Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins

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Direct one-step fluorescent labeling of O-GlcNAc modified proteins in live cells using metabolic intermediates Hong Yee Tan, Razieh Eskandari, David Shen, Yanping Zhu, Ta-Wei Liu, Lianne I. Willems, Matthew G. Alteen, Zarina Madden, and David J. Vocadlo J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08260 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 8, 2018

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Journal of the American Chemical Society

Direct one-step fluorescent labeling of O-GlcNAc modified proteins in live cells using metabolic intermediates Hong Yee Tan,† Razieh Eskandari, † David Shen,†,‡ Yanping Zhu, †,‡ Ta-Wei Liu, †,‡ Lianne Willems,†‡ Matthew G. Alteen,† Zarina Madden†,‡ and David J. Vocadlo*,†,‡ †Department ‡Department

of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A1S6, Canada. of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A1S6,

Canada. Supporting Information Placeholder ABSTRACT: The modification of proteins with O-linked Nacetylglucosamine (O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor OGlcNAcylation in cells has proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here we report on the creation of fluorescent uridine 5’-diphospho-N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosaminenitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time and dose dependent manner. Since no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-colour pulse chase experiments and monitoring OGT activity on specific protein substrate in live cells.

INTRODUCTION The modification of hundreds of nuclear, cytoplasmic, and mitochondrial proteins by N-acetylglucosamine (GlcNAc) residues O-linked to serine and threonine residues (O-GlcNAc)1 is emerging as a major regulator of cellular function. 2-3 Roles for O-GlcNAc have recently been uncovered in regulating gene transcription,4 nutrient sensing,5-6 and stress response7-8. Furthermore, various diseases are associated with changes in O-GlcNAc modification such as cancers,9 10 cardiovascular disease 11-12 and neurodegenerative disorders.13-14 Remarkably, despite the abundance of O-GlcNAc on hundreds of structurally diverse proteins within cells, only a single glycosyltransferase acts to install this modification. Uridine diphosphate-N-acetyl-Dglucosamine:polypeptidyl transferase (OGT, GT family 41) uses uridine-5’-diphospho-N-acetylglucosamine (UDP-GlcNAc) as a

donor substrate to modify its target proteins.15-16 The glycoside hydrolase N-acetyl--D-glucosaminidase (OGA, GH family 84) cleaves off O-GlcNAc residues and returns proteins to their unmodified state.17-18 Given the central regulatory role of OGT in regulating cellular O-GlcNAcylation, how it distinguishes its substrates, the basis for its substrate preference, and its regulation within cells are accordingly topics of great interest19-22. To address these and other related questions, considerable effort has been allocated to creating chemical strategies to aid in studying protein O-GlcNAcylation.23-24 For example, OGT has been found to be tolerant of subtle structural changes made to the donor substrate UDP-GlcNAc.25-28 This substrate tolerance has in turn enabled metabolic feeding of cells with analogues of GlcNAc, which are assimilated by cellular biosynthetic pathways, leading to labeling of O-GlcNAcylated proteins with analogues of GlcNAc bearing small bioorthogonal functional groups. Modification of the 2-acetamido group of GlcNAc, with azide,28 alkyne,29 cyclopropenyl,30 and N-propargyloxy-carbamate,31 as well as replacement of the 6-hydroxyl group with azide32 or alkyne33 functionalities has enabled subsequent chemoselective tagging of O-GlcNAcylated proteins with either affinity tags or fluorophores. These two-step metabolic feeding strategies have proven widely useful for inventorying O-GlcNAcylated proteins,29, 32-36 mapping O-GlcNAc to the genome,37 imaging O-GlcNAcylated proteins in situ,30, 34, 38 and to uncover fundamental biology33, 35, 39. While these metabolic labeling strategies have demonstrated value, these approaches, as well as alternative chemoenzymatic labeling methods,40-41 all rely on a two-step process, which limits their scope in live cell imaging. Notably, most, though not all,34, 42 GlcNAc precursor analogues give rise to conjugates that can be deglycosylated by OGA, which results in observed changes being an aggregate of both their installation and removal. Additionally, the potential for incomplete reactions for relatively slow ligations or non-specific reactions that may occur spontaneously when using excess of either metabolic probes or with various functionalities during some chemoselective ligations, 43-44 may limit potential applications that can be accessed using these strategies. Single step labeling methods that permit chemoenzymatic labelling of cell surface glycosylation have recently emerged.45 Inspired by these recent reports as well as work from the Kohler group, who showed metabolic feeding with a suitably protected, diazirine-modified, GlcNAc-1-phosphate analogue (GlcNDAz) enabled labelling of OGlcNAcylated proteins,42 we set out to develop a one-step metabolic labelling strategy that allows direct labeling of O-

