Glycoconjugate Journal

, Volume 29, Issue 1, pp 1–12 | Cite as

Design of a covalently bonded glycosphingolipid microarray

  • Emma Arigi
  • Ola Blixt
  • Karsten Buschard
  • Henrik Clausen
  • Steven B. LeveryEmail author
Glycoarray Section


Glycosphingolipids (GSLs) are well known ubiquitous constituents of all eukaryotic cell membranes, yet their normal biological functions are not fully understood. As with other glycoconjugates and saccharides, solid phase display on microarrays potentially provides an effective platform for in vitro study of their functional interactions. However, with few exceptions, the most widely used microarray platforms display only the glycan moiety of GSLs, which not only ignores potential modulating effects of the lipid aglycone, but inherently limits the scope of application, excluding, for example, the major classes of plant and fungal GSLs. In this work, a prototype “universal” GSL-based covalent microarray has been designed, and preliminary evaluation of its potential utility in assaying protein-GSL binding interactions investigated. An essential step in development involved the enzymatic release of the fatty acyl moiety of the ceramide aglycone of selected mammalian GSLs with sphingolipid N-deacylase (SCDase). Derivatization of the free amino group of a typical lyso-GSL, lyso-GM1, with a prototype linker assembled from succinimidyl-[(N-maleimidopropionamido)-diethyleneglycol] ester and 2-mercaptoethylamine, was also tested. Underivatized or linker-derivatized lyso-GSL were then immobilized on N-hydroxysuccinimide- or epoxide-activated glass microarray slides and probed with carbohydrate binding proteins of known or partially known specificities (i.e., cholera toxin B-chain; peanut agglutinin, a monoclonal antibody to sulfatide, Sulph 1; and a polyclonal antiserum reactive to asialo-GM2). Preliminary evaluation of the method indicated successful immobilization of the GSLs, and selective binding of test probes. The potential utility of this methodology for designing covalent microarrays that incorporate GSLs for serodiagnosis is discussed.


Glycosphingolipid Glycolipid Ganglioside Glycan Lectin Antibody Microarray Glycan array 



This work was supported by The Copenhagen Center for Glycomics at the University of Copenhagen, the Carlsberg Foundation, The Benzon Foundation, The Velux Foundation, The Danish Research Councils, NIH/NCI 5U01 CA128437 and 5U01 CA111294, EU FP7-HEALTH-2007-A 201381, and the University of Copenhagen Programme of Excellence. Early phases of the work were supported by an NIH grant (R21 RR20355) to S.B.L.


