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Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies

Nature Protocols volume 12, pages 2411–2422 (2017)Cite this article

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Abstract

Glycobiology, in particular the study of carbohydrate–protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments. The first stage is to add an oligo(ethylene glycol) linker (OEG-linker) that has a terminal triphenylmethanethiol group to the reducing end of the oligosaccharide by oxime formation catalyzed by aniline. The triphenylmethyl (trityl) tag is then removed from the linker to expose a thiol (stage 2) to allow a conjugation reaction at the thiol group (stage 3). There are many possible conjugation reactions, depending on the desired application. Examples shown in this protocol are as follows: (i) coupling of the oligosaccharide to a support for surface plasmon resonance (SPR) studies, (ii) fluorescence labeling for microscale thermophoresis (MST) or bioimaging, and (iii) biotinylation for biolayer interferometry (BLI) studies. This protocol starts from unprotected carbohydrates and provides glycoconjugates in milligram amounts in just 2 d.

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Figure 1: Overview of the general procedure.
Figure 2: Overview of the two-step carbohydrate-linker conjugation.
Figure 5: Chromatographic analysis of glycoconjugates (absorbance at 215 nm).
Figure 3: Fluorescence labeling of Nod factor by reaction between free thiol and maleimide-functionalized Alexa 546.
Figure 4: Biotinylation of EPS octasaccharide by a reaction between a free thiol and iodoacetamide-functionalized biotin.
Figure 6: Measurements of Nod factor binding to legume-plant receptors NFR1 and NFR5.
Figure 7: BLI experiments showing the interaction between the EPS fragment and the EPR3 receptor.

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Acknowledgements

Generous support by the Danish National Research Foundation (grant no. DNRF79) and by the Villum Foundation (grant no. VKR022710) is gratefully acknowledged. The authors thank C.W. Ronson and J.T. Sullivan for bacterial production of the EPS octa-saccharide and M. loti R7A Nod factors.

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Authors and Affiliations

  1. Centre for Carbohydrate Recognition and Signaling, Copenhagen University, Frederiksberg, Denmark

    Christian T Hjuler, Nicolai N Maolanon, Jørgen Sauer, Mikkel B Thygesen & Knud J Jensen

  2. Department of Chemistry, University of Copenhagen, Frederiksberg, Denmark

    Christian T Hjuler, Nicolai N Maolanon, Jørgen Sauer, Mikkel B Thygesen & Knud J Jensen

  3. Centre for Carbohydrate Recognition and Signaling, Aarhus University, Aarhus, Denmark

    Jens Stougaard

  4. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Jens Stougaard

Authors
  1. Christian T Hjuler
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  2. Nicolai N Maolanon
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Contributions

M.B.T., J. Sauer and K.J.J. designed the study. C.T.H., N.N.M., J. Sauer and M.B.T. performed the experiments. C.T.H., N.N.M., J. Stougaard, M.B.T. and K.J.J. wrote the manuscript.

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Correspondence to Mikkel B Thygesen or Knud J Jensen.

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The authors declare no competing financial interests.

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Hjuler, C., Maolanon, N., Sauer, J. et al. Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies. Nat Protoc 12, 2411–2422 (2017). https://doi.org/10.1038/nprot.2017.109

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