Generation of receptor-active, globotriaosyl ceramide/cholesterol lipid 'rafts' in vitro: A new assay to define factors affecting glycosphingolipid receptor activity

Abstract

Purified renal globotriaosyl ceramide (Gb₃)/cholesterol mixtures, sonicated and heated in a Triton-containing buffer and placed below a discontinuous sucrose gradient, form glycosphingolipid (GSL)-containing dense lipid structures at the 30/5% sucrose interface after centrifugation. Inclusion of fluorescein-labeled verotoxin 1 B subunit (FITC-VT1 B) within the most dense sucrose layer results in the fluorescent labeling of this Gb₃-containing raft structure. Alternatively, inclusion of ¹²⁵I-labeled VT1 and fractionation allows quantitation of binding. FITC-VT1 B effectively competes for ¹²⁵I-VT1/Gb₃ raft binding. This assay will allow the definition of the optimal raft composition for VT1 (or any other ligand) binding. The effect of several potential cellular raft components are reported. Increased cholesterol content increased VT1 binding. Addition of phosphatidylethanolamine had minimal effect while phosphatidylserine was inhibitory. Although inclusion of sphingomyelin increased the Gb₃ content of the 'raft', reduced VT1 binding was seen. Inclusion of other glycolipids can also be inhibitory. The addition of globotetraosyl ceramide had no effect; however, addition of sulfogalactosyl ceramide, but not sulfogalactoglycerolipid, inhibited VT1/Gb₃ raft binding. These results suggest that certain GSLs can disfavour the formation of the appropriate 'raft' structure for ligand binding and that this is dependent on both their carbohydrate and lipid structure. Such “deceptor” GSLs may provide an as yet, unappreciated mechanism for the regulation of cellular GSL receptor activity. This model is an effective tool to approach the dynamics and ligand-binding specificity of GSL/cholesterol-containing lipid microdomains. Published in 2004.