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Metabolic Labeling Enables Selective Photocrosslinking of O-Glcnac-Modified Proteins to Their Binding Partners

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Metabolic Labeling Enables Selective Photocrosslinking of O-Glcnac-Modified Proteins to Their Binding Partners Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners Seok-Ho Yua, Michael Boyceb, Amberlyn M. Wandsa, Michelle R. Bonda, Carolyn R. Bertozzib, and Jennifer J. Kohlera,1 aDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038 and bDepartment of Chemistry, University of California, Berkeley, CA 94720 Edited by Barbara Imperiali, Massachusetts Institute of Technology, Cambridge, MA, and approved January 30, 2012 (received for review August 31, 2011) O-linked β-N-acetylglucosamine (O-GlcNAc) is a reversible post- modified protein, although recent findings suggest that increased translational modification found on hundreds of nuclear and cyto- levels of O-GlcNAc can be associated with acquisition of function plasmic proteins in higher eukaryotes. Despite its ubiquity and (12–14). essentiality in mammals, functional roles for the O-GlcNAc modifi- Selectively observing the cellular behavior of O-GlcNAc- cation remain poorly defined. Here we develop a combined genetic modified proteins remains challenging. Most methods report and chemical approach that enables introduction of the diazirine on the bulk behavior of the protein of interest and do not distin- photocrosslinker onto the O-GlcNAc modification in cells. We engi- guish among the different posttranslationally modified forms. We neered mammalian cells to produce diazirine-modified O-GlcNAc reasoned that appending a small photoactivatable crosslinking by expressing a mutant form of UDP-GlcNAc pyrophosphorylase group to the O-GlcNAc modification would enable selectively and subsequently culturing these cells with a cell-permeable, induced covalent crosslinking between an O-GlcNAc-modified diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells protein and surrounding molecules. Because the crosslinker is on with UV light activated the crosslinker, resulting in formation of the O-GlcNAc residue, proteins that lack the modification will covalent bonds between O-GlcNAc-modified proteins and neigh- not engage in crosslinking and will be rendered essentially invi- boring molecules, which could be identified by mass spectrometry. sible in this assay. In this way, even proteins that are O-GlcNAc- We used this method to identify interaction partners for the modified at substoichiometric levels could be examined. O-GlcNAc-modified FG-repeat nucleoporins. We observed cross- To implement the photocrosslinking approach, we designed an linking between FG-repeat nucleoporins and nuclear transport fac- analog of GlcNAc, GlcNDAz, in which the N-acyl substituent was tors, suggesting that O-GlcNAc residues are intimately associated modified to include the diazirine photocrosslinker (Fig. 1). Using with essential recognition events in nuclear transport. Further, we a metabolic labeling approach, we induced cultured cells to pro- propose that the method reported here could find widespread use duce the modified nucleotide sugar donor UDP-GlcNDAz and to in investigating the functional consequences of O-GlcNAcylation. transfer GlcNDAz to proteins that are normally O-GlcNAc- modified. Subsequent photoirradiation resulted in the selective diazirine ∣ glycosylation ∣ metabolism ∣ nucleoporins ∣ covalent crosslinking of O-GlcNDAz-modified proteins. Mass posttranslational modification spectrometry analysis of purified complexes revealed crosslinking between O-GlcNAc-modified nucleoporins and nuclear transport -linked β-N-acetylglucosamine (O-GlcNAc) is a common factors. These results indicate that the O-GlcNAc modification Omodification of intracellular proteins in metazoa and higher is intimately associated with the recognition events that occur plants (1). Hundreds of O-GlcNAc-modified human nuclear and during nuclear transport. cytoplasmic proteins have been identified. Modified proteins fall into a variety of functional classes and include transcription fac- Results tors, ribosomal proteins and translational factors, signaling pro- In Vitro Production and Crosslinking of O-GlcNDAz-modified Peptides. teins, cytoskeletal proteins, and components of the nuclear pore We envisioned that introduction of a photocrosslinking group complex. Like phosphorylation, O-GlcNAcylation is reversible. directly onto an O-GlcNAc residue would enable the covalent Mammalian genomes encode a single O-GlcNAc transferase crosslinking between the O-GlcNAc modification and proximal (OGT) that transfers GlcNAc from the nucleotide sugar donor molecules. To test this idea, we assessed crosslinking between an UDP-GlcNAc to serine or threonine side chains of substrate pro- O-GlcNDAz-modified peptide and a monoclonal antibody, RL2 