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.

Fig. 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.

Fig. 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.

Fig. 3.

Derivatization of a synthetic O-GlcNAc-modified peptide by alkali-mediated β-elimination and Michael addition with a novel biotin-cystamine tag. A, shown is the strategy for replacement of the O-GlcNAc moiety on serine or threonine residues by the stable affinity tag BiCy. To discriminate between specific and unspecific reactions, differential isotopic tags (BiCy-d0 and BiCy-d4) were synthesized. B, MALDI-TOF/TOF-MS spectra of a synthetic O-GlcNAc-modified peptide before (upper lane) and after derivatization with BiCy-d0 (lower lane). The mass shift of 82 Da corresponds to the loss of O-GlcNAc (−203 Da) and water (−18 Da) and the addition of BiCy-d0 (303 Da). C, the MALDI-TOF/TOF-MS/MS spectrum of the BiCy-tagged O-GlcNAc peptide with precursor mass m/z 1927.0 is shown. Diagnostic ions at m/z 227.1 and 304.1 and the neutral loss of [M-303+H]⁺ confirm the tagging of the peptide. The site of modification is localized to Ser⁵.

Fig. 4.

Strategy for identifying O-GlcNAc sites by chemical tagging with the biotin-cystamine tag BiCy and relative quantification.

Fig. 5.

Representative mass spectra of specific and unspecific BiCy-tagged proteasomal peptides. A, the isotope pattern of the peptide ⁴G_BiCySSAGFDR¹¹ (α1, spleen) shows an L/H ratio of ≈1.7, which indicates a specific labeling of the O-GlcNAcylated serine residue. B, a typical spectrum of an unspecific tagged peptide with a BiCy-d0/d4 ratio of 1:1 (²⁹G _BiCySTAVGVR³⁶, α4 spleen) is depicted.