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GlcNAc modified proteins in vitro and within cells. We envisioned that this approach may ultimately facilitate studies focused on monitoring kinetics of protein-specific O-GlcNAc modification, intracellular trafficking of O-GlcNAc modified proteins within cells, as well as the cellular stability of O-GlcNAcylated proteins.

RESULTS AND DISCUSSION During the course of ongoing studies, we found the use of the standard radioactivity-based glycosyltransferase assay for OGT22 to be somewhat cumbersome for use as a frequently used primary assay. We therefore were interested in developing a more convenient fluorescent assay to monitor glycosyltransfer catalyzed by OGT. Analysis of the structure of human OGT, including a ternary complex with both donor and acceptor substrates bound,46 suggested that this enzyme might tolerate moieties appended to the 2-acetamido group that are significantly larger than the diazirinecontaining sidechain used by Kohler and coworkers42 or electrophilic derivatives used by Jiang and coworkers25. We hypothesized that the small, photochemically robust fluorophore, 4-nitro-2,1,3-benzoxadiazole (NBD, Figure 1) appended to GlcNAc might be tolerated by OGT. Looking toward the longer term goal of metabolic engineering, we reasoned that the low molecular weight of this fluorochrome, coupled with its amphiphilic physicochemical properties, should confer adequate solubility of downstream probes, yet not hinder their diffusion through the plasma membrane. We also recognized that positioning of this fluorochrome would be critical if it were to be tolerated by the enzymes of the hexosamine biosynthetic pathway (HBP, Figure 1), which also converts salvaged GlcNAc into UDPGlcNAc. In particular, previous studies showed that UDP-Nacetylglucosamine pyrophosphorylase 1(AGX1), which converts N-acetyl--D-glucosamine-1-phosphate into UDP-GlcNAc, represents a biosynthetic bottleneck for analogues of GlcNAc.47 Indeed, engineering of cell lines through site-directed mutagenesis of the active site of AGX1 was required to enable metabolic labeling using diazirine-containing GlcNAc precursors.42 Our examination of the AGX1 active site, however, suggested that linkers positioning the fluorophore away from the amide moiety might be better tolerated by this enzyme. Bearing these points in mind we designed a series of UDPGlcNAc analogues in which the NBD fluorochrome was attached to the 2-acetamido position of GlcNAc through linkers of increasing length (Figure 2A) including -alanine (-Ala, 1), 5aminovaleric acid (AVA, 2), glycine--alanine (Gly--Ala, 3), and tetraethyleneglycol (PEG4, 4). We accordingly prepared Nhydroxysuccinimidyl activated linkers appended to NBD using facile and general procedures (see supporting information). We then coupled these activated esters with either uridine 5’diphospho-2-amino-2-deoxy--D-glucopyranose (UDP-GlcNH2, 5) or uridine 5’-diphospho-N-aminoacetyl--D-glucosamine (UDP-GlcNAc-NH2, 6) to obtain the final compounds (Scheme 1). The reaction of UDP-GlcNH2 (5) with the N-hydroxysuccinimide esters (OSu) of NBD--alanine (7), NBD-aminovaleric acid (8) and NBD-PEG4 (9) in the presence of NaHCO3 in a mixture of MeCN:H2O afforded compounds 1, 2, and 4 accordingly. The reaction of UDP-GlcNAc-NH2 (6) with NBD--alanine (7) using the same conditions gave us the analogue containing a glycine-alanine linker (3). We expected that these various linkers should enable the NBD group to adopt a range of positions away from the OGT active site. Moreover, they also have varying solubility, polarity, and flexibility, variations that might lead to preferences by OGT and the enzymes of the HBP, including AGX1.

