CAREER: Investigating the Molecular Factors that Underlie Myelin-Associated Glycoprotein Binding

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Interactions between biomolecules govern all life sustaining processes. This project will investigate the interactions between biomolecules that are associated with the lipid membranes that form the boundaries of cells in the nervous system. Cholesterol is a major component of the lipid membranes, and it can alter the structure and function of other membrane-associated molecules, like lipids, carbohydrates, and proteins. Specifically, this project will investigate how cholesterol alters the binding of lipids that contain carbohydrates to proteins that regulate cell-cell interactions in the nervous system. The protein that is at the center of these studies, myelin-associated glycoprotein (MAG), is an inhibitor of neuron growth, and determining the molecular factors by which its interactions are modulated by cholesterol and the proteins it clusters with will illuminate the mechanisms through which it acts. Synergistically with research activities, the PI will design teaching laboratory modules that demonstrate the properties and analysis of lipids and membranes (liposome characterization, supported membrane formation, molecular diffusion), biosensors, and fluorescence microscopy. Additionally, an advanced graduate level course on biosensors will be developed to expand the reach of this project to the classroom as well as the laboratory. This course will rely heavily on the use of active learning teaching methods, and it will expose chemistry, biochemistry, biology, and engineering students to the wide variety of techniques that can be used to detect and characterize biomolecules.The overarching goal of this project is to determine, through quantitative analytical measurements, the factors that modulate biomolecular interactions responsible for the adhesion of lipid membranes to one another. The specific focus of the project is on the quantitative analysis of interactions between MAG on one membrane and its ganglioside and protein receptors on another. MAG is a sialic acid-binding immunoglobulin-type lectin (Siglec) found in the nervous system on myelin where one of its functions is to maintain adhesive interactions between myelin and axon membranes. Biding of MAG to gangliosides (GD1a and GT1b) and Nogo receptor 1 (NgR1) on neurons inhibits axonal growth and is a significant obstacle to nervous system repair. The first aim of this project will determine how cholesterol modulates MAG-ganglioside binding events and the membrane-membrane interactions they cause. The second aim will determine how membrane phase separation into lipid raft-like domains influences spatial and temporal aspects of MAG-ganglioside interactions. The final aim will determine how the stoichiometry of the MAG receptor complex (NgR1/p75/LINGO-1) influences the MAG’s binding affinity and thus membrane-membrane interactions. The mechanism by which anti-LINGO-1 antibodies disrupt MAG-NgR1 interactions will also be determined. This project will involve undergraduate and graduate students in the design, execution, and interpretation of data obtained with quartz crystal microbalance (QCM-D) biosensing, Förster resonance energy transfer (FRET), total internal refection fluorescence (TIRF) microscopy, and equilibrium fluctuation analysis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.