Glycoconjugate Journal

, Volume 29, Issue 1, pp 47–55 | Cite as

Asn54-linked glycan is critical for functional folding of intercellular adhesion molecule-5

  • Tomohiro Ohgomori
  • Tomohisa Nanao
  • Akinori Morita
  • Masahiko Ikekita


Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized type I membrane glycoprotein, and promotes dendritic filopodia formation. Although we have determined the N-glycan structures of ICAM-5 in a previous report, their function is unknown. Here, we produced fifteen ICAM-5 gene constructs, in which each potential N-glycosylation site was mutated, to elucidate the function of the N-glycans of ICAM-5, and observed the effects of transfection of them on a neuronal cell line, Neuro-2a (N2a). Only the N54Q mutant, which is the mutant for the most N-terminal glycosylation site, failed to induce filopodia-like protrusions in N2a cells. Immunofluorescence staining and cell surface biotinylation revealed that N54Q ICAM-5 was confined to the ER and also could not be expressed on the cell surface. This is further supported by the biochemical evidence that almost all N-glycans of N54Q ICAM-5 were digested by Endo glycosidase H and peptide:N-glycanase, indicating that almost all of them retain high-mannose-type structures in ER. In additon, it also failed to form disulfide bonds or functional protein complexes. The stable transformants of N54Q ICAM-5 showed retarded cell growth, but it was interesting that there was no apparent ER stress, because the mutant was sequentially degraded via ER associated degradation pathway by comparing the susceptibilities of the responses to various inhibitors of this pathway in wild-type and N54Q ICAM-5 transfectants. Taken together, the Asn54-linked glycan is necessary for normal trafficking and function of ICAM-5, but is unassociated with ER-associated degradation of it.


ICAM-5 N-glycan Site-directed mutagenesis Protein trafficking 

Supplementary material

10719_2011_9363_MOESM1_ESM.pdf (47 kb)
Fig. S1 DNA contents of parental N2a cells and, stable transfectannts expressing empty vector (mock), WT ICAM-5, or the N54Q mutant. (PDF 46 kb)
10719_2011_9363_MOESM2_ESM.pdf (39 kb)
Fig. S2 Amino-terminal N-glycan-deficient ICAM-1 is expressed on cell surfaces. (PDF 39 kb)
10719_2011_9363_MOESM3_ESM.pdf (35 kb)
Supplementary data 3 Supplemental materials and methods. (PDF 34 kb)