Figure 1. Metabolic labelling strategy for cellular biosynthesis of UDP-GlcN--Ala-NBD. Following diffusion into cells and deacetylation of Ac3GlcN--Ala-NBD-1-P(Ac-SATE)2 to form GlcN-Ala-NBD-1-P, this active -phosphate sugar readily enters into the biosynthetic pathway and is converted into UDP-GlcN--Ala-NBD (1), which can then be transferred by OGT onto a nucleocytoplasmic proteins.

To qualitatively screen these potential donor substrates (1-4) we used nucleoporin p62 (Nup62), which is a well-established protein acceptor substrate for OGT, 22, 28, 48 in reactions containing recombinant full length human OGT (hOGT). To confirm enzymatic transfer and more readily distinguish the best substrates we included uridine diphosphate (UDP), a competitive inhibitor of OGT having a low nanomolar Ki value,15 to partially antagonize OGT (Figure 2B). The resulting reactions were analyzed by SDSPAGE followed by laser fluorescence scanning of the acrylamide gel. Remarkably, all analogues were transferred by OGT onto Nup62, with probes 1, 3, and 4 appearing to be more efficient than probe 2. The presence of 200 µM UDP reduced modification of Nup62, indicating transfer was being catalyzed by OGT. Based on these results we selected probes 1 and 3 for more detailed kinetic analysis since these linkers were structurally related and could therefore be more readily compared in downstream assays. To establish quantitative measures of the kinetic parameters governing processing of these substrates by OGT, we next used the UDP-GlcNAc analogues 1 and 3 as sugar donors in OGT-catalyzed transfer reactions using the known O-GlcNAcylated protein acceptors: calcium/calmodulin-dependent kinase IV (CaMKIV)49 and Nup62 48. Previously established kinetic parameters for Nup62 (Km(app)1 = 5.8 ± 0.7 M; kcat(app)1 = 0.84 ± 0.04 nmol/min/mg hOGT) and CaMKIV (Km(app)1 = 2.1 ± 0.5 M; kcat(app)1 = 0.037 ± 0.002 nmol/min/mg hOGT) obtained using radioactively labelled UDP-GlcNAc, were used as the benchmark of OGT assay.22 To make quantitative measurements, we constructed a standard curve using lysozyme covalently modified with NBD as an in-gel standard (Supplementary Figures S1 and S2) in conjunction with quantitative in-gel laser fluorescence scanning (Supplementary Figure S3). We then assessed transfer of the NBD-sugar moiety of UDP-GlcNAc analogues (1 and 3) by first using a fixed concentration of UDP-GlcN-Gly--Ala-NBD (3) and varying


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Journal of the American Chemical Society CaMKIV and Nup62 concentrations. For CamKIV we found no saturation up to

We next varied the concentrations of donor substrates including UDP-GlcN-Gly--Ala-NBD (3), and UDP-GlcN--Ala-NBD (1), while keeping fixed the concentration of the acceptor protein substrates. In this way we determined that the Km(app)2 values for both unnatural sugars ranged between 40- to 150-fold higher than the value measured for UDP-GlcNAc (Figure 4), presumably due to the bulkier 2-N-acyl substituent. The kcat/Km values, which are the physiologically relevant rate constants since the cellular reactions are all in competition, are within 40-fold of the natural substrate UDP-GlcNAc. Interestingly, these differences in second order rate constants (kcat/Km) depended on the identity of the protein acceptor. In any event, these data show that hOGT processes both of these UDP-NBD-sugar conjugates (1 and 3) quite well, indicating they could potentially be useful substrates to replace the standard radioassay for directly measuring group transfer and thereby enable

