Direct In-Gel Fluorescence Detection and Cellular Imaging ofO-GlcNAc-Modified Proteins

Peter M. Clark,† Jessica F. Dweck,† Daniel E. Mason,‡ Courtenay R. Hart,§ Suzanne B. Buck,§

Eric C. Peters,‡ Brian J. Agnew,§ and Linda C. Hsieh-Wilson*,†

DiVision of Chemistry and Chemical Engineering and Howard Hughes Medical Institute, California Institute ofTechnology, Pasadena, California 91125, Genomics Institute of the NoVartis Research Foundation, San Diego,

California 92121, and InVitrogen Corporation, Eugene, Oregon 97402Received April 24, 2008; E-mail: lhw@caltech.edu

Understanding post-translational modifications to proteins iscritical for elucidating the functional roles of proteins within thedynamic environment of cells. O-Linked �-N-acetylglucosamine(O-GlcNAc) glycosylation has emerged as important for theregulation of diverse cellular processes, including transcription, celldivision, and glucose homeostasis.1 While new chemical tools haveprovided rapid, sensitive methods for detecting the modificationand enabled better control over the activity of O-GlcNAc enzymes,1a,2

significant challenges remain with regard to studying the functionsof O-GlcNAc in cells. For instance, a robust method for the directfluorescence detection of O-GlcNAc proteins in gels would facilitateproteomic studies and greatly extend the reach of existing technolo-gies. New tools for imaging O-GlcNAc-glycosylated proteins wouldenable the expression and dynamics of the modification to bemonitored in cells and tissues. Here, we report an advancedchemoenzymatic labeling strategy that addresses these importantneeds.

Previous studies have shown that an engineered �-1,4-galacto-syltransferase enzyme (Y289L GalT) efficiently transfers a keto-galactose moiety from an unnatural UDP substrate selectively ontoO-GlcNAc-modified proteins.2a However, treatment of cell lysateswith an aminooxy fluorescein derivative resulted in some non-specific labeling of proteins. We therefore investigated whetherY289L GalT would accept the UDP-azidogalactose substrate 1(UDP-GalNAz), which would allow for labeling of O-GlcNAcproteins using [3 + 2] azide-alkyne cycloaddition chemistry (Figure1A).3 In addition to providing alternative dyes and chemistry topotentially reduce nonspecific interactions, this Cu(I)-catalyzedcycloaddition reaction would have the advantage of being performedmore rapidly and at physiological pH.

We tested the approach using R-crystallin, a known O-GlcNAc-modified protein with a low extent (∼10%) of glycosylation.R-Crystallin was treated with 1 and Y289L GalT, followed byreaction with CuSO4, sodium ascorbate, and the biotin-alkynederivative 2 for 1 h at 25 °C. Analysis by gel electrophoresis andblotting with streptavidin conjugated to an IR680 dye showedrobust, selective labeling of R-crystallin, with no nonspecificlabeling in the absence of GalT, 1 or 2 (Figure 1B). Notably, aslittle as 250 fmol of protein (∼25 fmol of glycosylated protein)was detectable, highlighting the sensitivity of the approach. Incontrast, other methods such as O-GlcNAc antibodies or lectinsfailed to detect even 10 µg (500 pmol) of R-crystallin (SupportingInformation, Figure S1).2a

We next examined whether this approach could be used for directin-gel fluorescence detection and proteome-wide analyses ofO-GlcNAc-glycosylated proteins. Nuclear and cytosolic protein

fractions from rat forebrain were azide-labeled and then reactedwith the tetramethyl-6-carboxyrhodamine (TAMRA)-alkyne deriva-tive 3. The O-GlcNAc proteins were enriched by immunoprecipi-tation using an anti-TAMRA antibody to remove nonglycosylatedproteins, resolved by 1D or 2D gel electrophoresis, and visualizedby in-gel fluorescence imaging (Figures 2A, S2, and S3). Impor-tantly, minimal nonspecific labeling was detected with the TAMRA-alkyne dye (Figure 2A, -GalT control lanes), and we observedefficient capture and enrichment of the TAMRA-labeled proteins(+GalT, eluent and flow-through lanes).

To identify O-GlcNAc proteins, bands from the gel were excised,proteolytically digested, and subjected to nanoLC-MS/MS analysis.The data acquisition and subsequent database searching methodolo-gies employed are detailed in the Supporting Information. In total,we identified 213 proteins, representing 67 previously known and146 novel, putative O-GlcNAc-modified proteins (Table S1). Themajority of the proteins identified participate in neuronal signalingand synaptic function, suggesting important functional roles forO-GlcNAc in neuronal communication (Figure 2B). Surprisingly,in contrast to previous proteomic analyses of brain tissue,2b,4 weidentified many proteins involved in metabolism and biosynthesis,

† California Institute of Technology.‡ Genomics Institute of the Novartis Research Foundation.§ Invitrogen Corporation.

Figure 1. (A) Chemoenzymatic labeling of O-GlcNAc proteins using [3+ 2] cycloaddition chemistry. R ) biotin or TAMRA. (B) Selective labelingof R-Crystallin.

Figure 2. (A) Enrichment and in-gel fluorescence detection of O-GlcNAc-modified proteins. Fifteen µg of protein was loaded in the input and FTlanes; material captured from 470 µg of protein was loaded in the eluentlanes. FT ) flow-through. (B) Functional classification of O-GlcNAcproteins from rat brain identified by mass spectrometry.

