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Proceedings of the 32 nd European Peptide Symposium
George Kokotos, Violetta Constantinou-Kokotou, John Matsoukas (Editors)
European Peptide Society, 2012
Transduction of peptides, proteins and nucleotides into live
cells by cell penetrating peptides
Andrea-Anneliese Keller 1 , Reinhard Breitling 3 , Peter Hemmerich 2 ,
Franziska Mussbach 1 , Buerk Schaefer 3 , Stefan Lorkowski 1 ,
Siegmund Reissmann 1
1 Friedrich-Schiller-University Jena, Biological and Pharmaceutical Faculty; 2 Leibniz
Institute of Age Research; 3 Jena Bioscience GmbH
E-mail:siegmund.reissmann@uni-jena.de
Introduction
Cell membranes are permeable only for small hydrophobic compounds. While in-vitro
transfection studies can be performed with viral factors, electroporation, magneto-fection or
application of lipid detergents the internalization of cargos for medically relevant purposes
requires more gentle methods such as use of nano-particles or cell-penetrating peptides
(CPPs). Especially the formation and internalization of non-covalent complexes between
CPPs and cargos meets the requirements for application in diagnosis and therapy.
Results and Discussion
We were interested in consolidating knowledge about the very easy and convenient to
handle types of CPPs, which are able to form non-covalent complexes with different
cargos. We studied relationships between amphiphilicity and proteolytic stability of these
CPPs, properties of the cargos and of the cell types on the one hand and transport efficiency
into live cells as well as cytotoxicity on the other site. As cargos we used enzymes,
fluorescently labeled antibodies, bovine serum albumin and nucleoside triphosphates. Cells
differ in their membrane properties regarding lipid, protein and glycan composition,
surface-bound proteases as well as in signal pathways and metabolic activities. Cargos can
also strongly differ in molecular size, surface charge and other chemical properties. Only
certain structural types of CPPs are able to form sufficiently stable non-covalent complexes
with cargos. Thus, for each cell type and cargo the right CPP has to be estimated with
respect to uptake efficiency and cytotoxicity. Cocktails of CPPs provide a more universal
approach for internalization of cargos through compatibility with numerous cell types and
various membrane structures, triggering different mechanisms of transduction and allowing
complexation with structural different cargos.
For our studies we used cells from different sources, having different properties: HeLa-,
COS-7 and NIH 3T3 as adherent cells and Jurkat, NB-4 and Kasumi-1 as suspension cells
[2,3]. Additionally we investigated the behavior of the protozoa Leishmania tarentolae [4].
As CPPs we used the following peptides and proteins: HIV-TAT (YGRKKRRQRR),
histon H2A from calf, penetratin (RGIKWFGNRRM-KWKK), pentapeptide CPPP-2
(KLPVM), MPGα (AcGALFLAFLAAALSLMGLWSQPKKKRKV-NH-CH2-CH2-SH),
MPGβ (Ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-NH-CH2-CH2-SH) and CAD-2
(GLWRALWRLLRSLWRLLWKA-NH-CH2-CH2-SH). The latter three peptides were
particularly developed for formation of non-covalent complexes [1]. All cells have surface-
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ound proteolytic activities and hydrolyze the CPPs. Thus, intact HeLa-, NIH 3T3-cells and
Leishmania degrade penetratin completely within 60 minutes [4]. The used CPPs are
differently hydrophobic and show different proteolytic stabilities. CAD-2 is the most
hydrophobic peptide and is more resistant to proteolytic degradation than other used CPPs
[4]. We also found a cargo-dependent uptake. Thus, the CPPs trigger the uptake of the
high molecular weight enzyme β-galactosidase into HeLa-cells in the following rank order:
Proteoducin > MPGα ≥ MPGβ > CAD-2 >> CPPP-2. For the uptake of the fluorescently
labeled nucleotide ATTO488-dUTP into HeLa-cells we found another order: CAD-2 >
MPGα ≥ MPGβ > penetratin >> CPPP-2 [2,3]. Due to different membrane properties and
the transport mechanisms the uptake efficiency is cell-dependent [4]. CPPs are in many
cases able to transduce even very difficult to transfect cells such as Kasumi-1 cells [3].
Leishmania tarentolae can be transduced with β-galactosidase and with ATTO488-BSA
[4]. CPPs can have special localization sequences. Thus, MPGα, H2A and MPGβ are able
to transport the fluorescent protein ATTO488-BSA into the nucleus and the kinetoplast of
Leishmania. Quantitative uptake efficiencies were estimated by fluorescence
measurements and with calibrated SDS-PAGE. Intracellular amounts depend on the cell
volume and can reach the amole range. Intracellular concentrations come to the low
micromolar range [3,4]. Cytotoxic effects depends on the cell-type and CPP used [2,3,4].
Quantitative Uptake Efficiencies
Amount of added complex
Internalized amount
(0.3 x 10
per 1.6 ml serum-free medium
6 HeLa-cells per well)
amol per cell
Intracellular
concentration in µM
ATTO488-deoxyuridine triphosphate into HeLa-cells
1 µg + JBS-Nucleoducin, charge by charge 1:4 1.1 0.1
ATTO488-bovine serum albumin into HeLa-cells
10 µg + JBS-Proteoducin, molar ratio 1:10 20
25 µg + JBS-Proteoducin, molar ratio 1:10 50 4.3
FITC-antibody (secondary) into HeLa-cells
5 µg + JBS-Proteoducin, molar ratio 1:10 0.4
25 µg + JBS-Proteoducin, molar ratio 1:10 4.3 0.6
ATTO488-bovine serum albumin into Leishmania tarentolae
5 µg + MPGα, molar ratio 1:10 1.7 x 10 -2
0.1
10 µg + MPGα, molar ratio 1:10 3.3 x 10 -2
0.2
CPPs and cocktails of them allow cellular uptake of kinases, phosphatases, deacetylases,
proteases, small GTPases, activity modulators and as well as substrates and inhibitors of
enzymes as tools for signal pathway studies and for therapeutically use. Internalized
antibodies act more specifically than inhibitors of kinases or mono-valent ligands for
protein binding domains and can compete with RNA silencing techniques.
References
[1] Deshayes, S., Morris, M., Heitz, F., Divita, G. Adv. Drug Deliv. Rev. 2008, 60, 537.
[2] Mussbach, F.; Pietrucha, R.; Schaefer, B.; Reissmann, S. Meth. Mol. Biol. 2011, 683, 375.
[3] Mussbach, F.; Franke, M.; Zoch, A.; Schaefer, B.; Reissmann, S. J. Cell. Biol. 2011, 112, 3824.
[4] Keller, A.-A. et al., Pharmaceuticals, 2012, Issue:‖Cell-penetrating Peptides‖, submitted.
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