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

Galectin-3 和用于在膜表面聚集糖脂和糖蛋白的工程变体

• 批准号: 10652941
• 负责人: Crystal Marie Vander Zanden
• 金额: $ 42.99万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652941
• 关键词: Address; Apoptosis; Architecture; BODIPY; Back; Behavior; Binding; Biological; Carbohydrates; Cell Adhesion; Cell membrane; Cell model; Cell physiology; Cell surface; Chimera organism; Clinical Trials; Code; Coupled; Coupling; Disease; Drug Design; Dyes; Engineering; Event; Fluorescence; Fluorescence Microscopy; Foundations; Future; Galactose Binding Lectin; Galectin 3; Ganglioside GM1; Glycolipids; Glycoproteins; Goals; Human; Incidence; Knowledge; Label; Lectin; Ligands; Lipids; Liquid substance; Low Income Population; Malignant Neoplasms; Mediating; Membrane; Membrane Glycoproteins; Membrane Proteins; Methods; Military Personnel; Modeling; Molecular; Mucin 1 protein; N-terminal; Neoplasm Metastasis; Neurons; Outcome; Pattern; Pharmaceutical Preparations; Phase; Polysaccharides; Productivity; Protein Family; Proteins; Reader; Research; Roentgen Rays; Role; Signal Transduction; Site; Solid Neoplasm; Structural Models; Structure; Students; Surface; Synchrotrons; System; Tandem Repeat Sequences; Techniques; Variant; Work; X ray diffraction analysis; beta-galactoside; biological systems; cancer cell; cell behavior; cell growth; clinical application; dimer; drug development; druggable target; electron density; experimental study; flexibility; glycosylation; in silico; innovation; intermolecular interaction; membrane assembly; membrane model; molecular dynamics; neuron development; overexpression; receptor; self assembly; simulation; sugar; tumor; undergraduate student

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.

项目总结/摘要 细胞表面的分子聚集对于控制细胞信号传导和行为至关重要，约70%的药物 结合膜蛋白靶点。目前，大多数药物是围绕抑制特定的跨膜 受体，但靶向分子簇控制细胞行为的潜力尚未开发。半乳糖凝集素蛋白 调节分子聚集，因为它们结合并组装各种β-半乳糖苷修饰的脂质， proteins.例如，半乳糖凝集素-3（Gal-3）在转移性癌细胞中过表达高达30倍， 表明在细胞粘附中的作用。Gal-3的跨膜结合配偶体MUC 1在细胞表面异常表达， 约75%的人类实体瘤癌症。Gal-3还通过与神经元相互作用介导神经元细胞生长。 糖脂神经节苷脂GM 1。野生型（WT）半乳糖凝集素蛋白被组织成三种独特的分子 假设指导分子簇集的结构：同源二聚体，串联重复二聚体， 柔性接头和促进寡聚化的嵌合型。WT Gal-3是嵌合型蛋白。我们 假设Gal-3指导MUC 1和GM 1在膜表面聚集， 可以通过Gal-3分子结构（同源二聚体、串联重复或嵌合体）的工程化改变来指导。 如果这项研究成功，这将为通过表面控制细胞行为奠定基础。 介导的聚糖分子聚集。这广泛适用于许多细胞功能，包括 细胞凋亡、细胞粘附和神经元发育。我们提出两个目标：（1）确定 Gal-3结合膜表面上的MUC 1和GM 1，和（2）表征工程化Gal-3变体， 确定半乳糖凝集素分子结构如何指导聚糖聚集模式。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将表征WT Gal-3结合 糖脂GM 1和糖蛋白MUC 1的反应。这将使用同步加速器X- 射线散射和荧光显微镜。X射线反射率将用于确定电子密度 轮廓的膜，和掠入射X射线衍射将被用来表征半有序 例如脂质结构域或Gal-3组装体。荧光显微镜将用于表征脂质 使用相敏染料和BODIPY标记的聚糖进行相分离和糖蛋白/脂质组织。 目的2将确定工程化Gal-3变体如何聚集糖蛋白/脂质底物。述工程化 变体包括Gal-3的同源二聚体和串联重复形式，预期其引起不同的聚类 模式.这将通过与目标1相同的方法来实现。目标3支持目标1， 分子动力学模拟来模拟膜中Gal-3结合GM 1的原子结构。这些 模拟将确定分子簇大小、膜组织和分子间接触， 有助于Gal-3寡聚化。长期目标是了解半乳糖凝集素介导的中尺度 糖脂和糖蛋白在细胞表面的聚集，这是控制细胞行为的基础。