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. 2018 Oct 15:9:606.
doi: 10.3389/fendo.2018.00606. eCollection 2018.

A Novel Glycoproteomics Workflow Reveals Dynamic O-GlcNAcylation of COPγ1 as a Candidate Regulator of Protein Trafficking

Nathan J Cox  1 , Peter M Luo  1 , Timothy J Smith  1 , Brittany J Bisnett  1 , Erik J Soderblom  2 , Michael Boyce  1
Affiliations

A Novel Glycoproteomics Workflow Reveals Dynamic O-GlcNAcylation of COPγ1 as a Candidate Regulator of Protein Trafficking

Nathan J Cox et al. Front Endocrinol (Lausanne). .

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant and essential intracellular form of protein glycosylation in animals and plants. In humans, dysregulation of O-GlcNAcylation occurs in a wide range of diseases, including cancer, diabetes, and neurodegeneration. Since its discovery more than 30 years ago, great strides have been made in understanding central aspects of O-GlcNAc signaling, including identifying thousands of its substrates and characterizing the enzymes that govern it. However, while many O-GlcNAcylated proteins have been reported, only a small subset of these change their glycosylation status in response to a typical stimulus or stress. Identifying the functionally important O-GlcNAcylation changes in any given signaling context remains a significant challenge in the field. To address this need, we leveraged chemical biology and quantitative mass spectrometry methods to create a new glycoproteomics workflow for profiling stimulus-dependent changes in O-GlcNAcylated proteins. In proof-of-principle experiments, we used this new workflow to interrogate changes in O-GlcNAc substrates in mammalian protein trafficking pathways. Interestingly, our results revealed dynamic O-GlcNAcylation of COPγ1, an essential component of the coat protein I (COPI) complex that mediates Golgi protein trafficking. Moreover, we detected 11 O-GlcNAc moieties on COPγ1 and found that this modification is reduced by a model secretory stress that halts COPI trafficking. Our results suggest that O-GlcNAcylation may regulate the mammalian COPI system, analogous to its previously reported roles in other protein trafficking pathways. More broadly, our glycoproteomics workflow is applicable to myriad systems and stimuli, empowering future studies of O-GlcNAc in a host of biological contexts.

Keywords: COPI vesicle trafficking; O-GlcNAc; SILAC; click chemistry; glycoproteomics; protein secretion.

