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. 1988 Dec 1;107(6):2059–2073. doi: 10.1083/jcb.107.6.2059

Addition of carbohydrate side chains at novel sites on influenza virus hemagglutinin can modulate the folding, transport, and activity of the molecule

PMCID: PMC2115654  PMID: 2461945

Abstract

We have constructed and expressed a series of mutant influenza virus hemagglutinins, each containing a new consensus site for glycosylation in addition to the seven sites found on the wild-type protein. Oligosaccharide side chains were added with high efficiency at four of the five novel sites, located on areas of the protein's surface that are not normally shielded by carbohydrate. Investigations of the structure, intracellular transport, and biological activities of the mutant hemagglutinin molecules indicated that (a) supernumerary carbohydrate side chains can be used to shield or disrupt functional epitopes on the surface of hemagglutinin, and (b) the presence of an additional oligosaccharide may cause temperature-dependent defects in the transport of the glycoprotein. We discuss the addition of supernumerary oligosaccharides as a general tool for shielding chosen areas of the surface of proteins that enter or traverse the secretory pathway.

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Selected References

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  1. Air G. M., Laver W. G., Webster R. G. Antigenic variation in influenza viruses. Contrib Microbiol Immunol. 1987;8:20–59. [PubMed] [Google Scholar]
  2. Ash J. F., Louvard D., Singer S. J. Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5584–5588. doi: 10.1073/pnas.74.12.5584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bause E. Structural requirements of N-glycosylation of proteins. Studies with proline peptides as conformational probes. Biochem J. 1983 Feb 1;209(2):331–336. doi: 10.1042/bj2090331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blobel G. Intracellular protein topogenesis. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1496–1500. doi: 10.1073/pnas.77.3.1496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bole D. G., Hendershot L. M., Kearney J. F. Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J Cell Biol. 1986 May;102(5):1558–1566. doi: 10.1083/jcb.102.5.1558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Caton A. J., Brownlee G. G., Yewdell J. W., Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell. 1982 Dec;31(2 Pt 1):417–427. doi: 10.1016/0092-8674(82)90135-0. [DOI] [PubMed] [Google Scholar]
  7. Doms R. W., Helenius A. Quaternary structure of influenza virus hemagglutinin after acid treatment. J Virol. 1986 Dec;60(3):833–839. doi: 10.1128/jvi.60.3.833-839.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Doyle C., Roth M. G., Sambrook J., Gething M. J. Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport. J Cell Biol. 1985 Mar;100(3):704–714. doi: 10.1083/jcb.100.3.704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Doyle C., Sambrook J., Gething M. J. Analysis of progressive deletions of the transmembrane and cytoplasmic domains of influenza hemagglutinin. J Cell Biol. 1986 Oct;103(4):1193–1204. doi: 10.1083/jcb.103.4.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elbein A. D. Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annu Rev Biochem. 1987;56:497–534. doi: 10.1146/annurev.bi.56.070187.002433. [DOI] [PubMed] [Google Scholar]
  11. Fitting T., Kabat D. Evidence for a glycoprotein "signal" involved in transport between subcellular organelles. Two membrane glycoproteins encoded by murine leukemia virus reach the cell surface at different rates. J Biol Chem. 1982 Dec 10;257(23):14011–14017. [PubMed] [Google Scholar]
  12. Gething M. J., Doms R. W., York D., White J. Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus. J Cell Biol. 1986 Jan;102(1):11–23. doi: 10.1083/jcb.102.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  14. Gething M. J., Sambrook J. Cell-surface expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene. Nature. 1981 Oct 22;293(5834):620–625. doi: 10.1038/293620a0. [DOI] [PubMed] [Google Scholar]
  15. Gething M. J., Sambrook J. Construction of influenza haemagglutinin genes that code for intracellular and secreted forms of the protein. Nature. 1982 Dec 16;300(5893):598–603. doi: 10.1038/300598a0. [DOI] [PubMed] [Google Scholar]
  16. Gibson R., Schlesinger S., Kornfeld S. The nonglycosylated glycoprotein of vesicular stomatitis virus is temperature-sensitive and undergoes intracellular aggregation at elevated temperatures. J Biol Chem. 1979 May 10;254(9):3600–3607. [PubMed] [Google Scholar]
  17. Guan J. L., Cao H., Rose J. K. Cell-surface expression of a membrane-anchored form of the human chorionic gonadotropin alpha subunit. J Biol Chem. 1988 Apr 15;263(11):5306–5313. [PubMed] [Google Scholar]
  18. Guan J. L., Machamer C. E., Rose J. K. Glycosylation allows cell-surface transport of an anchored secretory protein. Cell. 1985 Sep;42(2):489–496. doi: 10.1016/0092-8674(85)90106-0. [DOI] [PubMed] [Google Scholar]
  19. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  20. Keil W., Niemann H., Schwarz R. T., Klenk H. D. Carbohydrates of influenza virus. V. Oligosaccharides attached to individual glycosylation sites of the hemagglutinin of fowl plague virus. Virology. 1984 Feb;133(1):77–91. doi: 10.1016/0042-6822(84)90427-6. [DOI] [PubMed] [Google Scholar]
