Polyamines pp 327-338 | Cite as

Heparan Sulfate Proteoglycan-Mediated Polyamine Uptake

  • Johanna Welch
  • Katrin Svensson
  • Paulina Kucharzewska
  • Mattias BeltingEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 720)


The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that efficient polyamine uptake depends on the presence of polyanionic, cell surface-associated heparan sulfate proteoglycans (HSPGs). In this chapter, we provide protocols for studying HSPG-mediated uptake of polyamines in various cell lines, and provide instructions for the use of two different genetic models of HSPG deficiency. We describe the enzymatic reduction of cell surface HSPG through Heparinase III lyase treatment as well as the use of phage display–derived single chain variable fragment (scFv) anti-HS antibodies to block HSPGs at the cell surface. Finally, we provide a protocol for the quantitative verification of loss or reduction of cell surface HSPGs and a detailed description of polyamine uptake measurement.

Key words

HSPG Polyamine uptake Flow cytometry analysis CHO-cells Anti-HS antibodies Heparinase III lyase treatment EXT-1 knock-out cells 



This work was supported by grants from The Swedish Cancer Fund; The Swedish Research Council; The Swedish Society of Medicine; The Physiographic Society, Lund; The Crafoordska, Gunnar Nilsson, and Kamprad Foundations; and the Lund University Hospital (ALF).


  1. 1.
    Gerner EW, Meyskens FL Jr (2004) Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 4:781–792PubMedCrossRefGoogle Scholar
  2. 2.
    Auvinen M, Paasinen A, Andersson LC, Hölttä E (1992) Ornithine decarboxylase activity is critical for cell transformation. Nature 360:355–358PubMedCrossRefGoogle Scholar
  3. 3.
    Clifford A, Morgan D, Yuspa SH, Soler AP, Gilmour S (1995) Role of ornithine decarboxylase in epidermal tumorigenesis. Cancer Res 55:1680–1686PubMedGoogle Scholar
  4. 4.
    Wallace HM, Fraser AV (2004) Inhibitors of polyamine metabolism. Amino Acids 26:353–365PubMedCrossRefGoogle Scholar
  5. 5.
    Igarashi K, Kashiwagi K (1999) Polyamine transport in bacteria and yeast. Biochem J 344:633–642PubMedCrossRefGoogle Scholar
  6. 6.
    Seiler N, Delcros JG, Moulinoux JP (1996) Polyamine transport in mammalian cells. An update. Int J Biochem Cell Biol 28:843–861PubMedCrossRefGoogle Scholar
  7. 7.
    Belting M, Persson S, Fransson LA (1999) Proteoglycan involvement in polyamine uptake. Biochem J 338:317–323PubMedCrossRefGoogle Scholar
  8. 8.
    Belting M, Borsig L, Fuster MM, Brown JR, Persson L, Fransson LA, Esko JD (2002) Tumor attenuation by combined heparan sulfate and polyamine depletion. Proc Natl Acad Sci U S A 99:371–376PubMedCrossRefGoogle Scholar
  9. 9.
    Belting M, Mani K, Jönsson M, Cheng F, Sandgren S, Jonsson S, Ding K, Delcros JG, Fransson LA (2003) Glypican-1 is a vehicle for polyamine uptake in mammalian cells: a pivotal role for nitrosothiol-derived nitric oxide. J Biol Chem 278:47181–47189PubMedCrossRefGoogle Scholar
  10. 10.
    Welch JE, Bengtson P, Svensson K, Wittrup A, Jenniskens GJ, Ten Dam GB, van Kuppevelt TH, Belting M (2008) Single chain fragment anti-heparan sulfate antibody targets the polyamine transport system and attenuates polyamine-dependent cell proliferation. Int J Oncol 32:749–756PubMedGoogle Scholar
  11. 11.
    Whitelock JM, Iozzo RV (2005) Heparan sulfate: a complex polymer charged with biological activity. Chem Rev 105:2745–2764PubMedCrossRefGoogle Scholar
  12. 12.
    Sandgren S, Cheng F, Belting M (2002) Nuclear targeting of macromolecular polyanions by an HIV-TAT derived peptide. Role for cell-surface proteoglycans. J Biol Chem 277:38877–38883PubMedCrossRefGoogle Scholar
  13. 13.
    Wittrup A, Sandgren S, Lilja J, Bratt C, Gustavsson N, Mörgelin M, Belting M (2007) Identification of proteins released by mammalian cells that mediate DNA internalization through proteoglycan-dependent macropinocytosis. J Biol Chem 282:27897–27904PubMedCrossRefGoogle Scholar
  14. 14.
    Lidholt K, Weinke JL, Kiser CS, Lugemwa FN, Bame KJ, Cheifetz S, Massagu’e J, Lindahl U, Esko JD (1992) A single mutation affects both N-acetylglucosaminyltransferase and glucuronsyltransferase activities in a Chinese hamster ovary cell mutant defective in heparan sulfate biosynthesis. Proc Natl Acad Sci U S A 89:2267–2271PubMedCrossRefGoogle Scholar
  15. 15.
    Inatani M, Irie F, Plump AS, Tessie-Lavigne M, Yamaguchi Y (2003) Mammalian brain morphogenesis and midline axon guidance require heparan sulfate. Science 302:1044–1046PubMedCrossRefGoogle Scholar
  16. 16.
    Dennissens MA, Jenniskens GJ, Pieffers M, Versteeg EM, Petitou M, Veerkamp JH, van Kuppevelt TH (2002) Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies. J Biol Chem 277:10982–10986CrossRefGoogle Scholar
  17. 17.
    Fritz TA, Esko JD (2001) Proteoglycans protocols. Humana, Totowa, NJGoogle Scholar
  18. 18.
    Humphries DE, Silbert JE (1988) Chlorate: a reversible inhibitor of proteoglycan sulfation. Biochem Biophys Res Commun 154:365–371PubMedCrossRefGoogle Scholar
  19. 19.
    Fransson LA (1985) Mammalian glycosaminoglycans. Academic, New YorkGoogle Scholar
  20. 20.
    Bai X, Crawford B, Esko JD (2001) Proteoglycan protocols. Humana, Totowa, NJGoogle Scholar
  21. 21.
    Forsberg E, Kjellén L (2001) Heparan sulfate: lessons from knockout mice. J Clin Invest 108:175–180PubMedGoogle Scholar
  22. 22.
    Whitelock JM, Iozzo RV (2002) Isolation and purification of proteoglycans. Methods Cell Biol 69:53–67PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Johanna Welch
    • 1
  • Katrin Svensson
    • 1
  • Paulina Kucharzewska
    • 1
  • Mattias Belting
    • 1
    Email author
  1. 1.Department of Clinical Sciences, Section of OncologyLund University and Lund University HospitalLundSweden

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