SpringerLink
Log in

Neoglycolipid-Based Oligosaccharide Microarray System: Preparation of NGLs and Their Noncovalent Immobilization on Nitrocellulose-Coated Glass Slides for Microarray Analyses

  • Protocol
  • First Online:
Carbohydrate Microarrays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 808))

Abstract

Carbohydrate microarrays, since their advent in 2002, are revolutionizing studies of the molecular basis of protein–carbohydrate interactions both in endogenous recognition systems and pathogen–host interactions. We have developed a unique carbohydrate microarray system based on the neoglycolipid (NGL) technology, a well-validated microscale approach for generating lipid-tagged oligosaccharide probes for use in carbohydrate recognition studies. This chapter provides an overview of the principles and key features of the NGL-based oligosaccharide microarrays, and describes in detail the basic techniques – from the preparation of NGL probes to the generation of microarrays using robotic arraying hardware, as well as a general protocol for probing the microarrays with carbohydrate-binding proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
GBP 34.95
Price includes VAT (United Kingdom)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
GBP 79.50
Price includes VAT (United Kingdom)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
GBP 99.99
Price includes VAT (United Kingdom)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info
Hardcover Book
GBP 129.99
Price includes VAT (United Kingdom)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free ship** worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Tang PW, Gooi HC, Hardy M, Lee YC, Feizi T (1985) Novel approach to the study of the antigenicities and receptor functions of carbohydrate chains of glycoproteins. Biochem Biophys Res Commun 132:474–480

    Article  PubMed  CAS  Google Scholar 

  2. Yuen C-T, Chai W, Loveless RW, Lawson AM, Margolis RU, Feizi T (1997) Brain contains HNK-1 immunoreactive O-glycans of the sulfoglucuronyl lactosamine series that terminate in 2-linked or 2,6-linked hexose (mannose). J Biol Chem 272:8924–8931

    Article  PubMed  CAS  Google Scholar 

  3. Mizuochi T, Loveless RW, Lawson AM, Chai W, Lachmann PJ, Childs RA, Thiel S, Feizi T (1989) A library of oligosaccharide probes (neoglycolipids) from N-glycosylated proteins reveals that conglutinin binds to certain complex type as well as high-mannose type oligosaccharide chains. J Biol Chem 264:13834–13839

    PubMed  CAS  Google Scholar 

  4. Leteux C, Chai W, Nagai K, Herbert CG, Lawson AM, and Feizi T (2001) 10E4 Antigen of scrapie lesions contains an unusual non­sulfated heparan motif. J Biol Chem 276:12539–12545

    Article  PubMed  CAS  Google Scholar 

  5. Fukui S, Feizi T, Galustian C, Lawson AM, Chai W (2002) Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nat Biotechnol 20:1011–1017

    Article  PubMed  CAS  Google Scholar 

  6. Feizi T, Chai W (2004) Oligosaccharide microarrays to decipher the glyco code. Nat Rev Mol Cell Biol 5:582–588

    Article  PubMed  CAS  Google Scholar 

  7. Galustian C, Park CG, Chai W, Kiso M, Bruening SA, Kang YS, Steinman RM, Feizi T (2004) High and low affinity carbohydrate ligands revealed for murine SIGN-R1 by carbohydrate array and cell binding approaches, and differing specificities for SIGN-R3 and langerin. Int Immunol 16:853–866

    Article  PubMed  CAS  Google Scholar 

  8. Reddy ST, Chai W, Childs RA, Page JD, Feizi T, Dahms NM (2004) Identification of a Low Affinity Mannose 6-Phosphate-binding Site in Domain 5 of the Cation-independent Mannose 6-Phosphate Receptor. J Biol Chem 279:38658–38667

    Article  PubMed  CAS  Google Scholar 

  9. Palma AS, Feizi T, Zhang Y, Stoll MS, Lawson AM, Diaz-Rodríguez E, Campanero-Rhodes AS, Costa J, Brown GD, Chai W (2006) Ligands for the beta-glucan receptor, Dectin-1, assigned using ‘designer’ microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides. J Biol Chem 281:5771–5779