  1. 1.
    Zhang, X., Kiechle, F.L.: Review: glycosphingolipids in health and disease. Ann. Clin. Lab. Sci. 34, 3–13 (2004)PubMedGoogle Scholar
  2. 2.
    Ariga, T., Miyatake, T., Yu, R.K.: Recent studies on the roles of antiglycosphingolipids in the pathogenesis of neurological disorders. J. Neurosci. Res. 65, 363–370 (2001)PubMedCrossRefGoogle Scholar
  3. 3.
    Fredman, P., Vedeler, C.A., Nyland, H., Aarli, J.A., Svennerholm, L.: Antibodies in sera from patients with inflammatory demyelinating polyradiculoneuropathy react with ganglioside LM1 and sulphatide of peripheral nerve myelin. J. Neurol. 238, 75–79 (1991)PubMedCrossRefGoogle Scholar
  4. 4.
    Hakomori, S.: Tumor-associated carbohydrate antigens defining tumor malignancy: basis for development of anti-cancer vaccines. Adv. Exp. Med. Biol. 491, 369–402 (2001)PubMedCrossRefGoogle Scholar
  5. 5.
    Buschard, K., Josefsen, K., Horn, T., Larsen, S., Fredman, P.: Sulphatide antigen in islets of Langerhans and in diabetic glomeruli, and anti-sulphatide antibodies in type 1 diabetes mellitus. APMIS 101, 963–970 (1993)PubMedCrossRefGoogle Scholar
  6. 6.
    Buschard, K., Josefsen, K., Horn, T., Fredman, P.: Sulphatide and sulphatide antibodies in insulin-dependent diabetes mellitus. Lancet 342, 840 (1993)PubMedCrossRefGoogle Scholar
  7. 7.
    Buschard, K., et al.: Sulphatide in islets of Langerhans and in organs affected in diabetic late complications: a study in human and animal tissue. Diabetologia 37, 1000–1006 (1994)PubMedCrossRefGoogle Scholar
  8. 8.
    Blomqvist, M., et al.: Sulfatide is associated with insulin granules and located to microdomains of a cultured beta cell line. Glycoconj. J. 19, 403–413 (2002)PubMedCrossRefGoogle Scholar
  9. 9.
    Andersson, K., et al.: Patients with insulin-dependent diabetes but not those with non-insulin-dependent diabetes have anti-sulfatide antibodies as determined with a new ELISA assay. Autoimmunity 35, 463–468 (2002)PubMedCrossRefGoogle Scholar
  10. 10.
    Fukui, S., Feizi, T., Galustian, C., Lawson, A.M., Chai, W.: Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat. Biotechnol. 20, 1011–1017 (2002)PubMedCrossRefGoogle Scholar
  11. 11.
    Willats, W.G., Rasmussen, S.E., Kristensen, T., Mikkelsen, J.D., Knox, J.P.: Sugar-coated microarrays: a novel slide surface for the high-throughput analysis of glycans. Proteomics 2, 1666–1671 (2002)PubMedCrossRefGoogle Scholar
  12. 12.
    Wang, D., Liu, S., Trummer, B.J., Deng, C., Wang, A.: Carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells. Nat. Biotechnol. 20, 275–281 (2002)PubMedCrossRefGoogle Scholar
  13. 13.
    Houseman, B.T., Mrksich, M.: Carbohydrate arrays for the evaluation of protein binding and enzymatic modification. Chem. Biol. 9, 443–454 (2002)PubMedCrossRefGoogle Scholar
  14. 14.
    Blixt, O., et al.: Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101, 17033–17038 (2004)PubMedCrossRefGoogle Scholar
  15. 15.
    Mamidyala, S.K., Ko, K.-S., Jaipuri, F.A., Park, G., Pohl, N.L.: Noncovalent fluorous interactions for the synthesis of carbohydrate microarrays. J. Fluorine Chem. 127, 571–579 (2006)CrossRefGoogle Scholar
  16. 16.
    Shin, I., Park, S., Lee, M.R.: Carbohydrate microarrays: an advanced technology for functional studies of glycans. Chemistry 11, 2894–2901 (2005)PubMedCrossRefGoogle Scholar
  17. 17.
    Larsen, K., Thygesen, M.B., Guillaumie, F., Willats, W.G., Jensen, K.J.: Solid-phase chemical tools for glycobiology. Carbohydr. Res. 341, 1209–1234 (2006)PubMedCrossRefGoogle Scholar
  18. 18.
    Horlacher, T., Seeberger, P.H.: Carbohydrate arrays as tools for research and diagnostics. Chem. Soc. Rev. 37, 1414–1422 (2008)PubMedCrossRefGoogle Scholar
  19. 19.
    Liu, Y., Palma, A.S., Feizi, T.: Carbohydrate microarrays: key developments in glycobiology. Biol. Chem. 390, 647–656 (2009)PubMedCrossRefGoogle Scholar
  20. 20.
    Lonardi, E., Balog, C.I., Deelder, A.M., Wuhrer, M.: Natural glycan microarrays. Exp. Rev. Proteomics. 7, 761–774 (2010)CrossRefGoogle Scholar
  21. 21.
    von Gunten, S., et al.: Intravenous immunoglobulin contains a broad repertoire of anticarbohydrate antibodies that is not restricted to the IgG2 subclass. J. Allergy Clin. Immunol. 123, 1268–1276 (2009)CrossRefGoogle Scholar
