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Studies of Proteoglycan Involvement in CPP-Mediated Delivery

  • Anders Wittrup
  • Si-He Zhang
  • Mattias BeltingEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 683)

Abstract

Cell-penetrating peptides (CPPs) are widely used to deliver macromolecular cargoes to intracellular sites of action. Many CPPs have been demonstrated to rely on cell surface heparan sulfate proteoglycans (HSPGs) for efficient cellular entry and delivery. In this chapter, we describe methods for the study of PG involvement in CPP uptake. We provide descriptions of how to determine whether uptake of a CPP of interest is dependent on PGs. We also provide detailed protocols for the purification of PGs by anion-exchange chromatography as well as the characterization of the HSPG core protein composition of a cell line of interest. Finally, we present methods for modulating the expression level of specific HSPG core proteins as a means to determine the core protein specificity in the uptake of a particular CPP.

Key words

Cell-penetrating peptides CHO cell mutants Heparan sulfate Glypican Proteoglycan siRNA Syndecan 

Notes

Acknowledgments

This book chapter is based on work supported by grants from the Swedish Cancer Fund; the Swedish Research Council; the Swedish Foundation for Strategic Research; the Swedish Society of Medicine; the Physiographic Society, Lund; the Crafoordska, Gunnar Nilsson, Lundbergs, and Kamprad Foundations; the Lund University Hospital donation funds; and the governmental funding of clinical research within the National Health Services.

