Akiyama Hisako, Sasaki Narie, Hanazawa Shuwa, Gotoh Mari, Kobayashi Susumu, Hirabayashi Yoshio, Murakami-Murofushi Kimiko
Graduate School of Humanities and Sciences, Department of Life Science, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan.
Biochim Biophys Acta. 2011 May;1811(5):314-22. doi: 10.1016/j.bbalip.2011.02.005. Epub 2011 Mar 17.
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 (UDP-glucose) 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-β-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.
胆固醇葡糖苷（CG）是一种膜糖脂，可调节热休克反应。在热休克转录因子1（HSF1）激活和热休克蛋白70（HSP70）产生之前，热休克可迅速诱导CG生成，而在人成纤维细胞中添加CG反过来又会诱导HSF1激活和HSP70产生；因此，合理的关联是CG在动物应激反应中作为一种关键的脂质介质发挥作用。在本研究中，我们聚焦于一种尚未被鉴定的CG合成酶——动物固醇葡糖基转移酶。在本研究中，我们描述了一种通过用荧光标记底物进行的灵敏检测法在猪胃和人成纤维细胞（TIG-3）中检测到的新型动物固醇葡糖基转移酶。研究了动物固醇葡糖基转移酶的阳离子需求、抑制剂抗性和底物特异性。有趣的是，动物固醇葡糖基转移酶不像植物和真菌中那样使用尿苷二磷酸葡萄糖（UDP-葡萄糖）作为直接的葡萄糖供体。在体外测试的糖脂中，葡糖神经酰胺（GlcCer）是动物组织和培养细胞中CG形成的最有效底物。使用化学合成的[U-（（13））C]Glc-β-Cer作为葡萄糖供体，我们通过质谱法证实了[U-（（13））C]CG在猪胃匀浆中合成。这些结果表明动物固醇葡糖基转移酶将葡萄糖部分从GlcCer转移到胆固醇上。此外，使用不表达神经酰胺葡糖基转移酶的突变B16黑色素瘤细胞系GM-95，我们表明GlcCer是细胞中动物固醇葡糖基转移酶的必需底物。
