Travel & Places

The Lipid Composition of a Cell Membrane Model Modulates the Action of an Antiparasitic Peptide at the Air-Water Interface

The antiparasitic property of peptides is believed to be associated with their interactions with the protozoan membrane, which calls for research on the identification of membrane sites capable of peptide binding. In this study we investigated the
of 6
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  The lipid composition of a cell membrane modulates the interaction of anantiparasitic peptide at the air – water interface Rondinelli D. Herculano a , Felippe J. Pavinatto b , Luciano Caseli c, ⁎ , Claudius D'Silva d , Osvaldo N. Oliveira Jr. b a Faculdade de Ciências e Letras de Assis, Universidade Estadual Paulista, Assis, SP, Brazil b Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil c Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP, Brazil d School of Biology, Chemistry & Health Science, Manchester Metropolitan University, Manchester, UK  a b s t r a c ta r t i c l e i n f o  Article history: Received 3 February 2011Received in revised form 16 March 2011Accepted 22 March 2011Available online 5 April 2011 Keywords: Cell membrane modelLangmuir monolayerRaftLipophilic peptideAfrican Sleeping Sickness The antiparasitic property of peptides is believed to be associated with their interactions with the protozoanmembrane, which calls for research on the identi fi cation of membrane sites capable of peptide binding. In thisstudyweinvestigatedtheinteractionofalipophilicglutathioinepeptideknowntobeeffectiveagainstthe  AfricanSleeping Sickness (  ASS  — African Trypanosomiasis ) and cell membrane models represented by Langmuirmonolayers.Itisshownthatevensmallamountsofthepeptideaffectthemonolayersofsomephospholipidsandother lipids, which points to a signi fi cant interaction. The latter did not depend on the electrical charge of themonolayer-forming molecules but the peptide action was particularly distinctive for cholesterol +sphingomyelinmonolayersthatroughlyresembleraftsonacellmembrane.Using insitu polarization-modulatedinfraredre fl ectionabsorptionspectroscopy (PM-IRRAS),we found thatthe orientationofthepeptideisaffectedbythephospholipidsanddioctadecyldimethylammoniumbromide(DODAB),butnotinmonolayerscomprisingcholesterol+sphingomyelin.Inthismixedmonolayerresemblingrafts,thepeptide stillinteractsandhassomeinducedorder,probablybecausethepeptidemoleculesare fi ttedtogetherintoacompactmonolayer.Therefore,the lipid composition of the monolayer modulates the interaction with the lipophilic glutathioine peptide, andthis may have important implications in understanding how the peptide acts on speci fi c sites of the protozoanmembrane.© 2011 Elsevier B.V. All rights reserved. 1. Introduction Thesynthesisandidenti fi cationofsmallpeptidestoserveasdrugsfor neglected diseases has received increased attention[1], as haveantiparasitic peptides[2 – 4]shown to act against the fl agellatedprotozoan, t. rhodescience, the causal agent of  African Sleeping Sickness (  ASS  — African Trypanosomiasis )[5].The latter peptides are being investigated as an alternative to the traditional therapeutic treat-ments using arsenical compounds[6], and have shown enhancedtrypanocidal activity due to esteri fi cation increasing hydrophobicity/surface activity and enhancing protozoan, cell membrane disruption.In a recent paper, we showed that antiparasitic peptides wereamenabletoformstableLangmuirmonolayersattheair/waterinterface,whose surface pressure isotherms displayed a region of negativecompression modulus, in the fi rst compression – decompression cycle[7]. Upon combining information from BAM and PM-IRRAS measure-ments, we concluded that this negative elasticity was ascribed toaggregation during compression caused by intermolecular associations.Also in ref.