  1. 1.
    Dustin, M.L., Rothlein, R., Bhan, A.K., Dinarello, C.A., Springer, T.A.: Introduction by IL-1 and interferon-gamma – tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J. Immunol. 137, 245–254 (1986)PubMedGoogle Scholar
  2. 2.
    Marlin, S.D., Springer, T.A.: Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 51, 813–819 (1987)PubMedCrossRefGoogle Scholar
  3. 3.
    Nortarmo, P., Salcedo, R., Timonen, T., Patarroyo, M., Gahmberg, C.G.: A monoclonal antibody to the human leukocyte adhesion molecule ICAM-2. Cellular distribution and molecular characterization of the antigen. J. Immunol. 176, 2530–2535 (1991)Google Scholar
  4. 4.
    Vazeux, R., Hoffman, P.A., Tomita, J.K., Dickinson, E.S., Jasman, R.L., Stjohn, T., Gallatin, W.M.: Cloning and characterization of a new intercellular-adhesion molecule ICAM-R. Nature 360, 485–488 (1992)PubMedCrossRefGoogle Scholar
  5. 5.
    Hernandezcaselles, T., Rubio, G., Campanero, M.R., Delpozo, M.A., Muro, M., Sanchezmadrid, F., Aparicio, P.: ICAM-3, The 3rd LFA-1 counterreceptor, is a costimulatory molecule for both resting and activated T-lymphocytes. Eur. J. Immunol. 23, 2799–2806 (1993)CrossRefGoogle Scholar
  6. 6.
    Fawcett, J., Holness, C.L.L., Needham, L.A., Turley, H., Gatter, K.C., Mason, D.Y., Simmons, D.L.: Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes. Nature 360, 481–484 (1992)PubMedCrossRefGoogle Scholar
  7. 7.
    Bailly, P., Tontti, E., Hermand, P., Cartron, J.P., Gahmberg, C.G.: The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/CD18 leukocyte integrins. Eur. J. Immunol. 25, 3316–3320 (1995)PubMedCrossRefGoogle Scholar
  8. 8.
    Mori, K., Fujita, S.C., Watanabe, Y., Obata, K., Hayaishi, O.: Telencephalon-specific antigen identified by monoclonal-antibody. Proc. Natl. Acad. Sci. USA 84, 3921–3925 (1987)PubMedCrossRefGoogle Scholar
  9. 9.
    Oka, S., Mori, K., Watanabe, Y.: Mammalian telencephalic neurons express a segment-specific membrane glycoprotein, telencephalin. Neuroscience 35, 93–103 (1990)PubMedCrossRefGoogle Scholar
  10. 10.
    Benson, D.L., Yoshihara, Y., Mori, K.: Polarized distribution and cell type-specific localization of telencephalin, an intercellular adhesion molecule. J. Neurosci. Res. 52, 43–53 (1998)PubMedCrossRefGoogle Scholar
  11. 11.
    Mitsui, S., Saito, M., Hayashi, K., Mori, K., Yoshihara, Y.: A novel phenylalanine-based targeting signal directs telencephalin to neuronal dendrites. J. Neurosci. 25, 1122–1131 (2005)PubMedCrossRefGoogle Scholar
  12. 12.
    Matsuno, H., Okabe, S., Mishina, M., Yanagida, T., Mori, K., Yoshihara, Y.: Telencephalin slows spine maturation. J. Neurosci. 26, 1776–1786 (2006)PubMedCrossRefGoogle Scholar
  13. 13.
    Furutani, Y., Matsuno, H., Kawasaki, Ma, Sasaki, T., Mori, K., Yoshihara, Y.: Interaction between telencephalin and ERM family proteins mediates dendritic filopodia formation. J. Neurosci. 27, 8866–8876 (2007)PubMedCrossRefGoogle Scholar
  14. 14.
    Bogoevska, V., Nollau, P., Lucka, L., Grunow, D., Klampe, B., Uotila, L.M., Samsen, A., Gahmberg, C.G., Wagener, C.: DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues. Glycobiology 17, 324–333 (2007)PubMedCrossRefGoogle Scholar
  15. 15.
    Jimenez, D., Roda-Navarro, P., Springer, T.A., Casasnovas, J.M.: Contribution of N-linked glycans to the conformation and function of intercellular adhesion molecules (ICAMs). J. Biol. Chem. 280, 5854–5861 (2005)PubMedCrossRefGoogle Scholar
  16. 16.
    Ohgomori, T., Funatsu, O., Nakaya, S.I., Morita, A., Ikekita, M.: Structural study of the N-glycans of intercellular adhesion molecule-5 (telencephalin). Biochim. Biophys. Acta Gen. Subj. 1790, 1611–1623 (2009)CrossRefGoogle Scholar
  17. 17.
    Bloom, J.W., Madanat, M.S., Ray, M.K.: Cell line and site specific comparative analysis of the N-linked oligosaccharides on human ICAM-1des454-532 by electrospray ionization mass spectrometry. Biochemistry 35, 1856–1864 (1996)PubMedCrossRefGoogle Scholar
  18. 18.
    Otto, V.I., Damoc, E., Cueni, L.N., Schürpf, T., Frei, R., Ali, S., Callewaert, N., Moise, A., Leary, J.A., Folkers, G., Przybylski, M.: N-glycan structures and N-glycosylation sites of mouse soluble intercellular adhesion molecule-1 revealed by MALDI-TOF and FTICR mass spectrometry. Glycobiology 16, 1033–1044 (2006)PubMedCrossRefGoogle Scholar
  19. 19.
    Bogoevska, V., Nollau, P., Lucka, L., Grunow, D., Klampe, B., Uotila, L.M., Samsen, A., Gahmberg, C.G., Wagener, C.: DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues. Glycobiology 17, 324–333 (2007)PubMedCrossRefGoogle Scholar
  20. 20.
    Yokoe, S., Takahashi, M., Asahi, M., Lee, S.H., Li, W., Osumi, D., Miyoshi, E., Taniguchi, N.: The Asn418-Linked N-Glycan of ErbB3 plays a crucial role in preventing spontaneous heterodimerization and tumor promotion. Cancer Res. 67, 1935–1942 (2007)PubMedCrossRefGoogle Scholar
