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. 1989 Apr 1;108(4):1387–1396. doi: 10.1083/jcb.108.4.1387

Analysis of the signal for attachment of a glycophospholipid membrane anchor

PMCID: PMC2115511  PMID: 2466848

Abstract

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophospholipid (GPI) membrane anchor. To define this signal we deleted portions of the DAF COOH terminus and expressed the mutant cDNAs it CV1 origin-deficient SV-40 cells. Our results show that the COOH-terminal hydrophobic domain (17 residues) is absolutely required for GPI anchor attachment. However, when fused to the COOH terminus of a secreted protein this hydrophobic domain is insufficient to direct attachment of a GPI anchor. Additional specific information located within the adjacent 20 residues appears to be necessary. We speculate that by analogy with signal sequences for membrane translocation, GPI anchor attachment requires both a COOH- terminal hydrophobic domain (the GPI signal) as well as a suitable cleavage/attachment site located NH2 terminal to the signal.

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Selected References

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  1. Anderson D. J., Blobel G. Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 1983;96:111–120. doi: 10.1016/s0076-6879(83)96012-3. [DOI] [PubMed] [Google Scholar]
  2. Bangs J. D., Andrews N. W., Hart G. W., Englund P. T. Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein. J Cell Biol. 1986 Jul;103(1):255–263. doi: 10.1083/jcb.103.1.255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Boothroyd J. C., Cross G. A., Hoeijmakers J. H., Borst P. A variant surface glycoprotein of Trypanosoma brucei synthesized with a C-terminal hydrophobic 'tail' absent from purified glycoprotein. Nature. 1980 Dec 11;288(5791):624–626. doi: 10.1038/288624a0. [DOI] [PubMed] [Google Scholar]
  6. Caras I. W., Davitz M. A., Rhee L., Weddell G., Martin D. W., Jr, Nussenzweig V. Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins. Nature. 1987 Feb 5;325(6104):545–549. doi: 10.1038/325545a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Cummings R. D., Kornfeld S., Schneider W. J., Hobgood K. K., Tolleshaug H., Brown M. S., Goldstein J. L. Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor. J Biol Chem. 1983 Dec 25;258(24):15261–15273. [PubMed] [Google Scholar]
  9. Davitz M. A., Low M. G., Nussenzweig V. Release of decay-accelerating factor (DAF) from the cell membrane by phosphatidylinositol-specific phospholipase C (PIPLC). Selective modification of a complement regulatory protein. J Exp Med. 1986 May 1;163(5):1150–1161. doi: 10.1084/jem.163.5.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eaton D. L., Wood W. I., Eaton D., Hass P. E., Hollingshead P., Wion K., Mather J., Lawn R. M., Vehar G. A., Gorman C. Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry. 1986 Dec 30;25(26):8343–8347. doi: 10.1021/bi00374a001. [DOI] [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. Ferguson M. A., Low M. G., Cross G. A. Glycosyl-sn-1,2-dimyristylphosphatidylinositol is covalently linked to Trypanosoma brucei variant surface glycoprotein. J Biol Chem. 1985 Nov 25;260(27):14547–14555. [PubMed] [Google Scholar]
  13. 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]
  14. Futerman A. H., Fiorini R. M., Roth E., Low M. G., Silman I. Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase from Torpedo electric organ. Effect of phosphatidylinositol-specific phospholipase C. Biochem J. 1985 Mar 1;226(2):369–377. doi: 10.1042/bj2260369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hemperly J. J., Edelman G. M., Cunningham B. A. cDNA clones of the neural cell adhesion molecule (N-CAM) lacking a membrane-spanning region consistent with evidence for membrane attachment via a phosphatidylinositol intermediate. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9822–9826. doi: 10.1073/pnas.83.24.9822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holder A. A., Cross G. A. Glycopeptides from variant surface glycoproteins of Trypanosoma Brucei. C-terminal location of antigenically cross-reacting carbohydrate moieties. Mol Biochem Parasitol. 1981 Feb;2(3-4):135–150. doi: 10.1016/0166-6851(81)90095-5. [DOI] [PubMed] [Google Scholar]