    Article  PubMed  CAS  Google Scholar 

  10. Komba S, Galustian C, Ishida H, Feizi T, Kannagi R, Kiso M (1999) The first total synthesis of 6-sulfo-de-N-acetylsialyl Lewisx ganglioside: A superior ligand for human L-selectin. Angew Chem Int Ed Engl 38:1131–1133

    Article  CAS  Google Scholar 

  11. Green PJ, Tamatani T, Watanabe T, Miyasaka M, Hasegawa A, Kiso M, Stoll MS, Feizi T (1992) High affinity binding of the leucocyte adhesion molecule L-selectin to 3′-sulphated-Lea and -Lex oligosaccharides and the predominance of sulphate in this interaction demonstrated by binding studies with a series of lipid-linked oligosaccharides. Biochem Biophys Res Commun 188:244–251

    Article  PubMed  CAS  Google Scholar 

  12. Feizi T, Stoll MS, Yuen C-T, Chai W, Lawson AM (1994) Neoglycolipids: probes of oligosaccharide structure, antigenicity and function. Methods Enzymol 230:484–519

    Article  PubMed  CAS  Google Scholar 

  13. Feizi T, Childs RA (1994) Neoglycolipids: probes in structure/function assignments to oligosaccharides. Methods Enzymol 242:205–217

    Article  PubMed  CAS  Google Scholar 

  14. Chai W, Stoll MS, Galustian C, Lawson AM, Feizi T (2003) Neoglycolipid technology - deciphering information content of glycome. Methods Enzymol 362:160–195

    Article  PubMed  CAS  Google Scholar 

  15. Yuen C-T, Lawson AM, Chai W, Larkin M, Stoll MS, Stuart AC, Sullivan FX, Ahern TJ, Feizi T (1992) Novel sulfated ligands for the cell adhesion molecule E-selectin revealed by the neoglycolipid technology among O-linked oligosaccharides on an ovarian cystadenoma glycoprotein. Biochemistry 31:9126–9131

    Article  PubMed  CAS  Google Scholar 

  16. Chai W, Yuen CT, Kogelberg H, Carruthers RA, Margolis RU, Feizi T, Lawson AM (1999) High prevalence of 2-mono- and 2,6-di-substituted Manol-terminating sequences among O-glycans released from brain glycopeptides by reductive alkaline hydrolysis. Eur J Biochem 263:879–888

    Article  PubMed  CAS  Google Scholar 

  17. Stoll MS, Feizi T, Loveless RW, Chai W, Lawson AM, Yuen CT (2000) Fluorescent neoglycolipids. Improved probes for oligosaccharide ligand discovery. Eur J Biochem 267:1795–1804

    Article  PubMed  CAS  Google Scholar 

  18. Liu Y, Chai W, Childs RA, Feizi T (2006) Preparation of Neoglycolipids with Ring-Closed Cores via Chemoselective Oxime-Ligation for Microarray Analysis of Carbohydrate-Protein Interactions. Methods Enzymol 415C:326–340

    Google Scholar 

  19. Liu Y, Feizi T, Campanero-Rhodes MA, Childs RA, Zhang Y, Mulloy B, Evans PG, Osborn HM, Otto D, Crocker PR, Chai W (2007) Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions. Chem Biol 14:847–859

    Article  PubMed  CAS  Google Scholar 

  20. Liu Y, Palma AS, Feizi T (2009) Carbohydrate microarrays: key developments in glycobiology. Biol Chem 390:647–656

    Article  PubMed  CAS  Google Scholar 

  21. Campanero-Rhodes MA, Childs RA, Kiso M, Komba S, Le Narvor C, Warren J, Otto D, Crocker PR, Feizi T (2006) Carbohydrate microarrays reveal sulphation as a modulator of siglec binding. Biochem Biophys Res Comm 344:1141–1146

