Skip to main content
SpringerLink
Log in

Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion

  • Research Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Among non-invasive cell delivery strategies, cell-penetrating peptide (CPP) vectors represent interesting new tools. To get fundamental knowledge about the still debated internalisation mechanisms of these peptides, we modified the membrane content of cells, typically by hydrolysis of sphingomyelin or depletion of cholesterol from the membrane outer leaflet. We quantified and visualised the effect of these viable cell surface treatments on the internalisation efficiency of different CPPs, among which the most studied Tat, R9, penetratin and analogues, that all carry the N-terminal biotin-Gly4 tag cargo. Under these cell membrane treatments, only penetratin and R6W3 underwent a massive glycosaminoglycan (GAG)-dependent entry in cells. Internalisation of the other peptides was only slightly increased, similarly in the absence or the presence of GAGs for R9, and only in the presence of GAGs for Tat and R6L3. Ceramide formation (or cholesterol depletion) is known to lead to the reorganisation of membrane lipid domains into larger platforms, which can serve as a trap and cluster receptors. These results show that GAG clustering, enhanced by formation of ceramide, is efficiently exploited by penetratin and R6W3, which contains Trp residues in their sequence but not Tat, R9 and R6L3. Hence, these data shed new lights on the differences in the internalisation mechanism and pathway of these peptides that are widely used in delivery of cargo molecules.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Bechara C, Sagan S (2013) Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett 587(12):1693–1702

    Article  CAS  PubMed  Google Scholar 

  2. Madani F, Lindberg S, Langel U, Futaki S, Graslund A (2011) Mechanisms of cellular uptake of cell-penetrating peptides. J Biophys 2011:414729

    PubMed Central  PubMed  Google Scholar 

  3. Jones AT, Sayers EJ (2012) Cell entry of cell penetrating peptides: tales of tails wagging dogs. J Control Release 161(2):582–591

    Article  CAS  PubMed  Google Scholar 

  4. van den Berg A, Dowdy SF (2011) Protein transduction domain delivery of therapeutic macromolecules. Curr Opin Biotechnol 22(6):888–893

    Article  PubMed  Google Scholar 

  5. Said Hassane F, Saleh AF, Abes R, Gait MJ, Lebleu B (2010) Cell penetrating peptides: overview and applications to the delivery of oligonucleotides. Cell Mol Life Sci 67(5):715–726

    Article  CAS  PubMed  Google Scholar 

  6. Jarver P, Mager I, Langel U (2010) In vivo biodistribution and efficacy of peptide mediated delivery. Trends Pharmacol Sci 31(11):528–535

    Article  PubMed  Google Scholar 

  7. Anderson RG, Jacobson K (2002) A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science (New York, NY) 296(5574):1821–1825

    Article  CAS  Google Scholar 

  8. Mayor S, Rao M (2004) Rafts: scale-dependent, active lipid organization at the cell surface. Traffic (Copenhagen, Denmark) 5(4):231–240

    Article  CAS  Google Scholar 

  9. Saalik P, Elmquist A, Hansen M, Padari K, Saar K, Viht K, Langel U, Pooga M (2004) Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjug Chem 15(6):1246–1253

    Article  PubMed  Google Scholar 

  10. Wadia JS, Stan RV, Dowdy SF (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10(3):310–315

    Article  CAS  PubMed  Google Scholar 

  11. Foerg C, Ziegler U, Fernandez-Carneado J, Giralt E, Rennert R, Beck-Sickinger AG, Merkle HP (2005) Decoding the entry of two novel cell-penetrating peptides in HeLa cells: lipid raft-mediated endocytosis and endosomal escape. Biochemistry 44(1):72–81

    Article  CAS  PubMed  Google Scholar 

  12. Fretz MM, Penning NA, Al-Taei S, Futaki S, Takeuchi T, Nakase I, Storm G, Jones AT (2007) Temperature-concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochem J 403(2):335–342

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Mager I, Langel K, Lehto T, Eiriksdottir E, Langel U (2012) The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides. Biochim Biophys Acta 1818(3):502–511

    Article  CAS  PubMed  Google Scholar 

  14. Mae M, Myrberg H, Jiang Y, Paves H, Valkna A, Langel U (2005) Internalisation of cell-penetrating peptides into tobacco protoplasts. Biochim Biophys Acta 1669(2):101–107

