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  3. A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency

A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency

  • J Biol Chem. 2009 Dec 25;284(52):36099-36108. doi: 10.1074/jbc.M109.036426.
Falk Duchardt 1,  Ivo R Ruttekolk 2,  Wouter P R Verdurmen 2,  Hugues Lortat-Jacob 3,  Jochen Bürck 4,  Hansjörg Hufnagel 1,  Rainer Fischer 1,  Maaike van den Heuvel 5,  Dennis W P M Löwik 5,  Geerten W Vuister 6,  Anne Ulrich 4,  Michel de Waard 7,  Roland Brock 8
Affiliations

Affiliations

  • 1 Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany.
  • 2 Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6525 GA Nijmegen, The Netherlands.
  • 3 Institut de Biologie Structurale, UMR 5075 CEA-CNRS, Université Joseph Fourier, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France.
  • 4 Karlsruhe Institute of Technology, Forschungszentrum Karlsruhe, IBG-2, 76131 Karlsruhe, Germany.
  • 5 Department of Bio-Organic Chemistry, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
  • 6 Department of Protein Biophysics, Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
  • 7 INSERM U836, Université Joseph Fourier, Grenoble Neuroscience Institute, Group 3, 38027 Grenoble Cedex 1, France.
  • 8 Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany; Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, 6525 GA Nijmegen, The Netherlands. Electronic address: R.Brock@ncmls.ru.nl.
PMID: 19858187 DOI: 10.1074/jbc.M109.036426
Abstract

The molecular events that contribute to the cellular uptake of cell-penetrating Peptides (CPP) are still a matter of intense research. Here, we report on the identification and characterization of a 22-amino acid CPP derived from the human milk protein, lactoferrin. The peptide exhibits a conformation-dependent uptake efficiency that is correlated with efficient binding to heparan sulfate and lipid-induced conformational changes. The peptide contains a disulfide bridge formed by terminal cysteine residues. At concentrations exceeding 10 mum, this peptide undergoes the same rapid entry into the cytoplasm that was described previously for the arginine-rich CPPs nona-arginine and Tat. Cytoplasmic entry strictly depends on the presence of the disulfide bridge. To better understand this conformation dependence, NMR spectroscopy was performed for the free peptide, and CD measurements were performed for free and lipid-bound peptide. In solution, the Peptides showed only slight differences in secondary structure, with a predominantly disordered structure both in the presence and absence of the disulfide bridge. In contrast, in complex with large unilamellar vesicles, the conformation of the oxidized and reduced forms of the peptide clearly differed. Moreover, surface plasmon resonance experiments showed that the oxidized form binds to heparan sulfate with a considerably higher affinity than the reduced form. Consistently, membrane binding and cellular uptake of the peptide were reduced when heparan sulfate chains were removed.

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