Azzouz N, Gerold P, Kedees M H, Shams-Eldin H, Werner R, Capdeville Y, Schwarz R T
Zentrum für Hygiene und Medizinische Mikrobiologie, Philipps-Universität Marburg, Robert-Koch-Strasse 17, 35037 Marburg, Germany.
Biochimie. 2001 Aug;83(8):801-9. doi: 10.1016/s0300-9084(01)01317-7.
A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Man alpha 1-2Man alpha 1-6(mannosylphosphate) Man alpha 1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTP gamma S (guanosine 5-O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTP gamma S inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory 'G' proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTP gamma S interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.
已经描述了一组糖基肌醇磷酸神经酰胺，它们属于在由自由生活的原生动物双小核草履虫制备的无细胞系统中合成的糖基磷脂酰肌醇（GPI）家族。最终的GPI前体被鉴定并进行了结构表征：乙醇胺磷酸-6甘露糖α1-2甘露糖α1-6（甘露糖磷酸）甘露糖α1-4葡糖胺-肌醇-磷酸-神经酰胺。在我们对酸不稳定修饰（额外的甘露糖磷酸取代）生物合成的研究中，我们观察到使用三磷酸核苷酸类似物GTPγS（鸟苷5'-O-（硫代三磷酸））会阻断甘露糖基化GPI糖脂的生物合成。我们表明GTPγS抑制了多萜醇磷酸甘露糖的合成，而多萜醇磷酸甘露糖是GPI生物合成中甘露糖残基的供体。因此，我们使用霍乱毒素和百日咳毒素以及细胞内第二信使cAMP类似物8-溴-cAMP研究了GTP结合调节“G”蛋白的作用。获得的所有数据表明经典异源三聚体G蛋白通过激活腺苷酸环化酶和蛋白质磷酸化，经多萜醇磷酸甘露糖合成参与GPI锚定生物合成的调节。此外，我们的数据表明GTPγS干扰了单磷酸多萜醇的合成，表明多萜醇激酶受异源三聚体G蛋白调节。