    Article  PubMed  Google Scholar 

  15. Lamaziere A, Wolf C, Lambert O, Chassaing G, Trugnan G, Ayala-Sanmartin J (2008) The homeodomain derived peptide Penetratin induces curvature of fluid membrane domains. PLoS One 3(4):e1938

    Article  PubMed Central  PubMed  Google Scholar 

  16. Caesar CE, Esbjorner EK, Lincoln P, Norden B (2006) Membrane interactions of cell-penetrating peptides probed by tryptophan fluorescence and dichroism techniques: correlations of structure to cellular uptake. Biochemistry 45(24):7682–7692

    Article  CAS  PubMed  Google Scholar 

  17. Verdurmen WP, Thanos M, Ruttekolk IR, Gulbins E, Brock R (2010) Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: implications for uptake. J Control Release 147(2):171–179

    Article  CAS  PubMed  Google Scholar 

  18. Bechara C, Pallerla M, Zaltsman Y, Burlina F, Alves ID, Lequin O, Sagan S (2013) Tryptophan within basic peptide sequences triggers glycosaminoglycan-dependent endocytosis. FASEB J 27(2):738–749

    Article  CAS  PubMed  Google Scholar 

  19. Poon GM, Gariepy J (2007) Cell-surface proteoglycans as molecular portals for cationic peptide and polymer entry into cells. Biochem Soc Trans 35(Pt 4):788–793

    CAS  PubMed  Google Scholar 

  20. Letoha T, Keller-Pinter A, Kusz E, Kolozsi C, Bozso Z, Toth G, Vizler C, Olah Z, Szilak L (2010) Cell-penetrating peptide exploited syndecans. Biochim Biophys Acta 1798(12):2258–2265

    Article  CAS  PubMed  Google Scholar 

  21. Imamura J, Suzuki Y, Gonda K, Roy CN, Gatanaga H, Ohuchi N, Higuchi H (2011) Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan cross-linkage activates Rac1 for internalization. J Biol Chem 286(12):10581–10592

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  22. Esko JD, Stewart TE, Taylor WH (1985) Animal cell mutants defective in glycosaminoglycan biosynthesis. Proc Natl Acad Sci 82(10):3197–3201

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Marty C, Meylan C, Schott H, Ballmer-Hofer K, Schwendener RA (2004) Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes. CMLS Cell Mol Life Sci 61(14):1785–1794

    Article  CAS  Google Scholar 

  24. Jiao CY, Delaroche D, Burlina F, Alves ID, Chassaing G, Sagan S (2009) Translocation and endocytosis for cell-penetrating peptide internalization. J Biol Chem 284(49):33957–33965

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Amand HL, Rydberg HA, Fornander LH, Lincoln P, Norden B, Esbjorner EK (2012) Cell surface binding and uptake of arginine- and lysine-rich penetratin peptides in absence and presence of proteoglycans. Biochim Biophys Acta 1818(11):2669–2678

    Article  PubMed  Google Scholar 

  26. Gump JM, June RK, Dowdy SF (2010) Revised role of glycosaminoglycans in TAT protein transduction domain-mediated cellular transduction. J Biol Chem 285(2):1500–1507

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Burlina F, Sagan S, Bolbach G, Chassaing G (2005) Quantification of the cellular uptake of cell-penetrating peptides by MALDI-TOF mass spectrometry. Angew Chem Int Ed 44(27):4244–4247

    Article  CAS  Google Scholar 

  28. Burlina F, Sagan S, Bolbach G, Chassaing G (2006) A direct approach to quantification of the cellular uptake of cell-penetrating peptides using MALDI-TOF mass spectrometry. Nat Protoc 1(1):200–205

    Article  CAS  PubMed  Google Scholar 

  29. Dupont E, Prochiantz A, Joliot A (2007) Identification of a signal peptide for unconventional secretion. J Biol Chem 282(12):8994–9000

    Article  CAS  PubMed  Google Scholar 

  30. Mathivet L, Cribier S, Devaux PF (1996) Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys J 70(3):1112–1121

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Holopainen JM, Subramanian M, Kinnunen PK (1998) Sphingomyelinase induces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane. Biochemistry 37(50):17562–17570

    Article  CAS  PubMed  Google Scholar 

  32. Fanani ML, Hartel S, Oliveira RG, Maggio B (2002) Bidirectional control of sphingomyelinase activity and surface topography in lipid monolayers. Biophys J 83(6):3416–3424

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  33. Lambaerts K, Wilcox-Adelman SA, Zimmermann P (2009) The signaling mechanisms of syndecan heparan sulfate proteoglycans. Curr Opin Cell Biol 21(5):662–669

