Membrane Binding and Lipid Extraction Studies of Gm2 Activator Protein (GM2AP)

Abstract

GM2AP is an accessory protein that solubilizes the ganglioside GM2 from intralysosomal vesicles for hydrolytic cleavage by HexA to form GM3. The precise molecular interactions and method of extraction of GM2 from lipid vesicles are unknown. GM2AP also functions as a lipid transfer protein. This non-enzymatic protein contains three tryptophan residues (W5, W63, W131) with two of these (W63, W131) located in putative membrane binding loops. In order to investigate the possible role of the tryptophan (TRP) residues in membrane binding and lipid extraction, gel filtration and resorcinol absorption assays were used to investigate the extraction efficiency of GM2 by GM2AP in a series of TRP to ALA substituted constructs. GM2AP is shown to have two distinct substrate binding modes, one for the GM2 ganglioside and another for phospholipids. Fluorescence experiments were used to determine the orientation of dansyl-DHPE in the binding pocket of GM2AP. Quenching results suggest that dansyl-DHPE is oriented such that the head group of the lipid is located in the hydrophobic pocket of the protein, consistent with the binding mode of other phospholipids which were previously studied. Dansyl-labeled lipids were used to monitor the changes in the rates of lipid extraction and transfer by GM2AP from liposomes as a function of both pH and the TRP to ALA substituted constructs. The ability of GM2AP to bind and/or extract dansyl-labeled lipids from liposomes was affected with increased pH of the lipid environment. Additionally, removal of TRP from the putative membrane binding loops resulted in slower lipid extraction rates, suggesting that these residues are relevant for membrane binding and/or extraction of GM2AP.