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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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Glucose concentration in culture medium influences proteasome activity and proteasome glycosylation.A, murine fibroblast cells (line C4) were incubated with 10 mm glucose in the culture medium for indicated time points in a 24-well plate. The cells were washed and lysed with 0.1% Nonidet P-40 in 20 mm Tris buffer, pH 7.2. The proteolytic activity within the lysates was monitored by hydrolysis of suc-LLVY-AMC (white bars). In parallel, the proteasome activity was inhibited by 10 μm MG132 (black bars). B, proteasomes were isolated from the C4 mouse fibroblast cell line cultured with 5 mm (lane 1) and 10 mm (lane 2) glucose for 2 h. In the left panel Coomassie staining of the separated proteasomes is shown, and in the right panel the detection of O-GlcNAc modifications by CTD110.6 is shown. Immunodetection of proteasome subunit α4 served as loading control. WB, Western blot.
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Figure 2: Glucose concentration in culture medium influences proteasome activity and proteasome glycosylation.A, murine fibroblast cells (line C4) were incubated with 10 mm glucose in the culture medium for indicated time points in a 24-well plate. The cells were washed and lysed with 0.1% Nonidet P-40 in 20 mm Tris buffer, pH 7.2. The proteolytic activity within the lysates was monitored by hydrolysis of suc-LLVY-AMC (white bars). In parallel, the proteasome activity was inhibited by 10 μm MG132 (black bars). B, proteasomes were isolated from the C4 mouse fibroblast cell line cultured with 5 mm (lane 1) and 10 mm (lane 2) glucose for 2 h. In the left panel Coomassie staining of the separated proteasomes is shown, and in the right panel the detection of O-GlcNAc modifications by CTD110.6 is shown. Immunodetection of proteasome subunit α4 served as loading control. WB, Western blot.

Mentions: When cells were pulsed with 10 mm glucose for 240 min, we observed a transient reduction of the proteasome activity within the first 60 min (Fig. 2A), corroborating earlier results of Zhang et al. (18). However, comparing the initial activity (0 min) with those after 240 min, a slight increase in proteasomal peptide hydrolyzing activity was observed. Furthermore, an incubation of cells with a high glucose concentration of 10 mm was accompanied by an O-glycosylation of 20S proteasome subunits (Fig. 2B). The change in glycosylation of proteasomal subunits was also demonstrated by immunoprecipitation of O-GlcNAc-modified proteasomes with the antibody CTD110.6 and the detection of O-GlcNAcylation in immunoprecipitated proteasomes (supplemental Fig. 1). The proteasome modification with O-GlcNAc was significantly enhanced upon increased glucose concentration in the culture medium.


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)

Glucose concentration in culture medium influences proteasome activity and proteasome glycosylation.A, murine fibroblast cells (line C4) were incubated with 10 mm glucose in the culture medium for indicated time points in a 24-well plate. The cells were washed and lysed with 0.1% Nonidet P-40 in 20 mm Tris buffer, pH 7.2. The proteolytic activity within the lysates was monitored by hydrolysis of suc-LLVY-AMC (white bars). In parallel, the proteasome activity was inhibited by 10 μm MG132 (black bars). B, proteasomes were isolated from the C4 mouse fibroblast cell line cultured with 5 mm (lane 1) and 10 mm (lane 2) glucose for 2 h. In the left panel Coomassie staining of the separated proteasomes is shown, and in the right panel the detection of O-GlcNAc modifications by CTD110.6 is shown. Immunodetection of proteasome subunit α4 served as loading control. WB, Western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Glucose concentration in culture medium influences proteasome activity and proteasome glycosylation.A, murine fibroblast cells (line C4) were incubated with 10 mm glucose in the culture medium for indicated time points in a 24-well plate. The cells were washed and lysed with 0.1% Nonidet P-40 in 20 mm Tris buffer, pH 7.2. The proteolytic activity within the lysates was monitored by hydrolysis of suc-LLVY-AMC (white bars). In parallel, the proteasome activity was inhibited by 10 μm MG132 (black bars). B, proteasomes were isolated from the C4 mouse fibroblast cell line cultured with 5 mm (lane 1) and 10 mm (lane 2) glucose for 2 h. In the left panel Coomassie staining of the separated proteasomes is shown, and in the right panel the detection of O-GlcNAc modifications by CTD110.6 is shown. Immunodetection of proteasome subunit α4 served as loading control. WB, Western blot.
Mentions: When cells were pulsed with 10 mm glucose for 240 min, we observed a transient reduction of the proteasome activity within the first 60 min (Fig. 2A), corroborating earlier results of Zhang et al. (18). However, comparing the initial activity (0 min) with those after 240 min, a slight increase in proteasomal peptide hydrolyzing activity was observed. Furthermore, an incubation of cells with a high glucose concentration of 10 mm was accompanied by an O-glycosylation of 20S proteasome subunits (Fig. 2B). The change in glycosylation of proteasomal subunits was also demonstrated by immunoprecipitation of O-GlcNAc-modified proteasomes with the antibody CTD110.6 and the detection of O-GlcNAcylation in immunoprecipitated proteasomes (supplemental Fig. 1). The proteasome modification with O-GlcNAc was significantly enhanced upon increased glucose concentration in the culture medium.

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