Phospholipid vesicle fusion induced by saposin C.
Saposin C is a small Trp-free, multifunctional glycoprotein that enhances the hydrolytic activity of acid beta-glucosidase in lysosomes. Saposin C's functions have been shown to include neuritogenic/neuroprotection effects and membrane fusion induction. Here, the mechanism and kinetics of saposin C's fusogenic activity were evaluated by fluorescence spectroscopic methods including dequenching, fluorescence resonance energy transfer, and stopped-flow analyses. Trp or dansyl groups were introduced as fluorescence reporters into selected sites of saposin C to serve as topological probes for protein-protein and protein-membrane interactions. Saposin C induction of liposomal vesicle enlargement was dependent upon anionic phospholipids and acidic pH. The initial fusion burst was completed in the timeframe of a few seconds to minutes and was dependent upon the unsaturated anionic phospholipid content. Two events were associated with saposin C-membrane interaction: membrane insertion of the saposin C terminal helices and reorientation of its central helical region. The latter conformational change likely exposed a binding site for saposins anchored on vesicles. Addition of selected saposin C peptides prior to intact saposin C in reaction mixtures abolished the liposomal fusion. These results indicated that saposin-membrane and saposin-saposin interactions are needed for the fusion process.
Wang
Ying
Y
Division and Program in Human Genetics, Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
Grabowski
Gregory A
GA
Qi
Xiaoyang
X
D031541:Fluorescence Resonance Energy Transfer; D006023:Glycoproteins / Q000737:chemistry*; D007700:Kinetics; D008081:Liposomes / Q000138:chemical synthesis / Q000737:chemistry*; D008560:Membrane Fluidity; D008561:Membrane Fusion; D008968:Molecular Conformation; D008970:Molecular Weight; D010316:Particle Size; D011485:Protein Binding; D049231:Saposins; D013050:Spectrometry, Fluorescence / Q000379:methods
D006023:Glycoproteins; D008081:Liposomes; D049231:Saposins