  22. 22.
    Oyelaran, O., McShane, L.M., Dodd, L., Gildersleeve, J.C.: Profiling human serum antibodies with a carbohydrate antigen microarray. J. Proteome Res. 8, 4301–4310 (2009)PubMedCrossRefGoogle Scholar
  23. 23.
    Springer, G.F.: Immunoreactive T and Tn epitopes in cancer diagnosis, prognosis, and immunotherapy. J. Mol. Med. 75, 594–602 (1997)PubMedCrossRefGoogle Scholar
  24. 24.
    Reis, C.A., et al.: Development and characterization of an antibody directed to an alpha-N-acetyl-D-galactosamine glycosylated MUC2 peptide. Glycoconj. J. 15, 51–62 (1998)PubMedCrossRefGoogle Scholar
  25. 25.
    Price, M.R., et al.: Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin. San Diego, Calif., November 17–23, 1996. Tumour. Biol. 19(Suppl 1), 1–20 (1998)PubMedCrossRefGoogle Scholar
  26. 26.
    Ryuko, K., et al.: Characterization of a new MUC1 monoclonal antibody (VU-2-G7) directed to the glycosylated PDTR sequence of MUC1. Tumour Biol. 21, 197–210 (2000)PubMedCrossRefGoogle Scholar
  27. 27.
    Fredman, P., et al.: Characterization of the binding epitope of a monoclonal antibody to sulphatide. Biochem. J. 251, 17–22 (1988)PubMedGoogle Scholar
  28. 28.
    Magnani, J.L., Smith, D.F., Ginsburg, V.: Detection of gangliosides that bind cholera toxin: direct binding of 125 I-labeled toxin to thin-layer chromatography. Anal. Biochem. 109, 399–402 (1980)PubMedCrossRefGoogle Scholar
  29. 29.
    Magnani, J.L., Brockhaus, M., Smith, D.F., Ginsburg, V.: Detection of glycolipid ligands by direct binding of carbohydrate-binding proteins to thin-layer chromatograms. Methods Enzymol. 83, 235–241 (1982)PubMedCrossRefGoogle Scholar
  30. 30.
    Holmgren, J., Svennerholm, A.M.: Enzyme-linked immunosorbent assays for cholera serology. Infect. Immun. 7, 759–763 (1973)PubMedGoogle Scholar
  31. 31.
    Holmgren, J., Elwing, H., Fredman, P., Svennerholm, L.: Immunoassays based on plastic-adsorbed gangliosides. Adv. Exp. Med. Biol. 125, 339–348 (1980)PubMedGoogle Scholar
  32. 32.
    Feizi, T., Chai, W.: Oligosaccharide microarrays to decipher the glyco code. Nat. Rev. Mol. Cell Biol. 5, 582–588 (2004)PubMedCrossRefGoogle Scholar
  33. 33.
    Wallner, F.K., Norberg, H.A., Johansson, A.I., Mogemark, M., Elofsson, M.: Solid-phase synthesis of serine-based glycosphingolipid analogues for preparation of glycoconjugate arrays. Org. Biomol. Chem. 3, 309–315 (2005)PubMedCrossRefGoogle Scholar
  34. 34.
    Kanter, J.L., et al.: Lipid microarrays identify key mediators of autoimmune brain inflammation. Nat. Med. 12, 138–143 (2006)PubMedCrossRefGoogle Scholar
  35. 35.
    Imamura, A., et al.: Design and synthesis of versatile ganglioside probes for carbohydrate microarrays. Glycoconj. J. 25, 269–278 (2008)PubMedCrossRefGoogle Scholar
  36. 36.
    Liang, P.H., et al.: Quantitative microarray analysis of intact glycolipid-CD1d interaction and correlation with cell-based cytokine production. J. Am. Chem. Soc. 130, 12348–12354 (2008)PubMedCrossRefGoogle Scholar
  37. 37.
    Yamazaki, V., et al.: Cell membrane array fabrication and assay technology. BMC. Biotechnol. 5, 18 (2005)PubMedCrossRefGoogle Scholar
  38. 38.
    Isobe, T., Naiki, M., Handa, S., Taki, T.: A simple assay method for bacterial binding to glycosphingolipids on a polyvinylidene difluoride membrane after thin-layer chromatography blotting and in situ mass spectrometric analysis of the ligands. Anal. Biochem. 236, 35–40 (1996)PubMedCrossRefGoogle Scholar
  39. 39.
    Guittard, J., Hronowski, X.L., Costello, C.E.: Direct matrix-assisted laser desorption/ionization mass spectrometric analysis of glycosphingolipids on thin layer chromatographic plates and transfer membranes. Rapid Commun. Mass Spectrom. 13, 1838–1849 (1999)PubMedCrossRefGoogle Scholar
  40. 40.
    Distler, U., et al.: Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer. Anal. Chem. 80, 1835–1846 (2008)PubMedCrossRefGoogle Scholar
  41. 41.
    Xia, B., et al.: Versatile fluorescent derivatization of glycans for glycomic analysis. Nat. Methods 2, 845–850 (2005)PubMedCrossRefGoogle Scholar
  42. 42.
    Blixt, O., et al.: Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a. Carbohydr. Res. 340, 1963–1972 (2005)PubMedCrossRefGoogle Scholar
  43. 43.