convenient monitoring of hOGT activity in vitro. Finally, we tested whether human OGA (hOGA) would remove this modification from proteins by incubating Nup62 that we had enzymatically glycosylated with GlcN-NBD with hOGA. Even over long incubation times, no decrease in fluorescence of the GlcN-NBD glycosylated Nup62 protein was observed (Supplementary Figure S4) indicating, as we expected,50 that this artificial GlcNAc sugar cannot be cleaved by OGA. More saliently for our objectives, based on these collective kinetic results, we expected that both UDPGlcN--Ala-NBD (1) and UDP-GlcN-Gly--Ala-NBD (3) would be competent substrates for OGT within the cellular milieu, with the fluorophore sugar modification accumulating on OGlcNAcylated proteins over time. Figure 2. UDP-GlcNAc analogues are accepted as substrates by OGT. (A) UDP-GlcNAc analogues (1-4), (B) Laser scanning of acrylamide gel assays show that UDP-GlcNAc-NBD analogues (14) can be used as donor sugars by OGT using Nup62 as an acceptor protein substrate. the limit of solubility of the protein (Figure 3A). Therefore, the kcat(app)1 and Km(app)1 values obtained when using CamKIV should be considered estimates. Nevertheless, on the basis of a linear regression of the second order region of the Michaelis-Menten plot we observe that the kcat/Km values for both CamKIV and Nup62 when using donor substrate 3 remained within approximately 3fold of the value observed when using UDP-GlcNAc (Figure 3A, 3B). Given that we saw no saturation for CaMKIV, we next used only Nup62 as an acceptor substrate when examining UDP-GlcN-Ala-NBD (1) as a donor substrate. With this substrate we found by standard non-linear fitting, that the kcat/Km value for Nup62 was again comparable with that measured for UDP-GlcNAc using the radioactivity-based assay (Figure 3C). Overall, these data indicate that these unnatural donor sugars do not greatly perturb binding and modification of protein substrates as assessed using these two model proteins.

Given these promising results, we accordingly set out to synthesize membrane permeable biosynthetic precursors of UDPGlcN--Ala-NBD (1) and UDP-GlcN-Gly--Ala-NBD (3) that could diffuse into the cell and be assimilated by cellular biosynthetic pathways. Given that a diazirine-modified GlcNAc analogue proved an incompetent substrate for the early steps of the HBP in which N-acetylglucosamine kinase (NAGK) phosphorylates the 6-hydroxyl of GlcNAc or N-acetylglucosaminephosphate mutase (AGM1) isomerizes GlcNAc-6-P to GlcNAc-1P,42 we considered using a suitably protected advanced intermediate within the HBP that would enable us to bypass these two enzymatic steps. Previous successes have been observed in metabolic feeding using per-O-acetylated 1--phospho-sugars in which the phosphate group is protected with either two Oacetoxymethyl51 or S-acetyl-2-thioethyl (SATE) groups42. These protecting groups mask both hydrogen bond donors and charged groups yet are sufficiently stable to enable passage across the cell membrane, after which the protecting groups are removed through the action of intracellular carboxyesterases and phosphodiesterases. Given our examination of the active site of AGX1 (Supplementary Figure S5), we were optimistic



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Scheme 1. Synthetic route to NBD-conjugates of UDP-GlcNAc analogues (1-4).

