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Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry

Abstract

The N-acetyl-D-mannosamine (ManNAc) analog Ac5ManNTGc, a non-natural metabolic precursor for the sialic acid biosynthetic pathway, can be used to display thiols on the cell surface. Sugar-expressed cell-surface thiols are readily accessible compared to their protein counterparts, making them ideal for exploitation in cell-adhesion and tissue-engineering applications. This report describes a protocol for the incubation of Jurkat (human acute T-cell leukemia) cells with Ac5ManNTGc and the quantitative estimation of the resulting sialic acid displayed thiols by flow cytometry after a reaction with a water-soluble biotin-conjugated maleimide reagent and fluorescein isothiocyanate-conjugated (FITC) avidin staining. These methods, with minimal optimization, are generally also applicable to other human cell lines. The labeling and flow cytometry steps of this protocol can be performed in five to eight hours.

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Figure 1: Metabolic expression of thiols on cell-surface sialic acids.
Figure 2: Metabolic incorporation, quantification and applications for cell-surface displayed, non-natural sialic acids.
Figure 3: Quantification of cell-surface thiols (CSTs) by flow cytometry.
Figure 4: Redox modification of Neu5TGc.
Figure 5: Dose dependency of cell-surface thiol expression and cell growth.
Figure 6: Applications of metabolic sialic acid engineering.
Figure 7: Effect of cell-surface thiol (CST) expression on morphology.

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References

  1. Angata, T. & Varki, A. Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective. Chem. Rev. 102, 439–469 (2002).

    CAS  PubMed  Google Scholar 

  2. Tanner, M.E. The enzymes of sialic acid biosynthesis. Bioorg. Chem. 33, 216–228 (2005).

    CAS  PubMed  Google Scholar 

  3. Kayser, H. et al. Biosynthesis of a nonphysiological sialic acid in different rat organs, using N-propanoyl-d-hexosamines as precursors. J. Biol. Chem. 267, 16934–16938 (1992).

    CAS  PubMed  Google Scholar 

  4. Keppler, O.T., Horstkorte, R., Pawlita, M., Schmidt, C. & Reutter, W. Biochemical engineering of the N-acyl side chain of sialic acid: biological implications. Glycobiology 11, 11R–18R (2001).

    CAS  PubMed  Google Scholar 

  5. Yarema, K.J. New directions in carbohydrate engineering: a metabolic substrate-based approach to modify the cell surface display of sialic acids. BioTechniques 31, 384–393 (2001).

    CAS  PubMed  Google Scholar 

  6. Goon, S. & Bertozzi, C.R. Metabolic substrate engineering as a tool for glycobiology. J. Carbohydr. Chem. 21, 943–977 (2002).

    CAS  Google Scholar 

  7. Dube, D.H. & Bertozzi, C.R. Metabolic oligosaccharide engineering as a tool for glycobiology. Curr. Opin. Chem. Biol. 7, 616–625 (2003).

    CAS  PubMed  Google Scholar 

  8. Mahal, L.K., Yarema, K.J. & Bertozzi, C.R. Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis. Science 276, 1125–1128 (1997).

    CAS  PubMed  Google Scholar 

  9. Jacobs, C.L. et al. Substrate specificity of the sialic acid biosynthetic pathway. Biochemistry 40, 12864–12874 (2001).

    CAS  PubMed  Google Scholar 

  10. Collins, B.E., Fralich, T.J., Itonori, S., Ichikawa, Y. & Schnaar, R.L. Conversion of cellular sialic acid expression from N-acetyl- to N-glycolylneuraminic acid using a synthetic precursor, N-glycolylmannosamine pentaacetate: inhibition of myelin-associated glycoprotein binding to neural cells. Glycobiology 10, 11–20 (2000).

    CAS  PubMed  Google Scholar 

  11. Saxon, E. & Bertozzi, C.R. Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010 (2000).

    CAS  PubMed  Google Scholar 

  12. Lemieux, G.A. & Bertozzi, C.R. Chemoselective ligation reactions with proteins, oligosaccharides and cells. Trends Biotechnol. 16, 506–513 (1998).

    CAS  PubMed  Google Scholar 

  13. Prescher, J.A. & Bertozzi, C.R. Chemistry in living systems. Nat. Chem. Biol. 1, 13–21 (2005).

    CAS  PubMed  Google Scholar 

  14. Nauman, D.A. & Bertozzi, C.R. Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting. Biochim. Biophys. Acta 1568, 147–154 (2001).

    CAS  PubMed  Google Scholar 

  15. Lee, J.H. et al. Engineering novel cell surface receptors for virus-mediated gene transfer. J. Biol. Chem. 274, 21878–21884 (1999).

    CAS  PubMed  Google Scholar 

  16. Iwasaki, Y., Tabata, E., Kurita, K. & Akiyoshi, K. Selective cell attachment to a biomimetic polymer surface through the recognition of cell-surface tags. Bioconjug. Chem. 16, 567–575 (2005).

