Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1992 Apr;89(4):1172–1177. doi: 10.1172/JCI115700

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

L J Thomas 1, M Urakaze 1, R DeGasperi 1, T Kamitani 1, E Sugiyama 1, H M Chang 1, C D Warren 1, E T Yeh 1
PMCID: PMC442976  PMID: 1532587

Abstract

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Full text

PDF
1172

Images in this article

1174Image
on p.1174
1174Image
on p.1174

Selected References

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

  1. Beck P. J., Orlean P., Albright C., Robbins P. W., Gething M. J., Sambrook J. F. The Saccharomyces cerevisiae DPM1 gene encoding dolichol-phosphate-mannose synthase is able to complement a glycosylation-defective mammalian cell line. Mol Cell Biol. 1990 Sep;10(9):4612–4622. doi: 10.1128/mcb.10.9.4612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berger J., Howard A. D., Brink L., Gerber L., Hauber J., Cullen B. R., Udenfriend S. COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein. J Biol Chem. 1988 Jul 15;263(20):10016–10021. [PubMed] [Google Scholar]
  3. Blaas P., Berger B., Weber S., Peter H. H., Hänsch G. M. Paroxysmal nocturnal hemoglobinuria. Enhanced stimulation of platelets by the terminal complement components is related to the lack of C8bp in the membrane. J Immunol. 1988 May 1;140(9):3045–3051. [PubMed] [Google Scholar]
  4. Brimijoin S., Hammond P. I., Petitt R. M. Paroxysmal nocturnal hemoglobinuria: erythrocyte acetylcholinesterase deficit analyzed by immunoassay and fluorescence-activated sorting. Mayo Clin Proc. 1986 Jul;61(7):522–529. doi: 10.1016/s0025-6196(12)61999-5. [DOI] [PubMed] [Google Scholar]
  5. Burroughs S. F., Devine D. V., Browne G., Kaplan M. E. The population of paroxysmal nocturnal hemoglobinuria neutrophils deficient in decay-accelerating factor is also deficient in alkaline phosphatase. Blood. 1988 Apr;71(4):1086–1089. [PubMed] [Google Scholar]
  6. Caras I. W., Weddell G. N., Davitz M. A., Nussenzweig V., Martin D. W., Jr Signal for attachment of a phospholipid membrane anchor in decay accelerating factor. Science. 1987 Nov 27;238(4831):1280–1283. doi: 10.1126/science.2446389. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Conzelmann A., Spiazzi A., Hyman R., Bron C. Anchoring of membrane proteins via phosphatidylinositol is deficient in two classes of Thy-1 negative mutant lymphoma cells. EMBO J. 1986 Dec 1;5(12):3291–3296. doi: 10.1002/j.1460-2075.1986.tb04642.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davignon D., Martz E., Reynolds T., Kürzinger K., Springer T. A. Lymphocyte function-associated antigen 1 (LFA-1): a surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4535–4539. doi: 10.1073/pnas.78.7.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. DeGasperi R., Thomas L. J., Sugiyama E., Chang H. M., Beck P. J., Orlean P., Albright C., Waneck G., Sambrook J. F., Warren C. D. Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene. Science. 1990 Nov 16;250(4983):988–991. doi: 10.1126/science.1978413. [DOI] [PubMed] [Google Scholar]
  11. Doering T. L., Masterson W. J., Hart G. W., Englund P. T. Biosynthesis of glycosyl phosphatidylinositol membrane anchors. J Biol Chem. 1990 Jan 15;265(2):611–614. [PubMed] [Google Scholar]
  12. Edberg J. C., Salmon J. E., Whitlow M., Kimberly R. P. Preferential expression of human Fc gamma RIIIPMN (CD16) in paroxysmal nocturnal hemoglobinuria. Discordant expression of glycosyl phosphatidylinositol-linked proteins. J Clin Invest. 1991 Jan;87(1):58–67. doi: 10.1172/JCI115001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Ferguson M. A., Williams A. F. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem. 1988;57:285–320. doi: 10.1146/annurev.bi.57.070188.001441. [DOI] [PubMed] [Google Scholar]