    Article  PubMed  CAS  Google Scholar 

  22. Campanero-Rhodes MA, Smith A, Chai W, Sonnino S, Mauri L, Childs RA, Zhang Y, Ewers H, Helenius A, Imberty A, Feizi T (2007) N-glycolyl GM1 ganglioside as a receptor for simian virus 40. J Virol 81:12846–12858

    Article  PubMed  CAS  Google Scholar 

  23. Schallus T, Jaeckh C, Feher K, Palma AS, Liu Y, Simpson JC, Mackeen M, Stier G, Gibson TJ, Feizi T, Pieler T, Muhle-Goll C (2008) Malectin - A Novel Carbohydrate-binding Protein of the Endoplasmic Reticulum and a Candidate Player in the Early Steps of Protein N-glycosylation. Mol Biol Cell 19:3404–3414

    Article  PubMed  CAS  Google Scholar 

  24. Palma AS, Liu Y, Muhle-Goll C, Butters TD, Zhang Y, Childs R, Chai W, Feizi T (2010) Multifaceted approaches including neoglycolipid oligosaccharide microarrays to ligand discovery for malectin. Methods Enzymol 478:265–286

    Article  PubMed  CAS  Google Scholar 

  25. Blumenschein TMA, Friedrich N, Childs RA, Saouros S, Carpenter EP, Campanero-Rhodes MA, Simpson P, Chai W, Koutroukides T, Blackman MJ, Feizi T, Soldati-Favre D, Matthews S (2007) Atomic resolution insight into host cell recognition by Toxoplasma gondii. EMBO J 26:2808–2820

    Article  PubMed  CAS  Google Scholar 

  26. Garnett JA, Liu Y, Leon E, Allman SA, Friedrich N, Saouros S, Curry S, Soldati-Favre D, Davis BG, Feizi T, Matthews S (2009) Detailed insights from microarray and crystallographic studies into carbohydrate recognition by microneme protein 1 (MIC1) of Toxoplasma gondii. Protein Sci 18:1935–1947

    Article  PubMed  CAS  Google Scholar 

  27. Allman SA, Jensen HH, Vijayakrishnan B, Garnett JA, Leon E, Liu Y, Anthony DC, Sibson NR, Feizi T, Matthews S, Davis BG (2009) Potent Fluoro-oligosaccharide Probes of Adhesion in Toxoplasmosis. Chembiochem 10:2522–2529

    Article  PubMed  CAS  Google Scholar 

  28. Friedrich N, Santos JM, Liu Y, Palma AS, Leon E, Saouros S, Kiso M, Blackman MJ, Matthews S, Feizi T, Soldati-Favre D (2010) Members of a novel protein family containing MAR domains act as sialic acid-binding lectins during host cell invasion by apicomplexan parasites. J Biol Chem 285:2064–2076

    Article  PubMed  CAS  Google Scholar 

  29. Childs RA, Palma AS, Wharton S, Matrosovich T, Liu Y, Chai W, Campanero-Rhodes MA, Zhang Y, Eickmann M, Kiso M, Hay A, Matrosovich M, Feizi T (2009) Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nat Biotechnol 27:797–799

    Article  PubMed  CAS  Google Scholar 

  30. Liu Y, Childs RA, Matrosovich T, Wharton S, Palma AS, Chai W, Daniels R, Gregory V, Uhlendorff J, Kiso M, Klenk HD, Hay A, Feizi T, Matrosovich M (2010) Altered receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus. J Virol 84:12069–12074

    Article  PubMed  Google Scholar 

  31. Capodicasa C, Chiani P, Bromuro C, De BF, Catellani M, Palma AS, Liu Y, Feizi T, Cassone A, Benvenuto E, Torosantucci A (2011) Plant production of anti-beta-glucan antibodies for immunotherapy of fungal infections in humans. Plant Biotechnol J. 9:776–787

