Skip to main content
SpringerLink
Log in
Book cover

The Nucleus pp 227–254Cite as

Detection and Analysis of (O-linked β-N-Acetylglucosamine)-Modified Proteins

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 464))

Abstract

Glycosylation is one of the most common and complex forms of posttranslational modifications of proteins in eukaryotes. Seven different protein-carbohydrate linkages have been characterized on nuclear and cytoplasmic glycoproteins, the most widespread of which is the modification of Ser/Thr residues with monosaccharides of O-linked β-N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification is concentrated in nuclear proteins. O-GlcNAc is thought to regulate protein function in a manner analogous to phosphorylation; and is implicated in the regulation of transcription, the proteasome, insulin and MAP kinase signaling, the cell cycle, and the cellular stress response. In this chapter we focus on methods for the detection of O-GlcNAc-modified proteins and discuss general techniques for the detection and subsequent analysis of other protein-carbohydrate conjugates.

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.99
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. Hart, G. W., Haltiwanger, R. S., Holt, G. D., and Kelly, W. G. (1989) Glycosylation in the nucleus and cytoplasm. Annu. Rev. Biochem. 58, 841-874.

    Article  PubMed  CAS  Google Scholar 

  2. Lomako, J., Lomako, W. M., and Whelan, W. J. (2004) Glycogenin: the primer for mammalian and yeast glycogen synthesis. Biochim. Biophys. Acta. 1673, 45-55.

    Article  PubMed  CAS  Google Scholar 

  3. Marchase, R, B., Bounelis, P., Brumley, L. M., Dey N., Browne, B., Auger, D., Fritz, T. A., Kulesza, P., and Bedwell, D. M. (1993) Phosphoglucomutase in Saccharomyces cerevisiae is a cytoplasmic glycoprotein and the acceptor for a Glc-phosphotransferase. J. Biol. Chem. 268, 8341-8349.

    PubMed  CAS  Google Scholar 

  4. Dey, N. B., Bounelis, P., Fritz, T. A., Bedwell, D. M., and Marchase, R. B. (1994) The glycosylation of phosphoglucomutase is modulated by carbon source and heat shock in Saccharomyces cerevisiae. J. Biol. Chem. 269, 27143-27148.

    PubMed  CAS  Google Scholar 

  5. Veyna, N. A., Jay, J. C., Srisomsap, C., Bounelis, P., and Marchase, R. B. (1994) The addition of glucose-1-phosphate to the cytoplasmic glycoprotein phosphoglucomutase is modulated by intracellular calcium in PC12 cells and rat cortical synaptosomes. J. Neurochem. 62, 456-464.

    Article  PubMed  CAS  Google Scholar 

  6. Schirmer, J. and Aktories, K. (2004) Large clostridial cytotoxins: cellular biology of Rho/Rasglucosylating toxins. Biochim. Biophys. Acta. 1673, 66-74.

    Article  PubMed  CAS  Google Scholar 

  7. West, C. M., Van Der Wel, H., Sassi, S., and Gaucher, E. A. (2004) Cytoplasmic glycosylation of protein-hydroxyproline and its relationship to other glycosylation pathways. Biochim. Biophys. Acta. 1673, 29-44.

    Article  PubMed  CAS  Google Scholar 

  8. Zachara, N. E. and Hart, G. W. (2004) O-GlcNAc a sensor of cellular state: the role of nucleo cytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim. Biophys. Acta. 1673, 13-28.

    Article  PubMed  CAS  Google Scholar 

  9. Zachara, N. E. and Hart, G. W. (2006) Cell signaling, the essential role of O-GlcNAc. Biochim. Biophys. Acta. 1761, 599-617.

    Article  PubMed  CAS  Google Scholar 

  10. O’Donnell, N., Zachara, N. E., Hart, G. W., and Marth, J. D. (2004) Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability. Mol. Cell. Biol. 24, 1680-1690.

    Article  PubMed  Google Scholar 

  11. Shafi, R., Iyer, S. P., Ellies, L. G., O’Donnell, N., Marek, K. W., Chui, D., Hart, G. W., and Marth, J. D. (2000) The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proc. Natl. Acad. Sci. USA. 97, 5735-5739.

    Article  PubMed  CAS  Google Scholar 

  12. Hartweck, L. M., Scott, C. L., and Olszewski, N. E. (2002) Free in PMC Two O-linked Nacetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development. Genetics. 161, 1279-1291.

