Skip to main content

Synthetic Glycan Microarrays

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1518))

Abstract

Structurally diverse glycans are expressed by all animate beings and exert diverse biological functions through specific interactions with glycan binding proteins (GBPs). In humans, glycan–GBP interactions are implicated in many disease-relevant processes in development, infection and immune response to bacterial and viral pathogens. Recent progress in chemical synthesis, including automated glycan assembly, has facilitated access to complex glycans that cannot be isolated from biological material. Glycan immobilization on microarrays allows rapid, multiplexed glycan–GBP interaction studies to reveal biological functions. Synthetic glycan microarrays have enabled, for instance, the identification of glycan ligands for lectins, the definition of vaccine antigens, revealed viral glycan receptors and can serve as diagnostic tools for human disease. Here, we describe the methods to fabricate custom glycan microarrays that are used to examine glycan–GBP binding specificities. Conjugation-ready synthetic glycans are covalently attached to microarray surfaces through nucleophilic linker moieties. Microarrays are incubated with GBPs, and binding events are quantitatively detected by fluorescent signals. These methods are readily adaptable to a multitude of purposes from basic research to biomedical applications.

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

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (eds) (2009) Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  2. Fuster MM, Esko JD (2005) The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 5:526–542

    Article  CAS  PubMed  Google Scholar 

  3. Vasta GR (2009) Roles of galectins in infection. Nat Rev Microbiol 7:424–438

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Dambuza IM, Brown GD (2015) C-type lectins in immunity: recent developments. Curr Opin Immunol 32:21–27

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Ströh LJ, Stehle T (2014) Glycan engagement by viruses: receptor switches and specificity. Annu Rev Virol 111:285–306

    Article  Google Scholar 

  6. Geissner A, Anish C, Seeberger PH (2014) Glycan arrays as tools for infectious disease research. Curr Opin Chem Biol 18:38–45

    Article  CAS  PubMed  Google Scholar 

  7. Astronomo RD, Burton DR (2010) Carbohydrate vaccines: developing sweet solutions to sticky situations? Nat Rev Drug Discov 9:308–324

    Article  CAS  PubMed  Google Scholar 

  8. Lepenies B, Seeberger PH (2010) The promise of glycomics, glycan arrays and carbohydrate-based vaccines. Immunopharmacol Immunotoxicol 32:196–207

    Article  CAS  PubMed  Google Scholar 

  9. Seeberger PH (2015) The logic of automated glycan assembly. Acc Chem Res 48:1450–1463

    Article  CAS  PubMed  Google Scholar 

  10. Calin O, Eller S, Seeberger PH (2013) Automated polysaccharide synthesis: assembly of a 30mer mannoside. Angew Chem Int Ed Engl 52:5862–5865

    Article  CAS  PubMed  Google Scholar 

  11. Eller S, Collot M, Yin J, Hahm HS, Seeberger PH (2013) Automated solid-phase synthesis of chondroitin sulfate glycosaminoglycans. Angew Chem Int Ed Engl 52:5858–5861

    Article  CAS  PubMed  Google Scholar 

  12. Kandasamy J, Schuhmacher F, Hahm HS, Klein JC, Seeberger PH (2014) Modular automated solid phase synthesis of dermatan sulfate oligosaccharides. Chem Commun (Camb) 50:1875–1877

    Article  CAS  Google Scholar 

  13. Fair RJ, Hahm HS, Seeberger PH (2015) Combination of automated solid-phase and enzymatic oligosaccharide synthesis provides access to α(2,3)-sialylated glycans. Chem Commun (Camb) 51:6183–6185

    Article  CAS  Google Scholar 

  14. Lai CH, Hahm HS, Liang CF, Seeberger PH (2015) Automated solid-phase synthesis of oligosaccharides containing sialic acids. Beilstein J Org Chem 11:617–621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Weishaupt MW, Matthies S, Seeberger PH (2013) Automated solid-phase synthesis of a β-(1,3)-glucan dodecasaccharide. Chemistry 19:12497–12503

    Article  CAS  PubMed  Google Scholar 

  16. Schmidt D, Schuhmacher F, Geissner A, Seeberger PH, Pfrengle F (2015) Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans. Chemistry 21:5709–5713

    Article  CAS  PubMed  Google Scholar 

  17. Lepenies B, Yin J, Seeberger PH (2010) Applications of synthetic carbohydrates to chemical biology. Curr Opin Chem Biol 14:404–411

    Article  CAS  PubMed  Google Scholar 

  18. de Paz JL, Seeberger PH (2008) Deciphering the glycosaminoglycan code with the help of microarrays. Mol Biosyst 4:707–711

    Article  PubMed  Google Scholar 

  19. Park S, Gildersleeve JC, Blixt O, Shin I (2013) Carbohydrate microarrays. Chem Soc Rev 42:4310–4326

    Article  CAS  PubMed  Google Scholar 

  20. Liang PH, Wu CY, Greenberg WA, Wong CH (2008) Glycan arrays: biological and medical applications. Curr Opin Chem Biol 12:86–92

    Article  CAS  PubMed  Google Scholar 

  21. Horlacher T, Seeberger PH (2008) Carbohydrate arrays as tools for research and diagnostics. Chem Soc Rev 37:1414–1422