Acceptor

Donor

Km(app)1 (M protein substrate)

kcat(app)1 (nmol/min/mg hOGT)

kcat / Km (nmol/min/mg hOGT/M acceptor)

A. CamKIV

UDP-GlcN-Gly--Ala-NBD, 3

35  17

0.72  0.19

24  20

B. NUP62


5.9  1.3

0.35  0.03

59  26

C. NUP62

UDP-GlcN--Ala-NBD, 1

0.70  0.15

0.22  0.01

314  16

Figure 3. Kinetic analysis of hOGT catalyzed transfer of UDP-Gly--Ala-NBD (3) and UDP--Ala-NBD (1) onto CamKIV and Nup62 at various protein concentrations at a fixed substrate concentration. that Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (10) and Ac3GlcNGly--Ala-NBD--1-P(Ac-SATE)2 (11) would be deacylated within cells to the corresponding -phosphate sugars GlcN--AlaNBD--1-P and GlcN-Gly--Ala-NBD--1-P, which would in turn, through the action of AGX, be converted within the cell to the donor sugars UDP-GlcN--Ala-NBD (1) and UDP-GlcN-Gly-Ala-NBD (3). Given our in vitro results described above, we reasoned that this strategy would lead to OGT modifying OGlcNAcmodified proteins within cells directly with the corresponding fluorescent sugar. For the synthesis of Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (10), we first attempted to couple per-O-acetylated glucosamine with NBD--Ala-OSu (7), followed by deprotection of anomeric acetate and installation of the protected phosphate moiety. Unfortunately, though the acylation and selective deprotection proceeded efficiently, we obtained only the undesired -phosphate that we attributed to steric hindrance by the NBD moiety. We therefore decided to install the phosphate group on 1,3,4,6-tetra-Oacetyl-2-azidoacetyl-2-deoxy-D-glucopyranose (15) and 1,3,4,6tetra-O-acetyl-2-azido-2-deoxy-D-glucopyranose (16) prior to the N-acyl group in order to enable formation of the stereochemically desired -glycosylphosphates (Scheme 2). Starting with glucosamine hydrochloride (12) we converted this to the corresponding 1,3,4,6-tetra-O-acetyl-2-azido-2-deoxyglucopyranoside (14) using imidazole-1-sulfonyl azide52 followed by per-O-acetylation. Selective deprotection of the anomeric hydroxyl afforded 3,4,6-tri-O-acetyl-2-azido-2-deoxy-Dglucopyranose (18), which was subsequently converted into the protected phosphite ester through the use of phosphoramidite S,S'((((diisopropylamino)phosphanediyl)bis(oxy))bis(ethane-2,1diyl)) diethanethioate in the presence of 1H-tetrazole. Low temperature oxidation of the phosphine intermediate using mchloroperbenzoic acid (mCPBA) afforded the protected 3,4,6-triO-acetyl-2-azido-2-deoxy-glucopyranosyl phosphate derivative (Ac3GlcN3--1-P(Ac-SATE)2, 20). We next chose to generate an intermediate iminophosphorane from azide (20) using tributyl phosphine, which readily reacted in-situ with NBD--Ala-OSu (7) to form the desired amide Ac3GlcN--Ala-NBD--1-P(AcSATE)2 (11). Spectral characterization of amide 11 to establish the stereochemistry at the anomeric position showed that H1 displayed

a large NOE with the vicinal proton H2, confirming that the product was, as desired, -configured (Spectra in Supplementary Material). Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10) was similarly synthesized (Spectra in Supplementary Material). With the two suitably protected candidate metabolic precursors of the HBP in hand (10 and 11), we evaluated whether treatment of wild-type HeLa cells with these probes used at concentrations ranging from 25 to 200 µM resulted in modification of proteins with the fluorescent NBD-sugar analogues. Notably, the smaller diazirine-containing precursor was reported as not being incorporated by WT HeLa cells, however, we felt that given the slightly longer linkers we used here in conjunction with analysis of the AGX1 structure, that these probes might be tolerated. Upon analysis of the lysates from treated cells using laser scanning fluorescence imaging of acrylamide gels, we were surprised to observe little, if any, increase in modification of proteins when using Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10), even when increasing probe concentrations up to 200 µM and incubating cells for up to 24 h (Supplementary Figure S6). Presumably, this failure to label proteins stems from limited or no conversion of precursor (10) into UDP-GlcN-Gly--Ala-NBD (3) by AGX1, though this idea remains speculative and it is possible this compound is simply catabolized within cells. However, upon incubation of cells with Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11), we observed fluorescent labelling of cellular proteins (Supplementary Figure S7). Cells treated for 24 h with 50 µM of probe 11 exhibited no visible signs of toxicity and continued to proliferate at a normal rate. The labelling efficiency of proteins in both MEF and HeLa cells treated for 8 hours at various doses of probe 11 showed a dose-dependent increase in protein labeling (Figure 5B) as assessed by fluorescence intensity. These data suggest that this labeling approach should prove general for use with various cell lines. We next sought to establish whether known O-GlcNAc modified proteins are labeled using this one step labeling strategy. We therefore immunoprecipitated FG-containing nucleoporins using monoclonal MAb414, which readily precipitates Nup62, Nup153, and Nup214.53 Imaging of fluorescence associated with the resulting immunoprecipitated proteins and simultaneous detection of Nup62, Nup153, and Nup214 using MAb414 revealed