  17. Hirose S., Ravi L., Hazra S. V., Medof M. E. Assembly and deacetylation of N-acetylglucosaminyl-plasmanylinositol in normal and affected paroxysmal nocturnal hemoglobinuria cells. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3762–3766. doi: 10.1073/pnas.88.9.3762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. LeClair K. P., Palfree R. G., Flood P. M., Hammerling U., Bothwell A. Isolation of a murine Ly-6 cDNA reveals a new multigene family. EMBO J. 1986 Dec 1;5(12):3227–3234. doi: 10.1002/j.1460-2075.1986.tb04633.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leo O., Foo M., Sachs D. H., Samelson L. E., Bluestone J. A. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1374–1378. doi: 10.1073/pnas.84.5.1374. [DOI] [PMC free article] [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. 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]
  24. Micanovic R., Kodukula K., Gerber L. D., Udenfriend S. Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7939–7943. doi: 10.1073/pnas.87.20.7939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moran P., Raab H., Kohr W. J., Caras I. W. Glycophospholipid membrane anchor attachment. Molecular analysis of the cleavage/attachment site. J Biol Chem. 1991 Jan 15;266(2):1250–1257. [PubMed] [Google Scholar]
  26. Nicholson-Weller A., Spicer D. B., Austen K. F. Deficiency of the complement regulatory protein, "decay-accelerating factor," on membranes of granulocytes, monocytes, and platelets in paroxysmal nocturnal hemoglobinuria. N Engl J Med. 1985 Apr 25;312(17):1091–1097. doi: 10.1056/NEJM198504253121704. [DOI] [PubMed] [Google Scholar]
  27. Parekh R. B., Tse A. G., Dwek R. A., Williams A. F., Rademacher T. W. Tissue-specific N-glycosylation, site-specific oligosaccharide patterns and lentil lectin recognition of rat Thy-1. EMBO J. 1987 May;6(5):1233–1244. doi: 10.1002/j.1460-2075.1987.tb02359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reiser H., Coligan J., Palmer E., Benacerraf B., Rock K. L. Cloning and expression of a cDNA for the T-cell-activating protein TAP. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2255–2259. doi: 10.1073/pnas.85.7.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rock K. L., Yeh E. T., Gramm C. F., Haber S. I., Reiser H., Benacerraf B. TAP, a novel T cell-activating protein involved in the stimulation of MHC-restricted T lymphocytes. J Exp Med. 1986 Feb 1;163(2):315–333. doi: 10.1084/jem.163.2.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rosse W. F. Paroxysmal nocturnal hemoglobinuria and decay-accelerating factor. Annu Rev Med. 1990;41:431–436. doi: 10.1146/annurev.me.41.020190.002243. [DOI] [PubMed] [Google Scholar]
  31. Selvaraj P., Dustin M. L., Silber R., Low M. G., Springer T. A. Deficiency of lymphocyte function-associated antigen 3 (LFA-3) in paroxysmal nocturnal hemoglobinuria. Functional correlates and evidence for a phosphatidylinositol membrane anchor. J Exp Med. 1987 Oct 1;166(4):1011–1025. doi: 10.1084/jem.166.4.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Singh N., Tartakoff A. M. Two different mutants blocked in synthesis of dolichol-phosphoryl-mannose do not add glycophospholipid anchors to membrane proteins: quantitative correction of the phenotype of a CHO cell mutant with tunicamycin. Mol Cell Biol. 1991 Jan;11(1):391–400. doi: 10.1128/mcb.11.1.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Stafford H. A., Tykocinski M. L., Lublin D. M., Holers V. M., Rosse W. F., Atkinson J. P., Medof M. E. Normal polymorphic variations and transcription of the decay accelerating factor gene in paroxysmal nocturnal hemoglobinuria cells. Proc Natl Acad Sci U S A. 1988 Feb;85(3):880–884. doi: 10.1073/pnas.85.3.880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. Waneck G. L., Stein M. E., Flavell R. A. Conversion of a PI-anchored protein to an integral membrane protein by a single amino acid mutation. Science. 1988 Aug 5;241(4866):697–699. doi: 10.1126/science.3399901. [DOI] [PubMed] [Google Scholar]
  37. Yeh E. T., Reiser H., Bamezai A., Rock K. L. TAP transcription and phosphatidylinositol linkage mutants are defective in activation through the T cell receptor. Cell. 1988 Mar 11;52(5):665–674. doi: 10.1016/0092-8674(88)90404-7. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES