All Title Author
Keywords Abstract

Effect of Repetitive Lysine-Tryptophan Motifs on the Eukaryotic Membrane

DOI: 10.3390/ijms14012190

Keywords: (KW)5, hemolytic peptide, eukaryotic membrane, phosphatidylcholine, cholesterol, sphingomyelin, aggregation

Full-Text   Cite this paper   Add to My Lib


In a previous study, we synthesized a series of peptides containing simple sequence repeats, (KW) n–NH 2 ( n = 2,3,4 and 5) and determined their antimicrobial and hemolytic activities, as well as their mechanism of antimicrobial action. However, (KW) 5 showed undesirable cytotoxicity against RBC cells. In order to identify the mechanisms behind the hemolytic and cytotoxic activities of (KW) 5, we measured the ability of these peptides to induce aggregation of liposomes. In addition, their binding and permeation activities were assessed by Trp fluorescence, calcein leakage and circular dichrorism using artificial phospholipids that mimic eukaryotic liposomes, including phosphatidylcholine (PC), PC/sphingomyelin (SM) (2:1, w/ w) and PC/cholesterol (CH) (2:1, w/ w). Experiments confirmed that only (KW) 5 induced aggregation of all liposomes; it formed much larger aggregates with PC:CH (2:1, w/ w) than with PC or PC:SM (2:1, w/ w). Longer peptide (KW) 5, but not (KW) 3 or (KW) 4, strongly bound and partially inserted into PC:CH compared to PC or PC:SM (2:1, w/ w). Calcein release experiments showed that (KW) 5 induced calcein leakage from the eukaryotic membrane. Greater calcein leakage was induced by (KW) 5 from PC:CH than from PC:SM (2:1, w/ w) or PC, whereas (KW) 4 did not induce calcein leakage from any of the liposomes. Circular dichroism measurements indicated that (KW) 5 showed higher conformational transition compared to (KW) 4 due to peptide-liposome interactions. Taken together, our results suggest that (KW) 5 reasonably mediates the aggregation and permeabilization of eukaryotic membranes, which could in turn explain why (KW) 5 displays efficient hemolytic activity.


[1]  Hoskin, D.W.; Ramamoorthy, A. Studies on anticancer activities of antimicrobial peptides. Biochim. Biophys. Acta 2008, 1778, 357–375.
[2]  Dürr, U.H.N.; Sudheendra, U.S.; Ramamoorthy, A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim. Biophys. Acta 2006, 1758, 1408–1425.
[3]  Dhople, V.; Krukemeyer, A.; Ramamoorthy, A. The human beta-defensin-3, an antibacterial peptide with multiple biological functions. Biochim. Biophys. Acta 2006, 1758, 1499–1512.
[4]  Abraham, T.; Lewis, R.N.; Hodges, R.S.; McElhaney, R.N. Isothermal titration calorimetry studies of the binding of the antimicrobial peptide gramicidin S to phospholipid bilayer membranes. Biochemistry 2005, 44, 11279–11285.
[5]  Verkleij, A.J.; Zwaal, R.F.; Roelofsen, B.; Comfurius, P.; Kastelijn, D.; van Deenen, L.L. The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. Biochim. Biophys. Acta 1973, 323, 178–193.
[6]  Vogel, H.J.; Schibli, D.J.; Jing, W.; Lohmeier-Vogel, E.M.; Epand, R.F.; Epand, R.M. Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and arginine-containing peptides. Biochem. Cell Biol 2002, 80, 49–63.
[7]  Matsuzaki, K. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochim. Biophys. Acta 1999, 1462, 1–10.
[8]  Lugtenberg, B.; van Alphen, L. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim. Biophys. Acta 1983, 737, 51–115.
[9]  Glukhov, E.; Stark, M.; Burrows, L.L.; Deber, C.M. Basis for selectivity of cationic antimicrobial peptides for bacterial versus mammalian membranes. J. Biol. Chem 2005, 280, 33960–33967.
[10]  Epand, R.F.; Schmitt, M.A.; Gellman, S.H.; Epand, R.M. Role of membrane lipids in the mechanism of bacterial species selective toxicity by two alpha/beta-antimicrobial peptides. Biochim. Biophys. Acta 2006, 1758, 1343–1350.
[11]  Shai, Y.; Fox, J.; Caratsch, C.; Shih, Y.L.; Edwards, C.; Lazarovici, P. Sequencing and synthesis of pardaxin, a polypeptide from the Red Sea Moses sole with ionophore activity. FEBS Lett 1988, 242, 161–166.
[12]  Oren, Z.; Shai, Y. Selective lysis of bacteria, but not mammalian cells by diastereomers of melittin: Structure-function study. Biochemistry 1997, 36, 1826–1835.
[13]  Johansson, J.; Gudmundsson, G.H.; Rottenberg, M.E.; Berndt, K.D.; Agerberth, B. Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37. J. Biol. Chem 1998, 273, 3718–3724.
[14]  Chen, Y.; Mant, C.T.; Farmer, S.W.; Hancock, R.E.; Vasil, M.L.; Hodges, R.S. Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index. J. Biol. Chem 2005, 280, 12316–12329.
[15]  Sengupta, D.; Leontiadou, H.; Mark, A.E.; Marrink, S.J. Toroidal pores formed by antimicrobial peptides show significant disorder. Biochim. Biophys. Acta 2008, 1778, 2308–2317.
[16]  Melo, M.N.; Ferre, R.; Castanho, M.A. Antimicrobial peptides: Linking partition, activity and high membrane-bound concentrations. Nat. Rev. Microbiol 2009, 7, 245–250.
[17]  Zhu, W.L.; Nan, Y.H.; Hahm, K.S.; Shin, S.Y. Cell selectivity of an antimicrobial peptide melittin diastereomer with D-amino acid in the leucine zipper sequence. J. Biochem. Mol. Biol 2007, 40, 1090–1094.
[18]  Matsuzaki, K. Control of cell selectivity of antimicrobial peptides. Biochim. Biophys. Acta 2009, 1788, 1687–1692.
[19]  Huang, Y.; Huang, J.; Chen, Y. Alpha-helical cationic antimicrobial peptides: Relationships of structure and function. Protein Cell 2010, 1, 143–152.
[20]  Wang, P.; Nan, Y.H.; Yang, S.T.; Kang, S.W.; Kim, Y.; Park, I.S.; Hahm, K.S.; Shin, S.Y. Cell selectivity and anti-inflammatory activity of a Leu/Lys-rich alpha-helical model antimicrobial peptide and its diastereomeric peptides. Peptides 2010, 31, 1251–1261.
[21]  Strom, M.B.; Haug, B.E.; Sker, M.L.; Stensen, W.; Stiberg, T.; Svendsen, J.S. The Pharmacophore of short cationic antibacterial peptides. J. Med. Chem 2003, 46, 1567–1570.
[22]  Liu, Z.; Brady, A.; Young, A.; Rasimick, B.; Chen, K.; Zhou, C.; Kallenbach, N.R. Length effects in antimicrobial peptides of the (RW)n series. Antimicrob. Agents Chemother 2007, 51, 597–603.
