PMID: 22556278

 

    Legend: Gene, Sites

Title : Mapping of O-GlcNAc sites of 20 S proteasome proteasome subunits and Hsp90 by a novel biotin-cystamine tag

Abstract :
  1. The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport
  2. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress
  3. O-Glycosylation of the 26 S proteasome proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation
  4. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome proteasome , the 20 S proteasome proteasome , and the impact on proteasome activity is very limited
  5. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes
  6. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies
  7. Here we describe an adapted β-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag
  8. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine
  9. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified
  10. The method was optimized using bovine α -crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver
  11. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90 β, of which one corresponds to a previously described phosphorylation site
Output (sent_index, trigger, protein, sugar, site):
  • 0. Hsp90, , Hsp90, O-GlcNAc sites, -
  • 0. subunits, , subunits, O-GlcNAc sites, -
  • 3. O-Glycosylation, , 26 S proteasome, -, -
  • 6. sites, , subunits, sites, -
Output(Part-Of) (sent_index, protein, site):
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX
20 S 27475 5,10
Hsp90 104434 0,10
20 S proteasome 27475 0,4,11
-crystallin 12954 10