  21. Machamer C. E., Florkiewicz R. Z., Rose J. K. A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface. Mol Cell Biol. 1985 Nov;5(11):3074–3083. doi: 10.1128/mcb.5.11.3074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Machamer C. E., Rose J. K. Influence of new glycosylation sites on expression of the vesicular stomatitis virus G protein at the plasma membrane. J Biol Chem. 1988 Apr 25;263(12):5948–5954. [PubMed] [Google Scholar]
  23. Machamer C. E., Rose J. K. Vesicular stomatitis virus G proteins with altered glycosylation sites display temperature-sensitive intracellular transport and are subject to aberrant intermolecular disulfide bonding. J Biol Chem. 1988 Apr 25;263(12):5955–5960. [PubMed] [Google Scholar]
  24. Matzuk M. M., Boime I. The role of the asparagine-linked oligosaccharides of the alpha subunit in the secretion and assembly of human chorionic gonadotrophin. J Cell Biol. 1988 Apr;106(4):1049–1059. doi: 10.1083/jcb.106.4.1049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miyazaki J., Appella E., Zhao H., Forman J., Ozato K. Expression and function of a nonglycosylated major histocompatibility class I antigen. J Exp Med. 1986 Apr 1;163(4):856–871. doi: 10.1084/jem.163.4.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nakamura K., Compans R. W. Host cell- and virus strain-dependent differences in oligosaccharides of hemagglutinin glycoproteins of influenza A viruses. Virology. 1979 May;95(1):8–23. doi: 10.1016/0042-6822(79)90397-0. [DOI] [PubMed] [Google Scholar]
  27. Owen M. J., Kissonerghis A. M., Lodish H. F. Biosynthesis of HLA-A and HLA-B antigens in vivo. J Biol Chem. 1980 Oct 25;255(20):9678–9684. [PubMed] [Google Scholar]
  28. Poruchynsky M. S., Tyndall C., Both G. W., Sato F., Bellamy A. R., Atkinson P. H. Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein. J Cell Biol. 1985 Dec;101(6):2199–2209. doi: 10.1083/jcb.101.6.2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Raymond F. L., Caton A. J., Cox N. J., Kendal A. P., Brownlee G. G. The antigenicity and evolution of influenza H1 haemagglutinin, from 1950-1957 and 1977-1983: two pathways from one gene. Virology. 1986 Jan 30;148(2):275–287. doi: 10.1016/0042-6822(86)90325-9. [DOI] [PubMed] [Google Scholar]
  30. Rodriguez Boulan E., Pendergast M. Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells. Cell. 1980 May;20(1):45–54. doi: 10.1016/0092-8674(80)90233-0. [DOI] [PubMed] [Google Scholar]
  31. Santos-Aguado J., Biro P. A., Fuhrmann U., Strominger J. L., Barbosa J. A. Amino acid sequences in the alpha 1 domain and not glycosylation are important in HLA-A2/beta 2-microglobulin association and cell surface expression. Mol Cell Biol. 1987 Mar;7(3):982–990. doi: 10.1128/mcb.7.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schuy W., Will C., Kuroda K., Scholtissek C., Garten W., Klenk H. D. Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus. EMBO J. 1986 Nov;5(11):2831–2836. doi: 10.1002/j.1460-2075.1986.tb04576.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Skehel J. J., Bayley P. M., Brown E. B., Martin S. R., Waterfield M. D., White J. M., Wilson I. A., Wiley D. C. Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc Natl Acad Sci U S A. 1982 Feb;79(4):968–972. doi: 10.1073/pnas.79.4.968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Skehel J. J., Stevens D. J., Daniels R. S., Douglas A. R., Knossow M., Wilson I. A., Wiley D. C. A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1779–1783. doi: 10.1073/pnas.81.6.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Webster R. G., Brown L. E., Jackson D. C. Changes in the antigenicity of the hemagglutinin molecule of H3 influenza virus at acidic pH. Virology. 1983 Apr 30;126(2):587–599. doi: 10.1016/s0042-6822(83)80015-4. [DOI] [PubMed] [Google Scholar]
  36. White J., Helenius A., Gething M. J. Haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion. Nature. 1982 Dec 16;300(5893):658–659. doi: 10.1038/300658a0. [DOI] [PubMed] [Google Scholar]
  37. Wiley D. C., Skehel J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem. 1987;56:365–394. doi: 10.1146/annurev.bi.56.070187.002053. [DOI] [PubMed] [Google Scholar]
  38. Wiley D. C., Wilson I. A., Skehel J. J. Structural identification of the antibody-binding sites of Hong Kong influenza haemagglutinin and their involvement in antigenic variation. Nature. 1981 Jan 29;289(5796):373–378. doi: 10.1038/289373a0. [DOI] [PubMed] [Google Scholar]
  39. Wilson I. A., Ladner R. C., Skehel J. J., Wiley D. C. The structure and role of the carbohydrate moieties of influenza virus haemagglutinin. Biochem Soc Trans. 1983 Apr;11(Pt 2):145–147. [PubMed] [Google Scholar]
  40. Wilson I. A., Skehel J. J., Wiley D. C. Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature. 1981 Jan 29;289(5796):366–373. doi: 10.1038/289366a0. [DOI] [PubMed] [Google Scholar]
  41. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. Methods Enzymol. 1983;100:468–500. doi: 10.1016/0076-6879(83)00074-9. [DOI] [PubMed] [Google Scholar]
  42. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. DNA. 1984 Dec;3(6):479–488. doi: 10.1089/dna.1.1984.3.479. [DOI] [PubMed] [Google Scholar]

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