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Journal of the American Chemical Society colocalization of fluorescence from NBD on three proteins with molecular weights corresponding to those of Nup62, Nup153, and Nup214 (Figure 6A). These data are consistent with Ac3GlcN-Ala-NBD--1-P(Ac-SATE)2 (11) being converted within cells to

Acceptor[a]

Donor

UDP-GlcN--Ala-NBD (1), which in turn is used as a substrate by OGT to modify target proteins with the NBD-sugar analogue. We then turned

Km(app)2 (M [b] )

kcat(app)2 (nmol/min/mg hOGT)

kcat / Km (nmol/min/mg hOGT/M acceptor)

A. CamKIV


3860  350

1.60  0.11

1.37  0.14

B. NUP62


51  14

0.43  0.03

8.3  2.1

C. NUP62

UDP-GlcN--Ala-NBD, 1

36  6

0.30  0.01

8.4  1.5

Figure 4. Kinetic analysis of hOGT catalyzed transfer of UDP-GlcN-Gly--Ala-NBD (3) and UDP-GlcN-Gly--Ala-NBD (1) onto CaMKIV and Nup62 at various substrate concentrations with fixed protein concentration. to establishing whether we could use this probe in conjunction with fluorescence microscopy. This is of particular interest since recent reports have shown that metabolic two-step labeling, in conjunction with fluorescence lifetime imaging (FLIM) using genetically encoded fluorescent fusion proteins, can be used to quantify the extent of O-GlcNAc modification of target proteins of interest within cells.30, 38 We therefore incubated MEF cells for 20 hours with 100 µM. Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11). After fixing and washing the cells, fluorescence microscopy revealed widespread labeling throughout the cytoplasm and nucleus (Figure 5C). Immunocytochemical imaging of MAb414 immunoreactivity, superimposed over NBD fluorescence, supports components of the nuclear pore being modified with GlcN--Ala-NBD. Notably, we observe significant NBD fluorescence associated with what may be the nucleolus within cells. The basis for this labeling is unclear but given that many nuclear proteins are O-GlcNAc modified, this may point to a specific set of proteins that are preferentially modified using this probe or, alternatively, some non-specific accumulation of the probe within this compartment.

To confirm that OGT is responsible for modification of the proteins in cells we used engineered mouse embryonic fibroblast (MEF) cells [MEF(OGTF/Y)] in which the OGT gene is flanked by loxP recombination sites and also harbor a transgene encoding the Cre recombinase from which expression of Cre can be induced by treatment of cells with tamoxifen or related analogues.54 Accordingly, these cells enable inducible deletion of the OGT gene. We incubated these cells with 4-hydroxytamoxifen (4-HT), or vehicle alone, for 56 hours prior to treatment with 50 µM Ac3GlcN-Ala- NBD--1-P(Ac-SATE)2 (11) for 16 hours. We confirmed that OGT protein levels were greatly decreased by immunoblot and also observed, consistent with this decrease in OGT levels, that OGA levels also decreased, as did overall O-GlcNAc levels (Figure 6B). Notably, control cells having normal levels of OGT showed labeling of proteins when treated with 11 whereas cells in which the OGT gene had been deleted showed much reduced protein labeling. These data indicate that OGT is the principle enzyme responsible for fluorescent labeling of proteins with Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11) within cells. As an additional test whether


