Dr. Shipley's overall objectives have been: (1) to define the structure, properties and intermolecular interactions of the complex polar lipids which constitute important structural and functional components of both the bilayer matrix of cell membranes and the monolayer surface of plasma lipoproteins; and (2) to study membrane receptor-ligand interactions as individual macromolecules, as two-dimensional arrays on lipid surfaces, as three dimensional crystals, and in detergent-solubilized or vesicle-reconstituted systems. The rationale was that the structural definition of lipids is a prerequisite for successful experiments with reconstituted membrane receptor-ligand systems. In this proposal, Dr. Shipley focuses on: (1) the structure and properties of glycosphingolipids (GSL), (2) the interactions of GSL with other membrane lipids, and (3) the role of GSL as receptors for bacterial toxins, with emphasis on the ganglioside GM1-cholera toxin system.
Aim 1 : Glycosphingolipid Structure and Interactions: to define the role of increasing oligosaccharide complexity on the structure, properties and interactions of GSL. The Shipley group's successful de novo synthesis of ceramides and cerebrosides will be extended to globosides and gangliosides. Dr. Shipley will define the structure and properties of these GSL and their interactions with phosphatidylcholine and cholesterol using x-ray diffraction, differential scanning calorimetry, and electron microscopy techniques. The hypothesis being tested is that increasing GSL oligosaccharide complexity will progressively alter their phase behavior and that this in turn will produce defects in host bilayer structure.
Aim 2 : Cholera Toxin/Ganglioside GM1 Interaction: to complete the structural studies of CTX, its B5-pentamer CTB, and their interaction with their receptor GM1. Dr. Shipley will focus on CTX and CTB bound to GM1-containing vesicles and will test the hypothesis that the membrane translocation step for the catalytic A1 domain is triggered by changes in pH. In parallel, crystallographic studies of CTX and CTB grown at low pH will be developed.
| Jeon, H; Shipley, G G (2000) Localization of the N-terminal domain of the low density lipoprotein receptor. J Biol Chem 275:30465-70 |
| Duclos Jr, R I (2000) The total synthesis of ganglioside GM3. Carbohydr Res 328:489-507 |
| Saxena, K; Zimmermann, P; Schmidt, R R et al. (2000) Bilayer properties of totally synthetic C16:0-lactosyl-ceramide. Biophys J 78:306-12 |
| Jeon, H; Shipley, G G (2000) Vesicle-reconstituted low density lipoprotein receptor. Visualization by cryoelectron microscopy. J Biol Chem 275:30458-64 |
| Woldin, C N; Hing, F S; Lee, J et al. (1999) Structural studies of the detergent-solubilized and vesicle-reconstituted insulin receptor. J Biol Chem 274:34981-92 |
| Saxena, K; Duclos, R I; Zimmermann, P et al. (1999) Structure and properties of totally synthetic galacto- and gluco-cerebrosides. J Lipid Res 40:839-49 |
| Saxena, K; Shipley, G G (1997) Structural studies of detergent-solubilized and vesicle-reconstituted low-density lipoprotein (LDL) receptor. Biochemistry 36:15940-8 |