References

  1. 1.
    Poon, G. M., and Gariepy, J. (2007) Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells, Biochem Soc Trans 35, 788–793.CrossRefPubMedGoogle Scholar
  2. 2.
    Mislick, K. A., and Baldeschwieler, J. D. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer, Proc Natl Acad Sci U S A 93, 12349–12354.CrossRefPubMedGoogle Scholar
  3. 3.
    Console, S., Marty, C., Garcia-Echeverria, C., Schwendener, R., and Ballmer-Hofer, K. (2003) Antennapedia and HIV transactivator of transcription (TAT) “protein transduction domains” promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans, J Biol Chem 278, 35109–35114.CrossRefPubMedGoogle Scholar
  4. 4.
    Tyagi, M., Rusnati, M., Presta, M., and Giacca, M. (2001) Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans, J Biol Chem 276, 3254–3261.CrossRefPubMedGoogle Scholar
  5. 5.
    Sandgren, S., Cheng, F., and Belting, M. (2002) Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans, J Biol Chem 277, 38877–38883.CrossRefPubMedGoogle Scholar
  6. 6.
    Sandgren, S., Wittrup, A., Cheng, F., Jonsson, M., Eklund, E., Busch, S., and Belting, M. (2004) The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis, J Biol Chem 279, 17951–17956.CrossRefPubMedGoogle Scholar
  7. 7.
    Tkachenko, E., Lutgens, E., Stan, R. V., and Simons, M. (2004) Fibroblast growth factor 2 endocytosis in endothelial cells proceed via syndecan-4-dependent activation of Rac1 and a Cdc42-dependent macropinocytic pathway, J Cell Sci 117, 3189–3199.CrossRefPubMedGoogle Scholar
  8. 8.
    Yanagishita, M., and Hascall, V. C. (1984) Metabolism of proteoglycans in rat ovarian granulosa cell culture. Multiple intracellular degradative pathways and the effect of chloroquine, J Biol Chem 259, 10270–10283.PubMedGoogle Scholar
  9. 9.
    Argyris, E. G., Kulkosky, J., Meyer, M. E., Xu, Y., Mukhtar, M., Pomerantz, R. J., and Williams, K. J. (2004) The perlecan heparan sulfate proteoglycan mediates cellular uptake of HIV-1 Tat through a pathway responsible for biological activity, Virology 330, 481–486.CrossRefPubMedGoogle Scholar
  10. 10.
    Nakase, I., Tadokoro, A., Kawabata, N., Takeuchi, T., Katoh, H., Hiramoto, K., Negishi, M., Nomizu, M., Sugiura, Y., and Futaki, S. (2007) Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis, Biochemistry 46, 492–501.CrossRefPubMedGoogle Scholar
  11. 11.
    Wittrup, A., Zhang, S. H., Ten Dam, G. B., van Kuppevelt, T. H., Bengtson, P., Johansson, M., Welch, J., Morgelin, M., and Belting, M. (2009) ScFv antibody-induced translocation of cell-surface heparan sulfate proteoglycan to endocytic vesicles: Evidence for heparan sulfate epitope specificity and role of both syndecan and glypican, J Biol Chem 284, 32959–32967.CrossRefPubMedGoogle Scholar
  12. 12.
    Landgraf, P., Wahle, P., Pape, H. C., Gundelfinger, E. D., and Kreutz, M. R. (2008) The survival-promoting peptide Y-P30 enhances binding of pleiotrophin to syndecan-2 and -3 and supports its neuritogenic activity, J Biol Chem 283, 25036–25045.CrossRefPubMedGoogle Scholar
  13. 13.
    Zhang, L., Lawrence, R., Frazier, B. A., and Esko, J. D. (2006) CHO glycosylation mutants: proteoglycans, Methods Enzymol 416, 205–221.CrossRefPubMedGoogle Scholar
  14. 14.
    Welch, J. E., Bengtson, P., Svensson, K., Wittrup, A., Jenniskens, G. J., Ten Dam, G. B., Van Kuppevelt, T. H., and 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, 749–756.PubMedGoogle Scholar
  15. 15.
    Dennissen, M. A., Jenniskens, G. J., Pieffers, M., Versteeg, E. M., Petitou, M., Veerkamp, J. H., and van Kuppevelt, T. H. (2002) Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies, J Biol Chem 277, 10982–10986.CrossRefPubMedGoogle Scholar
  16. 16.
    David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J., and Van den Berghe, H. (1992) Developmental changes in heparan sulfate expression: in situ detection with mAbs, J Cell Biol 119, 961–975.CrossRefPubMedGoogle Scholar
  17. 17.
    Belting, M., and Petersson, P. (1999) Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer of glycosaminoglycans to the nucleus, J Biol Chem 274, 19375–19382.CrossRefPubMedGoogle Scholar
  18. 18.
    Belting, M., and Petersson, P. (1999) Protective role for proteoglycans against cationic lipid cytotoxicity allowing optimal transfection efficiency in vitro, Biochem J 342(Pt 2), 281–286.CrossRefPubMedGoogle Scholar
  19. 19.
    Wittrup, A., and Belting, M. (2009) Characterizing peptide-mediated DNA internalization in human cancer cells, Methods Mol Biol 480, 101–112.PubMedGoogle Scholar
  20. 20.
    Iozzo, R. V. (2001) Proteoglycan Protocols, Humana Press, Totowa, New Jersey.CrossRefGoogle Scholar
  21. 21.
    Humphries, D. E., and Silbert, J. E. (1988) Chlorate: a reversible inhibitor of proteoglycan sulfation, Biochem Biophys Res Commun 154, 365–371.CrossRefPubMedGoogle Scholar
  22. 22.
    Inatani, M., Irie, F., Plump, A. S., Tessier-Lavigne, M., and Yamaguchi, Y. (2003) Mammalian brain morphogenesis and midline axon guidance require heparan sulfate, Science 302, 1044–1046.CrossRefPubMedGoogle Scholar
  23. 23.
    Bullock, S. L., Fletcher, J. M., Beddington, R. S., and Wilson, V. A. (1998) Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase, Genes Dev 12, 1894–1906.CrossRefPubMedGoogle Scholar
  24. 24.
    Wittrup, A., Sandgren, S., Lilja, J., Bratt, C., Gustavsson, N., Morgelin, M., and Belting, M. (2007) Identification of proteins released by mammalian cells that mediate DNA internalization through proteoglycan-dependent macropinocytosis, J Biol Chem 282,27897–27904.CrossRefPubMedGoogle Scholar
  25. 25.
    van den Born, J., Salmivirta, K., Henttinen, T., Ostman, N., Ishimaru, T., Miyaura, S., Yoshida, K.,and Salmivirta, M. (2005) Novel heparan sulfate structures revealed by monoclonal antibodies, J Biol Chem 280, 20516–20523.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Clinical Sciences, Section of OncologyLund UniversityLundSweden

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