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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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.).
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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.
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The University of Georgia Research Foundation, Inc. has non-exclusively licensed the antibodies to Millipore Corporation.
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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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DOI: https://doi.org/10.1038/nchembio.338
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