Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome

Nature Chemical Biology volume 8, pages 661–668 (2012)Cite this article

Subjects

Abstract

Despite the fundamental roles of sialyl- and fucosyltransferases in mammalian physiology, there are few pharmacological tools to manipulate their function in a cellular setting. Although fluorinated analogs of the donor substrates are well-established transition state inhibitors of these enzymes, they are not membrane permeable. By exploiting promiscuous monosaccharide salvage pathways, we show that fluorinated analogs of sialic acid and fucose can be taken up and metabolized to the desired donor substrate–based inhibitors inside the cell. Because of the existence of metabolic feedback loops, they also act to prevent the de novo synthesis of the natural substrates, resulting in a global, family-wide shutdown of sialyl- and/or fucosyltransferases and remodeling of cell-surface glycans. As an example of the functional consequences, the inhibitors substantially reduce expression of the sialylated and fucosylated ligand sialyl Lewis X on myeloid cells, resulting in loss of selectin binding and impaired leukocyte rolling.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Fluorinated monosaccharide analogs act as metabolic glycosyltransferase inhibitors.
Figure 2: Fluorinated fucose and sialic acid analogs act as fucosyl- and sialyltransferase inhibitors in cells.
Figure 3: MS analysis of N-glycans from inhibitor-treated cells.
Figure 4: Selectin binding and selectin-mediated leukocyte rolling.

Similar content being viewed by others

References

  1. Varki, A. et al. Essentials of Glycobiology (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2009).

  2. Becker, D.J. & Lowe, J. Fucose: biosynthesis and biological function in mammals. Glycobiology 13, 41R–53R (2003).

    Article  CAS  Google Scholar 

  3. Varki, A. Glycan-based interactions involving vertebrate sialic-acid–recognizing proteins. Nature 446, 1023–1029 (2007).

    Article  CAS  Google Scholar 

  4. Lowe, J.B. & Marth, J.D. A genetic approach to Mammalian glycan function. Annu. Rev. Biochem. 72, 643–691 (2003).

    Article  CAS  Google Scholar 

  5. Malý, P. et al. The α(1,3)fucosyltransferase Fuc-TVII controls leukocyte trafficking through an essential role in L-, E-, and P-selectin ligand biosynthesis. Cell 86, 643–653 (1996).

    Article  Google Scholar 

  6. Hennet, T., Chui, D., Paulson, J. & Marth, J. Immune regulation by the ST6Gal sialyltransferase. Proc. Natl. Acad. Sci. USA 95, 4504–4509 (1998).

    Article  CAS  Google Scholar 

  7. Priatel, J.J. et al. The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis. Immunity 12, 273–283 (2000).

    Article  CAS  Google Scholar 

  8. Grewal, P.K. et al. ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling. Mol. Cell Biol. 26, 4970–4981 (2006).

    Article  CAS  Google Scholar 

  9. Gitlin, J.M. et al. Disruption of tissue-specific fucosyltransferase VII, an enzyme necessary for selectin ligand synthesis, suppresses atherosclerosis in mice. Am. J. Pathol. 174, 343–350 (2009).

    Article  CAS  Google Scholar 

  10. Homeister, J.W., Daugherty, A. & Lowe, J.B. α(1,3)fucosyltransferases FucT-IV and FucT-VII control susceptibility to atherosclerosis in apolipoprotein E−/− mice. Arterioscler. Thromb. Vasc. Biol. 24, 1897–1903 (2004).

    Article  CAS  Google Scholar 

  11. Sarkar, A.K., Fritz, T.A., Taylor, W.H. & Esko, J.D. Disaccharide uptake and priming in animal cells: inhibition of sialyl Lewis X by acetylated Gal β1→4GlcNAc β-O-naphthalenemethanol. Proc. Natl. Acad. Sci. USA 92, 3323–3327 (1995).

    Article  CAS  Google Scholar 

  12. Fuster, M.M., Brown, J.R., Wang, L. & Esko, J.D. A disaccharide precursor of sialyl Lewis X inhibits metastatic potential of tumor cells. Cancer Res. 63, 2775–2781 (2003).

