The objective of the proposed studies is to elucidate kinetics and mechanistic bases of the interaction of saposins and lipid model membranes, We seek to define amino acid regions in saposins important to lipid bilayer binding and the residues critical to the binding activity of individual saposins for specific lipidl/ipid membrane. Saposins are small (80 aa) lysosomal proteins that have distinct mechanisms of action despite the high similarity of their primary sequences. These saposins or activator proteins are essential for normal function of several hydrolases involved in the glycosphingolipid/sphingolipid (GSL/JSL) catabolism pathway in lysosomes. Four (A, B, C, and D) derive from a single precursor, prosaposin. We hypothesize that the diverse functions of saposins are a result of distinct lipid/lipid membrane interaction properties of these proteins. The proposed studies focus on saposins B and C as prototypes since their mechanisms of actions with their respective enzyme appear distinct. Saposin B enhances arysulfatase A (ASA) activity by solubilizing the lipid substrate, sulfatide, and saposin C induced an acid beta- glucosidase conformational change in the presence of phosphatidylserine (pS) membranes at acidic pH. Saposin C also induces PS liposome fusion, while saposin B does not. The studies are designed to determine saposin- lipid and saposin-lipid-enzyme interactions by fluorescence and circular dichroism (CD) spectroscopic analyses. Intrinsic (Trp) and extrinsic (pyrene) fluorescent probes in proteins or lipids will be used as reporters for fluorescence resonance energy transfer, stopped-flow, and quenching analyses. Using biochemical and mutagenesis technologies, the location of key structural components for saposin functionality will be characterized. The availability of efficient expression systems and in vitro assay systems for saposins B and C provide the feasibility for the proposed studies. Transgenic """"""""rescue"""""""" systems based on the saposin precursor, prosaposin, deficient cell lines will provide ex vivo structure/function correlation of selected mutants of saposins B and C. The significance of the proposed studies relates to the delineation of the lipid-binding specificity of saposins that have major effects on the modulation of sphingolipid hydrolases and influence on the expression of lysosomal storage diseases.
Showing the most recent 10 out of 12 publications
