The human saposins are four homologous activator proteins that are essential for the lysosomal degradation of sphingolipids (SLs) with small headgroups. They function in part to increase the solvent accessibility of these SLs to specific acid hydrolases by two proposed modes of action. In the solubilizer model, saposins extract SLs into soluble saposin-lipid complexes, and in the liftase model, the saposins reorganize lipids within membranes. The four saposins, called saposins A, B, C and D, have dual characters and exist as soluble proteins and in membrane-associated states. In this thesis, I first present the crystal structure of saposin D in a lipid-free state. The structure exhibits a closed, monomeric fold as previously described for saposins A and C. Next, I examine the lipid interaction properties of saposin A and determine a crystal structure of SapA in a lipid-bound complex. The complex forms a discoidal lipoprotein particle composed of highly ordered bilayer-like hydrophobic core surrounded by a protein belt consisting of two copies of saposin A in an open conformation. The saposin A-lipid discs are most likely the effective substrate-presenting particles in galactosylceramide hydrolysis. Finally, I compare the lipid interaction properties of the four saposins and address membrane perturbation, liposome binding, lipid solubilization and lipoprotein particle formation for each protein. Each saposin displays a unique behavior in the presence of liposomes under conditions that mimic the lysosomal environment. In particular, saposin D reveals simultaneous formation of different sized protein-lipid complexes, which appear to be primarily dependent on the lipid to protein molar ratio. A comparison of the available structures of saposins A, B and C in the “closed” and “open” conformations reveals structural hinge regions that likely shape the different types of saposin self-association. These states are directly related to the protein-lipid solubilization and/or membrane association properties of the saposins. Collectively, these findings present a more complete understanding of the lipid interaction properties of the saposin proteins and provide new insights into their role as activator proteins.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31904 |
| Date | 11 January 2012 |
| Creators | Popovic, Konstantin |
| Contributors | Privé, Gilbert G. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
Page generated in 0.0024 seconds