    CAS  PubMed  Google Scholar 

  13. Brown, J.R. et al. A disaccharide-based inhibitor of glycosylation attenuates metastatic tumor cell dissemination. Clin. Cancer Res. 12, 2894–2901 (2006).

    Article  CAS  Google Scholar 

  14. Hosoguchi, K. et al. An efficient approach to the discovery of potent inhibitors against glycosyltransferases. J. Med. Chem. 53, 5607–5619 (2010).

    Article  CAS  Google Scholar 

  15. Lee, L.V. et al. A potent and highly selective inhibitor of human α-1,3-fucosyltransferase via click chemistry. J. Am. Chem. Soc. 125, 9588–9589 (2003).

    Article  CAS  Google Scholar 

  16. Burkart, M.D. et al. Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic probes for glycosyltransferases. Bioorg. Med. Chem. 8, 1937–1946 (2000).

    Article  CAS  Google Scholar 

  17. Lairson, L.L., Henrissat, B., Davies, G.J. & Withers, S.G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 77, 521–555 (2008).

    Article  CAS  Google Scholar 

  18. Gloster, T.M. et al. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat. Chem. Biol. 7, 174–181 (2011).

    Article  CAS  Google Scholar 

  19. Campbell, C.T., Sampathkumar, S.G. & Yarema, K.J. Metabolic oligosaccharide engineering: perspectives, applications, and future directions. Mol. Biosyst. 3, 187–194 (2007).

    Article  CAS  Google Scholar 

  20. Sullivan, F.X. et al. Molecular cloning of human GDP-mannose 4,6-dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. J. Biol. Chem. 273, 8193–8202 (1998).

    Article  CAS  Google Scholar 

  21. Hinderlich, S., Stäsche, R., Zeitler, R. & Reutter, W. A bifunctional enzyme catalyzes the first two steps in N-acetylneuraminic acid biosynthesis of rat liver. Purification and characterization of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. J. Biol. Chem. 272, 24313–24318 (1997).

    Article  CAS  Google Scholar 

  22. Burkart, M., Zhang, Z., Hung, S. & Wong, C. A new method for the synthesis of fluoro-carbohydrates and glycosides using selectfluor. J. Am. Chem. Soc. 119, 11743–11746 (1997).

    Article  CAS  Google Scholar 

  23. Weston, B.W. et al. A cloned CD15s-negative variant of HL60 cells is deficient in expression of FUT7 and does not adhere to cytokine-stimulated endothelial cells. Eur. J. Haematol. 63, 42–49 (1999).

    Article  CAS  Google Scholar 

  24. Nakayama, F. et al. CD15 expression in mature granulocytes is determined by α1,3-fucosyltransferase IX, but in promyelocytes and monocytes by α1,3-fucosyltransferase IV. J. Biol. Chem. 276, 16100–16106 (2001).

    Article  CAS  Google Scholar 

  25. Chiu, C.P. et al. Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog. Nat. Struct. Mol. Biol. 11, 163–170 (2004).

    Article  CAS  Google Scholar 

  26. Ni, L. et al. Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism. Biochemistry 46, 6288–6298 (2007).

    Article  CAS  Google Scholar 

  27. Marathe, D.D., Chandrasekaran, E.V., Lau, J.T., Matta, K.L. & Neelamegham, S. Systems-level studies of glycosyltransferase gene expression and enzyme activity that are associated with the selectin binding function of human leukocytes. FASEB J. 22, 4154–4167 (2008).

    Article  CAS  Google Scholar 

  28. Oetke, C. et al. Evidence for efficient uptake and incorporation of sialic acid by eukaryotic cells. Eur. J. Biochem. 268, 4553–4561 (2001).

    Article  CAS  Google Scholar 

  29. Mendla, K., Baumkötter, J., Rosenau, C., Ulrich-Bott, B. & Cantz, M. Defective lysosomal release of glycoprotein-derived sialic acid in fibroblasts from patients with sialic acid storage disease. Biochem. J. 250, 261–267 (1988).

    Article  CAS  Google Scholar 

  30. Keppler, O.T. et al. UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation. Science 284, 1372–1376 (1999).

    Article  CAS  Google Scholar 

  31. Ripka, J., Adamany, A. & Stanley, P. Two Chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose. Arch. Biochem. Biophys. 249, 533–545 (1986).

