Skip to main content
SpringerLink
Log in

Cellular Internalization of Human Calcitonin Derived Peptides in MDCK Monolayers: A Comparative Study with Tat(47-57) and Penetratin(43-58)

  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose. The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model.

Methods. Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58).

Results. Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed.

Conclusions. Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

REFERENCES

  1. A. M. Gewirtz, D. L. Sokol, and M. Z. Ratajczak. Nucleic acid therapeutics: state of the art and future prospects. Blood 92:712-736 (1998).

    Google Scholar 

  2. R. L. Juliano, S. Alahari, H. Yoo, R. Kole, and M. Cho. Antisense pharmacodynamics: critical issues in the transport and delivery of antisense oligonucleotides. Pharm. Res. 16:494-502 (1999).

    Google Scholar 

  3. E. Vives, P. Brodin, and B. Lebleu. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272:16010-16017 (1997).

    Google Scholar 

  4. S. R. Schwarze, A. Ho, A. Vocero-Akbani, and S. F. Dowdy. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285:1569-1572 (1999).

    Google Scholar 

  5. V. Polyakov, V. Sharma, J. L. Dahlheimer, C. M. Pica, G. D. Luker, and D. Piwnica-Worms. Novel Tat-peptide chelates for direct transduction of technetium-99m and rhenium into human cells for imaging and radiotherapy. Bioconjug. Chem. 11:762-771 (2000).

    Google Scholar 

  6. D. Derossi, S. Calvet, A. Trembleau, A. Brunissen, G. Chassaing, and A. Prochiantz. Cell internalization of the third helix of the antennapedia homeodomain is receptor-independent. J. Biol. Chem. 271:18188-18193 (1996).

    Google Scholar 

  7. G. Elliott and P. O'Hare. Intercellular trafficking of VP22-GFP fusion proteins. Gene Ther. 6:149-151 (1999).

    Google Scholar 

  8. N. Brewis, A. Phelan, J. Webb, J. Drew, G. Elliott, and P. O'Hare. Evaluation of VP22 spread in tissue culture. J. Virol. 74:1051-1056 (2000).

    Google Scholar 

  9. P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L. Steinman, and J. B. Rothbard. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc. Natl. Acad. Sci. USA 97:13003-13008 (2000).

    Google Scholar 

  10. S. Futaki, T. Suzuki, W. Ohashi, T. Yagami, S. Tanaka, K. Ueda, and Y. Sugiura. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276:5836-5840 (2001).

    Google Scholar 

  11. M. Pooga, U. Soomets, M. Hallbrink, A. Valkna, K. Saar, K. Rezaei, U. Kahl, J. X. Hao, X. J. Xu, Z. Wiesenfeld-Hallin, T. Hokfelt, T. Bartfai, and U. Langel. Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16:857-861 (1998).

    Google Scholar 

  12. A. Astriab-Fisher, D. Sergueev, M. Fisher, B. R. Shaw, and R. L. Juliano. Conjugates of antisense oligonucleotides with the Tat and antennapedia cell-penetrating peptides: effects on cellular uptake, binding to target sequences, and biologic actions. Pharm. Res. 19:744-754 (2002).

    Google Scholar 

  13. H. Nagahara, A. M. Vocero-Akbani, E. L. Snyder, A. Ho, D. G. Latham, N. A. Lissy, M. Becker-Hapak, S. A. Ezhevsky, and S. F. Dowdy. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4:1449-1452 (1998).

    Google Scholar 

  14. M. Lewin, N. Carlesso, C. H. Tung, X. W. Tang, D. Cory, D. T. Scadden, and R. Weissleder. Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol. 18:410-414 (2000).

    Google Scholar 

  15. V. P. Torchilin, R. Rammohan, V. Weissig, and T. S. Levchenko. TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc. Natl. Acad. Sci. USA 98:8786-8791 (2001).

