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

Overath T, Kuckelkorn U, Henklein P, Strehl B, Bonar D, Kloss A, Siele D, Kloetzel PM, Janek K - Mol. Cell Proteomics (2012)

Bottom Line: O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation.Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Charité-Universitätsmedizin Berlin, 13347 Berlin, Germany.

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.

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Detection of O-GlcNAcylation on proteasomes and Hsp90.A, detection of O-GlcNAc-modifications on diverse mouse tissue proteasomes with the O-GlcNAc antibody CTD110.6. Proteasomes were isolated from mouse liver (1), mouse spleen (2), and mouse brain (3). Three μg of each were separated by SDS-PAGE. The left panel shows the staining with Coomassie, the middle panel shows the detection of O-GlcNAc modifications with CTD110.6, and the right panel shows the competition of CTD110.6 with 500 mm GlcNAc. Proteasome subunit α4 was detected as loading control. B, detection of O-glycosylation on murine Hsp90. Co-purified Hsp90 was isolated from proteasomes, and both were separated by SDS-PAGE. The Coomassie staining of proteasomes (lane P), and Hsp90 is shown in the left panel. Hsp90 can be detected with CTD 110.6 (right panel, lane 1). The signal is diminished by saturation of the antibody with 500 mm GlcNAc (lane 2). The detection of Hsp90 with a monoclonal Hsp90 antibody is shown in lane 3. C, in vivo labeling of proteasomes with [14C]glucosamine. RMA cells (murine) were incubated with [14C]glucosamine for 1, 3, and 5 h. As control (lane co), [35S]Met-labeled 20 S proteasomes were used. The labeled proteasomes were precipitated with the polyclonal proteasome antibody K08. The precipitated protein was analyzed in SDS-PAGE and detected by autoradiography.
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Figure 1: Detection of O-GlcNAcylation on proteasomes and Hsp90.A, detection of O-GlcNAc-modifications on diverse mouse tissue proteasomes with the O-GlcNAc antibody CTD110.6. Proteasomes were isolated from mouse liver (1), mouse spleen (2), and mouse brain (3). Three μg of each were separated by SDS-PAGE. The left panel shows the staining with Coomassie, the middle panel shows the detection of O-GlcNAc modifications with CTD110.6, and the right panel shows the competition of CTD110.6 with 500 mm GlcNAc. Proteasome subunit α4 was detected as loading control. B, detection of O-glycosylation on murine Hsp90. Co-purified Hsp90 was isolated from proteasomes, and both were separated by SDS-PAGE. The Coomassie staining of proteasomes (lane P), and Hsp90 is shown in the left panel. Hsp90 can be detected with CTD 110.6 (right panel, lane 1). The signal is diminished by saturation of the antibody with 500 mm GlcNAc (lane 2). The detection of Hsp90 with a monoclonal Hsp90 antibody is shown in lane 3. C, in vivo labeling of proteasomes with [14C]glucosamine. RMA cells (murine) were incubated with [14C]glucosamine for 1, 3, and 5 h. As control (lane co), [35S]Met-labeled 20 S proteasomes were used. The labeled proteasomes were precipitated with the polyclonal proteasome antibody K08. The precipitated protein was analyzed in SDS-PAGE and detected by autoradiography.

Mentions: To study 20 S proteasomes modified with O-GlcNAc, 20 S proteasomes were isolated from murine tissues. O-GlcNAcylation was detected in Western blots using the monoclonal antibodies CTD110.6 (Fig. 1A) and MAI-076, as well as the ClickiT® system (data not shown). Proteasomes derived from liver, spleen, and brain were positively stained for O-linked glycosylation. In addition, co-purified Hsp90 separated from liver proteasomes was also shown to be O-GlcNAc-modified (Fig. 1B). To verify the glycosylation of 20 S proteasomes, murine RMA cells were incubated in presence of [14C]glucosamine. Within 3–5 h of incubation, multiple proteasomal subunits were labeled with [14C]glucosamine, i.e., modified with O-GlcNAc (Fig. 1C). Remarkably, in all experiments, several of the 17 different proteasomal subunits were shown to be O-GlcNAc-modified.


