Novel sterol glucosyltransferase in the animal tissue and cultured cells: Evidence that glucosylceramide as glucose donor

摘要

Cholesteryl glucoside (CG), a membrane glycolipid, regulates heat shock response. CG is rapidly induced by heat shock before the activation of heat shock transcription factor 1 (HSF1) and production of heat shock protein 70 (HSP70), and the addition of CG in turn induces HSF1 activation and HSP70 production in human fibroblasts; thus, a reasonable correlation is that CG functions as a crucial lipid mediator in stress responses in the animal. In this study, we focused on a CG-synthesizing enzyme, animal sterol glucosyltransferase, which has not yet been identified. In this study, we describe a novel type of animal sterol glucosyltransferase in hog stomach and human fibroblasts (TIG-3) detected by a sensitive assay with a fluorescence-labeled substrate. The cationic requirement, inhibitor resistance, and substrate specificity of animal sterol glucosyltransferase were studied. Interestingly, animal sterol glucosyltransferase did not use uridine diphosphate glucose (UDPglucose) as an immediate glucose donor, as has been shown in plants and fungi. Among the glycolipids tested in vitro, glucosylceramide (GlcCer) was the most effective substrate for CG formation in animal tissues and cultured cells. Using chemically synthesized [U-(13)C]Glc-beta-Cer as a glucose donor, we confirmed by mass spectrometry that [U-(13)C]CG was synthesized in hog stomach homogenate. These results suggest that animal sterol glucosyltransferase transfers glucose moiety from GlcCer to cholesterol. Additionally, using GM-95, a mutant B16 melanoma cell line that does not express ceramide glucosyltransferase, we showed that GlcCer is an essential substrate for animal sterol glucosyltransferase in the cell. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.