Assay for the transbilayer distribution of glycolipids: selective oxidation of glucosylceramide to glucuronylceramide by TEMPO nitroxyl radicals

Abstract

In the present study, 2,2,6,6-tetramethylpiperidinooxy nitroxide (TEMPO) has been applied successfully to discriminate between glucosylceramide in the outer and inner leaflets of closed membrane bilayers. The nitroxyl radicals TEMPO and carboxy-TEMPO, once oxidized to nitrosonium ions, are capable of oxidizing residues that contain primary hydroxyl and amino groups. When applied to radiolabeled glucosylceramide in liposomes, oxidation with TEMPO led to an oxidized product that was easily separated from the original lipid by thin-layer chromatography, and that was identified by mass spectrometric analysis as the corresponding acid glucuronylceramide. To test whether oxidation was confined to the external leaflet, TEMPO was applied to large unilamellar vesicles (LUVs) consisting of egg phosphatidylcholine-egg phosphatidylethanolamine-cholesterol 55:5:40 (mol/mol). TEMPO oxidized most radiolabeled phosphatidylethanolamine, whereas carboxy-TEMPO oxidized only half. Hydrolysis by phospholipase A 2 confirmed that 50% of the phosphatidylethanolamine was accessible in the external bilayer leaflet, suggesting that TEMPO penetrated the lipid bilayer and carboxy-TEMPO did not. When applied to LUVs containing Ͻ 1 mol% radiolabeled glucosylceramide or short-chain C 6 -glucosylceramide, carboxy-TEMPO oxidized half the glucosylceramide. However, if surface C 6 -glucosylceramide was first depleted by bovine serum albumin (BSA) (extracting 49 ؎ 1%), 94% of the remaining C 6 -glucosylceramide was resistant to oxidation. Carboxy-TEMPO oxidized glucosylceramide on the surface of LUVs without affecting inner leaflet glucosylceramide. At pH 9.5 and at 0 ؇ C, the reaction reached completion by 20 min.

Glycolipid Transfer Protein Mediated Transfer of Glycosphingolipids between Membranes: A Model for Action Based on Kinetic and Thermodynamic Analyses

“…Because ∆F ∞ max corresponds to the outer leaflet AV-GalCer in the LUV donors, the data are expressed as the fraction of total AV-GalCer present in the donor vesicles (Figure 2B). The maximum value of ∆F max (∆F max max ) is calculated as twice the value of ∆F max ∞ for LUVs, consistent with the transbilayer distributions reported by Sillence et al (38) for GlcCer derivatives in LUVs. The saturation value for LUV is half of the total calculated AV-GalCer pool (∆F max max ).…”

“…Thus, ∆F max ∞ reflects the AV-GalCer pool initially present in the outer leaflet of the donor vesicles. In LUVs, monohexosylceramides distribute uniformly (∼1:1) between the inner and outer leaflets of the POPC vesicle bilayer (38). Because ∆F ∞ max corresponds to the outer leaflet AV-GalCer in the LUV donors, the data are expressed as the fraction of total AV-GalCer present in the donor vesicles (Figure 2B).…”

The fate and function of glycosphingolipid glucosylceramide

“…It is a challenge to find out whether natural GlcCer is a substrate for these transporters. In addition to an assay for the sidedness of GlcCer based on chemical oxidation (Sillence et al 2000), an assay has now been developed making use of GlcCer hydrolysis by the lysosomal glucocerebrosidase and the activator protein saposin C (R. J. Raggers et al, unpublished data).…”