Phospholipid Membrane Interactions of Saposin C: In Situ Atomic Force Microscopic Study

Abstract

Saposin C (Sap C) is a small glycoprotein required for hydrolysis of glucosylceramidase in lysosomes. The full activity of glucosylceramidase requires the presence of both Sap C and acidic phospholipids. Interaction between Sap C and acidic phospholipid-containing membranes, a crucial step for enzyme activation, is not fully understood. In this study, the dynamic process of Sap C interaction with acidic phospholipid-containing membranes was investigated in aqueous buffer using atomic force microscopy. Sap C induced two types of membrane restructuring: formation of patch-like structural domains and the occurrence of membrane destabilization. The former caused thickness increase whereas the latter caused thickness reduction in the gel-phase membrane bilayer, possibly as a result of lipid loss or an interdigitating process. Patch-like domain formation was independent of acidic phospholipids, whereas membrane destabilization is dependent on the presence and concentration of acidic phospholipids. Sap C effects on membrane restructuring were further studied using synthetic peptides. Synthetic peptides corresponding to the amphipathic alpha-helical domains 1 (designated "H1 peptide") and 2 (H2 peptide) of Sap C were used. Our results indicated that H2 contributed to domain formation but not to membrane destabilization, whereas H1 induced neither type of membrane restructuring. However, H1 was able to mimic Sap C's destabilization effect in conjunction with H2, but only when H1 was present first and H2 was added afterwards. This study provides an approach to investigate the structure-function aspects of Sap C interaction with phospholipid membranes, with insights into the mechanism(s) of Sap C-membrane interaction.

Direct Visualization of Saposin Remodelling of Lipid Bilayers

“…One model suggests that the reduced membrane thickness may be due to partial lipid removal from the top leaflet by saposin molecules 12 leaving an intact lower leaflet. The hydrophobic tails of lower acyl chains are shielded from the aqueous environment by amphiphilic saposin molecules, possibly in an open conformation that permits amphiphilic shielding.…”

“…Here, we present the direct visualization by atomic force microscopy (AFM) imaging of model lipid bilayers subjected to saposins A, B or C. Previous AFM imaging results have shown that saposin C has the potential of restructuring phospholipid membranes. [11][12][13] Focusing on saposin C, we demonstrate stable association of protein with transformed membranes using simultaneous AFM and fluorescence imaging. Finally, we address the solubilizer and liftase mechanisms by following the kinetics of membrane lipid markers subjected to saposin C.…”

Imaging the Selective Binding of Synapsin to Anionic Membrane Domains

“…[15][16][17][18] Similarly, saposin, myelin, and cytochrome C all interact at least partially through electrostatic interactions with bilayers containing charged lipids. [10][11][12] This work provides information on the role of electrostatic and nonspecific interactions and membrane composition in controlling protein adsorption on lipid bilayers and also yields insight into protein-mediated membrane disruption. In order to provide more direct evidence for the interaction of synapsin with anionic membrane domains, we have used AFM and near-field scanning optical microscopy (NSOM) [19][20][21][22] to image supported lipid bilayers comprised of mixtures of neutral phosphatidylcholine (PC) and anionic PS lipids.…”

Cancer-Selective Targeting and Cytotoxicity by Liposomal-Coupled Lysosomal Saposin C Protein

“…A general property of saposins is their lipid membrane binding activity (9) because they play important roles in lipid transport (10), lipid microdomain assembly (11), and reorganization of biological membranes (12)(13)(14). Specifically, saposin C associates with lipid membranes by embedding into membrane leaflets.…”