[7], we noted that the peptide induced expansion in thesurface pressure isotherms of dipalmitoyl phosphatidyl choline (DPPC)monolayers used as a cell membrane model.In this paper, we investigate the possible generality of action of these antiparasitic glutathioine peptides on various types of mono-layermodelsthatmaymimiccellmembranesystems.Inparticular,weused zwitterionic phospholipids, such as dipalmitoyl phosphatidyl-ethanolamine (DPPE), in addition to the negatively charged dipalmi-toyl phosphatidic acid (DPPA) and dipalmitoyl phosphatidyl glycerol(DPPG), the positively charged dioctadecyldimethylammonium bro-mide (DODAB), and lipids known to form rafts in cell membranes(sphingomyelin and cholesterol). The motivation for using the rafts isthattheyarerelevantforvariouscellmembranephenomena[8],suchas signaling and cell transport. The methods employed to analyze themonolayers were surface pressure isotherms and polarization-modulated infrared re fl ection-absorption spectroscopy (PM-IRRAS).With the experiments reported here it is possible to extend theanalysisinref.[7]andshowthattheconformationofthepeptideattheair/water interface depends on several factors, including its relativeconcentration and the lipid composition. Biochimica et Biophysica Acta 1808 (2011) 1907 – 1912 ⁎ Corresponding author at: Rua Prof. Artur Riedel, 275-09972-270-Diadema, SP,Brazil. Tel.: +55 11 50843759; fax: +55 11 40436428. E-mail address: Caseli).0005-2736/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bbamem.2011.03.012 Contents lists available atScienceDirect Biochimica et Biophysica Acta  journal homepage:  2. Materials and methods The compound S-(2,4-dinitrophenyl)glutathioine di-2-propyl ester,whosechemicalstructureisshowninFig.1,wassynthesizedasdescribedin ref.[2]. The lipids DPPC, DPPE, DPPA, DPPG, DODAB, cholesterol andsphingomyelinwereacquiredfromAvantiPolarLipidsandused withoutfurther puri fi cation. For preparing Langmuir monolayers, the lipids andthe peptide were dissolved in chloroform (Merck) at a concentration of 0.5mg/mL. The solutions were spread on an aqueous subphase with pH7.5(consistingof0.1molL  − 1 NaCl(Merck),and0.001molL  − 1 phosphatebuffer (Na 2 HPO 4 :NaH 2 PO 4 =1:1, Merck)). Peptide – lipid mixed mono-layerswereobtainedbyspreadingthedrug-containing solutionafterthelipidmonolayerhadbeenformed,butatalargeareapermolecule(atzerosurface pressure). After 10 – 15min to allow for solvent evaporation, theair – waterinterfacewascompressedwithtwomovablebarriersatarateof 25cm 2 min − 1 .Areaspermoleculewerecalculatedassumingthatalldrugmolecules remained at the interface. For mixed drug-lipid monolayers,usewasmadeoftheareaperlipidmolecule(assumingthemoleculewasaloneattheinterface),inordertobetterevaluatethechangesinthelipidmonolayersinducedbythesmallamountsofthedrug(2%inmolorless).These experiments were performed in a NIMA trough (model 601M,subphase volume: 500mL).Polarization-ModulatedInfraredRe fl ection-AbsorptionSpectroscopy(PM-IRRAS)wasperformedusingaKSVPMI550instrument(KSV,BiolinScienti fi c Oy, Helsinki, Finland). The experimental setup was similar tothat described by Blaudez and co-workers[9].The Langmuir trough (mini KSV) was mounted so that the light beam reached the monolayerat a fi xed incidence angle of 80°, for which the upward-oriented bandsindicate a transition moment preferentially on the surface plane,whereas downward-oriented bands indicate preferential orientationperpendicular to the surface.All the experiments were carried out in a class 10,000 clean roomat the temperature of 23.0±0.2 °C. 3. Results and discussion The surface pressure-area isotherm for the pure peptide spread atthe air – water interface was reported in[7], showing a decrease inpressure upon compression between 70 and 60 Å 2 . This negativecompressibility modulus was associated with domain formation withincreased lateral fl uidity according to Brewster Angle Microscopyimages[7]. The possible reorganization of the peptide molecules atthe interface was discarded on the basis of the PM-IRRAS data, whichsupported the hypothesis of a pre-collapse of the monolayer[7].The set of surface pressure isotherms for the monolayers withmixed peptide – lipid for 2 mol% of the peptide are shown inFigs. 