Galectin-3 and engineered variants for clustering glycolipids and glycoproteinson membrane surfaces

项目摘要/摘要 细胞表面的分子聚集对于控制细胞信号和行为是至关重要的，而~70%的药物 结合膜蛋白靶标。目前，大多数药物都是围绕着抑制一种特定的跨膜而设计的 受体，但靶向分子聚集以控制细胞行为的潜力尚未开发。半乳凝集素蛋白 调节分子聚集，因为它们结合和组装各种β-半乳糖苷修饰的脂类和 蛋白质。例如，Galectin-3(Gal-3)在转移的癌细胞中过度表达高达30倍， 这表明它在细胞黏附中起作用。Gal-3‘S跨膜结合蛋白MUc1基因异常表达 约75%的人类实体瘤癌症。GAL-3还通过与神经细胞的相互作用来调节神经细胞的生长 糖脂神经节苷脂GM1。野生型(WT)Galectin蛋白被组织成三个独特的分子 假设直接分子聚集的结构：同源二聚体、串联重复二聚体和 灵活的连接体和促进齐聚的嵌合型。WT Gal-3是一种嵌合型蛋白。我们 假设Gal-3引导MUC1和GM1在膜表面聚集，并且该簇组织 可以通过对Gal-3分子结构(同源二聚体、串联重复或嵌合体)的工程改变来指导。 如果拟议的研究成功，这将为通过表面控制细胞行为奠定基础。 介导的葡聚糖分子的聚集。这广泛适用于许多蜂窝功能，包括 细胞凋亡、细胞黏附和神经元发育。我们提出了两个目标：(1)确定结构 Gal-3结合膜表面的MUC1和GM1以及(2)鉴定Gal-3工程变异体 确定半乳糖凝集素的分子结构如何引导聚糖簇模式。创新之处在于使用液体 表面同步加速器X射线散射和工程半乳糖凝集素变体。目标1将描述WT Gal-3结合的特征 糖脂GM1和糖蛋白MUC1在模型膜上。这将使用同步加速器X- 光线散射和荧光显微镜。将使用X射线反射率来确定电子密度 膜的轮廓和掠入射X射线衍射将被用来表征半有序 物种，如脂类结构域或Gal-3组装体。荧光显微镜将被用来表征脂质 使用相敏染料和BODIPY标记的葡聚糖进行相分离和糖蛋白/脂组织。 目标2将确定Gal-3工程变体如何聚集糖蛋白/脂底物。精心设计的 变异体包括Gal-3的同源二聚体和串联重复形式，预计它们会导致不同的聚集 模式。这将使用与目标1相同的方法来实现。 分子动力学模拟模拟Gal-3结合GM1在膜中的原子结构。这些 模拟将确定分子簇的大小、膜组织和分子间接触 有助于Gal-3齐聚。长期目标是理解半乳糖凝集素介导的中尺度 糖脂和糖蛋白在细胞表面聚集，这是控制细胞行为的基础。

PROJECT SUMMARY / ABSTRACT Molecule clustering at the cell surface is critical for controlling cell signaling and behavior, and ~70% of drugs bind membrane protein targets. Currently, most drugs are designed around inhibiting a specific transmembrane receptor, but there is untapped potential to target molecule clustering to control cell behavior. Galectin proteins regulate molecule clustering because they bind and assemble a variety of β-galactoside-decorated lipids and proteins. For example, galectin-3 (Gal-3) is overexpressed up to 30-fold in metastasizing cancer cells, suggesting a role in cell adhesion. Gal-3’s transmembrane binding partner, MUC1, is aberrantly expressed on ~75% of human solid tumor cancers. Gal-3 also mediates neuronal cell growth through interactions with the glycolipid ganglioside GM1. Wild type (WT) galectin proteins are organized into three unique molecular architectures which are hypothesized to direct molecule clustering: homodimers, tandem-repeat dimers with flexible linkers, and a chimeric-type that promotes oligomerization. WT Gal-3 is a chimeric-type protein. We hypothesize that Gal-3 directs MUC1 and GM1 clustering at the membrane surface and that cluster organization can be directed by engineered changes in Gal-3 molecular architecture (homodimer, tandem-repeat, or chimera). If the proposed research is successful, this will lay the foundation for controlling cell behavior through surface- mediated clustering of glycan molecules. This is broadly applicable to many cellular functions including apoptosis, cell adhesion, and neuronal development. We propose two goals: (1) determine the structure of Gal-3 binding MUC1 and GM1 on a membrane surface and (2) characterize engineered Gal-3 variants to determine how galectin molecular architecture directs glycan clustering patterns. The innovation is to use liquid surface synchrotron X-ray scattering and engineered galectin variants. Aim 1 will characterize WT Gal-3 binding to glycolipid GM1 and glycoprotein MUC1 on a model membrane. This will be performed using synchrotron X- ray scattering and fluorescence microcopy. X-ray reflectivity will be used to determine the electron density profile of the membrane, and grazing incidence X-ray diffraction will be used to characterize semi-ordered species such as lipid domains or Gal-3 assemblies. Fluorescence microscopy will be used to characterize lipid phase separation and glycoprotein/lipid organization using a phase-sensitive dye and BODIPY-labeled glycans. Aim 2 will determine how engineered Gal-3 variants cluster glycoprotein/lipid substrates. The engineered variants include homodimer and tandem-repeat forms of Gal-3, which are expected to cause different clustering patterns. This will be accomplished with the same methods as Aim 1. Aim 3 supports goal 1 by using molecular dynamics simulations to model the atomic structure of Gal-3 binding GM1 in a membrane. These simulations will determine the molecule cluster size, membrane organization, and intermolecular contacts that contribute to Gal-3 oligomerization. The long-term goal is to understand galectin-mediated mesoscale clustering of glycolipids and glycoproteins at the cell surface, which is foundational for controlling cell behavior.