Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants

• Published in: The Journal of biological chemistry Vol. 266; no. 16; pp. 10039 - 10042
• Main Authors: STEVENS, V. L, RAETZ, C. R. H
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.06.1991
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Autoradiography; Biological and medical sciences; Cell-Free System; Chromatography, Thin Layer; Fundamental and applied biological sciences. Psychology; Glycerolipids, phospholipids; Glycolipids - biosynthesis; glycosylphosphatidylinositol; Glycosylphosphatidylinositols; Lipids; Lymphoma - genetics; Lymphoma - metabolism; Lymphoma - pathology; Mutation; Other biological molecules; Phosphatidylinositols - biosynthesis; Phospholipids - pharmacology; Trypanosoma brucei brucei; Tumor Cells, Cultured; Cell culture; Rodentia; Biosynthesis; Glycosylation; In vitro; Lymphoma; Phosphatidylinositol; Vertebrata; Mammalia; Cell line; Mouse; Mutation; Mechanism of action
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)99182-9

The glycosyl phosphatidylinositol (GPI) anchors that attach certain proteins to membranes are preassembled by sequential addition of glycan components to phosphatidylinositol (PI) before being transferred to nascent polypeptide. A cell-free system consisting of trypanosome membranes has been reported to catalyze GPI biosynthesis (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800; Menon, A. K., Schwarz, R. T., Mayor, S., and Cross, G. A. M. (1990) J. Biol. Chem. 265, 9033-9042). We now describe conditions for studying the initial steps of GPI biosynthesis in extracts of murine lymphoma cells. Two chloroform-soluble products, tentatively identified as [6-3H]GlcNAc-PI and [6-3H]GlcN-PI were generated during incubations of EL4 cell lysates with UDP-[6-3H]GlcNAc. The involvement of PI in the reaction was established by the sensitivity of the products to hydrolysis by PI-specific phospholipase C and the finding that the addition of exogenous PI to the incubation stimulated the reaction. The minor, more polar product was sensitive to nitrous acid cleavage and was converted to the major product, as judged by TLC, after treatment with acetic anhydride. The glycolipids generated in lymphoma extracts appeared to be the same as the products produced in parallel incubations with trypanosome membranes. Analysis of available lymphoma mutants deficient in Thy-1 surface expression revealed that extracts of the class A, C, and H mutants are completely defective in synthesizing GlcNAc-PI and GlcN-PI.