2 – 5.Overall, there is a tendency of the isotherms to expansion at lowsurface pressures and in some cases a small condensation in the highsurfacepressures(forDPPEandDPPG).Therefore,thepeptideappearsto be incorporated in the monolayer at low densities, and be expelledfrom the monolayer at high pressures, in some cases even causing theneighboringlipidstobemoredenselypacked.Distinctiveresultswereobtained for DPPA, which was more expanded than the otherphospholipids (seeFig. 3B), and for cholesterol and cholesterol/sphingomyelin. For cholesterol, a large condensation was observed atthe condensed phase, but this did not occur when cholesterol wasmixed with sphingomyelin (seeFig. 5). The condensation induced bycholesterol is consistent with reports in the literature[10], and isexplained by cholesterol's ability to interact with hydrophobic chains[11,12]. We shall resume the discussion of the effects of addingsphingomyelin later in the paper. As for the results for DODAB, apositively charged lipid, the similarity with the data for thezwitterionic and negatively charged phospholipids means that thechargeofthemonolayer-formingmoleculesdoesnotappeartoexertagreat in fl uence on monolayer properties. This is not surprising sincethe esteri fi ed antiparasitic peptide is not charged and should not beexpected to participate in any type of ionic interaction.A general feature of the isotherms is that small amounts of thepeptide are suf  fi cient to affect the monolayer, which points tointeraction effects caused by the peptide – lipid interaction as alreadyobserved with other peptides[13,14].This is illustrated for DPPA in Fig. 6, whilethe datafor theother lipidsareomitted. Signi fi cantly, themonolayer condensation at high surface pressures for some lipidsdisappears as the amount of peptide is increased. The disappearancemay be caused by the increased area occupied by the drug itself ashigher proportions are used in the mixed monolayers. Fig. 1. Chemical structure of the peptide used in this work. 406080100120140010203040506070    S  u  r   f  a  c  e   P  r  e  s  s  u  r  e   (  m   N   /  m   ) Area per lipid (Å 2 ) 406080100 Area per lipid (Å 2 ) DPPCDPPC + drugDPPEDPPE + drug A 010203040506070    S  u  r   f  a  c  e   P  r  e  s  s  u  r  e   (  m   N   /  m   ) B Fig. 2. Surface pressure-area isotherm for zwitterionic lipids and mixed peptide (2% inmol)-lipid monolayer. For the x-axis, “ area per lipid ” means that only lipid moleculeswere taken into account in the calculation.1908 R.D. Herculano et al. / Biochimica et Biophysica Acta 1808 (2011) 1907  – 1912  In order to better visualize the effects of the peptide on the DPPAmonolayer, we compared the theoretical and experimental areas permolecule based on the surface pressure-area for each compound inthemixedmonolayer,forapressureof30 mN/m(whichisbelievedtocorrespond to the pressure in a real membrane[15]). For obtainingthe theoretical areas, we used the following expression:  A = x 1  A 1 + x 2  A 2 where x 1 and x 2 are the molar fractions of components 1 and 2,respectively, and A 1 and A 2 are the molecular area of each componentinferred from the isotherms of neat components.Fig. 6B showspositiveexcessareas,whichindicatesexpansionofthemonolayerdueto repulsive interactions or loose molecular accommodation at thesurface. We calculated the excess area for all mixed