    CAS  Google Scholar 

  13. Turner, J. R., Tartakoff, A. M., and Greenspan, N. S. (1990) Cytologic assessment of nuclear and cytoplasmic O-linked N-acetylglucosamine distribution by using anti-streptococcal monoclonal antibodies. Proc. Natl. Acad. Sci. USA. 87, 5608-5612.

    Article  PubMed  CAS  Google Scholar 

  14. Holt, G. D., Snow, C. M., Senior, A., Haltiwanger, R. S., Gerace, L., and Hart, G. W. (1987) Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J. Cell Biol. 104, 1157-1164.

    Article  PubMed  CAS  Google Scholar 

  15. Snow, C. M., Senior, A., and Gerace, L. (1987) Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol. 104, 1143-1156.

    Article  PubMed  CAS  Google Scholar 

  16. Comer, F. I., Vosseller, K., Wells, L., Accavitti, M. A., and Hart, G. W. (2001) Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal. Biochem. 293, 169-177.

    Article  PubMed  CAS  Google Scholar 

  17. Matsuoka, Y., Shibata, S., Yasuhara, N., and Yoneda, Y. (2002) MAb MY95, Anti-O-linked N-acetylglucosamine-modified proteins. Hybrid. Hybridomics. 21, 233-236.

    Article  PubMed  CAS  Google Scholar 

  18. Kamemura, K., Hayes, B. K., Comer, F. I., and Hart, G. W. (2002) Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: alternative glycosylation/phosphorylation of THR-58, a known mutational hot spot of c-Myc in lymphomas, is regulated by mitogens. J. Biol. Chem. 277, 19229-19235.

    Article  PubMed  CAS  Google Scholar 

  19. Ludemann, N., Clement, A., Hans, V. H., Leschik, J., Behl, C., and Brandt, R. (2005) O-glycosylation of the tail domain of neurofilament protein M in human neurons and in spinal cord tissue of a rat model of amyotrophic lateralsclerosis (ALS). J. Biol. Chem. 280, 31648-31658.

    Article  PubMed  Google Scholar 

  20. Roquemore, E. P., Chou, T. Y., and Hart, G. W. (1994) Detection of O-linked N-acetylglucosamine (O-GlcNAc) on cytoplasmic and nuclear proteins. Methods Enzymol. 230, 443-460.

    Article  PubMed  CAS  Google Scholar 

  21. Torres, C. R., and Hart, G. W. (1984) Topography and polypeptide distribution of terminal Nacetylglucosamine residues on the surfaces of intactlymphocytes.Evidence for O-linked GlcNAc. J. Biol. Chem. 259, 3308-3317.

    PubMed  CAS  Google Scholar 

  22. Khidekel, N., Arndt, S., Lamarre-Vincent, N., Lippert, A., Poulin-Kerstien, K.G., Ramakrishnan, B., Qasba, P. K., and Hsieh-Wilson, L. C. (2003) A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. J. Am. Chem. Soc. 125, 16162-16163.

    Article  PubMed  CAS  Google Scholar 

  23. Vocadlo, D. J., Hang, H. C., Kim, E. J., Hanover, J. A., and Bertozzi, C. R. (2003) A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc. Natl. Acad. Sci. USA. 100, 9116-9121.

    Article  PubMed  CAS  Google Scholar 

  24. Hayes, B. K., Greis, K. D., and Hart, G. W. (1995) Specific isolation of O-linked N-acetylglucosamine glycopeptides from complex mixtures. Anal. Biochem. 228, 115-122.

    Article  PubMed  CAS  Google Scholar 

  25. Greis, K. D., Hayes, B. K., Comer, F. I., Kirk, M., Barnes, S., Lowary, T. L., and Hart, G. W. (1996) Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by betaelimination and tandem electrospray mass spectrometry. Anal. Biochem. 234, 38-49.

    Article  PubMed  CAS  Google Scholar 

  26. Greis, K. D. and Hart, G. W. (1998) Analytical methods for the study of O-GlcNAc glycoproteins and glycopeptides. Methods Mol. Biol. 76, 19-33.

    PubMed  CAS  Google Scholar 

  27. Haynes, P. A., and Aebersold, R. (2000) Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography-tandem mass spectrometry. Anal. Chem. 72, 5402-5410.

    Article  PubMed  CAS  Google Scholar 

  28. Zachara, N. E. and Gooley, A. A. (2000) Identification of glycosylation sites in mucin peptides by edman degradation. Methods Mol. Biol. 125, 121-128.

    PubMed  CAS  Google Scholar 

  29. Vosseller, K., Hansen, K. C., Chalkley, R. J., Trinidad, J. C., Wells, L., Hart, G. W., and Burlingame, A. L. (2005) Quantitative analysis of both protein expression and serine/threonine post-translational modifications through stable isotope labeling with dithiothreitol. Proteomics. 5, 388-398.

