Skip to main content
SpringerLink
Log in

Biochemical characterization of human and murine isoforms of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE)

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the key enzyme for the biosynthesis of sialic acids, terminal components of glycoconjugates associated with a variety of physiological and pathological processes. Different protein isoforms of human and mouse GNE, deriving from splice variants, were predicted recently: GNE1 represents the GNE protein described in several studies before, GNE2 and GNE3 are proteins with extended and deleted N-termini, respectively. hGNE2, recombinantly expressed in insect and mamalian cells, displayed selective reduction of UDP-GlcNAc 2-epimerase activity by the loss of its tetrameric state, which is essential for full enzyme activity. hGNE3, which had to be expressed in Escherichia coli, only possessed kinase activity, whereas mGNE1 and mGNE2 showed no significant differences. Our data therefore suggest a role of GNE1 in basic supply of cells with sialic acids, whereas GNE2 and GNE3 may have a function in fine-tuning of the sialic acid pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

GNE:

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase

UDP-GlcNAc:

UDP-N-acetylglucosamine

ManNAc:

N-acetylmannosamine

Neu5Ac:

N-acetylneuraminic acid

References

  1. Corfield, A.P., Schauer, R.: Occurrence of sialic acids. In: Schauer, R. (ed.) Sialic Acids. Chemistry, Metabolism, and Function, pp. 5–50. Springer, Wien (1982)

    Google Scholar 

  2. Varki, A.: Glycan-based interactions involving vertebrate sialic-acid-recognizing proteins. Nature 446, 1023–1029 (2007). doi:10.1038/nature05816

    Article  PubMed  CAS  Google Scholar 

  3. Bhavanandan, V.P.: Cancer-associated mucins and mucin-type glycoproteins. Glycobiology 1, 493–503 (1991). doi:10.1093/glycob/1.5.493

    Article  PubMed  CAS  Google Scholar 

  4. Schauer, R., Kelm, S., Reuter, G., Roggentin, P., Shaw, L.: Biochemistry and role of sialic acids. In: Rosenberg, A. (ed.) Biology of the Sialic Acids, pp. 7–67. Plenum, New York (1995)

    Google Scholar 

  5. Rens-Domiano, S., Reisine, T.J.: Structural analysis and functional role of the carbohydrate component of somatostatin receptor. Biol. Chem. 266, 20094–20102 (1991)

    CAS  Google Scholar 

  6. Angata, T., Varki, A.: Chemical diversity in the sialic acids and related α-keto acids: an evolutionary perspective. Chem. Rev. 102, 439–469 (2002). doi:10.1021/cr000407m

    Article  PubMed  CAS  Google Scholar 

  7. Hinderlich, S., Oetke, C., Pawlita, M.: Biochemical engineering of sialic acids. In: Yarema, K.J. (ed.) Handbook of Carbohydrate Engineering, pp. 387–405. Taylor & Francis, Boca Raton (2005)

    Google Scholar 

  8. Kornfeld, S., Kornfeld, R., Neufeld, E.F., O’Brien, P.J.: The feedback control of sugar nucleotide biosynthesis in liver. Proc. Natl. Acad. Sci. USA 52, 371–379 (1964). doi:10.1073/pnas.52.2.371

    Article  PubMed  CAS  Google Scholar 

  9. Watson, D.R., Jourdian, G.W., Roseman, S.: The sialic acids. 8. Sialic acid 9-phosphate synthetase. J. Biol. Chem. 241, 5627–5636 (1966)

    PubMed  CAS  Google Scholar 

  10. Chen, H., Blume, A., Zimmermann-Kordmann, M., Reutter, W., Hinderlich, S.: Purification and characterization of N-acetylneuraminic acid-9-phosphate synthase from rat liver. Glycobiology 12, 65–71 (2002). doi:10.1093/glycob/12.2.65

    Article  PubMed  CAS  Google Scholar 

  11. Kean, E.L., Roseman, S.: The sialic acids. X. Purification and properties of cytidine 5′-monophosphosialic acid synthetase. J. Biol. Chem. 241, 5643–5650 (1966)

    PubMed  CAS  Google Scholar 

  12. Harduin-Lepers, A., Mollicone, R., Delannoy, P., Oriol, R.: The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach. Glycobiology 15, 805–817 (2005). doi:10.1093/glycob/cwi063

    Article  PubMed  CAS  Google Scholar 

  13. Keppler, O.T., Hinderlich, S., Langner, J., Schwartz-Albiez, R., Reutter, W., Pawlita, M.: UDP-GlcNAc 2-Epimerase: a regulator of cell surface sialylation. Science 284, 1372–1376 (1999). doi:10.1126/science.284.5418.1372

    Article  PubMed  CAS  Google Scholar 

  14. Schwarzkopf, M., Knobeloch, K.-P., Rohde, E., Hinderlich, S., Wiechens, N., Lucka, L., et al.: Sialylation is essential for early development in mice. Proc. Natl. Acad. Sci. U S A 99, 5267–5270 (2002). doi:10.1073/pnas.072066199

    Article  PubMed  CAS  Google Scholar 

  15. Seppala, R., Lehto, V.P., Gahl, W.A.: Mutations in the human UDP-N-acetylglucosamine 2-epimerase gene define the disease sialuria and the allosteric site of the enzyme. Am. J. Hum. Genet. 64, 1563–1569 (1999). doi:10.1086/302411

    Article  PubMed  CAS  Google Scholar 

  16. Eisenberg, I., Grabov-Nardini, G., Hochner, H., Korner, M., Sadeh, M., Bertorini, T., et al.: Mutations spectrum of GNE in hereditary inclusion body myopathy sparing the quadriceps. Hum. Mutat. 21, 99–105 (2002). doi:10.1002/humu.9100

    Article  Google Scholar 

  17. Eisenberg, I., Avidan, N., Potikha, T., Hochner, H., Chen, M., Olender, T., et al.: The UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene is mutated in recessive hereditary inclusion body myopathy. Nat. Genet. 29, 83–87 (2001). doi:10.1038/ng718

    Article  PubMed  CAS  Google Scholar 

  18. Watts, G.D.J., Thorne, M., Kovach, M.J., Pestronk, A., Kimonis, V.E.: Clinical and genetic heterogeneity in chromosome 9p associated hereditary inclusion body myopathy: exclusion of GNE and three other candidate genes. Neuromuscul. Disord. 13, 559–567 (2003). doi:10.1016/S0960-8966(03)00070-1

    Article  PubMed  Google Scholar 

  19. Lucka, L., Krause, M., Danker, K., Reutter, W., Horstkorte, R.: Primary structure and expression analysis of human UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase, the bifunctional enzyme in neuraminic acid biosynthesis. FEBS Lett. 454, 341–344 (1999). doi:10.1016/S0014-5793(99)00837-6

    Article  PubMed  CAS  Google Scholar 

  20. Effertz, K., Hinderlich, S., Reutter, W.: Selective loss of either the epimerase or kinase activity of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase due to site-directed mutagenesis based on sequence alignments. J. Biol. Chem. 274, 28771–28778 (1999). doi:10.1074/jbc.274.40.28771

    Article  PubMed  CAS  Google Scholar 

  21. Reinke, S.O., Hinderlich, S.: Prediction of three different isoforms of the human UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. FEBS Lett. 581, 3327–3331 (2007). doi:10.1016/j.febslet.2007.06.026

    Article  PubMed  CAS  Google Scholar 

  22. Hinderlich, S., Salama, I., Eisenberg, I., Potikha, T., Mantey, L.R., Yarema, K.J., et al.: The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy. FEBS Lett. 566, 105–109 (2004). doi:10.1016/j.febslet.2004.04.013

    Article  PubMed  CAS  Google Scholar 

  23. 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). doi:10.1074/jbc.272.39.24313

    Article  PubMed  CAS  Google Scholar 

  24. Hong, Y., Stanley, P.: Lec3 Chinese hamster ovary mutants lack UDP-N-acetylglucosamine 2-epimerase activity because of mutations in the epimerase domain of the Gne gene. J. Biol. Chem. 278, 3045–3054 (2003)

    Google Scholar 

  25. Blume, A., Ghaderi, D., Liebich, V., Hinderlich, S., Donner, P., Reutter, W., et al.: UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, functionally expressed in and purified from Escherichia coli, yeast, and insect cells. Protein Expr. Purif. 35, 387–396 (2004). doi:10.1016/j.pep.2004.02.013

    Article  PubMed  CAS  Google Scholar 

  26. Blume, A., Weidemann, W., Stelzl, U., Wanker, E.E., Lucka, L., Donner, P., et al.: Domain-specific characteristics of the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Biochem. J. 384, 599–607 (2004). doi:10.1042/BJ20040917

    Article  PubMed  CAS  Google Scholar 

  27. Ghaderi, D., Strauss, H.M., Reinke, S., Cirak, S., Reutter, W., Lucka, L., et al.: Evidence for dynamic interplay of different oligomeric states of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase by biophysical methods. J. Mol. Biol. 369, 746–758 (2007). doi:10.1016/j.jmb.2007.03.037

    Article  PubMed  CAS  Google Scholar 

  28. Wang, Z., Sun, Z., Li, A.V., Yarema, K.J.: Roles for UDP-GlcNAc 2-epimerase/ManNAc 6-kinase outside of sialic acid biosynthesis: modulation of sialyltransferase and BiP expression, GM3 and GD3 biosynthesis, proliferation, and apoptosis, and ERK1/2 phosphorylation. J. Biol. Chem. 281, 27016–27028 (2006). doi:10.1074/jbc.M604903200

    Article  PubMed  CAS  Google Scholar 

  29. Christmann, M., Tomicic, M.T., Roos, W.P., Kaina, B.: Mechanisms of human DNA repair: an update. Toxicology 193, 3–34 (2003). doi:10.1016/S0300-483X(03)00287-7

    Article  PubMed  CAS  Google Scholar 

  30. Bork, K., Reutter, W., Gerardy-Schahn, R., Horstkorte, R.: The intracellular concentration of sialic acid regulates the polysialylation of the neural cell adhesion molecule. FEBS Lett. 579, 5079–5083 (2005). doi:10.1016/j.febslet.2005.08.013

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the German-Israeli Foundation for Science Research & Development, Jerusalem, Israel. We thank Werner Reutter for helpful discussion and Christiane Kilian for excellent technical assistance. CHO Lec3 cells were kindly provided by Pamela Stanley.

Author information

Authors and Affiliations

  1. Institute of Biochemistry and Molecular Biology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Arnimallee 22, 14195, Berlin-Dahlem, Germany

    Stefan O. Reinke & Colin Eidenschink

  2. Department of Life Sciences and Technology, Laboratory of Biochemistry, University of Applied Sciences Berlin, Seestrasse 64, 13347, Berlin, Germany

    Stefan O. Reinke & Stephan Hinderlich

  3. Gradalis, Inc., 2545 Golden Bear Dr, suite 110, Carrollton, TX, 75006, USA

    Chris M. Jay

Authors
  1. Stefan O. Reinke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Colin Eidenschink
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chris M. Jay
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephan Hinderlich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stephan Hinderlich.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Reinke, S.O., Eidenschink, C., Jay, C.M. et al. Biochemical characterization of human and murine isoforms of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Glycoconj J 26, 415–422 (2009). https://doi.org/10.1007/s10719-008-9189-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-008-9189-6

Keywords

Search

Navigation