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

Falk Duchardt (1) , Ivo R. Ruttekolk (1) , 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 (1, 2)

1 Interfaculty Institute for Cell Biology
2 Department of Biochemistry
3 IBS - UMR 5075 - Institut de biologie structurale
4 KIT - Karlsruhe Institute of Technology
5 Bio-Organic Chemistry
6 Protein Biophysics
7 GIN - Grenoble Institut des Neurosciences

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.

Mots clés: Animals Cytoplasm/metabolism Disulfides/metabolism Dose-Response Relationship; Drug Hela Cells Heparitin Sulfate/metabolism Humans Lactoferrin/chemistry/*metabolism/*pharmacology Membrane Lipids/metabolism Nuclear Magnetic Resonance; Biomolecular Oxidation-Reduction Peptides/chemistry/*metabolism/*pharmacology Protein Structure; Secondary Rats Structure-Activity Relationship

Domaines: Biologie cellulaire; Neurosciences [q-bio.NC]

Falk Duchardt, Ivo R. Ruttekolk, Wouter P. R. Verdurmen, Hugues Lortat-Jacob, Jochen Bürck, et al.. A cell-penetrating peptide derived from human lactoferrin with conformation-dependent uptake efficiency.. Journal of Biological Chemistry, 2009, 284 (52), pp.36099-108. ⟨10.1074/jbc.M109.036426⟩. ⟨inserm-00515380⟩