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CMP-NeuAc:(NeuAcα2 → 8) n (colominic acid) sialyltransferase activity in rat brain and in tumour cells that express polysialic acid on neural cell adhesion molecules

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Abstract

A method for the assay of CMP-NeuAc:(NeuAcα2 → 8) n (colominic acid) sialyltransferase activity was developed. Using a 1-day-old rat brain membrane fraction as an enzyme preparation optimal activity was obtained at pH 6.5, 0.3% Triton X-100, and 5mm MnCl2. However, no absolute cation requirement was found as EDTA only partially inhibited the activity. Within a concentration range of 0.3–3 mg colominic acid (which consists of a mixture of oligomers of α2 → 8-linked sialic acid) per 50 µl aV of 0.61 nmol per mg protein h−1 was estimated while a half-maximal reaction velocity was obtained at a concentration of 1.75 mg per 50 µl. High performance anion-exchange chromatography of the radioactive products formed in the reaction showed that sialic acid oligomers ranging in size from a degree of polymerization (DP) of 2 up to at least DP 9 could serve as acceptor substrates. Comparison of the acceptor properties of DP 3 and DP 6 showed that the larger oligomer was acted upon with a 10-fold higher efficiency. Periodate oxidation of the products followed by reduction and hydrolysis yielded the C7 analogue of NeuAc as the only radioactive product, indicating that under the conditions of the assay only a single sialic acid residue was introduced into the acceptor molecules. Using the assay it appeared that in rat brain the activity of this sialyltransferase decreased six-fold during postnatal development to the adult stage. The assay method was also applied to lysates of several neuroblastoma and small cell lung tumour cell lines, which differ in the expression of polysialic acid as well as of the neural cell adhesion molecule NCAM, a major carrier of this polymer. Activity of the sialyltransferase appeared to be correlated with the expression of polysialic acid present on NCAM. These results indicate that this sialyltransferase might function in the process of poly-sialylation.

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Abbreviations

DP:

degree of polymerization

HPLC:

high-performance liquid chromatography

NeuAc:

N-acetylneuraminic acid

NCAM:

neural cell adhesion molecule

PSA:

polysialic acid

Sia:

sialic acid

References

  1. Finne J (1982)J Biol Chem 257: 11966–70.

    Google Scholar 

  2. Hoffman S, Sorkin BC, White PC, Brackenbury R, Mailhammer R, Rutishauser U, Cunningham BA, Edelman GM (1982)J Biol Chem 257: 7720–29.

    Google Scholar 

  3. Finne J, Finne U, Deagostini-Bazin H, Goridis C (1983)Biochem Biophys Res Commun 112: 482–87.

    Google Scholar 

  4. Edelman GM (1985)Annu Rev Biochem 54: 135–69.

    Google Scholar 

  5. Cunningham BA, Hemperly JJ, Murray BA, Prediger EA, Brackenbury R, Edelman GM (1987)Science 236: 799–806.

    Google Scholar 

  6. Rutishauser U, Acheson A, Hall AK, Mann DM, Sunshine J (1988)Science 240: 53–57.

    Google Scholar 

  7. Rothbard JB, Brackenbury R, Cunningham BA, Edelman GM (1982)J Biol Chem 257: 11064–69.

    Google Scholar 

  8. Hoffman S, Edelman GM (1983)Proc Natl Acad Sci USA 80: 5762–66.

    Google Scholar 

  9. Roth J, Taatjes DJ, Bitter-Suermann D, Finne J (1987)Proc Natl Acad Sci USA 84: 1969–73.

    Google Scholar 

  10. Rieger F, Grumet M, Edelman GM (1985)J Cell Biol 101: 285–93.

    Google Scholar 

  11. Figarella-Branger DF, Durbec PL, Rougon GN (1990)Cancer Res 50: 6364–70.

    Google Scholar 

  12. Roth J, Zuber C, Wagner P, Taatjes DJ, Weisgerber C, Heitz PU, Goridis C, Bitter-Suermann D (1988)Proc Natl Acad Sci USA 85: 2999–3003.

    Google Scholar 

  13. Livingston BD, Jacobs JL, Glick MC, Troy FA (1988)J Biol Chem 263: 9443–8.

    Google Scholar 

  14. Moolenaar CEC, Muller EJ, Schol DJ, Figdor C, Bock E, Bitter-Suermann D, Michalides RJAM (1990)Cancer Res 50: 1102–6.

    Google Scholar 

  15. Kibbelaar RE, Moolenaar CEC, Michalides RJAM, Bitter-Suermann D, Addis BJ, Mooi WJ (1989)J Pathol 159: 23–28.

    Google Scholar 

  16. Komminoth P, Roth J, Lackie PM, Bitter-Suermann D, Heitz PU (1991)Am J Pathol 139: 297–304.

    Google Scholar 

  17. Scheidegger EP, Lackie PM, Papay J, Roth J (1994)Lab Invest 70: 95–106.

    Google Scholar 

  18. Troy FA (1992)Glycobiology 2: 5–23.

    Google Scholar 

  19. McCoy RD, Vimr ER, Troy FA (1985)J Biol Chem 260: 12695–99.

    Google Scholar 

  20. Breen KC, Kelly PC, Regan CM (1987)J Neurochem 48: 1486–93.

    Google Scholar 

  21. Halberstadt JB, Flowers H, Glick MC (1993)Anal Biochem 209: 136–42.

    Google Scholar 

  22. Ito F, Inoue S, Inoue Y, Troy FA (1991)Glycoconjugate J 8: 153.

    Google Scholar 

  23. Kitazume S, Kitajama K, Inoue S, Inoue Y, Troy FA (1994)J Biol Chem 269: 10330–40.

    Google Scholar 

  24. Kundig FD, Aminoff D, Roseman S (1971)J Biol Chem 246: 2543–50.

    Google Scholar 

  25. Van den Eijnden DH, Van Dijk W (1972)Hoppe Seyler's Z Physiol Chem 353: 1817–20.

    Google Scholar 

  26. Frosch M, Goergen I, Boulnois GJ, Timmis KN, Bitter-Suermann D (1985)Proc Natl Acad Sci USA 82: 1194–98.

    Google Scholar 

  27. Schlesinger HR, Gerson JM, Moorhead PS, Maguire H, Hummeler K (1976)Cancer Res 36: 3094–100.

    Google Scholar 

  28. Hallenbeck PC, Yu F, Troy FA (1987)Anal Biochem 161: 181–86.

    Google Scholar 

  29. Manzi AE, Higa HH, Diaz S, Varki A (1994)J Biol Chem 269: 23617–24.

    Google Scholar 

  30. Rohr TE, Troy FA (1980)J Biol Chem 255: 2332–42.

    Google Scholar 

  31. Bergh MLE, Koppen P, Van den Eijnden DH (1981)Carbohydr Res 94: 225–29.

    Google Scholar 

  32. Easton EW, Schiphorst WECM, Van Drunen E, Van der Schoot CE, Van den Eijnden DH (1993)Blood 81: 2978–86.

    Google Scholar 

  33. Haverkamp J, Schauer R, Wember M, Kamerling JP, Vliegenthart JFG (1975)Hoppe-Seylers Z Physiol Chem 356: 1575–83.

    Google Scholar 

  34. Livingston BD, Paulson JC (1993)J Biol Chem 268: 11504–7.

    Google Scholar 

  35. Sasaki K, Kurata K, Kojima N, Kurosawa N, Ohta S, Hanai N, Tsuji S, Nishi T (1994)J Biol Chem 269: 15950–56.

    Google Scholar 

  36. Nara K, Watanabe Y, Maruyama K, Kasahara K, Nagai Y, Sanai Y (1994)Proc Natl Acad Sci USA 91: 7952–56.

    Google Scholar 

  37. Haraguchi M, Yamashiro S, Yamamoto A, Furukawa K, Takamiya K, Lloyd KO, Shiku H (1994)Proc Natl Acad Sci USA 91: 10455–59.

    Google Scholar 

  38. Eckhardt M, Mühlenhoff M, Bethe A, Koopman J, Frosch M, Gerardy-Schahn R (1995)Nature 373: 715–18.

    Google Scholar 

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Authors and Affiliations

  1. Department of Medical Chemistry, Vrije Universiteit, Van der Boechorststraat 7, NL-1081 BT, Amsterdam, The Netherlands

    Ellen W. Easton, Wietske E. C. M. Schiphorst, Carolien A. M. Koeleman & Dirk H. van den Eijnden

  2. Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, NL-1066 CX, Amsterdam, The Netherlands

    Rob J. A. M. Michalides

Authors
  1. Ellen W. Easton
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  2. Wietske E. C. M. Schiphorst
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  3. Carolien A. M. Koeleman
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  4. Rob J. A. M. Michalides
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  5. Dirk H. van den Eijnden
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Easton, E.W., Schiphorst, W.E.C.M., Koeleman, C.A.M. et al. CMP-NeuAc:(NeuAcα2 → 8) n (colominic acid) sialyltransferase activity in rat brain and in tumour cells that express polysialic acid on neural cell adhesion molecules. Glycoconjugate J 12, 829–837 (1995). https://doi.org/10.1007/BF00731245

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  • DOI: https://doi.org/10.1007/BF00731245

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