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

Overath T, Kuckelkorn U, Henklein P, Strehl B, Bonar D, Kloss A, Siele D, Kloetzel PM, Janek K - Mol. Cell Proteomics (2012)

Detection of O-GlcNAcylation on proteasomes and Hsp90.A, detection of O-GlcNAc-modifications on diverse mouse tissue proteasomes with the O-GlcNAc antibody CTD110.6. Proteasomes were isolated from mouse liver (1), mouse spleen (2), and mouse brain (3). Three μg of each were separated by SDS-PAGE. The left panel shows the staining with Coomassie, the middle panel shows the detection of O-GlcNAc modifications with CTD110.6, and the right panel shows the competition of CTD110.6 with 500 mm GlcNAc. Proteasome subunit α4 was detected as loading control. B, detection of O-glycosylation on murine Hsp90. Co-purified Hsp90 was isolated from proteasomes, and both were separated by SDS-PAGE. The Coomassie staining of proteasomes (lane P), and Hsp90 is shown in the left panel. Hsp90 can be detected with CTD 110.6 (right panel, lane 1). The signal is diminished by saturation of the antibody with 500 mm GlcNAc (lane 2). The detection of Hsp90 with a monoclonal Hsp90 antibody is shown in lane 3. C, in vivo labeling of proteasomes with [14C]glucosamine. RMA cells (murine) were incubated with [14C]glucosamine for 1, 3, and 5 h. As control (lane co), [35S]Met-labeled 20 S proteasomes were used. The labeled proteasomes were precipitated with the polyclonal proteasome antibody K08. The precipitated protein was analyzed in SDS-PAGE and detected by autoradiography.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3412975&req=5

Figure 1: Detection of O-GlcNAcylation on proteasomes and Hsp90.A, detection of O-GlcNAc-modifications on diverse mouse tissue proteasomes with the O-GlcNAc antibody CTD110.6. Proteasomes were isolated from mouse liver (1), mouse spleen (2), and mouse brain (3). Three μg of each were separated by SDS-PAGE. The left panel shows the staining with Coomassie, the middle panel shows the detection of O-GlcNAc modifications with CTD110.6, and the right panel shows the competition of CTD110.6 with 500 mm GlcNAc. Proteasome subunit α4 was detected as loading control. B, detection of O-glycosylation on murine Hsp90. Co-purified Hsp90 was isolated from proteasomes, and both were separated by SDS-PAGE. The Coomassie staining of proteasomes (lane P), and Hsp90 is shown in the left panel. Hsp90 can be detected with CTD 110.6 (right panel, lane 1). The signal is diminished by saturation of the antibody with 500 mm GlcNAc (lane 2). The detection of Hsp90 with a monoclonal Hsp90 antibody is shown in lane 3. C, in vivo labeling of proteasomes with [14C]glucosamine. RMA cells (murine) were incubated with [14C]glucosamine for 1, 3, and 5 h. As control (lane co), [35S]Met-labeled 20 S proteasomes were used. The labeled proteasomes were precipitated with the polyclonal proteasome antibody K08. The precipitated protein was analyzed in SDS-PAGE and detected by autoradiography.
Mentions: To study 20 S proteasomes modified with O-GlcNAc, 20 S proteasomes were isolated from murine tissues. O-GlcNAcylation was detected in Western blots using the monoclonal antibodies CTD110.6 (Fig. 1A) and MAI-076, as well as the ClickiT® system (data not shown). Proteasomes derived from liver, spleen, and brain were positively stained for O-linked glycosylation. In addition, co-purified Hsp90 separated from liver proteasomes was also shown to be O-GlcNAc-modified (Fig. 1B). To verify the glycosylation of 20 S proteasomes, murine RMA cells were incubated in presence of [14C]glucosamine. Within 3–5 h of incubation, multiple proteasomal subunits were labeled with [14C]glucosamine, i.e., modified with O-GlcNAc (Fig. 1C). Remarkably, in all experiments, several of the 17 different proteasomal subunits were shown to be O-GlcNAc-modified.

Bottom Line: O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation.Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies.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.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie, Charité-Universitätsmedizin Berlin, 13347 Berlin, Germany.

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.

Show MeSH
Related in: MedlinePlus