Journal of the American Chemical Society OH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O

HO HO HCl

OAc

OH

1. (ClCH2CO)2O, NaOMe, Et3N, MeOH, rt

O

HO HO

NH2 OH 2. NaN3, MeOH,

Ac2O pyridine, rt

R OH 13, R1 = NHCOCH3N3 14, R2 = N3

80oC

12

Page 6 of 9

AcO AcO

OAc

O

H2NNH2.HOAc

OAc

R 15, R1 = NHCOCH3N3

THF, rt

(44% over 3 steps) 16, R2 = N3 (87% over 2 steps)

Im-SO2-N3

AcO AcO

O

R OH 17, R1 = NHCOCH3N3 (67%) 18, R2 = N3 (93%)

CuSO45H2O, K2CO3, MeOH, 2h

AcO AcO R NH

OAc H2 O

H1

NH O O

P

O O 2N

N N O

OAc

nOe NBD--Ala-OSu PBu3, 0oC to rt, THF

O O

19, R1 = NHCOCH3N3 SAc

SAc

AcO AcO

(40% over 2 steps) 20, R2 = N3 (25% over 2 steps)

1. phosphoramidite, 1H-tetrazole, MeCN/THF, rt

O R

O

P

O

O

2. mCPBA, 40oC to rt,

O SAc

SAc

10, R1 = NHCO(CH2)2 (55%) 11, R2 = CH2 (37%)

Scheme 2. Synthetic route to Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10) and Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11). This strategy enables late-stage conjugation of NBD fluorophore using the Staudinger-Vilarrasa coupling.


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Journal of the American Chemical Society

Figure 6. Cellular proteins are modified by O-GlcN-NBD through the action of OGT. (A) Immunoprecipitation of nucleoporins. Left lane: Immunoblotting using mAB414 antibody indicates nucleopore proteins that are immunoprecipitated; Middle lane: Fluorescent bands indicate proteins that were glycosylated with the GlcN-NBD residue; Right lane: Merger of left and middle lane indicating overlapping immunoreactivity and fluorophore. (B) Left blot: Treatment of MEF cells with 4-HT leads to decreased OGT. Middle blot: GlcN-NBD labeled proteins in the nuclear extract of control and OGT KO MEF cells obtained from incubating cells with compound 11. Right blot: Fluorescence signal from immunoprecipitated Nup62 is diminished after knocking out OGT. Figure 5. (A) Metabolic precursors Ac3GlcN-Gly--Ala-NBD--1P(Ac-SATE)2 (10) and Ac3GlcN--Ala-NBD--1-P(Ac-SATE)2 (11) to donor sugars UDP-GlcN--Ala-NBD (1) and UDP-GlcN-Gly-Ala-NBD (3). (B) Dose-dependence (0 to 200 µM) of protein labeling in mouse embryonic fibroblasts (left) and HeLa (right) cells; WCL: whole cell lysate (C) top left: HeLa cell lines stained with mAB414; top middle: HeLa cells after incubation with compound (11); top right: merged image depicting HeLa nucleus after treatment of compound (11); bottom left: CTD110.6 stained HeLa cells indicating OGlcNAcylated proteins; bottom middle: HeLa cells after treatment with compound (11); bottom right: merged image of depicting HeLa cells treated with compound (11) and the O-GlcNAcylated proteins in the cytoplasmic region.

OGT function is required for modification of O-GlcNAcylated proteins with unnatural metabolic precursor (11), we examined modification of nuclear pore protein Nup62 using these same cell lines. Immunoprecipitation of Nup62 from these cells revealed that only in control cells containing normal levels of OGT did we observe significant GlcN--Ala-NBD-labeled Nup62. Collectively, these data provided strong evidence for OGT being responsible for fluorescent labeling of O-GlcNAcylated proteins by 11 within cells.