    Article  CAS  PubMed  Google Scholar 

  22. Rillahan CD, Paulson JC (2011) Glycan microarrays for decoding the glycome. Annu Rev Biochem 80:797–823

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Eriksson M, Serna S, Maglinao M, Schlegel MK, Seeberger PH, Reichardt NC, Lepenies B (2014) Biological evaluation of multivalent lewis X-MGL-1 interactions. Chembiochem 15:844–851

    Article  CAS  PubMed  Google Scholar 

  24. Maglinao M, Eriksson M, Schlegel MK, Zimmermann S, Johannssen T, Götze S, Seeberger PH, Lepenies B (2014) A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation. J Control Release 175:36–42

    Article  CAS  PubMed  Google Scholar 

  25. Martin CE, Broecker F, Oberli MA, Komor J, Mattner J, Anish C, Seeberger PH (2013) Immunological evaluation of a synthetic Clostridium difficile oligosaccharide conjugate vaccine candidate and identification of a minimal epitope. J Am Chem Soc 135:9713–9722

    Article  CAS  PubMed  Google Scholar 

  26. Martin CE, Broecker F, Eller S, Oberli MA, Anish C, Pereira CL, Seeberger PH (2013) Glycan arrays containing synthetic Clostridium difficile lipoteichoic acid oligomers as tools toward a carbohydrate vaccine. Chem Commun (Camb) 49:7159–7161

    Article  CAS  Google Scholar 

  27. Reinhardt A, Yang Y, Claus H, Pereira CL, Cox AD, Vogel U, Anish C, Seeberger PH (2015) Antigenic potential of a highly conserved Neisseria meningitidis lipopolysaccharide inner core structure defined by chemical synthesis. Chem Biol 22:38–49

    Article  CAS  PubMed  Google Scholar 

  28. Mietzsch M, Broecker F, Reinhardt A, Seeberger PH, Heilbronn R (2014) Differential adeno-associated virus serotype-specific interaction patterns with synthetic heparins and other glycans. J Virol 88:2991–3003

    Article  PubMed  PubMed Central  Google Scholar 

  29. Tamborrini M, Werz DB, Frey J, Pluschke G, Seeberger PH (2006) Anti-carbohydrate antibodies for the detection of anthrax spores. Angew Chem Int Ed Engl 45:6581–6582

    Article  CAS  PubMed  Google Scholar 

  30. Oberli MA, Tamborrini M, Tsai YH, Werz DB, Horlacher T, Adibekian A, Gauss D, Möller HM, Pluschke G, Seeberger PH (2010) Molecular analysis of carbohydrate-antibody interactions: case study using a Bacillus anthracis tetrasaccharide. J Am Chem Soc 132:10239–10241

    Article  CAS  PubMed  Google Scholar 

  31. Tamborrini M, Holzer M, Seeberger PH, Schürch N, Pluschke G (2010) Anthrax spore detection by a luminex assay based on monoclonal antibodies that recognize anthrose-containing oligosaccharides. Clin Vaccine Immunol 17:1446–1451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Anish C, Guo X, Wahlbrink A, Seeberger PH (2013) Plague detection by anti-carbohydrate antibodies. Angew Chem Int Ed Engl 52:9524–9528

    Article  CAS  PubMed  Google Scholar 

  33. Broecker F, Aretz J, Yang Y, Hanske J, Guo X, Reinhardt A, Wahlbrink A, Rademacher C, Anish C, Seeberger PH (2014) Epitope recognition of antibodies against a Yersinia pestis lipopolysaccharide trisaccharide component. ACS Chem Biol 9:867–873

    Article  CAS  PubMed  Google Scholar 

  34. Götze S, Azzouz N, Tsai YH, Groß U, Reinhardt A, Anish C, Seeberger PH, Varón Silva D (2014) Diagnosis of toxoplasmosis using a synthetic glycosylphosphatidylinositol glycan. Angew Chem Int Ed Engl 53:13701–13705

    Article  PubMed  Google Scholar 

  35. Palma AS, Feizi T, Childs RA, Chai W, Liu Y (2014) The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome. Curr Opin Chem Biol 18:87–94

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Song X, Heimburg-Molinaro J, Cummings RD, Smith DF (2014) Chemistry of natural glycan microarrays. Curr Opin Chem Biol 18:70–77

    Article  CAS  PubMed  Google Scholar 

  37. Martin CE, Weishaupt MW, Seeberger PH (2011) Progress toward developing a carbohydrate-conjugate vaccine against Clostridium difficile ribotype 027: synthesis of the cell-surface polysaccharide PS-I repeating unit. Chem Commun (Camb) 47:10260–10262

    Article  CAS  Google Scholar 

  38. Oberli MA, Hecht ML, Bindschädler P, Adibekian A, Adam T, Seeberger PH (2011) A possible oligosaccharide-conjugate vaccine candidate for Clostridium difficile is antigenic and immunogenic. Chem Biol 18:580–588