    Google Scholar 

  16. T. Suzuki, S. Futaki, M. Niwa, S. Tanaka, K. Ueda, and Y. Sugiura. Possible existence of common internalization mechanisms among arginine-rich peptides. J. Biol. Chem. 277:2437-2443 (2002).

    Google Scholar 

  17. U. Koppelhus, S. K. Awasthi, V. Zachar, H. U. Holst, P. Ebbesen, and P. E. Nielsen. Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates. Antisense Nucleic Acid Drug Dev. 12:51-63 (2002).

    Google Scholar 

  18. J. P. Richard, K. Melikov, E. Vives, C. Ramos, B. Verbeure, M. J. Gait, L. V. Chernomordik, and B. Lebleu. Cell-penetrating peptides: A re-evaluation of the mechanism of cellular uptake. J. Biol. Chem. 30:585-590 (2003).

    Google Scholar 

  19. C. R. Gaush, W. L. Hard, and T. F. Smith. Characterization of an established line of canine kidney cells (MDCK). Proc. Soc. Exp. Biol. Med. 122:931-935 (1966).

    Google Scholar 

  20. B. Rothen-Rutishauser, S. D. Kramer, A. Braun, M. Gunthert, and H. Wunderli-Allenspach. MDCK cell cultures as an epithelial in vitro model: cytoskeleton and tight junctions as indicators for the definition of age-related stages by confocal microscopy. Pharm. Res. 15:964-971 (1998).

    Google Scholar 

  21. M. Lindgren, M. Hallbrink, A. Prochiantz, and U. Langel. Cell-penetrating peptides. Trends Pharmacol. Sci. 21:99-103 (2000).

    Google Scholar 

  22. M. C. Schmidt, B. Rothen-Rutishauser, B. Rist, A. Beck-Sickinger, H. Wunderli-Allenspach, W. Rubas, W. Sadee, and H. P. Merkle. Translocation of human calcitonin in respiratory nasal epithelium is associated with self-assembly in lipid membrane. Biochemistry 37:16582-16590 (1998).

    Google Scholar 

  23. Z. Machova, C. Muhle, U. Krauss, R. Trehin, A. Koch, H. P. Merkle, and A. G. Beck-Sickinger. Cellular internalization of enhanced green fluorescent protein ligated to a human calcitonin-based carrier peptide. Chembiochem. 3:672-677 (2002).

    Google Scholar 

  24. B. Rist, M. Entzeroth, and A. G. Beck-Sickinger. From micromolar to nanomolar affinity: A systematic approach to identify the binding site of CGRP at the human calcitonin gene-related peptide 1 receptor. J. Med. Chem. 41:117-123 (1998).

    Google Scholar 

  25. P. J. A. Weber, J. E. Bader, G. Folkers, and A. G. Beck-Sickinger. A fast and inexpensive method for N-terminal fluorescein-labeling of peptides. Bioorg. Med. Chem. Lett. 8:597-600 (1998).

    Google Scholar 

  26. N. Courret, C. Frehel, N. Gouhier, M. Pouchelet, E. Prina, P. Roux, and J. C. Antoine. Biogenesis of Leishmania-harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the parasites. J. Cell Sci. 115:2303-2316 (2002).

    Google Scholar 

  27. J. Moran, W. Hunziker, and J. A. Fischer. Calcitonin and calcium ionophores: cyclic AMP responses in cells of a human lymphoid line. Proc. Natl. Acad. Sci. USA 75:3984-3988 (1978).

    Google Scholar 

  28. S. R. Lang, W. Staudenmann, P. James, H. J. Manz, R. Kessler, B. Galli, H. P. Moser, A. Rummelt, and H. P. Merkle. Proteolysis of human calcitonin in excised bovine nasal mucosa: Elucidation of the metabolic pathway by liquid secondary ionization mass spectrometry (LSIMS) and matrix assisted laser desorption ionization mass spectrometry (MALDI). Pharm. Res. 13:1679-1685 (1996).