[23]  Strom, M.B.; Rekdal, O.; Svendsen, J.S. Antimicrobial activity of short arginine- and tryptophan-rich peptides. J. Pept. Sci 2002, 8, 431–437.
[24]  Jing, W.; Hunter, H.N.; Hagel, J.; Vogel, H.J. The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers. J. Pept. Res 2003, 61, 219–229.
[25]  Dathe, M.; Nikolenko, H.; Klose, J.; Bienert, M. Cyclization increases the antimicrobial activity and selectivity of arginine- and tryptophan- containing hexapeptides. Biochemistry 2004, 43, 9140–9150.
[26]  Gopal, R.; Seo, C.H.; Song, P.I.; Park, Y. Effect of repetitive lysine-tryptophan motifs on the bactericidal activity of antimicrobial peptides. Amino Acids 2012. [Epub ahead of print].
[27]  Chen, Y.; Guarnieri, M.T.; Vasil, A.I.; Vasil, M.L.; Mant, C.T.; Hodges, R.S. Role of peptide hydrophobicity in the mechanism of action of alpha-helical antimicrobial peptides. Antimicrob. Agents Chemother 2007, 51, 1398–1406.
[28]  Ramamoorthy, A.; Lee, D.K.; Narasimhaswamy, T.; Nanga, R.P.R. Cholesterol reduces pardaxin’s dynamics—a barrel—stave mechanism of membrane disruption investigated by solid-state NMR. Biochim. Biophys. Acta 2010, 1798, 223–227.
[29]  McHenry, A.J.; Sciacca, M.F.M.; Brender, J.R.; Ramamoorthy, A. Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes? Biochim. Biophys. Acta 2012, 1818, 3019–3024.
[30]  Bhattacharjya, S.; Ramamoorthy, A. Multifunctional host defense peptides: Functional and mechanistic insights from NMR structures of potent antimicrobial peptides. FEBS J 2009, 276, 6465–6473.
[31]  Ramamoorthy, A. Beyond NMR spectra of antimicrobial peptides: Dynamical images at atomic resolution and functional insights. Solid State Nucl. Magn. Reson 2009, 35, 201–207.
[32]  Gottler, L.M.; Ramamoorthy, A. Structure, membrane orientation, mechanism and function of pexiganan—A highly potent antimicrobial peptide designed from magainin. Biochim. Biophys. Acta 2009, 1788, 1680–1686.
[33]  March, E.N.; Buer, B.C.; Ramamoorthy, A. Fluorine—A new element in the design of membrane-active peptides. Mol. Biosyst 2009, 5, 1143–1147.
[34]  Matsuyama, K.; Natori, S. Mode of action of sapecin, a novel antibacterial protein of Sarcophaga peregrina (flesh fly). J. Biochem 1990, 108, 128–132.
[35]  Dhople, V.M.; Nagaraj, R. Generation of analogs having potent antimicrobial and hemolytic activities with minimal changes from an inactive 16-residue peptide corresponding to the helical region of Staphylococcus aureus δ-toxin. Protein Eng. 1995, 8, 315–318.
[36]  Blondelle, S.E.; Lohner, K.; Aguilar, M. Lipid induced conformation and lipid-binding properties of cytolytic and antimicrobial peptides: Determination and biological specificity. Biochim. Biophys. Acta 1999, 1462, 89–108.
[37]  Som, A.; Vemparala, S.; Ivanov, I.; Tew, G.N. Synthetic mimics of antimicrobial peptides. Biopolymers 2008, 90, 83–93.
[38]  Gopal, R.; Park, S.C.; Ha, K.J.; Cho, S.J.; Kim, S.W.; Song, P.I.; Nah, J.W.; Park, Y.; Hahm, K.S. Effect of leucine and lysine substitution on the antimicrobial activity and evaluation of the mechanism of the HPA3NT3 analog peptide. J. Pept. Sci 2009, 15, 589–594.
[39]  Rathinakumar, R.; Walkenhorst, W.F.; Wimley, W.C. Broad-spectrum antimicrobial peptides by rational combinatorial design and high-throughput screening: The importance of interfacial activity. J. Am. Chem. Soc 2009, 131, 7609–7617.
[40]  Zhu, W.L.; Shin, S.Y. Effects of dimerization of the cell-penetrating peptide Tat analog on antimicrobial activity and mechanism of bactericidal action. J. Pept. Sci 2009, 15, 345–352.
[41]  Torrent, M.; de la Torre, B.G.; Nogués, V.M.; Andreu, D.; Boix, E. Bactericidal and membrane disruption activities of the eosinophil cationic protein are largely retained in an N-terminal fragment. Biochem. J 2009, 421, 425–434.
[42]  Javadpour, M.M.; Barkley, M.D. Self-assembly of designed antimicrobial peptides in solution and micelles. Biochemistry 1997, 36, 9540–9549.