Published on Web 08/07/2008

10.1021/ja8030467 CCC: $40.75 2008 American Chemical Society11576 9 J. AM. CHEM. SOC. 2008, 130, 11576–11577

consistent with roles for O-GlcNAc in nutrient sensing and cellsurvival observed in other tissues.1 Interestingly, the metabolicproteins included 9 of the 10 enzymes required for glycolysis,suggesting a previously unidentified level of control by O-GlcNAcof this pathway. Thus, the approach enables the identification of alarge number of unique O-GlcNAc-modified proteins and has theadvantages of ease and accessibility (e.g., short incubation times,simple gel-based detection and separation versus multiple chroma-tography steps, high-throughput analyses, commercially availablereagents).

Understanding the cellular dynamics of O-GlcNAc glycosylationwill be critical for elucidating its functional roles in both physi-ological and diseased states. However, few methods exist forquantifying changes in O-GlcNAc glycosylation in response tocellular stimuli.4b,5 Glycosylation levels are typically monitored byimmunoblotting with a general O-GlcNAc antibody,5 which detectsonly a limited number of O-GlcNAc proteins and affords littleopportunity to identify proteins undergoing changes in glycosyl-ation. We examined whether our chemoenzymatic approach couldovercome such limitations.

HeLa cells were stimulated with PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate), an in-hibitor of the �-N-acetylglucosaminidase enzyme that removesO-GlcNAc, and the O-GlcNAc-modified proteins were labeled andanalyzed as before. PUGNAc treatment resulted in a 163 ( 3%increase in overall O-GlcNAc glycosylation levels as measured byin-gel fluorescence analysis, and interestingly, the levels rangedfrom 136-176%, depending on the specific protein (Figure 3A).The varying extent to which O-GlcNAc is induced upon cellularstimulation may indicate complex regulatory control of the modi-fication. Thus, this approach provides a new method to visualizeand quantify dynamic changes in protein O-GlcNAc glycosylationwhich, when coupled with in-gel digestion and MS analyses asdescribed above, will enable the identification of specific proteinsundergoing those changes.

Finally, we examined whether O-GlcNAc-modified proteinscould be chemoenzymatically tagged and imaged in cells. HeLacells and cultured cortical neurons were fixed, permeabilized, andlabeled with 1 and Y289L GalT, followed by biotin-alkyne 2 orTAMRA-alkyne 3. The biotin-treated cells were further incubatedwith a streptavidin-AlexaFluor 488 conjugate. Notably, additionof exogenous GalT and 1 to the cells led to robust labeling of

O-GlcNAc-glycosylated proteins (Figure 3B). Although the TAMRA-alkyne 3 produced background labeling in the absence of 1 (datanot shown), strong staining and minimal background labeling wereobserved using biotin-alkyne 2. Consistent with the reportedlocalization of O-GlcNAc enzymes,1a,c O-GlcNAc-glycosylatedproteins were found in both the nucleus and cytoplasm. Moreover,we observed robust staining of proteins along neuronal processes,corroborating our mass spectrometric identification of many O-GlcNAc proteins involved in synaptic signaling. This is the firstexample of exploiting chemical tagging methods to image O-GlcNAc-modified proteins within cells.

In summary, we describe an advanced chemoenzymatic approachthat exploits [3 + 2] cycloaddition chemistry to attach fluorescentand biotin tags to O-GlcNAc residues. Unlike other strategies suchas metabolic labeling using GlcNAz sugars,2c this method affordsnear quantitative labeling, does not perturb signaling pathways, andis amenable to all cell types and tissues. We show that the approachenables studies of O-GlcNAc glycosylation that were previouslyinaccessible. The ability to tag proteins selectively with a fluorescentreporter group permits rapid in-gel detection of O-GlcNAc proteins,facilitating proteomic analyses and providing a new method toquantify dynamic changes in glycosylation. Covalent labeling ofproteins allows for cellular imaging of O-GlcNAc proteins in theirnative biological environment. Finally, this approach was developedin conjunction with researchers at Invitrogen with the goal ofproviding commercially available reagents that are now accessibleto the wider research community. We anticipate that this newapproach will be a powerful tool for advancing our understandingof the physiological functions and dynamic regulation of O-GlcNAcglycosylation within cells.

Acknowledgment. We thank Dr. P. Qasba for providing themutant GalT plasmid, J. Rexach for providing neurons and helpfuldiscussions, T. Nyberg for helpful discussions, and Prof. N. Piercefor use of the FujiFilm imager. This work was supported by theNIH (RO1 GM084724) and Howard Hughes Medical Institute.

Supporting Information Available: Experimental procedures andsupplemental figures. This material is available free of charge via theInternet at http://pubs.acs.org.

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JA8030467

Figure 3. (A) Direct detection of changes in O-GlcNAc glycosylation levelsupon cellular stimulation. Tubulin controls indicate equal loading of proteinin each lane. (B) Fluorescence imaging of O-GlcNAc proteins (green) inHeLa cells (left) or cortical neurons (right). Nuclei were stained with DAPI(blue). Scale bars ) 10 µm (HeLa) and 25 µm (neurons).

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