    Google Scholar 

  29. S. Lang, B. Rothen-Rutishauser, J. C. Perriard, M. C. Schmidt, and H. P. Merkle. Permeation and pathways of human calcitonin (hCT) across excised bovine nasal mucosa. Peptides 19:599-607 (1997).

    Google Scholar 

  30. R. Muff, W. Born, and J. A. Fischer. Calcitonin, calcitonin gene-related peptide, adrenomedullin and amylin: homologous peptides, separate receptors and overlapping biological actions. Eur. J. Endocrinol. 133:17-20 (1995).

    Google Scholar 

  31. A. Eguchi, T. Akuta, H. Okuyama, T. Senda, H. Yokoi, H. Inokuchi, S. Fujita, T. Hayakawa, K. Takeda, M. Hasegawa, and M. Nakanishi. Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells. J. Biol. Chem. 276:26204-26210 (2001).

    Google Scholar 

  32. M. Lindgren, X. Gallet, U. Soomets, M. Hallbrink, E. Brakenhielm, M. Pooga, R. Brasseur, and U. Langel. Translocation properties of novel cell penetrating transportan and penetratin analogues. Bioconjug. Chem. 11:619-626 (2000).

    Google Scholar 

  33. L. A. Kueltzo and C. R. Middaugh. Potential use of non-classical pathways for the transport of macromolecular drugs. Expert Opin. Invest. Drugs. 9:2039-2050 (2000).

    Google Scholar 

  34. H. H. Bauer, U. Aebi, M. Haner, R. Hermann, M. Muller, and H. P. Merkle. Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin. J. Struct. Biol. 115:1-15 (1995).

    Google Scholar 

  35. S. Violini, V. Sharma, J. L. Prior, M. Dyszlewski, and D. Piwnica-Worms. Evidence for a plasma membrane-mediated permeability barrier to tat basic domain in well-differentiated epithelial cells: lack of correlation with heparan sulfate. Biochemistry 41:12652-12661 (2002).

    Google Scholar 

  36. S. D. Krämer and H. Wunderli-Allenspach. No entry for TAT(44-57) into liposomes and intact MDCK cells: novel approach to study membrane permeation of cell-penetrating peptides. Biochim. Biophys. Acta 1609:161-169 (2003).

    Google Scholar 

  37. H. Xia, Q. Mao, and B. L. Davidson. The HIV Tat protein transduction domain improves the biodistribution of beta-glucuronidase expressed from recombinant viral vectors. Nat. Biotechnol. 19:640-644 (2001).

    Google Scholar 

  38. M. Lundberg and M. Johansson. Positively charged DNA-binding proteins cause apparent cell membrane translocation. Biochem. Biophys. Res. Commun. 291:367-371 (2002).

    Google Scholar 

  39. C. Pichon, M. Monsigny, and A. C. Roche. Intracellular localization of oligonucleotides: influence of fixative protocols. Antisense Nucleic Acid Drug Dev. 9:89-93 (1999).

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry and Applied BioSciences, Swiss Federal Institute of Technology Zurich (ETH Zurich), Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Rachel Tréhin, Martina Meinecke & Hans P. Merkle

  2. Institute of Biochemistry, University of Leipzig, 04103, Leipzig, Germany

    Ulrike Krauss & Annette G. Beck-Sickinger

  3. Departments of Orthopaedic Surgery and Medicine, University of Zurich, Klinik Balgrist, Forchstrasse 340, 8008, Zurich, Switzerland

    Roman Muff

Authors
  1. Rachel Tréhin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ulrike Krauss
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roman Muff
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martina Meinecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Annette G. Beck-Sickinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hans P. Merkle
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hans P. Merkle.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tréhin, R., Krauss, U., Muff, R. et al. Cellular Internalization of Human Calcitonin Derived Peptides in MDCK Monolayers: A Comparative Study with Tat(47-57) and Penetratin(43-58). Pharm Res 21, 33–42 (2004). https://doi.org/10.1023/B:PHAM.0000012149.83119.bf

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/B:PHAM.0000012149.83119.bf

Search

Navigation