  17. Kinoshita T., Medof M. E., Nussenzweig V. Endogenous association of decay-accelerating factor (DAF) with C4b and C3b on cell membranes. J Immunol. 1986 May 1;136(9):3390–3395. [PubMed] [Google Scholar]
  18. Lodish H. F. Transport of secretory and membrane glycoproteins from the rough endoplasmic reticulum to the Golgi. A rate-limiting step in protein maturation and secretion. J Biol Chem. 1988 Feb 15;263(5):2107–2110. [PubMed] [Google Scholar]
  19. Low M. G. Biochemistry of the glycosyl-phosphatidylinositol membrane protein anchors. Biochem J. 1987 May 15;244(1):1–13. doi: 10.1042/bj2440001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Low M. G., Kincade P. W. Phosphatidylinositol is the membrane-anchoring domain of the Thy-1 glycoprotein. Nature. 1985 Nov 7;318(6041):62–64. doi: 10.1038/318062a0. [DOI] [PubMed] [Google Scholar]
  21. Low M. G., Saltiel A. R. Structural and functional roles of glycosyl-phosphatidylinositol in membranes. Science. 1988 Jan 15;239(4837):268–275. doi: 10.1126/science.3276003. [DOI] [PubMed] [Google Scholar]
  22. Medof M. E., Kinoshita T., Nussenzweig V. Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J Exp Med. 1984 Nov 1;160(5):1558–1578. doi: 10.1084/jem.160.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Medof M. E., Walter E. I., Roberts W. L., Haas R., Rosenberry T. L. Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid. Biochemistry. 1986 Nov 4;25(22):6740–6747. doi: 10.1021/bi00370a003. [DOI] [PubMed] [Google Scholar]
  24. Micanovic R., Bailey C. A., Brink L., Gerber L., Pan Y. C., Hulmes J. D., Udenfriend S. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1398–1402. doi: 10.1073/pnas.85.5.1398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nicholson-Weller A., Burge J., Fearon D. T., Weller P. F., Austen K. F. Isolation of a human erythrocyte membrane glycoprotein with decay-accelerating activity for C3 convertases of the complement system. J Immunol. 1982 Jul;129(1):184–189. [PubMed] [Google Scholar]
  26. Roberts W. L., Rosenberry T. L. Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase. Biochemistry. 1986 Jun 3;25(11):3091–3098. doi: 10.1021/bi00359a004. [DOI] [PubMed] [Google Scholar]
  27. Russell D. W., Schneider W. J., Yamamoto T., Luskey K. L., Brown M. S., Goldstein J. L. Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell. 1984 Jun;37(2):577–585. doi: 10.1016/0092-8674(84)90388-x. [DOI] [PubMed] [Google Scholar]
  28. Stahl N., Borchelt D. R., Hsiao K., Prusiner S. B. Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell. 1987 Oct 23;51(2):229–240. doi: 10.1016/0092-8674(87)90150-4. [DOI] [PubMed] [Google Scholar]
  29. Stroynowski I., Soloski M., Low M. G., Hood L. A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa-2 antigen: anchoring of the product by a phospholipid tail. Cell. 1987 Aug 28;50(5):759–768. doi: 10.1016/0092-8674(87)90334-5. [DOI] [PubMed] [Google Scholar]
  30. Tse A. G., Barclay A. N., Watts A., Williams A. F. A glycophospholipid tail at the carboxyl terminus of the Thy-1 glycoprotein of neurons and thymocytes. Science. 1985 Nov 29;230(4729):1003–1008. doi: 10.1126/science.2865810. [DOI] [PubMed] [Google Scholar]
  31. Waneck G. L., Sherman D. H., Kincade P. W., Low M. G., Flavell R. A. Molecular mapping of signals in the Qa-2 antigen required for attachment of the phosphatidylinositol membrane anchor. Proc Natl Acad Sci U S A. 1988 Jan;85(2):577–581. doi: 10.1073/pnas.85.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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