title: Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.
abstract: The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted β-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. 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. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90β, of which one corresponds to a previously described phosphorylation site.
authors.affiliation: Institut für Biochemie, Charité-Universitätsmedizin Berlin, 13347 Berlin, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Overath
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kuckelkorn
authors.forename: Petra
authors.initials: P
authors.lastname: Henklein
authors.forename: Britta
authors.initials: B
authors.lastname: Strehl
authors.forename: David
authors.initials: D
authors.lastname: Bonar
authors.forename: Alexander
authors.initials: A
authors.lastname: Kloss
authors.forename: Dagmar
authors.initials: D
authors.lastname: Siele
authors.forename: Peter-Michael
authors.initials: PM
authors.lastname: Kloetzel
authors.forename: Katharina
authors.initials: K
authors.lastname: Janek
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002250:Carbon Radioisotopes / Q000378:metabolism; D002417:Cattle; D002853:Chromatography, Liquid; D003538:Cystamine / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D006031:Glycosylation / Q000187:drug effects; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D038201:alpha-Crystallins / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D018841:HSP90 Heat-Shock Proteins; D010455:Peptides; D038201:alpha-Crystallins; D001710:Biotin; D046988:Proteasome Endopeptidase Complex; D005947:Glucose; D003538:Cystamine; D000117:Acetylglucosamine