[43]  Feder, R.; Dagan, A.; Mor, A. Structure-activity relationship study of antimicrobial dermaseptin S4 showing the consequences of peptide oligomerization on selective cytotoxicity. J. Biol. Chem 2000, 275, 4230–4238.
[44]  Chongsiriwatana, N.P.; Barron, A.E. Comparing bacterial membrane interactions of antimicrobial peptides and their mimics. Methods Mol. Biol 2010, 618, 171–182.
[45]  Subbalakshmi, C.; Krishnakumari, V.; Sitaram, N.; Nagaraj, R. Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes. J. Biosci 1998, 23, 9–13.
[46]  Takahashi, D.; Shukla, S.K.; Prakash, O.; Zhang, G. Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity. Biochimie 2010, 92, 1236–1241.
[47]  Schmidtchen, A.; Pasupuleti, M.; M?rgelin, M.; Davoudi, M.; Alenfall, J.; Chalupka, A.; Malmsten, M. Boosting antimicrobial peptides by hydrophobic oligopeptide end tags. J. Biol. Chem 2009, 284, 17584–17594.
[48]  William, C.W. Describing the mechanism of antimicrobial peptide action with the interfacial activity model. ACS Chem. Biol 2010, 5, 905–917.
[49]  Wimley, W.C.; Hristova, K.; Ladokhin, A.S.; Silvestro, L.; Axelsen, P.H.; White, S.H. Folding of beta-sheet membrane proteins: A hydrophobic hexapeptide model. J. Mol. Biol 1998, 277, 1091–1110.
[50]  Ladokhin, A.S.; White, S.H. Folding of amphipathic alpha-helices on membranes: Energetics of helix formation by melittin. J. Mol. Biol 1999, 285, 1363–1369.
[51]  Wimley, W.C.; White, S.H. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struc. Biol 1996, 3, 842–848.
[52]  Thennarasu, S.; Huang, R.; Lee, D.K.; Yang, P.; Maloy, L.; Chen, Z.; Ramamoorthy, A. Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: A case study of MSI-367. Biochemistry 2010, 49, 10595–10605.
[53]  Andr?, J.; Monreal, D.; Martinez de Tejada, G.; Olak, C.; Brezesinski, G.; Gomez, S.S.; Goldmann, T.; Bartels, R.; Brandenburg, K.; Moriyon, I. Rationale for the design of shortened derivatives of the NK-lysin-derived antimicrobial peptide NK-2 with improved activity against Gram-negative pathogens. J. Biol. Chem 2007, 282, 14719–14728.
[54]  Hawrani, A.; Howe, R.A.; Walsh, T.R.; Dempsey, C.E. Origin of low mammalian cell toxicity in a class of highly active antimicrobial amphipathic helical peptides. J. Biol. Chem 2008, 283, 18636–18645.
[55]  Zhao, H.; Sood, R.; Jutila, A.; Bose, S.; Fimland, G.; Nissen-Meyer, J.; Kinnunen, P.K. Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action. Biochim. Biophys. Acta 2006, 1758, 1461–1474.
[56]  Pandey, B.K.; Ahmad, A.; Asthana, N.; Azmi, S.; Srivastava, R.M.; Srivastava, S.; Verma, R.; Vishwakarma, A.L.; Ghosh, J.K. Cell-selective lysis by novel analogues of melittin against human red blood cells and Escherichia coli. Biochemistry 2010, 49, 7920–7929.
[57]  Lee, J.K.; Park, S.C.; Hahm, K.S.; Park, Y. Antimicrobial HPA3NT3 peptide analogs: Placement of aromatic rings and positive charges are key determinants for cell selectivity and mechanism of action. Biochim. Biophys. Acta 2012, 1828, 443–454.
[58]  Brender, J.R.; McHenry, A.J.; Ramamoorthy, A. Does cholesterol role in the bacterial selectivity of antimicrobial peptides? Front. Immunol 2012, 3, 195–198.
[59]  Steward, J.C. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal. Biochem 1980, 104, 10–14.
[60]  Oren, Z.; Lerman, J.C.; Gudmundsson, G.H.; Agerberth, B.; Shai, Y. Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: Relevance to the molecular basis for its non-cell-selective activity. Biochem. J 1999, 341, 501–513.
[61]  Mao, D.; Wallace, B.A. Differential light scattering and adsorption flattening optical effects are minimal in the circular dichroism spectra of small unilamellar vesicles. Biochemistry 1984, 23, 2667–2673.
[62]  Matsuzaki, K.; Sugishita, K.; Miyajima, K. Interactions of an antimicrobial peptide, magainin 2, with lipopolysaccharide containing liposomes as a model for outer membranes of gram-negative bacteria. FEBS Lett 1999, 449, 221–224.


comments powered by Disqus