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Hao M, Mukherjee S, Sun Y, Maxfield FR (2004) Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes. J Biol Chem 279(14):14171–14178

    Article  CAS  PubMed  Google Scholar 

  35. Kaplan IM, Wadia JS, Dowdy SF (2005) Cationic TAT peptide transduction domain enters cells by macropinocytosis. J Control Release 102(1):247–253

    Article  CAS  PubMed  Google Scholar 

  36. Letoha T, Gaal S, Somlai C, Venkei Z, Glavinas H, Kusz E, Duda E, Czajlik A, Petak F, Penke B (2005) Investigation of penetratin peptides. Part 2. In vitro uptake of penetratin and two of its derivatives. J Pept Sci 11(12):805–811

    Article  CAS  PubMed  Google Scholar 

  37. Conner SD, Schmid SL (2003) Regulated portals of entry into the cell. Nature 422(6927):37–44

    Article  CAS  PubMed  Google Scholar 

  38. Subtil A, Gaidarov I, Kobylarz K, Lampson MA, Keen JH, McGraw TE (1999) Acute cholesterol depletion inhibits clathrin-coated pit budding. Proc Natl Acad Sci USA 96(12):6775–6780

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  39. Irie T, Uekama K (1999) Cyclodextrins in peptide and protein delivery. Adv Drug Deliv Rev 36(1):101–123

    Article  CAS  PubMed  Google Scholar 

  40. Rodal SK, Skretting G, Garred O, Vilhardt F, van Deurs B, Sandvig K (1999) Extraction of cholesterol with methyl-beta-cyclodextrin perturbs formation of clathrin-coated endocytic vesicles. Mol Biol Cell 10(4):961–974

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  41. Bishop JR, Stanford KI, Esko JD (2008) Heparan sulfate proteoglycans and triglyceride-rich lipoprotein metabolism. Curr Opin Lipidol 19(3):307–313

    Article  CAS  PubMed  Google Scholar 

  42. Bishop JR, Passos-Bueno MR, Fong L, Stanford KI, Gonzales JC, Yeh E, Young SG, Bensadoun A, Witztum JL, Esko JD, Moulton KS (2010) Deletion of the basement membrane heparan sulfate proteoglycan type XVIII collagen causes hypertriglyceridemia in mice and humans. PLoS One 5(11):e13919

    Article  PubMed Central  PubMed  Google Scholar 

  43. Veatch SL, Keller SL (2005) Miscibility phase diagrams of giant vesicles containing sphingomyelin. Phys Rev Lett 94(14):148101

    Article  PubMed  Google Scholar 

  44. Gulbins E, Grassme H (2002) Ceramide and cell death receptor clustering. Biochim Biophys Acta 1585(2–3):139–145

    Article  CAS  PubMed  Google Scholar 

  45. Gulbins E, Dreschers S, Wilker B, Grassme H (2004) Ceramide, membrane rafts and infections. J Mol Med (Berlin, Germany) 82(6):357–363

    Article  CAS  Google Scholar 

  46. Grassme H, Jendrossek V, Riehle A, von Kurthy G, Berger J, Schwarz H, Weller M, Kolesnick R, Gulbins E (2003) Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts. Nat Med 9(3):322–330

    Article  CAS  PubMed  Google Scholar 

  47. Zha X, Pierini LM, Leopold PL, Skiba PJ, Tabas I, Maxfield FR (1998) Sphingomyelinase treatment induces ATP-independent endocytosis. J Cell Biol 140(1):39–47

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Holopainen JM, Angelova MI, Kinnunen PK (2000) Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophys J 78(2):830–838

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Chatterjee S (1994) Neutral sphingomyelinase action stimulates signal transduction of tumor necrosis factor-alpha in the synthesis of cholesteryl esters in human fibroblasts. J Biol Chem 269(2):879–882

    CAS  PubMed  Google Scholar 

  50. Ridgway ND (2000) Interactions between metabolism and intracellular distribution of cholesterol and sphingomyelin. Biochim Biophys Acta 1484(2–3):129–141

    Article  CAS  PubMed  Google Scholar 

  51. Ridgway ND, Lagace TA, Cook HW, Byers DM (1998) Differential effects of sphingomyelin hydrolysis and cholesterol transport on oxysterol-binding protein phosphorylation and Golgi localization. J Biol Chem 273(47):31621–31628