    Google Scholar 

  32. Torosantucci A, Chiani P, Bromuro C, De Bernardis F, Palma AS, Liu Y, Mignogna G, Maras B, Colone M, Stringaro A, Zamboni S, Feizi T, Cassone A (2009) Protection by anti-beta-glucan antibodies is associated with restricted beta-1,3 glucan binding specificity and inhibition of fungal growth and adherence. PLoS ONE 4:e5392

    Article  PubMed  Google Scholar 

  33. Dunlop C, Bonomelli C, Mansab F, Vasiljevic S, Doores KJ, Wormald MR, Palma AS, Feizi T, Harvey DJ, Dwek RA, Crispin M, Scanlan CN (2010) Polysaccharide mimicry of the epitope of the broadly neutralising anti-HIV antibody, 2G12, induces enhanced antibody responses to self oligomannose glycans. Glycobiology 20:812–823

    Article  PubMed  CAS  Google Scholar 

  34. Stoll MS, Mizuochi T, Childs RA, Feizi T (1988) Improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities from reducing oligosaccharides. Biochem J 256:661–664

    PubMed  CAS  Google Scholar 

  35. Stoll, Feizi T (2009) Software tools for storing, processing and displaying carbohydrate microarray data. In: Kettner C (ed) Proceeding of the Beilstein Symposium on Glyco-Bioinformatics, 4–8 October, 2009, Potsdam, Germany. pp 123–140 (available online at http://www.beilstein-institut.de/en/symposia/overview/proceedings/2009-glycobioin­formatics/)

Download references

Acknowledgments

We gratefully acknowledge collaborations of other members (past and present) in the Glycosciences Laboratory, in particular Alex Lawson, Yibing Zhang, and Colin Herbert. The Glycosciences Laboratory acknowledges with gratitude collaborators over the years with whom our microarray probes were studied. We also acknowledge UK Research Councils’ Basic Technology Initiative “Glycoarrays” (GRS/79268) and follow on EPSRC Translational Grant (EP/G037604/1), MRC and Wellcome Trust and NCI Alliance of Glycobiologists for Detection of Cancer and Cancer Risk (U01 CA128416) for financial support. A.S.P. is a fellow of the Fundação para a Ciência e Tecnologia (SFRH/BPD/26515/2006, Portugal) and M.A.C. of the Ministerio de Ciencia e Innovación (JCI-2007-123-160, Spain).

Author information

Authors and Affiliations

  1. The Glycosciences Laboratory, Department of Medicine, Imperial College London, Hammersmith Campus, London, UK

    Yan Liu, Robert A. Childs, Angelina S. Palma, Maria A. Campanero-Rhodes, Mark S. Stoll, Wengang Chai & Ten Feizi

  2. Chemistry and Technology Network Laboratory (REQUIMTE), Department of Chemistry, Faculty of Science and Technology, New University of Lisbon, Caparica, Portugal

    Angelina S. Palma

  3. Department of Biological Physical Chemistry, Instituto de Química-Física “Rocasolano”, CSIC and CIBERES, Madrid, Spain

    Maria A. Campanero-Rhodes

Authors
  1. Yan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert A. Childs
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Angelina S. Palma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maria A. Campanero-Rhodes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mark S. Stoll
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wengang Chai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ten Feizi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yan Liu .

Editor information

Editors and Affiliations

  1. CNRS UMR 5270, Inst. Nanotechnologies de Lyon, Université Lyon 1, av. Guy de Collongue 36, Ecully, 69134, France

    Yann Chevolot

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Liu, Y. et al. (2012). Neoglycolipid-Based Oligosaccharide Microarray System: Preparation of NGLs and Their Noncovalent Immobilization on Nitrocellulose-Coated Glass Slides for Microarray Analyses. In: Chevolot, Y. (eds) Carbohydrate Microarrays. Methods in Molecular Biology, vol 808. Humana Press. https://doi.org/10.1007/978-1-61779-373-8_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-373-8_8

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-372-1

  • Online ISBN: 978-1-61779-373-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation