Synthetic amphipathic helical peptides that
mimic apolipoprotein A-I in clearing cellular
A J Mendez, … , J P Segrest, J F Oram
J Clin Invest.
Clearance of excess cholesterol from cells by HDL is facilitated by the interaction of HDL
apolipoproteins with cell-surface binding sites or receptors, a process that may be important
in preventing atherosclerosis. In this study, synthetic peptides containing 18-mer
amphipathic helices of the class found in HDL apolipoproteins (class A) were tested for their
abilities to remove cholesterol and phospholipid from cultured sterol-laden fibroblasts and
macrophages and to interact with cell-surface HDL binding sites. Lipid-free peptides
containing two identical tandem repeats of class A amphipathic helices promoted
cholesterol and phospholipid efflux from cells and depleted cellular cholesterol accessible
for esterification by acyl CoA/cholesterol acyltransferase, similar to what was observed for
purified apolipoprotein A-I. Peptide-mediated removal of plasma membrane cholesterol and
depletion of acyl CoA/cholesterol acyltransferase-accessible cholesterol appeared to occur
by separate mechanisms, as the latter process was less dependent on extracellular
phospholipid. The dimeric amphipathic helical peptides also competed for high-affinity HDL
binding sites on cholesterol-loaded fibroblasts and displayed saturable high-affinity binding
to the cell surface. In contrast, peptides with a single helix had little or no ability to remove
cellular cholesterol and phospholipid, or to interact with HDL binding sites, suggesting that
cooperativity between two or more helical repeats is required for these activities. Thus,
synthetic peptides comprising dimers of a structural motif common to exchangeable
apolipoproteins can mimic apolipoprotein […]
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Helical Peptides That
Mimic Apolipoprotein A-I
Clearing Cellular Cholesterol
Armando J. Mendez,* G. M. Anantharamaiah,t Jere P. Segrest,*and John F.Oram* *Department of Medicine RG-26, University of Washington, Seattle, Washington 98195; and
*Departmentof Medicine and Biochemistry, and theAtherosclerotic Research Unit,
Universityof AlabamaatBirmingham Medical Center,Birmingham, Alabama 35294
Clearance of excesscholesterol from cells by HDL is facili-tatedby the interaction ofHDL apolipoproteins with
cell-surface binding sites or receptors, a process that may be important in preventing atherosclerosis. In this study,
syn-theticpeptides containing18-meramphipathichelices of the class found in HDL apolipoproteins (class A) were tested for their abilities to remove cholesterol and phospholipid from culturedsterol-ladenfibroblasts and macrophages and to interact with cell-surface HDL binding sites. Lipid-free
peptides containingtwoidenticaltandem repeats of class A
amphipathichelicespromoted cholesterol and phospholipid efflux from cells and depleted cellular cholesterol accessible for esterification by acyl CoA/cholesterol acyltransferase, similartowhatwasobserved forpurified apolipoprotein A-I. Peptide-mediated removal ofplasma membrane choles-terolanddepletionofacyl CoA/cholesterol
acyltransferase-accessiblecholesterolappearedtooccurby separate mecha-nisms,asthe latter processwaslessdependenton
extracellu-larphospholipid. Thedimeric amphipathichelicalpeptides alsocompetedfor
high-affinityHDLbindingsiteson choles-terol-loadedfibroblasts anddisplayedsaturable
high-affin-itybinding tothecell surface. In contrast,peptides with a singlehelixhadlittleor no
abilitytoremovecellular choles-terol and phospholipid, or to interact with HDL binding sites, suggesting that
cooperativitybetween two or more helical repeats is required for these activities. Thus, syn-theticpeptides comprisingdimersofastructural motif com-mon toexchangeable
apolipoproteinscanmimic apolipopro-tein A-I in both binding toputative cell-surface receptors and clearing cholesterol from cells. (J. Clin. Invest. 1994.
94:1698-1705.) Key words: synthetic peptides * amphi-pathichelices-
high-densitylipoprotein-cellularcholesterol efflux * HDLreceptors
The roleofHDLinpromotingthe removal ofexcesscholesterol fromcultured cellshasbeen wellestablished; however,
mecha-AddresscorrespondencetoJohn F.Oram, Departmentof Medicine RG-26, UniversityofWashington, Seattle,WA98195.
Receivedfor publication27April1993 andinrevisedform2 June 1994.
nisms by which this occurs remain poorly understood. HDL appears to remove cholesterol from cells by both passive and active processes. Cholesterol that is synthesized by cells or ingested by receptor-mediated endocytosis of lipoproteins can be transported rapidly to the plasma membrane where it pas-sively desorbs into the extracellular fluid and reabsorbs to phos-pholipid-containing particles ( 1, 2). When cells are overloaded with sterol, however, cholesterol accumulates within an intracel-lular pool that is accessible to the enzyme acyl CoA/cholesterol acyltransferase
(ACAT),'thus allowing cells to store excess cholesterol as ester-rich lipid droplets. Under this condition, excretion ofexcessACAT-accessible cholesterolcanbe facili-tatedbyanactive process (3, 4) that is stimulated by the interac-tionof HDL apolipoproteins with cell-surfacebinding sites or receptors (5).
A common secondary structural motif described for the plasma lipoprotein-associated apolipoproteins is the
amphi-pathic helix (reviewed in 6 and 7). Basedontheproperties of
amphipathichelices found in the exchangeableapolipoproteins,
asynthetic peptide, designated 18A, wasdesigned and synthe-sized(8-10).This classAamphipathic helical peptide has been showntoform stablecomplexes withphospholipidssimilarto thoseformedbyapolipoproteinA-I, the major protein constit-uentofHDL (8-10). A dimer of 18A linked with aproline residue (37pA) has greater lipid affinity than 18A,presumably
dueto cooperativity provided by twocovalently linked lipid-bindingpeptides. Incontrast, an analogue of 18A(18R), with theposition of charged residues reversed along the polar face of the helix, can only marginally interact with lipid (8-10). Synthetic amphipathichelicescanmimic several of the
biologi-cal effects of apo A-I, and the potencies of these effects are related tothelipid binding affinities ofthe synthetic peptides
(11-15).The present study examines whether these synthetic
amphipathic peptides could also mimicthe abilityoflipid-free
apoA-IandHDL topromote efflux ofexcess cholesterol from cultured cells andtointeract withHDLbindingsitesonthecell surface.
Lipoprotein andapolipoprotein isolation. LDL and HDL3 (hereafter referredto asHDL)werepreparedbysequential ultracentrifugationin thedensityintervals 1.019to1.063and 1.125to1.21g/ml, respectively. LDLwasacetylated bythemethod ofGoldsteinetal.(16)and labeled
1.Abbreviationused in thispaper:ACAT, acylCoA/cholesterol
acyl-transferase. J. Clin. Invest.
©The AmericanSocietyfor ClinicalInvestigation,Inc. 0021-9738/94/10/1698/08 $2.00
with [3H]cholesteryl linoleate asdescribed (17).HDL wasiodinated by theiodine monochloride method describedbyBilheimer(18).apo A-I waspurifiedfromHDL aspreviously described (4). Purified apo A-Iexhibited a single band when examined by sodiumdodecylsulfate polyacrylamide gel electrophoresis with Coomassie bluestaining.
Phospholipidvesicles. Vesicles werepreparedbythe cholate dial-ysis method (19). For cell cultureexperiments,the vesicles were added tothemediumimmediatelyprior toaddingthemedium to cells.
Peptides. Peptides were synthesized andpurifiedasdescribed pre-viously (9). The primary sequences of the peptides used in this study are shown in Table I.
Cellculture. Human skin fibroblastswerecultured and maintained as previously described (4, 5, 18). Usually 70,000 cells/2 ml medium in 35 mm dishes or 15,000cells/l ml medium in 16mmwells were seededand grown to confluencebeforeuseinexperiments. Cellswere loaded with nonlipoproteincholesterolby incubation for 48 h with DME containing 2 mg/ml fatty acid-free BSA (Sigma Chemical Co., St. Louis,MO) and 30
Msg/mlcholesterol. Cellswerethenincubated over-night (16-18 h) in DME containing 1 mg/ml BSA and0.2 sCi/ml [3H]cholesterol(40-60Ci/mmol,AmershamCorp.,Arlington Heights, IL) tolabel plasma membrane cholesterol. Forexperimentscontaining [3H]choline-labeled phospholipids (see below), cells were labeled with0.1 uCi/ml[14C]cholesterol(50-60 Ci/mmol,Amersham Corp.). Choline-containing phospholipids werelabeled by including 1 tCi/ml [3H]choline chloride (75-85 Ci/mmol, Amersham Corp.) duringthe overnight incubation offibroblasts. After incubation with radiolabel, celllayerswererinsed five times withPBS containing 0.1%BSA(PBS/ BSA) before the additionof experimental media.
Resident mouse peritoneal macrophage cultures wereestablished from unstimulated Swiss Webster micebythe method of Edelson and Cohn(20). Peritonealcells were harvestedfrom 20-25mice inPBS, pooled at4°C, collected by centrifugation (400g, 10min, 4°C), and washedoncein DME. Cellswereplated in DMEcontaining20%(vol/ vol)FBS at 1 x 106cells/0.5ml/16mmwell. After incubation for2 h at37°C, nonadherent cells were removed by washing three times with DME.Adherent cells were maintained in culture with DMEcontaining 20% total bovineserumfor 5 dbeforeuseinexperiments. Macrophages werecholesterol loaded by incubation with 50
jig/mlof[3H]cholesteryl linoleate-labeledacetylated-LDL for 24 h. Cells were then washed two times with serum-free DME and incubatedovernightwith serum-free DMEtoallow equilibration ofcholesterol pools.
Cholesterol andphospholipid effluxfrom cells.After theappropriate labeling protocol,cellswereincubatedat37°Cwith DMEcontaining1 mg/ml BSA and the indicated additions.At the indicated times, the efflux media were collected and celllayerswererinsed three times with PBS. Mediaandcells were stored frozen at-20°Cuntil extraction for lipid andproteincontent. Efflux mediawere extractedbythe method of Folchetal.(21)and,in someexperiments (labeled with cholesterol), counteddirectly afterabriefcentrifugation to remove cell debris. Cho-lesterol-labeledcell layerswereextracted withhexane/isopropanol(3:2, vol/vol) asdescribed (5, 17). Cells containing labeled phospholipids wereextracted with 1 ml ofisopropanolfor 1 h, and then with hexane/ isopropanolasabove.
Sterolspecieswereseparatedbythinlayer chromatographyonsilica gel Gplates developed in hexane/diethyl ether/methanol/acetic acid (120:30:10:1.5, vol:vol:vol:vol). Choline-containing phospholipids were separated by thin layer chromatography on silica gel H plates developedinchloroform/methanol/water (65:35:4, vol:vol:vol). Lipid spots were identified by staining with 12 vapor and comigration with standards. After allowing 12 stainto dissipate, appropriate spots were taken for determination ofsterol mass and/or scintillation counting. Labeledcholesterol efflux and labeledphospholipidefflux were calcu-lated as the percent of label appearing in the media, relative to the total (media+ cell)radioactivityfor each lipid.
Cell cholesterol esterification. To assess the relative activity of ACATfollowing incubationwithtestmedium, cellswerewashed once with PBS and incubated for 1 hat 370Cwith DMEcontaining9qM '4C-oleate (50-60mCi/mmol,AmershamCorp.)boundto3
(5). Cellswerechilled onice and washed twice with PBS/BSA and twice withPBS. Cellswerestoredfrozenat-20'Cuntil extraction for lipidandproteinasdescribedabove. Celllipidswereseparatedbythin layer chromatographyasdescribed abovetodeterminecholesterylester radioactivity. Incorporationof radiolabel into cholesterol esters
repre-sentscholesterolesterification by ACAT, and results werequantitated as picomoles of ['4C]oleate incorporated into cholesteryl esters per milligramof cellprotein.
high-affinityHDLbindingsitesonfibroblastswastestedusingasequential competitive binding assay (22). Cholesterol-loaded fibroblasts were washed twice with PBS/BSA and chilled oniceduring athird wash. Cellswerethen incubated at0C (on ice) with Hepes-buffered DME containing1mg/mlBSA and theindicated concentration ofcompetitor. After3h,the cellswererapidlywashed three times with ice-cold PBS/ BSA and incubated for 1 hat0CwithHepes-bufferedDMEcontaining 1 mg/mlBSAand 5
/sg/mlof'"I-HDL. Specific activityof'25I-HDL ranged from200 to 300cpm/ngHDLproteinwith less than 2% of label associated withlipids.Cellswerewashed five times with ice-cold PBS/ BSAandtwice with PBS. Celllayersweredissolved in 0.1 MNaOH, andaliquotsweretakentoquantitate radioactivityandprotein.
To measure directbinding ofpeptides 18A and 37pA to the cell surface, tyrosylresidues were labeled with1251 bytheiodine monochlo-ride method (18, 23),and radiolabeled peptideswere extensively dia-lyzed.Thespecific activities ofthepeptides rangedfrom 800 to 2,200 cpm/pmol.Fibroblasts were incubated with 10 nM to 2jMradiolabeled peptide for 3 h at 0°C as described above for binding of
lodination of thepeptideshad no effect onbiological activitybasedon results showing that iodination of37pA did not change its ability to inhibit cellular cholesterol esterification and that unlabeled 37pA blocked cell-surfacebindingofiodinated37pA (datanotshown).
Othermethods.Proteinconcentrations wereestimated bythe method ofLowryetal.(24) usingBSA as the standard. Statistical differences werecalculated byStudent's ttest. Kinetic analyses of peptidebinding and lipid efflux were performed using the SAAM30 computer program.
Theeffects of HDL,apo A-I,andsynthetic amphipathic helical peptides on cholesterol clearance from cultured human skin fibroblasts were determined by two independent assays. First,
as an index of cholesterol efflux from the plasma membrane,
the appearance of radiolabeled cholesterol in the medium was measured after cholesterol-loaded cells were labeledovernight
with trace quantities of [3H]cholesterol. Second, depletion of cellular pools accessible for esterification by ACAT was esti-mated by the decrease in incorporation of
[14C]oleate into cholesteryl esters.Results show thatHDLand lipid-freeapo A-I can promote [3H]cholesterol efflux and decrease cholesterol esterification (Fig. 1,A and B). Both apo A-I and HDL had maximal effects on cholesterol esterification at - 400 nM,
al-thoughHDLhada greatercapacity to promote [3H] cholesterol efflux,presumably because of exchange between HDL choles-terol and plasma membrane tracer (1, 2).
l1000 rAc18A-NH2 60
400 2 200
0 3 6 9 0 2 4
Table 1. Amino Acid Sequence of Synthetic Peptides
_ + +_
+ -+ - +
Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-_ - +
Glu-Leu-Glu-Lys-Ala-Phe 37pA 18A-Pro-18A
37aA 18A-Ala-18A Ac-18A-NH2 Acetyl-18A-NH2
Figure1. Effect of HDL, apo A-I, and synthetic amphipathic helical peptidesoncellular cholesterol effluxand esterification. Cholesterol-loaded human skin fibroblasts, labeled with[3H]cholesterol,were incu-bated with serum-free DME containing 1 mg/ml BSA and the indicated concentrations of HDL or peptide. After 6 h, the medium was removed and cells werefurtherincubatedfor 1 h with medium containing 14C-oleateto measurecholesterolesterification byACAT.Celland medium lipids were extracted and separated by thin-layer chromatography, and cholesterol andcholesterylesterradioactivitywasquantitated. Choles-terolefflux(opensymbols)wascalculatedasthe percent of total(cell andmedium) [3H]cholesterolappearingin the medium. Total
[3H]-cholesterolcounts were18,605±1,211 cpm/dish.Cholesterol esterifica-tion(closedsymbols)wascalculatedaspicomolesof'4C-oleate incorpo-ratedintocholesterylestersnormalized forcellprotein. The resultsare themeansofduplicatedishesrepresentative oftwo tofive experiments. Thecoefficientsofvariationforcholesterol efflux and cholesterol esteri-fication assayswere 5.9% and6.9%, respectively.
shown). None of the other peptides tested had any effect on
incorporation of oleate intotriglycerides comparedtocontrols. Thus, even thoughAc-18A-NH2 appeared toperturb thelipid
environment of the plasma membrane, itwas no more active than 18A or18R in promotingcholesterol efflux.
In contrast to the monomers, dimers of 18A linked with proline (37pA) oralanine (37aA) wereabletoinhibit choles-terol esterification and promote[3H]cholesterol efflux (Fig. 1, Dand F). The maximumextentofdepletion of ACAT substrate for thesepeptideswascomparabletothatforHDLand apo A-I.Time course studiesatsaturating concentrations of HDL, apo A-I, and 37pA demonstrated equivalentrates for thedecrease incholesterol esterification over16 h ofincubation, while18A remained withouteffect (datanotshown).
Thesynthetic peptides werecompared to HDLand apo A-Ifor theirabilitytopromote clearance of cholesterol from sterol-laden mouseperitonealmacrophages,acultured cell model with features common tofoam cells of atherosclerotic lesions(16).
