Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells - PubMed (original) (raw)

Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells

Tracey M Gloster et al. Nat Chem Biol. 2011 Mar.

Abstract

Glycosyltransferases are ubiquitous enzymes that catalyze the assembly of glycoconjugates throughout all kingdoms of nature. A long-standing problem is the rational design of probes that can be used to manipulate glycosyltransferase activity in cells and tissues. Here we describe the rational design and synthesis of a nucleotide sugar analog that inhibits, with high potency both in vitro and in cells, the human glycosyltransferase responsible for the reversible post-translational modification of nucleocytoplasmic proteins with O-linked N-acetylglucosamine residues (O-GlcNAc). We show that the enzymes of the hexosamine biosynthetic pathway can transform, both in vitro and in cells, a synthetic carbohydrate precursor into the nucleotide sugar analog. Treatment of cells with the precursor lowers O-GlcNAc in a targeted manner with a single-digit micromolar EC(50). This approach to inhibition of glycosyltransferases should be applicable to other members of this superfamily of enzymes and enable their manipulation in a biological setting.

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Figures

Figure 1

Figure 1. The mammalian hexosamine biosynthetic pathway (HBP), GlcNAc salvage pathway, and the structures of compounds studied here

(a) Structures of some of the molecules used in this study (X = O or S as indicated, R = H or Ac as indicated). (b) The end product of the HBP is UDP-GlcNAc (2), the donor substrate used by OGT. The first step in the de novo pathway, where fructose-6-phosphate is converted to glutamine-6-phosphate, is catalyzed by glutamine: fructose-6-phosphate amidotransferase (GFAT). Glutamine-6-phosphate is transformed into GlcNAc-6-phosphate by acetyl-CoA:D-glucosamine-6-phosphate _N_-acetyltransferase (GAT). The GlcNAc salvage pathway recycles cellular GlcNAc (1), which is converted into GlcNAc-6-phosphate by GlcNAc kinase (GNK). GlcNAc-6-phosphate is converted into GlcNAc-1-phosphate by GlcNAc mutase (AGM), and then to the end product, UDP-GlcNAc, by UDP-GlcNAc pyrophosphorylase (AGX1). The endocyclic heteroatom is denoted X (O in nature and S in the synthetic compounds depicted in panel a). 5SGlcNAc (3) is converted via the salvage pathway to generate intracellular UDP-5SGlcNAc (4). Ac-5SGlcNAc (5) is deacetylated by cellular esterases.

Figure 2

Figure 2. Ac-5SGlcNAc (5) acts in cells to decrease global _O_-GlcNAc levels in a dose and time dependent manner

(a) Western blots of COS-7 cell lysates following Ac-5SGlcNAc (5) administration at different doses (0-1000 μM) for 24 h. Upper panel, probed with anti-_O_-GlcNAc antibody (CTD110.6); lower panel, probed with anti-actin antibody. Densitometry analysis (see Supplementary Figure 6a) yields an EC50 value of 5 μM. (b) Western blots of COS-7 cell lysates following Ac-5SGlcNAc (5) administration at 50 μM for various times. Upper panel, probed with CTD110.6; lower panel, probed with anti-actin antibody. Densitometry analysis (see Supplementary Figure 6e) shows how _O_-GlcNAc levels diminish over time. (c) Western blots of COS-7 cell lysates following Ac-5SGlcNAc (5) administration at 50 μM for different amounts of time. Probed with (from top to bottom) anti-_O_-GlcNAc antibody (CTD110.6), anti-OGA antibody, anti-OGT antibody, and anti-actin antibody. Full versions of the blots are shown in Supplementary Figure 9b. (d) Western blots of COS-7 cell lysates administered specified agents at 50 μM for 24 h; vehicle only (C), Ac-GlcNAc (8) or Ac-5SGlcNAc (5). Probed with (from top to bottom) anti-_O_-GlcNAc antibody (CTD110.6), anti-OGA antibody, anti-OGT antibody, and anti-actin antibody. Full versions of the blots are shown in Supplementary Figure 9a. (e) Immunocytochemistry of COS-7 cells treated with no, 50 μM or 250 μM Ac-5SGlcNAc (5) for 24 hours. Immunoreactivity from anti-_O_-GlcNAc antibody CTD110.6 is shown in green and DAPI (which stains DNA in the nucleus) in blue. The white bar corresponds to a distance of 25 μM.

