Thomas L J, DeGasperi R, Sugiyama E, Chang H M, Beck P J, Orlean P, Urakaze M, Kamitani T, Sambrook J F, Warren C D
Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston 02114.
J Biol Chem. 1991 Dec 5;266(34):23175-84.
The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.
糖基磷脂酰肌醇（GPI）锚定物可能能够产生多种第二信使，如二酰基甘油、磷脂酸和磷酸肌醇聚糖，据推测它参与包括T细胞在内的多种细胞类型的信号转导。我们已经鉴定出一组在GPI锚定物生物合成的各个步骤存在缺陷的T细胞杂交瘤突变体。由于它们源自一个功能性的T - T杂交瘤，我们能够确定GPI锚定物在T细胞活化中的精确作用。为此分析选择了两个突变体。第一个突变体在GPI锚定物生物合成的第一步存在缺陷，即在将N - 乙酰葡糖胺转移到磷脂酰肌醇受体上存在缺陷。因此，它不能形成任何GPI前体或GPI样化合物。有趣的是，该突变体可以被抗原、超抗原和伴刀豆球蛋白A以与野生型杂交瘤相当的方式激活。这些数据强烈表明，GPI锚定物、其前体或其潜在的裂解产物磷酸肌醇聚糖在T细胞活化的早期阶段不是必需的。第二个突变体能够合成前两个GPI前体，但由于磷酸多萜醇 - 甘露糖（Dol - P - Man）生物合成缺陷而不能向它们添加甘露糖残基。出乎意料的是，尽管T细胞受体表达正常，但所有Dol - P - Man突变体在被抗原、超抗原和伴刀豆球蛋白A激活方面都存在缺陷。在这里，我们表明激活缺陷是由于多效性糖基化异常，因为Dol - P - Man是GPI锚定物和N - 连接寡糖生物合成所必需的。当酵母Dol - P - Man合酶基因稳定转染到突变体中时，表面GPI锚定蛋白的完全表达得以恢复。然而，在不同的转染子中，N - 连接糖基化要么部分要么完全得到纠正。激活缺陷的恢复与N - 连接糖基化的状态密切相关，但与GPI锚定蛋白的表达无关。因此，这些结果揭示了N - 连接糖基化在T细胞活化中的意想不到的作用。