teins, and one O-GlcNAcase (nuclear cytoplasmic O-GlcNAcase (15), that specifically recognizes the O-GlcNAc modification. and acetyltransferase; NCOAT) that hydrolytically removes First, we prepared the diazirine-modified nucleotide-sugar donor GlcNAc residues from modified proteins. The O-GlcNAc mod- UDP-GlcNDAz (Fig. 1) (16) and incubated it with recombinant ification is essential in mammals (2) and O-GlcNAc is critical to human OGT and a biotinylated substrate peptide derived from cells’ ability to tolerate a variety of forms of stress (3, 4). casein kinase II (CKII) (17). The product mixture was analyzed While characterization of O-GlcNAc-modification sites has by MALDI mass spectrometry, revealing a mass consistent with advanced dramatically (5), a comprehensive understanding of transfer of the GlcNDAz residue to the CKII peptide (Fig. S1A). the functional consequences of O-GlcNAc modification remains Next, the O-GlcNDAz-modified peptide was incubated with more elusive (6). How does O-GlcNAc affect a protein’s activity, RL2 and UV irradiated to induce photocrosslinking (Fig. S2A). stability, and ability to engage in binding interactions? O-GlcNAc is often found at sites that can be alternatively phosphorylated, leading to a reciprocal relationship between these two modifica- Author contributions: S.-H.Y., M.B., C.R.B., and J.J.K. designed research; S.-H.Y. and M.B. performed research; S.-H.Y., A.M.W., and M.R.B. contributed new reagents/analytic tools; tions. This relationship led to the hypothesis that a key role for S.-H.Y., M.B., C.R.B., and J.J.K. analyzed data; and S.-H.Y., M.B., C.R.B., and J.J.K. wrote O-GlcNAc is to interfere with phosphorylation (7). In fact, ex- the paper. perimental evidence suggests that the interplay between these The authors declare no conflict of interest. two modifications is complex, involving both negative and posi- This article is a PNAS Direct Submission. – tive associations (8 10). Another documented function for 1To whom correspondence should be addressed: E-mail: jennifer.kohler@utsouthwestern. O-GlcNAc is to disrupt binding interactions of the modified pro- edu. teins, presumably by steric interference (11). Less clear is whether This article contains supporting information online at www.pnas.org/lookup/suppl/ the O-GlcNAc modification can impart a novel activity to the doi:10.1073/pnas.1114356109/-/DCSupplemental. 4834–4839 ∣ PNAS ∣ March 27, 2012 ∣ vol. 109 ∣ no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1114356109 Downloaded by guest on October 1, 2021 Fig. 1. Diazirine-modified GlcNAc derivatives used in this study. Immunoblot analysis revealed a biotinylated species migrating with an apparent molecular weight identical to that of the light chain of RL2 (Fig. S2B). Observation of the biotinylated species depended on inclusion of the crosslinker (UDP-GlcNDAz) and on UV irradiation. We concluded that this biotinylated species represented the O-GlcNDAz-modified peptide crosslinked to the light chain of the RL2 antibody. These results indicate that the addition of the diazirine modification to GlcNAc does not Fig. 2. Metabolic labeling strategy for cellular biosynthesis of O-GlcNDAz- preclude transfer of the sugar by OGT nor does it abrogate the modified proteins. Cellular GlcNAc can be converted to UDP-GlcNAc via three binding interaction between O-GlcNAc-modified CKII and RL2. enzymatic steps catalyzed by N-acetylglucosamine kinase (NAGK), N-acetyl- Having established that human OGT is capable of accepting glucosamine-phosphate mutase (AGM1), and UDP-GlcNAc pyrophosphory- UDP-GlcNDAz, we conducted competition experiments to lase (AGX1 or AGX2). HeLa cells expressing AGX1(F383G) and cultured with Ac3GlcNDAz-1-PðAc-SATEÞ efficiently produced UDP-GlcNDAz. determine whether OGT would transfer GlcNDAz from UDP- 2 GlcNDAz when UDP-GlcNAc was also available. Lysates phate is protected with two S-acetyl-2-thioethyl (Ac-SATE) prepared from HeLa cells overexpressing human OGT were in- groups. We predicted that Ac3GlcNDAz-1-PðAc-SATEÞ2 would cubated with UDP-GlcNAc, UDP-GlcNDAz or an equimolar be capable of diffusing across the plasma membrane, after which mixture of the two, along with an OGT acceptor peptide (+P2) α the protecting groups would be removed by chemical hydrolysis derived from human -A crystallin (18) (Fig. S1B). Lysates incu- or the action of intracellular esterases (20). Indeed, HPAEC analysis bated with UDP-GlcNAc produced a product with the mass of HeLa lysates revealed that cells cultured with Ac3GlcNDAz- (m∕z ¼ 1; 241) expected for an O-GlcNAc-modified peptide. 1-PðAc-SATEÞ2 readily accumulated intracellular GlcNDAz-1-P; Lysates incubated with UDP-GlcNDAz produced a product however, UDP-GlcNDAz was not detected (Fig. S3A). These data whose mass corresponded to the intact O-GlcNDAz-modified m∕z ¼ 1; 309 suggested that GlcNDAz-1-P is a poor
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