title: 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.
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, 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.
authors.affiliation: Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Boston 02114.
authors.forename: L J
authors.initials: LJ
authors.lastname: Thomas
authors.forename: R
authors.initials: R
authors.lastname: DeGasperi
authors.forename: E
authors.initials: E
authors.lastname: Sugiyama
authors.forename: H M
authors.initials: HM
authors.lastname: Chang
authors.forename: P J
authors.initials: PJ
authors.lastname: Beck
authors.forename: P
authors.initials: P
authors.lastname: Orlean
authors.forename: M
authors.initials: M
authors.lastname: Urakaze
authors.forename: T
authors.initials: T
authors.lastname: Kamitani
authors.forename: J F
authors.initials: JF
authors.lastname: Sambrook
authors.forename: C D
authors.initials: CD
authors.lastname: Warren
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D017252:CD3 Complex; D002240:Carbohydrate Sequence; D002460:Cell Line; D005434:Flow Cytometry; D005800:Genes, Fungal; D006017:Glycolipids / Q000096:biosynthesis*; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols; D006649:Histocompatibility Antigens / Q000378:metabolism; D006825:Hybridomas; D017493:Leukocyte Common Antigens; D008213:Lymphocyte Activation; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D011233:Precipitin Tests; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
chemical_list: D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017252:CD3 Complex; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D006649:Histocompatibility Antigens; D010716:Phosphatidylinositols; D011948:Receptors, Antigen, T-Cell; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D017493:Leukocyte Common Antigens