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J. Lipid Res.
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Originally published In Press as doi:10.1194/jlr.M800385-JLR200 on December 13, 2008 Originally published In Press as doi:10.1194/jlr.M800385-JLR200 on August 20, 2008

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Journal of Lipid Research, Vol. 50, 1744-1755, September 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Fatty acid-dependent globotriaosyl ceramide receptor function in detergent resistant model membranes

Radhia Mahfoud*, Adam Manis*,§ and Clifford A. Lingwood1,*,{dagger},**

* Division of Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada
{dagger} Department of Pediatric Laboratory Medicine, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada
§ Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, Canada
** Department of Biochemistry, University of Toronto, Toronto, Canada

1 To whom correspondence should be addressed. email: cling{at}sickkids.ca

Glycosphingolipid (GSL) fatty acid strictly regulates verotoxin 1 (VT1) and the HIV adhesin, gp120 binding to globotriaosyl ceramide within Gb3/cholesterol detergent resistant membrane (DRM) vesicle constructs and in Gb3 water-air interface monolayers in a similar manner. VT2 bound Gb3/cholesterol vesicles irrespective of fatty acid composition, but VT1 bound neither C18 nor C20Gb3vesicles. C18/C20Gb3 were dominant negative in mixed Gb3 fatty acid isoform vesicles, but including C24:1Gb3 gave maximal binding. VT1 bound C18Gb3 vesicles after cholesterol removal, but C20Gb3vesicles required sphingomyelin in addition for binding. HIV-1gp120 also bound C16, C22, and C24, but neither C18 nor C20Gb3 vesicles. C18 and C20Gb3 were, in mixtures without C24:1Gb3, dominant negative for gp120 vesicle binding. Gp120/VT1bound C18 and C24:1Gb3 mixtures, although neither isoform bound alone. Monolayer surface pressure measurement showed VT1, but not VT2, bound Gb3 at cellular DRM surface pressures, and confirmed loss of VT1 and gp120 (but not VT2) specific C18Gb3 binding. We conclude fatty-acid mediated fluidity within simple model GSL/cholesterol DRM can selectively regulate GSL carbohydrate-ligand binding.

Supplementary key words cholesterol • DRM • HIVgp120 • Langmuir trough • lipid raft • sucrose gradient • verotoxin 1 • verotoxin 2

Abbreviations: Chol, cholesterol; DRM, detergent resistant membrane; Gb3, globotriaosyl ceramide; GSL, glycosphingolipid; SM, sphingomyelin; VT, verotoxin


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