CONCLUSIONS We show that OGT tolerates unnatural analogues of the donor substrate UDP-GlcNAc in which the 2-acetamido group is replaced by relatively large substituents including, as shown here, by fluorophores such as NBD. This observation should open the door to convenient new direct transfer assays for OGT that do not rely on the use of radioactivity, which should prove useful for the wider community. We build on these observations to show that one step labeling of O-GlcNAcylated proteins within live cells can be realized by feeding cells with a suitable metabolic precursor that can be assimilated at a late stage by the hexosamine biosynthetic pathway. In particular, we find that feeding cells with suitably protected 1-phosphosugar analogues, exemplified here by Ac3GlcN-Gly--Ala-NBD--1-P(Ac-SATE)2 (10), leads to labeling of known O-GlcNAcylated proteins such as nucleoporins with a GlcN-NBD residue. We find the structure of the N-acyl linker moiety plays a key role in governing the utility of such probes and suggests that further tuning of the structure may enable more efficient labeling. Notably, genetic engineering of cells to express mutant AGX1 tolerant of modifications to the N-acyl group



is unnecessary and does not lead to more efficient labeling in cells (Supplementary Figure S8). Accordingly, we expect that this approach will prove useful in a wide range of cells to explore the physiological roles and regulation of O-GlcNAc in different cellular models. Though we find that OGT is clearly the principle transferase mediating the labeling of cells with GlcN-NBD, it is possible that other transferases may tolerate this unnatural donor sugar, though given our observations this would likely be at a lower level of efficiency than OGT-catalyzed transfer. An obvious limitation to this one-step approach, shared with two-step metabolic labeling strategies, is that the kinetics of OGT-catalyzed transfer of these unnatural GlcNAc analogues do not match the kinetics of transfer of GlcNAc itself. Nevertheless, such one step metabolic in cell labeling presents the possibility of performing a range of experiments. One can envision for example, generating additional probes that incorporate photophysically distinct fluorochromes that could enable two colour pulse-chase experiments. Additionally, we expect that engineering these metabolic precursors should permit delivery of other moieties of interest, such as biotin, which could enable simple proteomic strategies to studying O-GlcNAcylation. Perhaps most notably, this one step strategy may be used in conjunction with approaches described recently in which fluorescence lifetime imaging (FLIM) to monitor the extent of modification of specific proteins of interest directly within cells.30, 38 These FLIM studies made use of two-step chemoselective ligation chemistry, which does not permit continuous imaging of protein modification in cells. Accordingly, this one-step metabolic feeding strategy, when used in conjunction with genetically engineered proteins of interest fused to suitable fluorescent proteins, could permit monitoring the spatiotemporal action of OGT activity on specific recombinant target proteins of interest within live cells in real time using FLIM.

ASSOCIATED-CONTENT Supporting figures, Experimental methods, NMR characterization, and kinetic data figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ORCID: David J. Vocadlo: 0000-0001-6897-5558 Hong Yee Tan: 0000-0003-3530-9350 Notes: The authors declare no competing financial interest.

ACKNOWLEDGEMENTS Financial support through a Discovery grant from the Natural Sciences and Engineering Research (D.V., NSERC, RGPIN/-201505426) and Project grant from the Canadian Institutes of Health Research (D.V., CIHR PJT-148732) is gratefully acknowledged. H.Y.T. was supported by the NSERC CREATE program ChemNet. The authors thank J. Kohler (UT Southwestern) for the AGX mutant cell line and N. Zachara (Johns Hopkins) for the inducible OGT KO MEF cells. D.V. acknowledges the kind support of the Canada Research Chairs program for a Tier I Canada Research Chair in Chemical Glycobiology and NSERC for support as an E.W.R. Steacie Memorial Fellow.

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Table of contents (TOC) figure.


Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins

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