    Article  PubMed  CAS  Google Scholar 

  30. Wells, L., Vosseller, K., Cole, R. N., Cronshaw, J. M., Matunis, M. J., and Hart, G. W. (2002) Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine posttranslational modifications. Mol. Cell. Proteomics. 1, 791-804.

    Article  PubMed  CAS  Google Scholar 

  31. Chalkley, R. J., and Burlingame, A. L. (2003) Identification of novel sites of O-N-acetylglucosamine modification of serum response factor using quadrupole time-of-flight mass spectrometry. Mol. Cell. Proteomics. 2, 182-190.

    Article  PubMed  CAS  Google Scholar 

  32. Chalkley, R. J. and Burlingame, A. L. (2001) Identification of GlcNAcylation sites of peptides and alpha-crystallin using Q-TOF mass spectrometry. J. Am. Soc. Mass. Spectrom. 12, 1106-1113.

    Article  PubMed  CAS  Google Scholar 

  33. Reason, A. J., Morris, H. R., Panico, M., Marais, R., Treisman, R. H., Haltiwanger, R. S., Hart, G. W., Kelly, W. G., and Dell, A. (1992) Localization of O-GlcNAc modification on the serum response transcription factor. J. Biol. Chem. 267, 16911-16021.

    PubMed  CAS  Google Scholar 

  34. Reason, A. J., Blench, I. P., Haltiwanger, R. S., Hart, G. W., Morris, H. R., Panico, M., and Dell, A. (1991) High-sensitivity FAB-MS strategies for O-GlcNAc characterization. Glycobiology. 1, 585-594.

    Article  PubMed  CAS  Google Scholar 

  35. Reason, A., Dell, A., Morris, H. R., Panico, M., Treisman, R., Marais, R., Hart, G. W., and Haltiwanger, R. S. (1991) Identification of O-GlcNAc attachment sites in transcription factors. Glycoconjugate J. 8, 211.

    Google Scholar 

  36. Nandi, A., Sprung, R., Barma, D. K., Zhao, Y., Kim, S. C., Falck, J. R., and Zhao, Y. (2006) Global identification of O-GlcNAc-modified proteins. Anal. Chem. 78, 452-458.

    Article  PubMed  CAS  Google Scholar 

  37. Sprung, R., Nandi, A., Chen, Y., Kim, S. C., Barma, D., Falck, J. R., and Zhao, Y. (2005) Taggingvia-substrate strategy for probing O-GlcNAc modified proteins. J. Proteome Res. 4, 950-957.

    Article  PubMed  CAS  Google Scholar 

  38. Hart, G. W., Cole, R. N., Kreppel, L. K., Arnold, C. S., Comer, F. I., Iyer, S., Cheng, X., Carroll, J. and Parker, G. J. (2000) Glycosylation of proteins—a major challenge in mass spectrometry and proteomics. In: Proceedings of the 4th international symposium on mass spectrometry in the health and life sciences (Burlingame, A., Carr, S., and Baldwin, M., eds), Humana Press, Totowa, New Jersey, pp. 365-382

    Google Scholar 

  39. Bakhtiar, R. and Guan, Z. (2006) Electron capture dissociation mass spectrometry in characterization of peptides and proteins. Biotechnol. Lett. 28, 1047-1059.

    Article  PubMed  CAS  Google Scholar 

  40. Dong, D. L. and Hart, G. W. (1994) Purification and characterization of an O-GlcNAc selective N-acetyl-beta-d-glucosaminidase from rat spleen cytosol. J. Biol. Chem. 269, 19321-19330.

    PubMed  CAS  Google Scholar 

  41. Haltiwanger, R. S., Grove, K., and Philipsberg, G. A. (1998) Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. J. Biol. Chem. 273, 3611-3617.

    Article  PubMed  CAS  Google Scholar 

  42. Kim, E. J., Perreira, M., Thomas, C. J., and Hanover, J. A. (2006) An O-GlcNAcase-specific inhibitor and substrate engineered by the extension of the N-acetyl moiety. J. Am. Chem. Soc. 128, 4234-4235.

    Article  PubMed  CAS  Google Scholar 

  43. Lee, T. N., Alborn, W. E., Knierman, M. D., and Konrad, R. J. (2006) Alloxan is an inhibitor of O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase. Biochem. Biophys. Res. Commun. 350, 1038-1043.