    Article  CAS  PubMed  Google Scholar 

  52. Al-Makdissy N, Younsi M, Pierre S, Ziegler O, Donner M (2003) Sphingomyelin/cholesterol ratio: an important determinant of glucose transport mediated by GLUT-1 in 3T3-L1 preadipocytes. Cell Signal 15(11):1019–1030

    Article  CAS  PubMed  Google Scholar 

  53. Megha London E (2004) Ceramide selectively displaces cholesterol from ordered lipid domains (rafts): implications for lipid raft structure and function. J Biol Chem 279(11):9997–10004

    Article  CAS  PubMed  Google Scholar 

  54. Nyholm TK, Grandell PM, Westerlund B, Slotte JP (2010) Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length. Biochim Biophys Acta 1798(5):1008–1013

    Article  CAS  PubMed  Google Scholar 

  55. Mahammad S, Dinic J, Adler J, Parmryd I (2010) Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation. Biochim Biophys Acta (BBA) Mol Cell Biol Lipids 1801(6):625–634

    Article  CAS  Google Scholar 

  56. Quinn PJ (2010) A lipid matrix model of membrane raft structure. Prog Lipid Res 49(4):390–406

    Article  CAS  PubMed  Google Scholar 

  57. López-Montero I, Monroy F, Vélez M, Devaux PF (2010) Ceramide: from lateral segregation to mechanical stress. Biochim Biophys Acta (BBA) Biomembr 1798(7):1348–1356

    Article  Google Scholar 

  58. Last NB, Schlamadinger DE, Miranker AD (2013) A common landscape for membrane-active peptides. Protein Sci 22(7):870–882

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  59. Rothbard JB, Jessop TC, Lewis RS, Murray BA, Wender PA (2004) Role of membrane potential and hydrogen bonding in the mechanism of translocation of guanidinium-rich peptides into cells. J Am Chem Soc 126(31):9506–9507

    Article  CAS  PubMed  Google Scholar 

  60. Christianson HC, Belting M (2013) Heparan sulfate proteoglycan as a cell-surface endocytosis receptor. Matrix Biol 35(4):51–55

  61. Christianson HC, Svensson KJ, van Kuppevelt TH, Li JP, Belting M (2013) Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity. Proc Natl Acad Sci USA 110(43):17380–17385

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

Support for this research was provided by the Université Pierre et Marie Curie (UPMC; Sorbonne Universités), by ANR BLAN2010-ParaHP (postdoctoral position for M.P.), by the École Normale Supérieure (ENS), the Centre National de la Recherche Scientifique (CNRS), and the French Ministère de l’Enseignement Supérieur et de la Recherche (PhD fellowship for C.B.).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Author notes

  1. Chérine Bechara

    Present address: Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, UK

Authors and Affiliations

  1. Sorbonne Universités, UPMC Univ Paris 06, LBM, 4 Place Jussieu, 75005, Paris, France

    Chérine Bechara, Manjula Pallerla, Fabienne Burlina, Françoise Illien, Sophie Cribier & Sandrine Sagan

  2. Département de Chimie, Ecole Normale Supérieure-PSL Research University, 24, rue Lhomond, 75005, Paris, France

    Chérine Bechara, Manjula Pallerla, Fabienne Burlina, Françoise Illien, Sophie Cribier & Sandrine Sagan

  3. CNRS, UMR 7203 LBM, 75005, Paris, France

    Chérine Bechara, Manjula Pallerla, Fabienne Burlina, Françoise Illien, Sophie Cribier & Sandrine Sagan

  4. Laboratoire des Biomolécules, Université Pierre et Marie Curie, UMR 7203 CNRS-ENS, cc182, 4 place Jussieu, 75252, Paris cedex 05, France

    Chérine Bechara, Manjula Pallerla, Fabienne Burlina, Françoise Illien, Sophie Cribier & Sandrine Sagan

Authors
  1. Chérine Bechara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Manjula Pallerla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fabienne Burlina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Françoise Illien
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sophie Cribier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sandrine Sagan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sandrine Sagan.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 39,356 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bechara, C., Pallerla, M., Burlina, F. et al. Massive glycosaminoglycan-dependent entry of Trp-containing cell-penetrating peptides induced by exogenous sphingomyelinase or cholesterol depletion. Cell. Mol. Life Sci. 72, 809–820 (2015). https://doi.org/10.1007/s00018-014-1696-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-014-1696-y

Keywords

Search

Navigation