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Figures

Figure 1
Figure 1
Glycoproteomics workflow. First, cells are stably labeled with either “heavy” (red) or “light” (blue) arginine and lysine to create two distinctly labeled populations, per standard SILAC protocols. Next, cells are treated with GalNAz for a brief period to allow incorporation into endogenous OGT substrates, while limiting the labeling of “housekeeping” proteins. One cell population is treated with a stimulus, inducing changes in O-GlcNAcylation, while the other remains an untreated control. Both cell populations are mixed, fractionated to remove secretory pathway glycans, and covalently ligated to an alkyne-functionalized agarose bead via a click reaction. After extremely stringent washing, the covalently captured glycoproteins are trypsinized on-bead and the resulting peptides are analyzed by quantitative SILAC MS proteomics.
Figure 2
Figure 2
XL4.1 cells were treated with either DMSO vehicle or 100 μM GalNAz for 24 h and processed via the glycoproteomics workflow. To confirm enrichment, proteins with fewer than 25 spectral counts were excluded, and the remaining proteins were rank-ordered by the ratio of spectral counts from the vehicle and GalNAz samples (low to high). Table displays the top 25 proteins ranked this way. Commonly O-GlcNAcylated substrates (e.g., nucleoporins, HCF1) were identified exclusively in the GalNAz-treated samples, confirming the selective enrichment of endogenous OGT substrates by the workflow. Complete proteomics datasets are available as Supplemental Material.
Figure 3
Figure 3
(A) XL4.1 cells were treated with DMSO vehicle or 100 μM GalNAz for the indicated times. Cell lysates were subjected to click reactions with an alkyne-biotin probe and analyzed by IB. GalNAz incorporation is evident as soon as 2 h after treatment. Tubulin is a loading control. (B,C) Parental FL5.12 N6 cells were treated with the indicated doses of BFA for 4 or 24 h and mitochondrial function (B) (n = 2) and ATP levels (C) (n = 3) were measured. Marked decreases in mitochondrial function and ATP content occurred with ≥500 ng/ml BFA treatment for 24 h, but no changes were observed at those doses after 4 h. In each assay, values were normalized to vehicle-treated control. Error bars are standard error of the mean. *p < 0.05 compared to control (DMSO) by Tukey's HSD.
Figure 4
Figure 4
Glycoproteomics workflow detects global BFA-induced changes in O-GlcNAcylated proteins. Data for nuclear and cytoplasmic proteins are displayed as a log2 transform of the ratio of detected intensities from the DMSO and BFA samples, as described previously (–68). (A) In the first biological replicate, 1,252 nuclear and 792 cytoplasmic proteins were identified. (B) In the second biological replicate, 1,703 nuclear and 1,262 cytoplasmic proteins were identified. (C) The nuclear and cytoplasmic proteins exhibiting concordant BFA-dependent changes (up or down) across biological replicates were rank-ordered by the magnitude of the fold-change between DMSO and BFA samples, as described previously (–68). Tables list the top 15 nuclear and cytoplasmic proteins from each biological replicate by this ranking. Some proteins appear in both tables but at different positions, reflecting rank-order in each case. (D) Representative MS spectra from one SILAC biological replicate, depicting light and heavy versions of the COPγ1 peptide SIATLAITTLLK. Complete proteomics datasets are available as Supplemental Material.
Figure 5
Figure 5
(A) Ramos cells were treated with 100 μM GalNAz or DMSO vehicle for 6 h, followed by 500 ng/ml BFA or DMSO vehicle for an additional 4 h, and then harvested. Nuclear and cytoplasmic fractions were prepared, subjected to click reactions with an alkyne-biotin probe and incubated with streptavidin beads for enrichment. Beads were washed and eluted proteins were analyzed by IB. Nuclear fraction IBs from a representative experiment are shown. Nucleoporin-62, a heavily O-GlcNAcylated protein, is a control for equal loading (input lanes) and biotin enrichment (pulldown lanes). Band intensities in the COPγ1 IB were quantified in ImageJ, normalized to control (DMSO, input lane) and listed below. The ratio of pulldown:input for each treatment was calculated as a measure of COPγ1GalNAz modification and is given in the graph at the right. Consistent with the glycoproteomics results, COPγ1 levels were reduced after BFA treatment, indicating a reduction in O-GlcNAcylation. Similar results were obtained with XL4.1 cells (not shown). (B) Ramos (left) and XL4.1 (right) cells were treated with 50 μM Thiamet-G or DMSO vehicle for 8 h and lysates were subjected to anti-COPγ1IP and analyzed via IB.
Figure 6
Figure 6
(A, left) Myc-6xHis-tagged human COPγ1 was tandem-purified to homogeneity from transfected 293T cells and analyzed via ETD/HCD-MS. Peptide sequence, number of O-GlcNAc moieties detected, and number of serine and threonine residues are displayed for tryptic peptides exhibiting glycosylation. (A, right) Alignment of human and mouse COPγ1 regions with unambiguously assigned O-GlcNAc modification sites, denoted in red. Human and mouse COPγ1 are 97.3% identical and are 99.4% similar. All O-GlcNAc sites identified on human COPγ1 are conserved in the mouse ortholog. (B) Schematic depicting candidate O-GlcNAc sites on the COPγ1 domain structure. The C-terminal appendage domain contains an ARFGAP2-interacting region and binds the α/β/ε COPI subcomplex. (C) Interface of COPγ1 (green) and COPβ1 (blue), with potential O-GlcNAc sites in red, from the previously reported structure of the COPI triad (PDB: 5A1U) (117). (D) Zoomed view from (C) (black box) of the COPγ1/COPβ1 interface. T552 and S554, two unambiguously assigned O-GlcNAc sites on COPγ1, lie in close proximity to COPβ1. Complete proteomics datasets are available as Supplemental Material.
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