  21. 21.
    Markkanen, P.M.H., Petäjä-Repo, U.E.: N-Glycan-mediated quality control in the endoplasmic reticulum is required for the expression of correctly folded δ-opioid receptors at the cell surface. J. Biol. Chem. 283, 29086–29098 (2008)PubMedCrossRefGoogle Scholar
  22. 22.
    Petäjä-Repo, U.E., Hogue, M., Laperriere, A., Walker, P., Bouvier, M.: Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human δ opioid receptor. J. Biol. Chem. 275, 13727–13736 (2000)PubMedCrossRefGoogle Scholar
  23. 23.
    Ellgaard, L., Molinari, M., Helenius, A.: Setting the standards: Quality control in the secretory pathway. Science 286, 1882–1888 (1999)PubMedCrossRefGoogle Scholar
  24. 24.
    Hebert, D.N., Zhang, J.X., Chen, W., Foellmer, B., Helenius, A.: The number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin. J. Cell Biol. 139, 613–623 (1997)PubMedCrossRefGoogle Scholar
  25. 25.
    Nyman-Huttunen, H., Tian, L., Ning, L., Gahmberg, C.G.: α-actinin-dependent cytoskeletal anchorage is important for ICAM-5-mediated neuritic outgrowth. J. Cell Sci. 119, 3057–3066 (2006)PubMedCrossRefGoogle Scholar
  26. 26.
    Mitchell, W.B., Li, J., French, D.L., Coller, B.S.: alphaIIbbeta3 biogenesis is controlled by engagement of alphaIIb in the calnexin cycle via the N15-linked glycan. Blood 107, 2713–2719 (2006)PubMedCrossRefGoogle Scholar
  27. 27.
    Kaushal, S., Ridge, K.D., Khorana, H.G.: Structure and function in rhodopsin: the role of asparagine-linked glycosylation. Proc. Natl. Acad. Sci. USA 91, 4024–4028 (1994)PubMedCrossRefGoogle Scholar
  28. 28.
    Rands, E., Candelore, M.R., Cheung, A.H., Hill, W.S., Strader, C.D., Dixon, R.A.: Mutational analysis of beta-adrenergic receptor glycosylation. J. Biol. Chem. 265, 10759–10764 (1990)PubMedGoogle Scholar
  29. 29.
    Servant, G., Dudley, D.T., Escher, E., Guillemette, G.: Analysis of the role of N-glycosylation in cell-surface expression and binding properties of angiotensin II type-2 receptor of rat pheochromocytoma cells. Biochem. J. 313, 297–304 (1996)PubMedGoogle Scholar
  30. 30.
    Fan, G., Goldsmith, P.K., Collins, R., Dunn, C.K., Krapcho, K.J., Rogers, K.V., Spiegel, A.M.: N-Linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface. Endocrinology 138, 1916–1922 (1997)PubMedCrossRefGoogle Scholar
  31. 31.
    el Battari, A., Forget, P., Fouchier, F., Pic, P.: Effect of inhibiting N-glycosylation or oligosaccharide processing on vasoactive intestinal peptide receptor binding activity and structure. Biochem. J. 278, 527–533 (1991)PubMedGoogle Scholar
  32. 32.
    Liu, X., Davis, D., Segaloff, D.L.: Disruption of potential sites for N-linked glycosylation does not impair hormone binding to the lutropin/choriogonadotropin receptor if Asn-173 is left intact. J. Biol. Chem. 268, 1513–1516 (1993)PubMedGoogle Scholar
  33. 33.
    García Rodríguez, C., Cundell, D.R., Tuomanen, E.I., Kolakowski Jr., L.F., Gerard, C., Gerard, N.P.: The role of N-glycosylation for functional expression of the human platelet-activating factor receptor. Glycosylation is required for efficient membrane trafficking. J. Biol. Chem. 270, 25178–25184 (1995)PubMedCrossRefGoogle Scholar
  34. 34.
    Zhou, F., Su, J., Fu, L., Yang, Y., Zhang, L., Wang, L., Zhao, H., Zhang, D., Li, Z., Zha, X.: Unglycosylation at Asn-633 made extracellular domain of E-cadherin folded incorrectly and arrested in endoplasmic reticulum, then sequentially degraded by ERAD. Glycoconjugate J. 25, 727–740 (2008)CrossRefGoogle Scholar
  35. 35.
    Isaji, T., Sato, Y., Zhao, Y., Miyoshi, E., Wada, Y., Taniguchi, N., Gu, J.: N-glycosylation of the β-propeller domain of the integrin α5 subunit is essential for α5β1 heterodimerization, expression on the cell surface, and its biological function. J. Biol. Chem. 281, 33258–33267 (2006)PubMedCrossRefGoogle Scholar
  36. 36.
    Isaji, T., Sato, Y., Fukuda, T., Gu, J.: N-glycosylation of the I-like domain of β1 integrin is essential for β1 integrin expression and biological function – Identification of the minimal N-glycosylation requirement for α5β1. J. Biol. Chem. 284, 12207–12216 (2009)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Applied Biological Science, Faculty of Science and TechnologyTokyo University of ScienceNodaJapan
  2. 2.Department of BiochemistryNagoya University Graduate School of MedicineNagoyaJapan
  3. 3.Department of Cell Biology and NeuroscienceJuntendo University Graduate School of MedicineBunkyo-kuJapan
  4. 4.Department of Radiation Medicine, Research Institute for Radiation Biology and MedicineHiroshima UniversityMinami-kuJapan

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