Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Jan 1;313(Pt 1):253–258. doi: 10.1042/bj3130253

Isolation and characterization of a Chinese hamster ovary (CHO) mutant defective in the second step of glycosylphosphatidylinositol biosynthesis.

V L Stevens 1, H Zhang 1, M Harreman 1
PMCID: PMC1216891  PMID: 8546692

Abstract

Mutant cell lines defective in the biosynthesis of glycosylphosphatidylinositol (GPI) described to date were isolated by selecting cells which no longer expressed one or more endogenous GPI-anchored proteins on their surface. In this study, a new mutant in this pathway was isolated from ethylmethane-sulphonate-mutagenized Chinese hamster ovary cells stably transfected with human placental alkaline phosphatase (PLAP) as a marker of GPI-anchored proteins. A three-step protocol was employed. In the first step, cells with decreased surface expression of PLAP were selected by four rounds of complement-mediated lysis with an anti-(alkaline phosphatase) antibody. The surviving cells were cloned by limiting dilution and those with low levels of total alkaline phosphatase activity were selected in the second step. Finally, the ability of each clone to synthesize the first three intermediates in GPI biosynthesis in vitro was assessed to determine which cells with low alkaline phosphatase activity harboured a defect in one of these reactions. Of 230 potential mutants, one was defective in the second step of GPI biosynthesis. Microsomes from this mutant, designated G9PLAP.85, were completely unable to deacetylate either endogenous GlcNAc-phosphatidylinositol (PI) synthesized from UDP[6-3H]GlcNAc or exogenous GlcNAc-PI added directly to the membranes. Complementation analysis with the Thy-1-deficient murine lymphoma cells demonstrated that G9PLAP.85 has a molecular defect distinct from these previously described mutants. Therefore, these results suggest that mutants in GPI biosynthesis could be selected from almost any cell line expressing a GPI-anchored marker protein.