    Article  CAS  PubMed  Google Scholar 

  39. Yang Y, Oishi S, Martin CE, Seeberger PH (2013) Diversity-oriented synthesis of inner core oligosaccharides of the lipopolysaccharide of pathogenic Gram-negative bacteria. J Am Chem Soc 135:6262–6271

    Article  CAS  PubMed  Google Scholar 

  40. Anish C, Martin CE, Wahlbrink A, Bogdan C, Ntais P, Antoniou M, Seeberger PH (2013) Immunogenicity and diagnostic potential of synthetic antigenic cell surface glycans of Leishmania. ACS Chem Biol 8:2412–2422

    Article  CAS  PubMed  Google Scholar 

  41. Hewitt MC, Seeberger PH (2001) Solution and solid-support synthesis of a potential leishmaniasis carbohydrate vaccine. J Org Chem 66:4233–4243

    Article  CAS  PubMed  Google Scholar 

  42. Hanashima S, Seeberger PH (2007) Total synthesis of sialylated glycans related to avian and human influenza virus infection. Chem Asian J 2:1447–1459

    Article  CAS  PubMed  Google Scholar 

  43. Hanashima S, Castagner B, Esposito D, Nokami T, Seeberger PH (2007) Synthesis of a sialic acid alpha(2-3) galactose building block and its use in a linear synthesis of sialyl Lewis C. Org Lett 9:1777–1779

    Article  CAS  PubMed  Google Scholar 

  44. Boonyarattanakalin S, Liu X, Michieletti M, Lepenies B, Seeberger PH (2008) Chemical synthesis of all phosphatidylinositol mannoside (PIM) glycans from Mycobacterium tuberculosis. J Am Chem Soc 130:16791–16799

    Article  CAS  PubMed  Google Scholar 

  45. Hölemann A, Stocker BL, Seeberger PH (2006) Synthesis of a core arabinomannan oligosaccharide of Mycobacterium tuberculosis. J Org Chem 71:8071–8088

    Article  PubMed  Google Scholar 

  46. Seeberger PH, Soucy RL, Kwon YU, Snyder DA, Kanemitsu T (2004) A convergent, versatile route to two synthetic conjugate anti-toxin malaria vaccines. Chem Commun (Camb) 15:1706–1707

    Article  Google Scholar 

  47. Kwon YU, Soucy RL, Snyder DA, Seeberger PH (2005) Assembly of a series of malarial glycosylphosphatidylinositol anchor oligosaccharides. Chemistry 11:2493–2504

    Article  CAS  PubMed  Google Scholar 

  48. Stowell SR, Arthur CM, McBride R, Berger O, Razi N, Heimburg-Molinaro J, Rodrigues LC, Gourdine JP, Noll AJ, von Gunten S, Smith DF, Knirel YA, Paulson JC, Cummings RD (2014) Microbial glycan microarrays define key features of host-microbial interactions. Nat Chem Biol 10:470–476

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Julien JP, Lee JH, Cupo A, Murin CD, Derking R, Hoffenberg S, Caulfield MJ, King CR, Marozsan AJ, Klasse PJ, Sanders RW, Moore JP, Wilson IA, Ward AB (2013) Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9. Proc Natl Acad Sci U S A 110:4351–4356

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Silva-Martin N, Schauer JD, Park CG, Hermoso JA (2009) Crystallization and preliminary X-ray diffraction studies of the carbohydrate-recognition domain of SIGN-R1, a receptor for microbial polysaccharides and sialylated antibody on splenic marginal zone macrophages. Acta Crystallogr Sect F: Struct Biol Cryst Commun 65:1264–1266

    Article  CAS  Google Scholar 

  51. Haji-Ghassemi O, Blackler RJ, Young NM, Evans SV (2015) Antibody recognition of carbohydrate epitopes. Glycobiology (Epub ahead of print)

    Google Scholar 

  52. Moller IE, Pettolino FA, Hart C, Lampugnani ER, Willats WG, Bacic A (2012) Glycan profiling of plant cell wall polymers using microarrays. J Vis Exp 70:e4238

    Google Scholar 

  53. Wang CC, Huang YL, Ren CT, Lin CW, Hung JT, Yu JC, Yu AL, Wu CY, Wong CH (2008) Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer. Proc Natl Acad Sci U S A 105:11661–11666

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank the Max Planck Society, the Körber foundation (Körber Prize to PHS) and the German Federal Ministry of Education and Research (grant No. 0315447) for generous financial support. This work was supported by the Deutsche Forschungsgemeinschaft (SFB-TR84 to PHS). We acknowledge careful and critical reviewing of the manuscript by Mr. Andreas Geissner, Ms. Anika Reinhardt, and Dr. Sebastian Götze. We are grateful to Dr. Sebastian Götze, Dr. You Yang, Dr. Xiaoquang Guo, and Dr. Shinya Hanashima for supplying the synthetic glycans used in this study.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Peter H. Seeberger .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Broecker, F., Seeberger, P.H. (2017). Synthetic Glycan Microarrays. In: Uttamchandani, M., Yao, S. (eds) Small Molecule Microarrays. Methods in Molecular Biology, vol 1518. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6584-7_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6584-7_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6582-3

  • Online ISBN: 978-1-4939-6584-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics