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

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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.