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Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc

Nature Chemical Biology volume 6, pages 338–343 (2010)Cite this article

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Abstract

Studies of post-translational modification by β-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc–specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc–specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc–modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.

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Figure 1: Structures of fully synthetic three-component immunogens 1 and 2.
Figure 2: Immunoblots of three monoclonal antibodies.
Figure 3: Application of monoclonal antibodies for O-GlcNAc-omics in mammalian tissue.

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Acknowledgements

We thank R. Davis (University of Georgia) for monoclonal antibody production, J.-M. Lim and L. Zhao for expert assistance with mass spectrometry, E.G. El-Karim for assistance with molecular biology and T. Buskas for assistance with glycopeptide synthesis and protein conjugation. We also thank BioInquire, Inc. for access to the beta version of ProteoIQ that was used in the evaluation of the mass spectrometry data. We thank G.W. Hart (Johns Hopkins School of Medicine) for CTD110.6 and AL28 (anti-OGT antibodies) and S.W. Whiteheart (University of Kentucky) for the anti-OGA antibody. This research was supported by a grant from the US National Institute of Diabetes and Digestive and Kidney disorders (NIH RO1 DK075069 to L.W.), the Research Resource for Integrated Glycotechnology (NIH/NCRR P41R005351 to G.-J.B.) and the National Cancer Institute of the US National Institutes of Health (NIH/NCI R01CA088986 to G.-J.B.). C.F.T. was supported by a predoctoral fellowship from the American Heart Association (Southeast Affiliation). This work was further supported by grants NIH HL067464 and HL079364 (to J.C.C.).

Author information

Author notes

  1. Laszlo G Nöt

    Present address: Present address: Department of Traumatology and Hand Surgery, Institute of Musculoskeletal Surgery, University of Pécs, Pécs, Hungary.,

  2. Chin Fen Teo, Sampat Ingale and Margreet A Wolfert: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, USA

    Chin Fen Teo & Lance Wells

  2. Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, USA

    Chin Fen Teo, Sampat Ingale, Margreet A Wolfert, Galal A Elsayed, Lance Wells & Geert-Jan Boons

  3. Department of Chemistry, University of Georgia, Athens, Georgia, USA

    Sampat Ingale, Lance Wells & Geert-Jan Boons

  4. Department of Chemistry, Faculty of Science, Ain Shams University, Cairo, Egypt

    Galal A Elsayed

  5. Department of Cell Biology, University of Alabama, Birmingham, Alabama, USA

    Laszlo G Nöt & John C Chatham

  6. Division of Cardiovascular Disease, Department of Medicine, University of Alabama, Birmingham, Alabama, USA

    John C Chatham

  7. Center for Cardiovascular Biology, University of Alabama, Birmingham, Alabama, USA

    John C Chatham

  8. Center for Free Radical Biology, University of Alabama, Birmingham, Alabama, USA

    John C Chatham

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Contributions

S.I. and G.A.E. performed the chemical synthesis. M.A.W. performed, analyzed and directed the immunological experiments. C.F.T. performed and analyzed the western blots and MS experiments. L.G.N. and J.C.C. were responsible for the rat model. L.W. and G.-J.B. were responsible for the overall experimental design and wrote the paper. G.-J.B. was responsible for compound design.

Corresponding authors

Correspondence to Lance Wells or Geert-Jan Boons.

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Competing interests

The University of Georgia Research Foundation, Inc. has non-exclusively licensed the antibodies to Millipore Corporation.

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Supplementary Methods, Supplementary Figures 1–6 and Supplementary Tables 1–7 (PDF 3767 kb)

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Teo, C., Ingale, S., Wolfert, M. et al. Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6, 338–343 (2010). https://doi.org/10.1038/nchembio.338

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