Membrane surface associated helices promote lipid interactions and cellular uptake of human calcitonin-derived cell penetrating peptides
Michael E Herbig 1, Kathrin Weller 1, Ulrike Krauss 2, Annette G Beck-Sickinger 2, Hans-Peter Merkle 1 and Oliver Zerbe 3*
1 Institute of Pharmaceutical Sciences of the ETH Zurich
2 Institute of Biochemistry, University of Leipzig
3 Institute of Organic Chemistry of the University of Zurich
* To whom correspondence should be addressed. E-mail: oliver.zerbe{at}oci.unizh.ch.
Submitted on June 14, 2005
Revised on July 20, 2005
Accepted on 8 September 2005
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Abstract |
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hCT(9-32) is a human calcitonin (hCT) derived cell penetrating peptide (CPP) that has been shown to translocate the plasma membrane of mammalian cells. It has been suggested as a cellular carrier for drugs, green fluorescent protein, and plasmid DNA. Because of its temperature dependent cellular translocation resulting in punctuated cytoplasmatic distribution, its uptake is likely to follow an endocytic pathway. To gain insight into the molecular orientation of hCT(9-32) when interacting with lipid models, and to learn more about its mode of action, various biophysical techniques from liposome partitioning to high-resolution NMR spectroscopy were utilized. Moreover, to establish the role of individual residues for the topology of its association with the lipid membrane, two mutants of hCT(9-32), i.e. W30-hCT(9-32) and A23-hCT(9-32), were also investigated. Whereas unstructured in aqueous solution, hCT(9-32) adopted two short helical stretches when bound to dodecylphosphocholine (DPC) micelles, extending from Thr10 to Asn17 and from Gln24 to Val29. A23-hCT(9-32), in which the helix-breaking Pro23 was replaced by Ala, displayed a continuous a-helix extending from residue 12 to 26. Probing with the spin-label 5-doxylstearate revealed that association with DPC micelles was such that the helix engaged in parallel orientation to the micelle surface. Moreover, the Gly to Trp exchange in W30-hCT(9-32) resulted in a more stable anchoring of the C-terminal segment close to the interface, as reflected by a twofold increase in the partition coefficient in liposomes. Interestingly, tighter binding to model membranes was associated with an increase in the in vitro uptake in HeLa cells. Liposome leakage studies excluded pore formation, and the punctuated fluorescence pattern of internalized peptide indicated vesicular localization and, in conclusion, strongly suggested an endocytic pathway of translocation.
Key Words:
cell penetrating peptides, cellular uptake, liposome buffer partitioning, membrane models, nuclear magnetic resonance, peptide-lipid interactions