    CAS  PubMed  Google Scholar 

  17. Prescher, J.A., Dube, D.H. & Bertozzi, C.R. Chemical remodelling of cell surfaces in living animals. Nature 430, 873–877 (2004).

    CAS  PubMed  Google Scholar 

  18. Sampathkumar, S.-G., Li, A.V., Jones, M.B., Sun, Z. & Yarema, K.J. Metabolic installation of thiols into sialic acid modulates adhesion and stem cell biology. Nat. Chem. Biol. 2, 149–152 (2006).

    CAS  PubMed  Google Scholar 

  19. Sampathkumar, S.-G., Li, A.V. & Yarema, K.J. Synthesis of non-natural ManNAc analogs for the expression of thiols on cell surface sialic acids. Nat. Protocols (in the press).

  20. Howarth, M. & Ting, A.Y. Giving cells a new sugar-coating. Nat. Chem. Biol. 2, 127–128 (2006).

    CAS  PubMed  Google Scholar 

  21. 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 β1,4GalcNAc β-Onaphthalenemethanol. Proc. Natl. Acad. Sci. USA 92, 3323–3327 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Jones, M.B. et al. Characterization of the cellular uptake and metabolic conversion of acetylated N-acetylmannosamine (ManNAc) analogues to sialic acids. Biotechnol. Bioeng. 85, 394–405 (2004).

    CAS  PubMed  Google Scholar 

  23. Yarema, K.J., Mahal, L.K., Bruehl, R.E., Rodriguez, E.C. & Bertozzi, C.R. Metabolic delivery of ketone groups to sialic acid residues. Application to cell surface glycoform engineering. J. Biol. Chem. 273, 31168–31179 (1998).

    CAS  PubMed  Google Scholar 

  24. Jacobs, C.L. et al. Metabolic labeling of glycoproteins with chemical tags through unnatural sialic acid biosynthesis. Meth. Enzymol. 327, 260–275 (2000).

    CAS  Google Scholar 

  25. Sahaf, B., Heydari, K., Herzenberg, L.A. & Herzenberg, L.A. Lymphocyte surface thiol levels. Proc. Natl. Acad. Sci. USA 100, 4001–4005 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Getz, E.B., Xiao, M., Chakrabarty, T., Cooke, R. & Selvin, P.R. A comparison between the sulfhydryl reductants tris(2-carboxyethy)phosphine and dithiothreitol for use in protein biochemistry. Anal. Biochem. 273, 73–80 (1999).

    CAS  PubMed  Google Scholar 

  27. Kim, E.J., Jones, M.B., Rhee, J.K., Sampathkumar, S.-G. & Yarema, K.J. Establishment of N-acetylmannosamine (ManNAc) analogue-resistant cell lines as improved hosts for sialic acid engineering applications. Biotechnol. Prog. 20, 1674–1682 (2004).

    PubMed  Google Scholar 

  28. Kim, E.J . et al. Characterization of the metabolic flux and apoptotic effects of O-hydroxyl- and N-acetylmannosamine (ManNAc) analogs in Jurkat (human T-lymphoma-derived) cells. J. Biol. Chem. 279, 18342–18352 (2004).

    CAS  PubMed  Google Scholar 

  29. Martin, M.J., Muotri, A., Gage, F. & Varki, A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228–232 (2005).

    CAS  PubMed  Google Scholar 

  30. Barbosa, J.N., Barbosaa, M.A. & Águas, A.P. Inflammatory responses and cell adhesion to self-assembled monolayers of alkanethiolates on gold. Biomaterials 25, 2557–2563 (2004).

    CAS  PubMed  Google Scholar 

  31. Nam, Y., Chang, J.C., Wheeler, B.C. & Brewer, G.J. Gold-coated microelectrode array with thiol linked self-assembled monolayers for engineering neuronal cultures. IEEE Trans. Biomed. Eng. 51, 158–165 (2004).

    PubMed  Google Scholar 

  32. Turner, N., Armitage, M., Butler, R. & Ireland, G. An in vitro model to evaluate cell adhesion to metals used in implantation shows significant differences between palladium and gold or platinum. Cell Biol. Int. 28, 541–547 (2004).

    CAS  PubMed  Google Scholar 

  33. Yousaf, M.N., Houseman, B.T. & Mrksich, M. Using electroactive substrates to pattern the attachment of two different cell populations. Proc. Natl. Acad. Sci. USA 98, 5992–5996 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Mrksich, M. What can surface chemistry do for cell biology? Curr. Opin. Chem. Biol. 6, 794–797 (2002).

    CAS  PubMed  Google Scholar 

  35. Liu, T., Guo, Z., Yang, Q., Sad, S. & Jennings, H.J. Biochemical engineering of surface α2,8 polysialic acid for immunotargeting tumor cells. J. Biol. Chem. 275, 32832–32836 (2000).

    CAS  PubMed  Google Scholar 

  36. Lemieux, G.A., Yarema, K.J., Jacobs, C.L. & Bertozzi, C.R. Exploiting differences in sialoside expression for selective targeting of MRI contrast reagents. J. Am. Chem. Soc. 121, 4278–4279 (1999).

