力学 将由ITC确定。由于受体-配体络合物的机械性能不能 仅从热力学测量获得，而不是从导致络合物的反应路径获得 分离后，将使用单分子原子力显微镜来确定相互作用的结合动力学。一个重要的 这项建议的组成部分是让学生积极参与跨学科研究。我们将为您提供亲身体验 本科生和研究生在化学和生物界面的研究经验，给出 学生独特的体验，汇集了合成有机化学、糖化学的许多方面 和糖生物学，在最近来自美国国家研究委员会的报告中强调 学院。我们的目标是扩大未来的研究人员社区，重点是增加 妇女和少数群体参与化学和其他STEM学科。总而言之，这个项目将 提供新的和独特的合成工具来研究多糖对蛋白质结构的影响并破译 免疫系统调节中糖-蛋白质相互作用的生化基础。

PROJECT SUMMARY Carbohydrate recognition is one of the most sophisticated recognition processes in biological systems. The exceptional capacity of oligosaccharides for high density information coding is essential for cells to mediate many important aspects of cell-cell recognition. The endogenous glycan-binding proteins, known as lectins, have been increasingly recognized as “decoders” of the carbohydrate-encoded biological information based on their ability to distinguish between closely related glycan structures. This inherent complexity constitutes a major barrier to understanding the functional significance of these modifications on the molecular level. To help address this knowledge gap, the proposed research aims to characterize the interaction between Tn/sTn glycans of tumor-associated MUC1 with macrophage galactose-specific lectin (MGL). To date, the significance of the diversity in the type and positions of tumor-associated O-glycans on modulation of immune responses by MGL is still unclear. Thus, mono- and multiple-glycosylated MUC1 peptide models and MUC1-derived glycopeptide positional scanning combinatorial libraries displaying native-like heterogeneous and aberrant Tn/sTn O-glycan epitopes will be prepared to examine epitope heterogeneity, the glycoside cluster effect, and steric hindrance effect of neighboring glycans on binding to lectins (Aim 1). Individual glycopeptides with enhanced ability to bind MGL will be synthesized in a multivalent ligand display to mimic biological polyvalent systems (Aim 2). The effect of the central scaffold (linear or branched), presenting multiple copies of the recognition elements, on the lectin binding specificities will be evaluated by isothermal titration calorimetry (ITC) and atomic force spectroscopy (AFM) (Aim 3). The thermodynamics of ligand-receptor binding interaction will be determined by ITC. Since the mechanical properties of the receptor-ligand complexes cannot be obtained solely from thermodynamic measurements, but rather from the reaction path that leads to complex separation, single-molecule AFM will be used to determine the binding kinetics of the interaction. An important component of this proposal is to engage students actively in interdisciplinary research. We will offer a hands-on research experience for undergraduate and graduate students on the interface of chemistry and biology, giving students a unique experience that brings together many aspects of synthetic organic chemistry, glycochemistry and glycobiology as emphasized in the recent report from the National Research Council of the U.S. National Academies. Our goal is to broaden the future community of researchers with an emphasis on increasing the participation of women and minorities in chemistry and other STEM disciplines. In summary, this project will provide novel and unique synthetic tools to study the effects of glycans on protein structure and to decipher the biochemical basis of glycan–protein interactions in regulation of the immune system.