Figure 3

Figure 3. Evaluation of the effects of Ac-5SGlcNAc (5) treatment of cells on the _O_-GlcNAc modification state of nup62

(a) Western blots of immunoprecipitated nup62 from cell lysates following vehicle (C) or 250 μM Ac-5SGlcNAc (5) treatment for 24 h. Following immunoprecipitation, nup62 was incubated with UDP-GalNAz in the absence (−) or presence (+) of GalT1 and chemoselectively labelled. Upper panel, probed with anti-nup62 antibody; lower panel, probed with streptavidin. (b) Western blots of immunoprecipitated nup62 from cell lysates following vehicle (C) or 250 μM Ac-5SGlcNAc (5) treatment for 24 h. Following immunoprecipitation, nup62 was incubated with buffer (−) or with _Bt_GH84 (+) for 2 h to remove _O_-GlcNAc. Upper panel, probed with anti-nup62 antibody; middle panel, probed with anti-_O_-GlcNAc antibody (CTD110.6) (long exposure); bottom panel probed with anti-_O_-GlcNAc antibody (CTD110.6) (short exposure). (c) Western blots of COS-7 cell lysates following Ac-5SGlcNAc (5) administration at different doses (0-1000 μM) for 24 h. Blots are probed with (from top to bottom) anti-nup62 antibody, anti-_O_-GlcNAc antibody (CTD110.6) and anti-actin antibody. Full versions of all of these blots are shown in Supplementary Figure 11.

Figure 4

Figure 4. Metabolic feeding of Ac-5SGlcNAz (14) to cells causes a decrease in _O_-GlcNAc levels, but chemoselecctive ligation demonstrates there is no accumulation of 5SGlcNAz (13) on proteins

(a) Structures of the molecules used or generated during the chemoselective ligation study (X = O or S as indicated, R = H or Ac as indicated). (b) Western blots of COS-7 cell lysates administered specified agents at 50 μM for 24 h; vehicle only (C), Ac-GlcNAz (15) or Ac-5SGlcNAz (14). Cells were harvested and then underwent the Staudinger ligation with biotin phosphine. Blots are probed with (from top to bottom) streptavidin-HRP, anti _O_-GlcNAc (CTD110.6) antibody and anti-actin antibody. (c) Western blot of immunoprecipitated nup62 from cell lysates following vehicle (C), 50 μM Ac-GlcNAz (15) or Ac-5SGlcNAz (14) treatment for 24 h. Following immunoprecipitation, nup62 was incubated with buffer (−) or with _Bt_GH84 (+) for 2 h to remove _O_-GlcNAc, and then underwent the Staudinger ligation with biotin phosphine. Blots are probed with streptavidin-HRP. A full version of this western blot is shown in Supplementary Figure 13.

Figure 5

Figure 5. Ac-5SGlcNAc (5) is converted in cells to generate intracellular UDP-5SGlcNAc (4), causing perturbations in UDP-sugar nucleotide pools

(a) Analysis of UDP-sugar pools of COS-7 cells treated with different concentrations of Ac-5SGlcNAc (0-1000 μM from bottom to top) for 24 h; the CE trace shows absorbance at 254 nm as a function of retention time. Peak A, GDP-Glc (internal standard); peak B, UDP-GlcNAc; peak C, UDP-Glc; peak D, UDP-5SGalNAc; peak E, UDP-5SGlcNAc; peak F, UDP-GalNAc; peak G, UDP-Gal. UDP-5SGlcNAc (4) and UDP-GalNAc co-elute, but the amount of each is estimated using the epimeric ratios determined from the standards (Supplementary Figure 14b). (b) Bar chart showing relative concentrations of UDP-GlcNAc (2), UDP-5SGlcNAc (4), UDP-Gal, UDP-5SGal, UDP-Glc, and UDP-Gal following treatment with 0-1000 μM Ac-5SGlcNAc (5).

Figure 6

Figure 6. Treatment of cells with Ac-5SGlcNAc (5) has no apparent effects on global _N_-glycosylation or _N_-glycosylation of a secreted IgG as evaluated by lectin blot analysis

(a) Western blots of COS-7 cell lysates following Ac-5SGlcNAc (5) administration at different doses (0-1000 μM) for 24 h. C+ denotes untreated cell lysate incubated with PNGase F and C- denotes untreated cell lysate incubated with vehicle. Blots are probed with (from top to bottom) anti-_O_-GlcNAc antibody (CTD110.6), anti-actin antibody, ConA lectin (recognizes α-

d

-mannose, α-

d

-glucose and branched mannose), GNA lectin (recognizes mannose), PHA-L (recognizes complex branched chain oligosaccharide structure), SNA lectin (recognizes NeuAcα(2,6)Gal/GalNAc) and MAA lectin (recognizes NeuAcα(2,3)Gal). Full versions of these blots are shown in Supplementary Figure 15a. (b) Western blots of mouse hybridoma cell lysates (_O_-GlcNAc and actin) and immunoprecipitated mouse hybridoma antibody (IgG, ConA and GNA) following administration of Ac-5SGlcNAc (5) at different doses (0-1000 μM) for 24 h. C+, untreated cell lysate incubated with PNGase F; C-, untreated cell lysate incubated with vehicle. Blots are probed with (from top to bottom) anti-_O_-GlcNAc antibody (CTD110.6), anti-actin antibody, anti-IgG antibody, ConA lectin and GNA lectin. Full versions of these blots are shown in Supplementary Figure 15b.

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