    Article  CAS  Google Scholar 

  32. Beyer, T.A. et al. Biosynthesis of mammalian glycoproteins. Glycosylation pathways in the synthesis of the nonreducing terminal sequences. J. Biol. Chem. 254, 12531–12534 (1979).

    CAS  PubMed  Google Scholar 

  33. Sperandio, M., Gleissner, C.A. & Ley, K. Glycosylation in immune cell trafficking. Immunol. Rev. 230, 97–113 (2009).

    Article  CAS  Google Scholar 

  34. Lowe, J.B. Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr. Opin. Cell Biol. 15, 531–538 (2003).

    Article  CAS  Google Scholar 

  35. Mestas, J. & Ley, K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc. Med. 18, 228–232 (2008).

    Article  CAS  Google Scholar 

  36. Leppänen, A., White, S.P., Helin, J., McEver, R.P. & Cummings, R.D. Binding of glycosulfopeptides to P-selectin requires stereospecific contributions of individual tyrosine sulfate and sugar residues. J. Biol. Chem. 275, 39569–39578 (2000).

    Article  Google Scholar 

  37. Rillahan, C.D., Brown, S.J., Register, A.C., Rosen, H. & Paulson, J.C. High-throughput screening for inhibitors of sialyl- and fucosyltransferases. Angew. Chem. Int. Edn Engl. 50, 12534–12537 (2011).

    Article  CAS  Google Scholar 

  38. Gross, B.J., Kraybill, B. & Walker, S. Discovery of O-GlcNAc transferase inhibitors. J. Am. Chem. Soc. 127, 14588–14589 (2005).

    Article  CAS  Google Scholar 

  39. Gross, B.J., Swoboda, J.G. & Walker, S. A strategy to discover inhibitors of O-linked glycosylation. J. Am. Chem. Soc. 130, 440–441 (2008).

    Article  CAS  Google Scholar 

  40. Chiaramonte, M. et al. Inhibition of CMP-sialic acid transport into Golgi vesicles by nucleoside monophosphates. Biochemistry 40, 14260–14267 (2001).

    Article  CAS  Google Scholar 

  41. Han, S., Collins, B.E., Bengtson, P. & Paulson, J.C. Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking. Nat. Chem. Biol. 1, 93–97 (2005).

    Article  CAS  Google Scholar 

  42. Luchansky, S.J., Goon, S. & Bertozzi, C.R. Expanding the diversity of unnatural cell-surface sialic acids. ChemBioChem 5, 371–374 (2004).

    Article  CAS  Google Scholar 

  43. Alley, S.C.J. et al. Methods and compositions for making antibodies and antibody derivatives with reduced core fucosylation. US Patent Application. 200903178691 (2009).

  44. Frantom, P.A., Coward, J.K. & Blanchard, J.S. UDP-(5F)-GlcNAc acts as a slow-binding inhibitor of MshA, a retaining glycosyltransferase. J. Am. Chem. Soc. 132, 6626–6627 (2010).

    Article  CAS  Google Scholar 

  45. Hayashi, T., Murray, B.W., Wang, R. & Wong, C.H. A chemoenzymatic synthesis of UDP-(2-deoxy-2-fluoro)-galactose and evaluation of its interaction with galactosyltransferase. Bioorg. Med. Chem. 5, 497–500 (1997).

    Article  CAS  Google Scholar 

  46. Barthel, S.R. et al. Peracetylated 4-fluoro-glucosamine reduces the content and repertoire of N- and O-glycans without direct incorporation. J. Biol. Chem. 286, 21717–21731 (2011).

    Article  CAS  Google Scholar 

  47. Dimitroff, C.J., Kupper, T.S. & Sackstein, R. Prevention of leukocyte migration to inflamed skin with a novel fluorosugar modifier of cutaneous lymphocyte-associated antigen. J. Clin. Invest. 112, 1008–1018 (2003).

    Article  CAS  Google Scholar 

  48. Gainers, M.E. et al. Skin-homing receptors on effector leukocytes are differentially sensitive to glyco-metabolic antagonism in allergic contact dermatitis. J. Immunol. 179, 8509–8518 (2007).