drug-lipidmonolayers, and the behavior is the same, pointing again to repulsiveinteractions between the peptide and the lipid.Fig. 7shows the PM-IRRAS spectrum for a Langmuir monolayer of the pure peptide, which features inverted amide I bands at 1577 cm − 1 sincetheC=Ogroupslieparalleltotheair – waterinterface,asreportedin[7].The direction of the band is preserved upon compression, thus indicating no reorientation. The band at 1677cm − 1 is assigned todisordered structures, which is reasonable considering the lack of secondary structure of the small peptide. The amide II band (C – Nvibration) appears at 1521 cm − 1 . 6080100120140160010203040506070    S  u  r   f  a  c  e   P  r  e  s  s  u  r  e   (  m   N   /  m   ) Area per lipid (Å 2 )Area per lipid (Å 2 ) DPPGDPPG + drugDPPADPPA + drug A 3035404550556065707580859001020304050607080    S  u  r   f  a  c  e   P  r  e  s  s  u  r  e   (  m   N   /  m   ) B Fig. 3. Surface pressure-area isotherm for negatively charged lipids, and mixed peptide(2% in mol)-lipid monolayer. For the x-axis, “ area per lipid ” means that only lipidmolecules were taken into account in the calculation. 6080100120140160010203040506070    S  u  r   f  a  c  e   P  r  e  s  s  u  r  e   (  m   N   /  m   ) DODABDODAB + drug Area per lipid (Å 2 ) Fig. 4. Surface pressure-area isotherm for a positively charged lipid (DODAB), andmixedpeptide(2%inmol)-lipidmonolayer.Forthex-axis,theareaperlipidmeansthatonly lipid molecules were taken into account in the calculation. 303540455055600102030405060    S  u  r   f  a  c  e   P  r  e  s  s  u  r  e   (  m   N   /  m   ) CholesterolCholesterol + drug A 80120160200240081624324048    S  u  r   f  a  c  e   P  r  e  s  s  u  r  e   (  m   N   /  m   ) sphingomyelin + cholesterol (1:1 mol)sphingomyelin + cholesterol (1:1 mol)+ drug B Area per lipid (Å 2 )Mean Molecular Area (Å 2 ) Fig. 5. Surface pressure-area isotherm for cholesterol and sphingomyelin + cholesterolmixed monolayer, andmixed peptide(2% inmol)-lipid monolayer. Forthex-axis, “ areaper lipid ” means that only lipid molecules were taken into account in the calculation.1909 R.D. Herculano et al. / Biochimica et Biophysica Acta 1808 (2011) 1907  – 1912  Consistentwiththesurfacepressureresults,thePM-IRRASinFig.8shows practically the same behavior for the mixed monolayerscontaining2 mol%ofthepeptideandDPPC,DPPGorDODAB (thedatafor DPPEandDPPA wereomittedbecausetheyaresimilar).Again,thechargestateofthephospholipidhasnoeffectontheinteractionofthemonolayer with the peptide. The bands appearing in the fi gure areassignedtoamidebands.Now,theamideIbandisdirectedupward,incontrast to the downward direction of the monolayer of the neatpeptide. Therefore, the dipole moment of the amide vibration wasreorientedfromaparalleltoaperpendicularpositioninrelationtotheair – waterinterface.Thisisincontrastwiththe fi ndinginourpreviouspaper[7], for which the peptide appeared not to be reoriented byDPPC. The discrepancy may be explained by the fact that in theprevious paper the PM-IRRAS measurement was performed for amixed monolayer containing 10 mol% of the peptide, and thereforethe peptide molecules in excess maintained their geometricalorientation as in a pure monolayer. Also, the amide I band has beensplit in two peaks at 1640 and 1690 cm − 1 . The fi rst one results fromthe re-arrangements of the peptide upon interaction with the lipidmolecules,whilethatat1690 cm − 1 representsthedisorderedamideIband.Thelatterisshiftedtohigherenergiesuponinteractionwiththelipid, which means that accommodation of the peptide into the lipidmembrane leads to stable freedom for the C=O vibration moment.The amide II also appears shifted, at ca. 1555 cm − 1 , owing tointeraction of C – N vibration with side chains[16].Signi fi cant differences were observed in the spectrum for thepeptide-containing cholesterol in the monolayer, as shown inFig. 