    Bohorov, O., Andersson-Sand, H., Hoffmann, J., Blixt, O.: Arraying glycomics: a novel bi-functional spacer for one-step microscale derivatization of free reducing glycans. Glycobiology. 16, 21C–27C (2006)PubMedCrossRefGoogle Scholar
  44. 44.
    Lester, R.L., Dickson, R.C.: Sphingolipids with inositolphosphate-containing head groups. Adv. Lipid Res. 26, 253–274 (1993)PubMedGoogle Scholar
  45. 45.
    Song, X., et al.: Shotgun glycomics: a microarray strategy for functional glycomics. Nat. Methods 8, 85–90 (2011)PubMedCrossRefGoogle Scholar
  46. 46.
    Ito, M., Kurita, T., Kita, K.: A novel enzyme that cleaves the N-acyl linkage of ceramides in various glycosphingolipids as well as sphingomyelin to produce their lyso forms. J. Biol. Chem. 270, 24370–24374 (1995)PubMedCrossRefGoogle Scholar
  47. 47.
    Ito, M., Kita, K., Kurita, T., Sueyoshi, N., Izu, H.: Enzymatic N-deacylation of sphingolipids. Methods Enzymol. 311, 297–303 (2000)PubMedCrossRefGoogle Scholar
  48. 48.
    Kita, K., Kurita, T., Ito, M.: Characterization of the reversible nature of the reaction catalyzed by sphingolipid ceramide N-deacylase. A novel form of reverse hydrolysis reaction. Eur. J. Biochem. 268, 592–602 (2001)PubMedCrossRefGoogle Scholar
  49. 49.
    Kurita, T., Izu, H., Sano, M., Ito, M., Kato, I.: Enhancement of hydrolytic activity of sphingolipid ceramide N-deacylase in the aqueous-organic biphasic system. J. Lipid Res. 41, 846–851 (2000)PubMedGoogle Scholar
  50. 50.
    Li, Y., Arigi, E., Eichert, H., Levery, S.B.: Mass spectrometry of fluorocarbon-labeled glycosphingolipids. J. Mass Spectrom. 45, 504–519 (2010)PubMedCrossRefGoogle Scholar
  51. 51.
    Cuatrecasas, P.: Gangliosides and membrane receptors for cholera toxin. Biochemistry 12, 3558–3566 (1973)PubMedCrossRefGoogle Scholar
  52. 52.
    Cuatrecasas, P.: Interaction of Vibrio cholerae enterotoxin with cell membranes. Biochemistry 12, 3547–3558 (1973)PubMedCrossRefGoogle Scholar
  53. 53.
    Kuziemko, G.M., Stroh, M., Stevens, R.C.: Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance. Biochemistry 35, 6375–6384 (1996)PubMedCrossRefGoogle Scholar
  54. 54.
    MacKenzie, C.R., Hirama, T., Lee, K.K., Altman, E., Young, N.M.: Quantitative analysis of bacterial toxin affinity and specificity for glycolipid receptors by surface plasmon resonance. J. Biol. Chem. 272, 5533–5538 (1997)PubMedCrossRefGoogle Scholar
  55. 55.
    Momoi, T., Tokunaga, T., Nagai, Y.: Specific interaction of peanut agglutinin with the glycolipid asialo GM1. FEBS Lett. 141, 6–10 (1982)PubMedCrossRefGoogle Scholar
  56. 56.
    Månsson, J.E., Olofsson, S.: Binding specificities of the lectins from Helix pomatia, soybean and peanut against different glycosphingolipids in liposome membranes. FEBS Lett. 156, 249–252 (1983)CrossRefGoogle Scholar
  57. 57.
    Ito, M., Yamagata, T.: A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccharide and ceramide of neutral and acidic glycosphingolipids. J. Biol. Chem. 261, 14278–14282 (1986)PubMedGoogle Scholar
  58. 58.
    Zhou, B., Li, S.C., Laine, R.A., Huang, R.T., Li, Y.T.: Isolation and characterization of ceramide glycanase from the leech, Macrobdella decora. J. Biol. Chem. 264, 12272–12277 (1989)PubMedGoogle Scholar
  59. 59.
    Linman, M.J., Yu, H., Chen, X., Cheng, Q.: Fabrication and characterization of a sialoside-based carbohydrate microarray biointerface for protein binding analysis with surface plasmon resonance imaging. ACS Appl. Mater. Interfaces 1, 1755–1762 (2009)PubMedCrossRefGoogle Scholar
  60. 60.
    Ke, B.B., Wan, L.S., Xu, Z.K.: Controllable construction of carbohydrate microarrays by site-directed grafting on self-organized porous films. Langmuir 26, 8946–8952 (2010)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Emma Arigi
    • 1
    • 3
  • Ola Blixt
    • 1
  • Karsten Buschard
    • 2
  • Henrik Clausen
    • 1
  • Steven B. Levery
    • 1
    Email author
  1. 1.Department of Cellular and Molecular MedicineUniversity of CopenhagenCopenhagen NDenmark
  2. 2.Rigshospitalet, Bartholin InstituteCopenhagen OEDenmark
  3. 3.Department of Biological Sciences, The Border Biomedical Research CenterUniversity of Texas at El PasoEl PasoUSA

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