    Article  PubMed  CAS  Google Scholar 

  44. Macauley, M. S., Whitworth, G. E., Debowski, A. W., Chin, D., and Vocadlo, D. J. (2005) O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J. Biol. Chem. 280, 25313-25322.

    Article  PubMed  CAS  Google Scholar 

  45. Stubbs, K. A., Zhang, N., and Vocadlo, D. J. (2006) A divergent synthesis of 2-acyl derivatives of PUGNAc yields selective inhibitors of O-GlcNAcase. Org. Biomol. Chem. 4, 839-845.

    Article  PubMed  CAS  Google Scholar 

  46. Whitworth, G. E., Macauley, M. S., Stubbs, K. A., Dennis, R. J., Taylor, E. J., Davies, G. J., Greig, I. R., and Vocadlo, D. J. (2007) Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise. J. Am. Chem. Soc. 129, 635-644.

    Article  PubMed  CAS  Google Scholar 

  47. Okuyama, R. and Yachi, M. (2001) Cytosolic O-GlcNAc accumulation is not involved in beta cell death in HIT-T15 or Min6. Biochem. Biophys. Res. Commun. 287, 366-371.

    Article  PubMed  CAS  Google Scholar 

  48. Gao, Y., Parker, G. J., and Hart, G. W. (2000) Streptozotocin-induced beta-cell death is independent of its inhibition of O-GlcNAcase in pancreatic Min6 cells. Arch. Biochem. Biophys. 383, 296-302.

    Article  PubMed  CAS  Google Scholar 

  49. Gross, B. J., Kraybill, B. C., and Walker, S. (2005) Discovery of O-GlcNAc transferase inhibitors. J. Am. Chem. Soc. 127, 14588-14589.

    Article  PubMed  CAS  Google Scholar 

  50. Fiordaliso, F., Leri, A., Cesselli, D., Limana, F., Safai, B., Nadal-Ginard, B., Anversa, P., and Kajstura, J. (2001) Hyperglycemia activates p53 and p53-regulated genes leading to myocyte cell death. Diabetes. 50, 2363-2375.

    Article  PubMed  CAS  Google Scholar 

  51. James, L. R., Tang, D., Ingram, A., Ly, H., Thai, K., Cai, L., and Scholey, J. W. (2002) Flux through the hexosamine pathway is a determinant of nuclear factor kappaB- dependent promoter activation. Diabetes. 51, 1146-1156.

    Article  PubMed  CAS  Google Scholar 

  52. Konrad, R. J., Zhang, F., Hale, J. E., Knierman, M. D., Becker, G. W., and Kudlow, J. E. (2002) Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase. Biochem. Biophys. Res. Commun. 293, 207-212.

    Article  PubMed  CAS  Google Scholar 

  53. Marshall, S., Bacote, V., and Traxinger, R. R. (1991) Complete inhibition of glucose-induced desensitization of the glucose transport system by inhibitors of mRNA synthesis. Evidence for rapid turnover of glutamine:fructose-6-phosphate amidotransferase. J. Biol. Chem. 266, 4706-4712.

    PubMed  CAS  Google Scholar 

  54. Greis, K. D., Hayes, B. K., Comer, F. I., Kirk, M., Barnes, S., Lowary, T. L., and Hart, G. W. (1996) Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by betaelimination and tandem electrospray mass spectrometry. Anal. Biochem. 234, 38-49.

    Article  PubMed  CAS  Google Scholar 

  55. Myllyharju, J. and Nokkala, S. (1998) Localization and identification of galactose/N-acetyl galactosamine and sialic acid-containing proteins in Chinese hamster metaphase chromosomes. Cell. Biol. Int. 22, 85-89.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The author acknowledges Prof. Gerald W. Hart (Johns Hopkins University School of Medicine) for comments on this manuscript, and the technical help of Katie Zoey Ho (Johns Hopkins Singapore). NEZ was supported by an A*Star Research grant to Johns Hopkins Singapore.

Author information

Authors and Affiliations

  1. The Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Natasha E. Zachara

Authors
  1. Natasha E. Zachara
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Hôtel-Dieu Hospital, Laval University Cancer Research Centre, Québec, Canada

    Ronald Hancock

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science + Business Media, LLC

About this protocol

Cite this protocol

Zachara, N.E. (2008). Detection and Analysis of (O-linked β-N-Acetylglucosamine)-Modified Proteins. In: Hancock, R. (eds) The Nucleus. Methods in Molecular Biology, vol 464. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-461-6_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-461-6_13

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-460-9

  • Online ISBN: 978-1-60327-461-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation