Glycobiology Advance Access published online on November 22, 2008
Glycobiology, doi:10.1093/glycob/cwn129
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Published by Oxford University Press 2008.
Glycosidase-induced fusion of isoprenoid gentiobiosyl lipid membranes at acidic pH
1 Institute for Biological Sciences, National Research Council, 100 Sussex Drive, Ottawa, Ont., Canada K1A OR6
3 To whom correspondence should be addressed: Tel.: +1-613-998-7891; Fax.: +1-613-952-9092. e-mail: dennis.sprott{at}nrc-cnrc.gc.ca
Received on July 31, 2008; accepted on November 15, 2008
A difficulty in explaining the mechanism whereby archaeal lipid membrane vesicles (archaeosomes) deliver entrapped protein antigens to the MHC class I cytosolic pathway from phagolysosomes of antigen-presenting cells, has been the observation that they tend not to fuse. Here, we determine that archaeosomes, composed of archaeal isoprenoid mixtures of glyco and phospholipids, can be highly fusogenic when exposed to the pH and enzymes found in late phagolysosomes. Fusions were strictly dependent on acidic pH and the presence of 
- or β-glucosidase. RET assays demonstrated that fusion conditions induced lipid mixing of archaeosome lipids with self-unlabeled archaeosomes. Because PC/PG/cholesterol liposomes by themselves did not fuse, it was possible to unequivocally show fusion of rhodamine-labeled liposomes with archaeosomes by fluorescence microscopy, and to demonstrate lipid mixing between labeled liposomes and archaeosomes by the RET assay. Radiotracer and 1NMR studies revealed that glycolipids in fused archaeosomes were not degraded significantly by glucosidase treatment during fusion. Rather, the glucosidases dramatically induced small archaeosomes to rapidly and visually aggregate at pH 4.8, but not 6.8, thus bringing membranes together appropriately as a first step in the fusion process. 1NMR was used to demonstrate that conditions causing aggregation correlated with binding of glucosidase to the archaeosomes. Binding at acidic pH occurred by electrostatic interaction of positively charged glucosidase with the anionic phospholipids, although interaction also occurred with the gentiobiosyl lipids. The data indicate a mechanism of membrane-membrane fusion for archaeal glycolipid membranes induced by glycosidase, and illustrate importance for inclusion of glycolipids in compositions of-vesicles designed to deliver protein antigens to the cytosol for MHC class I presentation.
Key words: fusion / phagolysosome / glycosidase / glycosyl isoprenoids / glycolipid membranes
2 Current address: Université de Montréal, Faculté de Médecine Vétérinaire, St.-Hyacinthe, 3200 Sicotte, St.-Hyacinthe, Québec J2S 7C6, Canada