Advertisement

Analyzing the Posttranslational Modification Status of Notch Using Mass Spectrometry

  • Shinako Kakuda
  • Robert S. HaltiwangerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1187)

Abstract

Notch is modified by multiple types of posttranslational modifications, most of which are known to affect Notch function. The extracellular domain (ECD) is modified with N-glycosylation and at least three types of O-glycosylation (O-fucose, O-glucose, and O-GlcNAc), while the intracellular domain is hydroxylated, phosphorylated, and ubiquitinated. In order to analyze the structure and function of the O-glycans decorating the ECD, we have developed semiquantitative mass spectral methods for identifying modifications at individual sites on Notch that are generally applicable to most posttranslational modifications. Here we describe the expression and purification of Notch ECD fragments, digestion of the fragments with proteases to prepare for mass spectral analysis, and identification of peptides modified with O-glycans using mass spectrometry.

Key words

Notch EGF repeats O-fucose O-glucose O-GlcNAc Mass spectral analysis 

Notes

Acknowledgements

We would like to thank Haltiwanger lab members for helpful comments. Primary work introduced here was supported by NIH grants GM061126 and CA12307101.

References

  1. 1.
    Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Rana NA, Haltiwanger RS (2011) Fringe benefits: functional and structural impacts of O-glycosylation on the extracellular domain of Notch receptors. Curr Opin Struct Biol 21: 583–589PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Moloney DJ, Shair LH, Lu FM et al (2000) Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. J Biol Chem 275:9604–9611PubMedCrossRefGoogle Scholar
  4. 4.
    Shao L, Moloney DJ, Haltiwanger RS (2003) Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region. J Biol Chem 278:7775–7782PubMedCrossRefGoogle Scholar
  5. 5.
    Matsuura A, Ito M, Sakaidani Y et al (2008) O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors. J Biol Chem 283:35486–35495PubMedCrossRefGoogle Scholar
  6. 6.
    Rana NA, Nita-Lazar A, Takeuchi H et al (2011) O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1. J Biol Chem 286: 31623–31637PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Foltz DR, Santiago MC, Berechid BE et al (2002) Glycogen synthase kinase-3beta modulates notch signaling and stability. Curr Biol 12:1006–1011PubMedCrossRefGoogle Scholar
  8. 8.
    Gupta-Rossi N, Six E, LeBail O et al (2004) Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor. J Cell Biol 166:73–83PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Coleman ML, McDonough MA, Hewitson KS et al (2007) Asparaginyl hydroxylation of the Notch ankyrin repeat domain by factor inhibiting hypoxia-inducible factor. J Biol Chem 282:24027–24038PubMedCrossRefGoogle Scholar
  10. 10.
    Campbell ID, Bork P (1993) Epidermal Growth Factor-like Modules. Curr Opin Struct Biol 3:385–392CrossRefGoogle Scholar
  11. 11.
    Sakaidani Y, Nomura T, Matsuura A et al (2011) O-linked-N-acetylglucosamine on extracellular protein domains mediates epithelial cell-matrix interactions. Nat Commun 2:583PubMedCrossRefGoogle Scholar
  12. 12.
    Acar M, Jafar-Nejad H, Takeuchi H et al (2008) Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling. Cell 132: 247–258PubMedCentralPubMedGoogle Scholar
  13. 13.
    Sethi MK, Buettner FF, Ashikov A et al (2012) Molecular cloning of a xylosyltransferase that transfers the second xylose to O-glucosylated epidermal growth factor repeats of notch. J Biol Chem 287:2739–2748PubMedCentralPubMedGoogle Scholar
  14. 14.
    Sethi MK, Buettner FF, Krylov VB et al (2010) Identification of glycosyltransferase 8 family members as xylosyltransferases acting on O-glucosylated notch epidermal growth factor repeats. J Biol Chem 285:1582–1586PubMedCentralPubMedGoogle Scholar
  15. 15.
    Wang Y, Shao L, Shi S et al (2001) Modification of epidermal growth factor-like repeats with O-fucose. Molecular cloning of a novel GDP-fucose protein O-fucosyltransferase J Biol Chem 276:40338–40345Google Scholar
  16. 16.
    Moloney DJ, Panin VM, Johnston SH et al (2000) Fringe is a glycosyltransferase that modifies Notch. Nature 406:369–375PubMedGoogle Scholar
  17. 17.
    Sakaidani Y, Ichiyanagi N, Saito C et al (2012) O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1. Biochem Biophys Res Commun 419:14–19PubMedGoogle Scholar
  18. 18.
    Shi S, Stanley P (2003) Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc Natl Acad Sci USA 100:5234–5239PubMedCentralPubMedGoogle Scholar
  19. 19.
    Fernandez-Valdivia R, Takeuchi H, Samarghandi A et al (2011) Regulation of mammalian Notch signaling and embryonic development by the protein O-glucosyltransferase Rumi. Development 138:1925–1934PubMedCentralPubMedGoogle Scholar
  20. 20.
    Lee TV, Sethi MK, Leonardi J et al (2013) Negative regulation of notch signaling by xylose. PLoS Genet 9:e1003547PubMedCentralPubMedGoogle Scholar
  21. 21.
    Panin VM, Papayannopoulos V, Wilson R et al (1997) Fringe modulates Notch-ligand interactions. Nature 387:908–912PubMedGoogle Scholar
  22. 22.
    Yamamoto S, Charng W-L, Rana NA et al (2012) A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands. Science 338:1229–1232PubMedCentralPubMedGoogle Scholar
  23. 23.
    Liebler DC (2002) Introduction to proteomics tools for the new biology. Humana Press, TotowaGoogle Scholar
  24. 24.
    Unwin RD, Evans CA, Whetton AD (2006) Relative quantification in proteomics: new approaches for biochemistry. Trends Biochem Sci 31:473–484PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biochemistry and Cell BiologyStony Brook UniversityStony BrookUSA

Personalised recommendations