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Heparan Sulfate Structure: Methods to Study N-Sulfation and NDST Action

  • Anders Dagälv
  • Anders Lundequist
  • Beata Filipek-Górniok
  • Tabea Dierker
  • Inger Eriksson
  • Lena KjellénEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1229)

Abstract

Heparan sulfate proteoglycans are important modulators of cellular processes where the negatively charged polysaccharide chains interact with target proteins. The sulfation pattern of the heparan sulfate chains will determine the proteins that will bind and the affinity of the interactions. The N-deacetylase/N-sulfotransferase (NDST) enzymes are of key importance during heparan sulfate biosynthesis when the sulfation pattern is determined. In this chapter, metabolic labeling of heparan sulfate with [35S]sulfate or [3H]glucosamine in cell cultures is described, in addition to characterization of polysaccharide chain length and degree of N-sulfation. Methods to measure NDST enzyme activity are also presented.

Key words

Heparan sulfate biosynthesis N-deacetylase/N-sulfotransferase N-sulfation ELISA JM-403 

References

  1. 1.
    Bishop JR, Schuksz M, Esko JD (2007) Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446:1030–1037PubMedCrossRefGoogle Scholar
  2. 2.
    Kreuger J, Kjellen L (2012) Heparan sulfate biosynthesis: regulation and variability. J Histochem Cytochem 60:898–907PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Li F, Shi W, Capurro M, Filmus J (2011) Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling. J Cell Biol 192:691–704PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Ledin J, Staatz W, Li JP, Gotte M, Selleck S, Kjellen L et al (2004) Heparan sulfate structure in mice with genetically modified heparan sulfate production. J Biol Chem 279:42732–42741PubMedCrossRefGoogle Scholar
  5. 5.
    Carlsson P, Presto J, Spillmann D, Lindahl U, Kjellen L (2008) Heparin/heparan sulfate biosynthesis: processive formation of N-sulfated domains. J Biol Chem 283:20008–20014PubMedCrossRefGoogle Scholar
  6. 6.
    Kreuger J, Spillmann D, Li JP, Lindahl U (2006) Interactions between heparan sulfate and proteins: the concept of specificity. J Cell Biol 174:323–327PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Humphries DE, Silbert CK, Silbert JE (1986) Glycosaminoglycan production by bovine aortic endothelial cells cultured in sulfate-depleted medium. J Biol Chem 261:9122–9127PubMedGoogle Scholar
  8. 8.
    Pikas DS, Eriksson I, Kjellen L (2000) Overexpression of different isoforms of glucosaminyl N-deacetylase/N-sulfotransferase results in distinct heparan sulfate N-sulfation patterns. Biochemistry 39:4552–4558PubMedCrossRefGoogle Scholar
  9. 9.
    Ledin J, Ringvall M, Thuveson M, Eriksson I, Wilen M, Kusche-Gullberg M et al (2006) Enzymatically active N-deacetylase/N-sulfotrans-ferase-2 is present in liver but does not contribute to heparan sulfate N-sulfation. J Biol Chem 281:35727–35734PubMedCrossRefGoogle Scholar
  10. 10.
    van den Born J, Pikas DS, Pisa BJ, Eriksson I, Kjellen L, Berden JH (2003) Antibody-based assay for N-deacetylase activity of heparan sulfate/heparin N-deacetylase/N-sulfotransferase (NDST): novel characteristics of NDST-1 and -2. Glycobiology 13:1–10PubMedCrossRefGoogle Scholar
  11. 11.
    Shively JE, Conrad HE (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry 15:3932–3942PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Anders Dagälv
    • 1
  • Anders Lundequist
    • 1
  • Beata Filipek-Górniok
    • 1
  • Tabea Dierker
    • 1
  • Inger Eriksson
    • 1
  • Lena Kjellén
    • 1
    Email author
  1. 1.Department of Medical Biochemistry and Microbiology, Science for Life LaboratoryUppsala UniversityUppsalaSweden

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