    Article  CAS  Google Scholar 

  49. Nishimura, S.I., Hato, M., Hyugaji, S., Feng, F. & Amano, M. Glycomics for drug discovery: metabolic perturbation in androgen-independent prostate cancer cells induced by unnatural hexosamine mimics. Angew. Chem. Int. Edn Engl. (2012).

  50. Chesnutt, B.C. et al. Induction of LFA-1-dependent neutrophil rolling on ICAM-1 by engagement of E-selectin. Microcirculation 13, 99–109 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by US National Institutes of Health grants to J.C.P. (R01AI050143 and P01HL107151), C.D.R. (T32AI007606), K.L. (R01HL111969), C.T.L. (T32AI060536) and the Complex Carbohydrate Research Center (1 P41 RR018502-01) as well as by funding from the Biotechnology and Biological Sciences Research Council to A.D. and S.M.H. (BBF0083091).

Author information

Authors and Affiliations

  1. Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA

    Cory D Rillahan & James C Paulson

  2. Division of Molecular Biosciences, Faculty of Natural Sciences, Imperial College–London, London, UK

    Aristotelis Antonopoulos, Anne Dell & Stuart M Haslam

  3. Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Craig T Lefort & Klaus Ley

  4. Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia, USA

    Roberto Sonon & Parastoo Azadi

Authors
  1. Cory D Rillahan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aristotelis Antonopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Craig T Lefort
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roberto Sonon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Parastoo Azadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Klaus Ley
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Anne Dell
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stuart M Haslam
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. James C Paulson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.D.R. conceived of the idea, synthesized the inhibitors, designed the experiments and performed biochemical assays. A.A. performed the N- and O-linked glycan MS analysis. C.T.L. performed the rolling assays. R.S. performed the nucleotide sugar analysis. J.C.P., S.M.H., A.D., K.L. and P.A. supervised the research. C.D.R and J.C.P. wrote the manuscript.

Corresponding author

Correspondence to James C Paulson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 2406 kb)

Supplementary Video 1

Rolling Velocity Measurement of control (DMSO) treated HL-60 cells on E-Selectin at 3 dynes/cm2 (MOV 2837 kb)

Supplementary Video 2

Rolling Velocity Measurement of 2F-Fuc (2) treated HL-60 cells on ESelectin at 3 dynes/cm2 (MOV 3188 kb)

Supplementary Video 3

Rolling Velocity Measurement of 3Fax-Neu5Ac (8) treated HL-60 cells on E-Selectin at 3 dynes/cm2 (MOV 3169 kb)

Supplementary Video 4

Rolling Velocity Measurement of control (DMSO) treated HL-60 cells on P-Selectin at 3 dynes/cm2 (MOV 3596 kb)

Supplementary Video 5

Rolling Velocity Measurement of 2F-Fuc (2) treated HL-60 cells on PSelectin at 3 dynes/cm2 (MOV 3855 kb)

Supplementary Video 6

Rolling Velocity Measurement of 3Fax-Neu5Ac (8) treated HL-60 cells on P-Selectin at 3 dynes/cm2 (MOV 3516 kb)

Supplementary Video 7

Tethering Analysis of Control (DMSO) treated HL-60 cells on E-Selectin (MOV 2935 kb)

Supplementary Video 8

Tethering Analysis of 2F-Fuc (2) treated HL-60 cells on E-Selectin (MOV 2803 kb)

Supplementary Video 9

Tethering Analysis of 3Fax-Neu5Ac (8) treated HL-60 cells on E-Selectin (MOV 3301 kb)

Supplementary Video 10

Tethering Analysis of Control (DMSO) treated HL-60 cells on PSelectin (MOV 3605 kb)

Supplementary Video 11

Tethering Analysis of 2F-Fuc (2) treated HL-60 cells on P-Selectin (MOV 3842 kb)

Supplementary Video 12

Tethering Analysis of 3Fax-Neu5Ac (8) treated HL-60 cells on PSelectin (MOV 3778 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rillahan, C., Antonopoulos, A., Lefort, C. et al. Global metabolic inhibitors of sialyl- and fucosyltransferases remodel the glycome. Nat Chem Biol 8, 661–668 (2012). https://doi.org/10.1038/nchembio.999

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.999

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing