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High-Specific-Activity 35S-Labeled Heparan Sulfate Prepared from Cultured Cells

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Part of the Methods in Molecular Biology™ book series (MIMB,volume 171)

Abstract

Cultured cells are a facile reagent for elucidating the molecular mechanisms that regulate the biosynthesis of heparan sulfate (HS) (13). However, a typical confluent flask (∼20 million cells) produces only a small amount of HS (1–2 µg), which is at or below the detection limit of many nonradioisotopic techniques. Fortunately, this limitation can be circumvented by the metabolic labeling of cells with Na2 35SO4. Sulfate from the culture medium is transported into the cytoplasm, where it is incorporated into the biosynthetic sulfate donor adenosine 3′-phosphate 5′-phosphosulfate (PAPS), which is transported into the Golgi apparatus (4). Specific biosynthetic enzymes transfer a sulfonyl group from PAPS onto maturing glycosaminoglycan chains.

Keywords

  • Heparan Sulfate
  • Equilibration Buffer
  • Radioactive Contamination
  • Metabolic Label
  • Label Medium

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Bame, K. J. and Esko, J. D. (1989) Undersulfated heparan sulfate in a chinese hamster ovary cell mutant defective in heparan sulfate N-sulfotransferase. J. Biol. Chem. 264, 8059–8065.

    PubMed  CAS  Google Scholar 

  2. Bame, K. J., Lidholt, K., Lindahl, U., and Esko, J. D. (1991) Biosynthesis of heparan sulfate. Coordination of polymer-modification reactions in a chinese hamster ovary cell mutant defective in N-sulfotransferase. J. Biol. Chem. 266, 10,287–10,293.

    PubMed  CAS  Google Scholar 

  3. Shworak, N. W., Shirakawa, M., Colliec-Jouault, S., Liu, J., Mulligan, R. C., Birinyi, L. K., and Rosenberg, R. D. (1994) Pathway-specific regulation of the synthesis of anticoagulantly active heparan sulfate. J. Biol. Chem. 269, 24,941–24,952.

    PubMed  CAS  Google Scholar 

  4. Ozeran, J. D., Westley, J., and Schwartz, N. B. (1996) Kinetics of PAPS translocase: evidence for an antiport mechanism. Biochemistry 35, 3685–3694.

    PubMed  CrossRef  CAS  Google Scholar 

  5. Klaassen, C. D. and Boles, J. W. (1997) Sulfation and sulfotransferases 5: the importance of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) in the regulation of sulfation. Faseb J. s11, 404–418.

    Google Scholar 

  6. Sjoberg, I. and Malmstrom, A. (1982) Biosynthesis of dermatan sulphate in cultured fibroblasts. Characterization of newly synthesized glycans from cells and microsomes. Eur. J. Biochem. 128, 29–34

    PubMed  CrossRef  CAS  Google Scholar 

  7. Kimura, J. H., Caputo, C. B., and Hascall, V. C. (1981) The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma. J. Biol. Chem. 256, 4368–4376

    PubMed  CAS  Google Scholar 

  8. Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) Cell-free synthesis of anticoagulant heparan sulfate reveals a limiting activity which modifies a nonlimiting precursor pool. J. Biol. Chem. 271, 27,063–27,071.

    PubMed  CrossRef  CAS  Google Scholar 

  9. Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) Purification of heparan sulfate D-glucosaminyl 3-O-sulfotransferase. J. Biol. Chem. 271, 27,072–27,082.

    PubMed  CrossRef  CAS  Google Scholar 

  10. Liu, J., Shworak, N. W., Sinay, P., Schwartz, J. J., Zhang, L., Fritze, L. M., and Rosenberg, R. D. (1999) Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities. J. Biol. Chem. 274, 5185–5192.

    PubMed  CrossRef  CAS  Google Scholar 

  11. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, MA.

    Google Scholar 

  12. Humphries, D. E., Silbert, C. K., and Silbert, J. E. (1986) Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium. J. Biol. Chem. 261, 9122–9127.

    PubMed  CAS  Google Scholar 

  13. Keller, J. M. and Keller, K. M. (1987) Amino acid sulfur as a source of sulfate for sulfated proteoglycans produced by Swiss mouse 3T3 cells. Biochim. Biophys. Acta 926, 139–144.

    PubMed  CAS  Google Scholar 

  14. Esko, J. D., Elgavish, A., Prasthofer, T., Taylor, W. H., and Weinke, J. L. (1986) Sulfate transport-deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine. J. Biol. Chem. 261, 15,725–15,733.

    PubMed  CAS  Google Scholar 

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© 2001 Humana Press Inc., Totowa, NJ

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Shworak, N.W. (2001). High-Specific-Activity 35S-Labeled Heparan Sulfate Prepared from Cultured Cells. In: Iozzo, R.V. (eds) Proteoglycan Protocols. Methods in Molecular Biology™, vol 171. Humana Press. https://doi.org/10.1385/1-59259-209-0:079

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  • DOI: https://doi.org/10.1385/1-59259-209-0:079

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-759-5

  • Online ISBN: 978-1-59259-209-8

  • eBook Packages: Springer Protocols