Full Text

The Full Text of this article is available as a PDF (372.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Bangs J. D., Hereld D., Krakow J. L., Hart G. W., Englund P. T. Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein. Proc Natl Acad Sci U S A. 1985 May;82(10):3207–3211. doi: 10.1073/pnas.82.10.3207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chapman A., Fujimoto K., Kornfeld S. The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose. J Biol Chem. 1980 May 25;255(10):4441–4446. [PubMed] [Google Scholar]
  4. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  5. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  6. Conzelmann A., Spiazzi A., Bron C., Hyman R. No glycolipid anchors are added to Thy-1 glycoprotein in Thy-1-negative mutant thymoma cells of four different complementation classes. Mol Cell Biol. 1988 Feb;8(2):674–678. doi: 10.1128/mcb.8.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Costello L. C., Orlean P. Inositol acylation of a potential glycosyl phosphoinositol anchor precursor from yeast requires acyl coenzyme A. J Biol Chem. 1992 Apr 25;267(12):8599–8603. [PubMed] [Google Scholar]
  8. Doering T. L., Masterson W. J., Englund P. T., Hart G. W. Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine. J Biol Chem. 1989 Jul 5;264(19):11168–11173. [PubMed] [Google Scholar]
  9. Fatemi S. H., Tartakoff A. M. Hydrophilic anchor-deficient Thy-1 is secreted by a class E mutant T lymphoma. Cell. 1986 Aug 29;46(5):653–657. doi: 10.1016/0092-8674(86)90340-5. [DOI] [PubMed] [Google Scholar]
  10. Fatemi S. H., Tartakoff A. M. The phenotype of five classes of T lymphoma mutants. Defective glycophospholipid anchoring, rapid degradation, and secretion of Thy-1 glycoprotein. J Biol Chem. 1988 Jan 25;263(3):1288–1294. [PubMed] [Google Scholar]
  11. Ferguson M. A., Duszenko M., Lamont G. S., Overath P., Cross G. A. Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor. J Biol Chem. 1986 Jan 5;261(1):356–362. [PubMed] [Google Scholar]
  12. Fuhlbrigge R. C., Fine S. M., Unanue E. R., Chaplin D. D. Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5649–5653. doi: 10.1073/pnas.85.15.5649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hirose S., Mohney R. P., Mutka S. C., Ravi L., Singleton D. R., Perry G., Tartakoff A. M., Medof M. E. Derivation and characterization of glycoinositol-phospholipid anchor-defective human K562 cell clones. J Biol Chem. 1992 Mar 15;267(8):5272–5278. [PubMed] [Google Scholar]
  14. Hirose S., Prince G. M., Sevlever D., Ravi L., Rosenberry T. L., Ueda E., Medof M. E. Characterization of putative glycoinositol phospholipid anchor precursors in mammalian cells. Localization of phosphoethanolamine. J Biol Chem. 1992 Aug 25;267(24):16968–16974. [PubMed] [Google Scholar]
  15. Inoue N., Kinoshita T., Orii T., Takeda J. Cloning of a human gene, PIG-F, a component of glycosylphosphatidylinositol anchor biosynthesis, by a novel expression cloning strategy. J Biol Chem. 1993 Apr 5;268(10):6882–6885. [PubMed] [Google Scholar]
  16. Kamitani T., Chang H. M., Rollins C., Waneck G. L., Yeh E. T. Correction of the class H defect in glycosylphosphatidylinositol anchor biosynthesis in Ltk- cells by a human cDNA clone. J Biol Chem. 1993 Oct 5;268(28):20733–20736. [PubMed] [Google Scholar]
  17. Kamitani T., Menon A. K., Hallaq Y., Warren C. D., Yeh E. T. Complexity of ethanolamine phosphate addition in the biosynthesis of glycosylphosphatidylinositol anchors in mammalian cells. J Biol Chem. 1992 Dec 5;267(34):24611–24619. [PubMed] [Google Scholar]
  18. Kinoshita T., Takahashi M., Inoue N., Miyata T., Takeda J. Expression cloning of genes for GPI-anchor biosynthesis. Braz J Med Biol Res. 1994 Feb;27(2):127–132. [PubMed] [Google Scholar]
  19. Krakow J. L., Hereld D., Bangs J. D., Hart G. W., Englund P. T. Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein. J Biol Chem. 1986 Sep 15;261(26):12147–12153. [PubMed] [Google Scholar]
  20. Masterson W. J., Doering T. L., Hart G. W., Englund P. T. A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell. 1989 Mar 10;56(5):793–800. doi: 10.1016/0092-8674(89)90684-3. [DOI] [PubMed] [Google Scholar]
  21. Mayor S., Menon A. K., Cross G. A., Ferguson M. A., Dwek R. A., Rademacher T. W. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. I. Can structure of the phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids. J Biol Chem. 1990 Apr 15;265(11):6164–6173. [PubMed] [Google Scholar]
  22. Mayor S., Menon A. K., Cross G. A. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids. J Biol Chem. 1990 Apr 15;265(11):6174–6181. [PubMed] [Google Scholar]
  23. McConville M. J., Ferguson M. A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J. 1993 Sep 1;294(Pt 2):305–324. doi: 10.1042/bj2940305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Menon A. K., Eppinger M., Mayor S., Schwarz R. T. Phosphatidylethanolamine is the donor of the terminal phosphoethanolamine group in trypanosome glycosylphosphatidylinositols. EMBO J. 1993 May;12(5):1907–1914. doi: 10.1002/j.1460-2075.1993.tb05839.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Menon A. K., Mayor S., Ferguson M. A., Duszenko M., Cross G. A. Candidate glycophospholipid precursor for the glycosylphosphatidylinositol membrane anchor of Trypanosoma brucei variant surface glycoproteins. J Biol Chem. 1988 Feb 5;263(4):1970–1977. [PubMed] [Google Scholar]
  26. Menon A. K., Mayor S., Schwarz R. T. Biosynthesis of glycosyl-phosphatidylinositol lipids in Trypanosoma brucei: involvement of mannosyl-phosphoryldolichol as the mannose donor. EMBO J. 1990 Dec;9(13):4249–4258. doi: 10.1002/j.1460-2075.1990.tb07873.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Menon A. K., Schwarz R. T., Mayor S., Cross G. A. Cell-free synthesis of glycosyl-phosphatidylinositol precursors for the glycolipid membrane anchor of Trypanosoma brucei variant surface glycoproteins. Structural characterization of putative biosynthetic intermediates. J Biol Chem. 1990 Jun 5;265(16):9033–9042. [PubMed] [Google Scholar]
  28. Milne K. G., Field R. A., Masterson W. J., Cottaz S., Brimacombe J. S., Ferguson M. A. Partial purification and characterization of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes. J Biol Chem. 1994 Jun 10;269(23):16403–16408. [PubMed] [Google Scholar]
  29. Miyata T., Takeda J., Iida Y., Yamada N., Inoue N., Takahashi M., Maeda K., Kitani T., Kinoshita T. The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science. 1993 Feb 26;259(5099):1318–1320. doi: 10.1126/science.7680492. [DOI] [PubMed] [Google Scholar]
  30. Miyata T., Yamada N., Iida Y., Nishimura J., Takeda J., Kitani T., Kinoshita T. Abnormalities of PIG-A transcripts in granulocytes from patients with paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1994 Jan 27;330(4):249–255. doi: 10.1056/NEJM199401273300404. [DOI] [PubMed] [Google Scholar]
  31. Mohney R. P., Knez J. J., Ravi L., Sevlever D., Rosenberry T. L., Hirose S., Medof M. E. Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants. J Biol Chem. 1994 Mar 4;269(9):6536–6542. [PubMed] [Google Scholar]
  32. Nair B. C., Majeska R. J., Rodan G. A. Rat alkaline phosphatase. I. Purification and characterization of the enzyme from osteosarcoma: generation of monoclonal and polyclonal antibodies. Arch Biochem Biophys. 1987 Apr;254(1):18–27. doi: 10.1016/0003-9861(87)90076-2. [DOI] [PubMed] [Google Scholar]
  33. Orlean P. Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O mannosylation, and N glycosylation of protein in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Nov;10(11):5796–5805. doi: 10.1128/mcb.10.11.5796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Puoti A., Conzelmann A. Characterization of abnormal free glycophosphatidylinositols accumulating in mutant lymphoma cells of classes B, E, F, and H. J Biol Chem. 1993 Apr 5;268(10):7215–7224. [PubMed] [Google Scholar]
  35. Puoti A., Conzelmann A. Structural characterization of free glycolipids which are potential precursors for glycophosphatidylinositol anchors in mouse thymoma cell lines. J Biol Chem. 1992 Nov 5;267(31):22673–22680. [PubMed] [Google Scholar]
  36. Puoti A., Desponds C., Fankhauser C., Conzelmann A. Characterization of glycophospholipid intermediate in the biosynthesis of glycophosphatidylinositol anchors accumulating in the Thy-1-negative lymphoma line SIA-b. J Biol Chem. 1991 Nov 5;266(31):21051–21059. [PubMed] [Google Scholar]
  37. Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. [DOI] [PubMed] [Google Scholar]
  38. Stevens V. L., Raetz C. R. Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants. J Biol Chem. 1991 Jun 5;266(16):10039–10042. [PubMed] [Google Scholar]
  39. Stevens V. L. Regulation of glycosylphosphatidylinositol biosynthesis by GTP. Stimulation of N-acetylglucosamine-phosphatidylinositol deacetylation. J Biol Chem. 1993 May 5;268(13):9718–9724. [PubMed] [Google Scholar]
  40. Stevens V. L., Zhang H. Coenzyme A dependence of glycosylphosphatidylinositol biosynthesis in a mammalian cell-free system. J Biol Chem. 1994 Dec 16;269(50):31397–31403. [PubMed] [Google Scholar]
  41. Sugiyama E., DeGasperi R., Urakaze M., Chang H. M., Thomas L. J., Hyman R., Warren C. D., Yeh E. T. Identification of defects in glycosylphosphatidylinositol anchor biosynthesis in the Thy-1 expression mutants. J Biol Chem. 1991 Jul 5;266(19):12119–12122. [PubMed] [Google Scholar]
  42. Takeda J., Miyata T., Kawagoe K., Iida Y., Endo Y., Fujita T., Takahashi M., Kitani T., Kinoshita T. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993 May 21;73(4):703–711. doi: 10.1016/0092-8674(93)90250-t. [DOI] [PubMed] [Google Scholar]
  43. Trowbridge I. S., Hyman R., Mazauskas C. The synthesis and properties of T25 blycoprotein in Thy-1-negative mutant lymphoma cells. Cell. 1978 May;14(1):21–32. doi: 10.1016/0092-8674(78)90297-0. [DOI] [PubMed] [Google Scholar]
  44. Urakaze M., Kamitani T., DeGasperi R., Sugiyama E., Chang H. M., Warren C. D., Yeh E. T. Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells. J Biol Chem. 1992 Apr 5;267(10):6459–6462. [PubMed] [Google Scholar]
  45. Worton R. G. Karyotypic heterogeneity in CHO cell lines. Cytogenet Cell Genet. 1978;21(1-2):105–110. doi: 10.1159/000130883. [DOI] [PubMed] [Google Scholar]
  46. Yoon K., Thiede M. A., Rodan G. A. Alkaline phosphatase as a reporter enzyme. Gene. 1988 Jun 15;66(1):11–17. doi: 10.1016/0378-1119(88)90220-x. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES