Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation

L. J. Thomas, R. DeGasperi, E. Sugiyama, H. M. Chang, P. J. Beck, P. Orlean, M. Urakaze, T. Kamitani, J. F. Sambrook, C. D. Warren, E. T.H. Yeh

Research output: Contribution to journalArticlepeer-review

Abstract

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, superantigen, 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.

Original languageEnglish (US)
Pages (from-to)23175-23184
Number of pages10
JournalJournal of Biological Chemistry
Volume266
Issue number34
StatePublished - 1991
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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