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Glycosidase-induced fusion of isoprenoid gentiobiosyl lipid membranes at acidic pH

Author:
Sprott, G. Dennis, Côté, Jean-Philippe, Jarrell, Harold C.
Source:
Glycobiology 2009 v.19 no.3 pp. 267-276
ISSN:
0959-6658
Subject:
Archaea, antigen-presenting cells, antigens, beta-glucosidase, cholesterol, cytosol, electrostatic interactions, energy transfer, fluorescence microscopy, glycolipids, isoprenoids, major histocompatibility complex, mixing, nuclear magnetic resonance spectroscopy, pH, phagosomes, phospholipids
Abstract:
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 α- or β-glucosidase. Resonance energy transfer (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 a 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 ¹H NMR 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. ¹H NMR was used to demonstrate that conditions causing aggregation correlated with binding of glucosidase to the archaeosomes. Binding at acidic pH occurred by the electrostatic interaction of positively charged glucosidase with the anionic phospholipids, although the 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 the importance for inclusion of glycolipids in compositions of vesicles designed to deliver protein antigens to the cytosol for MHC class I presentation.
Agid:
2122086