    CAS  Google Scholar 

  37. De Bank, P.A., Kellam, B., Kendall, D.A. & Shakesheff, K.M. Surface engineering of living myoblasts via selective periodate oxidation. Biotechnol. Bioeng. 81, 800–808 (2003).

    CAS  PubMed  Google Scholar 

  38. Yarema, K.J. in Cell Engineering 3. Glycosylation Vol. 3 (ed. Al-Rubeai, M.) 171–196 (Kluwer Academic Publishers, Dordrecht, 2002).

    Google Scholar 

  39. Garige, M., Gong, M., Rao, M.N., Zhang, Y. & Lakshmana, M.R. Mechanism of action of ethanol in the down-regulation of Galβ1,4GlcNAc α2,6-sialyltransferase messenger RNA in human liver cell lines. Metabolism 54, 729–734 (2005).

    CAS  PubMed  Google Scholar 

  40. Marmillot, P., Rao, M.N., Liu, Q.H. & Lakshman, M.R. Chronic ethanol increases ganglioside sialidase activity in rat leukocytes, erythrocytes, and brain synaptosomes. Alcohol Clin. Exp. Res. 23, 376–380 (1999).

    CAS  PubMed  Google Scholar 

  41. dos Santos, A., Rodrigues, M., Alviano, C. & de Araujo Soares, R. Changes of sialomolecules during the dimethylsulfoxide-induced differentiation of Herpetomonas samuelpessoai. Parasitol. Res. 88, 951–955 (2002).

    PubMed  Google Scholar 

  42. Mantey, L.R., Keppler, O.T., Pawlita, M., Reutter, W. & Hinderlich, S. Efficient biochemical engineering of cellular sialic acids using an unphysiological sialic acid precursor in cells lacking UDP-N-acetylglucosamine 2-epimerase. FEBS Lett. 503, 80–84 (2001).

    CAS  PubMed  Google Scholar 

  43. Yarema, K.J., Goon, S. & Bertozzi, C.R. Metabolic selection of glycosylation defects in human cells. Nat. Biotechnol. 19, 553–558 (2001).

    CAS  PubMed  Google Scholar 

  44. Luchansky, S.J., Yarema, K.J., Takahashi, S. & Bertozzi, C.R. GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism. J. Biol. Chem. 278, 8036–8042 (2003).

    Google Scholar 

  45. Villavicencio-Lorini, P., Laabs, S., Danker, K., Reutter, W. & Horstkorte, R. Biochemical engineering of the acyl side chain of sialic acids stimulates integrin-dependent adhesion of HL60 cells to fibronectin. J. Mol. Med. 80, 671–677 (2002).

    CAS  PubMed  Google Scholar 

  46. Editorial. Biology and brimstone. Nat. Chem. Biol. 2, 169 (2006).

  47. Hogg, P.J. Disulfide bonds as switches for protein function. Trends Biochem. Sci. 28, 210–214 (2003).

    CAS  PubMed  Google Scholar 

  48. Nakashima, I. et al. Redox-linked signal transduction pathways for protein tyrosine kinase activation. Antioxid. & Redox Signal. 4, 517–531 (2002).

    CAS  Google Scholar 

  49. Sahaf, B., Heydari, K., Herzenberg, L.A. & Herzenberg, L.A. The extracellular microenvironment plays a key role in regulating the redox status of cell surface proteins in HIV-infected subjects. Arch. Biochem. Biophys. 434, 26–32 (2005).

    CAS  PubMed  Google Scholar 

  50. Ghetie, V. & Vitetta, E.S. Chemical construction of immunotoxins. Mol. Biotechnol. 18, 251–268 (2001).

    CAS  PubMed  Google Scholar 

  51. Hakomori, S.-I. The glycosynapse. Proc. Natl. Acad. Sci. USA 99, 225–232 (2002).

    CAS  PubMed Central  Google Scholar 

  52. Han, S., Collins, B.E., Bengtson, P. & Paulson, J.C. Homo-multimeric complexes of CD22 revealed by in situ photoaffinity protein-glycan crosslinking. Nat. Chem. Biol. 1, 93–97 (2005).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to K. Konstantopoulos for flow cytometer access, P. Pawar for technical assistance in FACS experiments, J.D. Gearhart and M. J. Shamblott for the kind gift of hEBD-LVEC cells, T.H. Wang for a kind gift of QD655–streptavidin conjugate, and J.M. McCaffery and E. Perkins of Integrated Imaging Facility (Johns Hopkins University, Department of Biology) for help with confocal microscopy. Funding was provided by the Arnold and Mabel Beckman Foundation, the US National Institutes of Health (1R01CA112314-01A1) and the National Science Foundation (QSB-0425668).

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Correspondence to Kevin J Yarema.

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Sampathkumar, SG., Jones, M. & Yarema, K. Metabolic expression of thiol-derivatized sialic acids on the cell surface and their quantitative estimation by flow cytometry. Nat Protoc 1, 1840–1851 (2006). https://doi.org/10.1038/nprot.2006.252

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