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Photocrosslinking of glycoconjugates using metabolically incorporated diazirine-containing sugars

Nature Protocols volume 4, pages 1044–1063 (2009)Cite this article

Abstract

Transient interactions among glycoconjugates underlie developmental, immunological and metastatic recognition. Glycan-mediated interactions have low binding affinities and rapid dissociation rates. As a result, these complexes dissociate when removed from their cellular context, complicating characterization. Photocrosslinkers introduce a covalent bond between glycoconjugates and their binding partners, allowing physiologically relevant complexes to be isolated. This protocol describes metabolic incorporation of a diazirine photocrosslinker into sialic acids in cellular glycoconjugates. Subsequent irradiation results in photocrosslinking of sialic acid to neighboring macromolecules, providing a photochemical 'snapshot' of binding events. As photocrosslinking sugars are light activated, these reagents have the potential to be used for temporally and/or spatially restricted crosslinking. We provide instructions for the synthesis of photocrosslinking sugar precursors, cell culture for metabolic incorporation, flow cytometry to evaluate metabolic incorporation, photoirradiation and analysis of the crosslinked complexes. Synthesis of photocrosslinking sugars requires 4–6 d, and photocrosslinking experiments can be completed in an additional 6 d.

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Figure 1: Synthesis of reagents for metabolic labeling of cell-surface glycoconjugates.
Figure 2: Metabolic incorporation of photocrosslinking sialic acid.
Figure 3: Optimal ManNDAz concentration for maximizing cell growth and photocrosslinking sugar incorporation.
Figure 4: Strategy for identification of glycoconjugate interaction partners using photocrosslinking sugars.
Figure 5: Cell surface display of SiaDAz measured in UDP-GlcNAc 2-epimerase-deficient cells.
Figure 6: SiaDAz-mediated photocrosslinking of CD22 oligomers.

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Acknowledgements

We thank Michael Pawlita (German Cancer Research Center) and James Paulson (The Scripps Research Institute) for sharing BJAB K20, BJAB K88, HL60-I and HL60-II cells, and Ellen Vitetta (UT Southwestern Medical Center) for sharing Daudi cells. We thank Yan Li (UT Southwestern Medical Center Protein Chemistry Technology Center) for mass spectrometry analysis of sugars and Angela Mobley (UT Southwestern Medical Center Flow Cytometry Core Facility) for help with flow cytometry. We thank Dr Yoshihito Tanaka and Dr Seokho Yu for advice on synthesis. M.R.B. acknowledges the support of an NSF graduate fellowship and Stanford University. This work was supported by funds from a March of Dimes Basil O'Connor Starter Scholar Award, the Welch Foundation and the University of Texas Southwestern Medical Center.

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Authors and Affiliations

  1. Department of Chemistry, Stanford University, Stanford, California, USA

    Michelle R Bond

  2. Division of Translational Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Michelle R Bond, Haochi Zhang, Peter D Vu & Jennifer J Kohler

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Correspondence to Jennifer J Kohler.

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Bond, M., Zhang, H., Vu, P. et al. Photocrosslinking of glycoconjugates using metabolically incorporated diazirine-containing sugars. Nat Protoc 4, 1044–1063 (2009). https://doi.org/10.1038/nprot.2009.85

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