8B, 1520253035404501020304050607080    S  u  r   f  a  c  e   P  r  e  s  s  u  e   (  m   N   /  m   ) DPPADPPA + drug 0.25%DPPA + drug 0.50%DPPA + drug 0.75%DPPA + drug 1.00%DPPA + drug 1.25%DPPA + drug 2.00% A drug fraction % Ideal AreaExperimental Area B Mean Molecular Area (Å 2 )    M  o   l  e  c  u   l  a  r  a  r  e  a   (    Å    2    ) Fig. 6. Surface pressure-area isotherm for DPPA and mixed peptide (% in mol indicatedin the insert)-DPPA monolayer. Panel B shows the comparison between the averagemolecular areas of the mixed monolayer at 30 mN/m obtained experimentally andtheoretically. The areas from both the lipid and peptide were taken into account. Theexcess area may be calculated subtracting the experimental from theoretical values. 1750170016501600155015001450    P   M  -   I   R   R   A   S  s   i  g  n  a   l   (  a .  u .   ) wavenumber (cm -1 ) 16771521 Fig. 7. PM-IRRAS spectrum for the peptide monolayer at 0 mN/m. At higher surfacepressures, the bands have their intensity increased with no signi fi cant change in thewavenumber position, in agreement with ref.[7]. 180017001600150014001527    P   M  -   I   R   R   A   S  s   i  g  n  a   l   (  a .  u .   ) DODABDPPCDPPG169016401555 A 18001700160015001400    P   M  -   I   R   R   A   S  s   i  g  n  a   l   (  a .  u .   ) wavenumber (cm -1 )wavenumber (cm -1 ) CholesterolRaft168816251665 B Fig. 8. PM-IRRAS spectra for peptide – lipid (as indicated in the inset) monolayer at30 mN/m. Raft indicates a mixture of 1:1 cholesterol + sphingomyelin.1910 R.D. Herculano et al. / Biochimica et Biophysica Acta 1808 (2011) 1907  – 1912  where the amide I band is not deconvoluted. It appears as a singleband at 1688 cm − 1 with a shoulder at ca. 1650 cm − 1 , pointing to lessunfolding of the peptide when interacting with cholesterol. It is likelythat interaction with cholesterol makes it easier for the peptide to beincorporated in the lipid membrane with further accommodation.Also, for the monolayer simulating roughly a “ raft ” (sphingomyelin +cholesterol), the spectra show even more signi fi cant changes: theamideIbandisdownward,withthemainpeakassignedtodisorderedstructures shifted to lower energies (1665 cm − 1 ). Also, a band at1625 cm − 1 due toturnsfor amidebondsappears.Thismeansthatthepeptide is not reoriented when incorporated in the “ raft ” monolayer,in contrast to the other types of monolayer. Nevertheless, there is stillpeptide – lipid interaction leading to ordered structures. This isassociated with the peptide molecules being fi tted into a compactmonolayer, which does not allow for peptide reorientation in themembrane.Overall,themostimportantresult – consideringthesurfacepressureisotherms and PM-IRRAS spectra – is associated with the fact that aspeci fi c composition of the monolayer, namely that prepared withcomponents roughly simulating rafts, provides signi fi cantly differentresults from the other model membrane systems. Not only theorientation of the peptide at the air/water interface was affected butalso the folding of the peptide was changed leading to the existence of turns. A scheme depicting the conformation of the peptide is given inFig.9,whichdependsonthelipidcompositionofthemonolayer,ontherelative peptide concentration and on the surface pressure. Fig.9. Schematicmodelforthemolecularorientationforthepeptidein:(A)neatmonolayer;(B)monolayermadewithphospholipidspossessingtwoaliphaticchainsandcontainingup to 2% of peptide; (C) sphingomyelin-cholesterol monolayer containing up to 2% of peptide. The relative sizes of molecules are only illustrative and out-of-scale.1911 R.D. Herculano et al. / Biochimica et Biophysica Acta 1808 (2011) 1907  – 1912
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!