Cellswereoverloaded with cholesterol by incubation with
ace-tylated-LDL containing [3H] cholesteryl linoleate to radiolabel cellularpools of free and esterified cholesterol by the scavenger receptor pathway (16, 25). HDL and apo A-I were able to promote effluxof [3H]cholesterol into themedium and decrease the amount ofcholesterolavailable foresterification by ACAT (Fig. 2,AandB). Peptides37pA and 37aAwerealso effective inpromoting[3H cholesterolefflux and decreasing [ 4C]oleate
20- A Figure 2. Effect of synthetic
20 A amphipathic peptides on
15 cholesteroleffluxfrom mouseperitoneal
macro-CiF( De D D D * phages.5-d-oldmacrophage
-cultures were incubated for 24 h withserum-freeDME 4 containing 0.5 mg/ml BSA
eX23 * * * and 501Lg/ml
acetylated-@B** * LDL,whichhadbeen
la-? t E 2
.c- * g g g g g linoleate. Cellswerewashed
and incubated with medium o
50 C for 18 htoallow equilibra-_40 _ - _ * tion of cholesterol pools. *§ 30 _, * * * * S Cellswereincubated with 8jz - - * * * - mediumcontaining 0.5 mg/
*20 ml BSA alone (control) or
I 10 * with the following additions: 60 - ~-D 0.36jAMHDL, 0.36 ,M apo
_ X40 37pA, and 4.5
8zcz30-+ * --- After6h,theefflux medium I=ut20 - * --- wasremoved and cells were 10- pulse-labeled with "C-oleate
o0_ to determine cholesterol es-i -r<; terificationbyACAT. Media
Iandcelllipidswereextracted 0 0 andsubjectedtothin-layer chromatographytodetermine medium(A)andcellular(D)[3H]
-cholesterol,cellular[3H]cholesterylesters(B),and'4C-oleate incorpo-rated into cellularcholesterylesters(C).Resultsarethe mean±SD of threedishes. Total[3H]cholesterol radioactivity was5,770±157 cpm/
dishforall dishes. Values marked withanasteriskaresignificantly different from controlsbyStudent'sttest;P< 0.005 in(B),P
<0.05in(C)and(D).In(A),all values except thosefor 18Aare
:0 5 0
0 5 10 0 0.2 0.4 0.6 0.8
Figure3. Comparison of the effects ofHDL, apoA-I,andsynthetic peptidesoncholesterol and
phosphatidylcholineeffiuxfrom cholesterol-loaded fibroblasts. Cholesterol-cholesterol-loadedfibroblastswereincubated for 18 hwith serum-free mediumcontaining1mg/mlBSA, 0.1 MCi/ml[14C] -cholesterol,and1.0,.sOi/ml[3H ]choline chloride(tolabel cellular phos-phatidylcholine). Labeled cellswerethenincubated with the indicated concentrations of HDLorpeptidesfor 6 h. Cell and mediumlipidswere
extractedandseparated by thin-layer chromatography toquantitate
['4C]cholesterol (AandB) and[
(asdescribed inMethods). Effiuxwascalculatedasthepercentof total (cellandmedium) labelappearinginthemedium. For[3H]
ofduplicatedishes. Thecoefficient of variationwas 10.0% and 9.6% forphospholipidandcholesteroleffiux assays, respectively.
incorporationinto cholesterol esters.
without effect. The appearance of
H]cholesterol in the
me-dium couldbe accounted for
bydecreases inboth cellfree and esterified cholesterol
(Fig.2, C and D). apo
A-I, 37pA,and 37aAcauseda
significantdecrease inesterified cholesterol con-tent. HDLwas less effectiveat
cholesterylesterclearance, and effiux,wasdue
mostlytoaloss of freecholesterol. Itis
notewor-thythat the decrease in cell[
stronglycorrelated (r = 0.794) with the decrease in ACAT-mediated cholesterol esterification. These data demonstrate that apo, A-I,
37pA,and 37aA have similar abilitiestopromote clearance ofexcesscholesterol from
comparedto HDL and apo A-I for their
abilityto promote cholesterol and
effiux from cholesterol-loaded fibroblasts
re-sponsecurvesshowed thatHDL, apo
effiux ofbothradiolabeled cholesteroland
saturatingconcentrations, all three acceptors were able to promote cholesterol effiuxto
nearlythesameextent.Incontrast, HDLwasmuch lesseffectiveat
Peptide18A did notpromoteeffiux of either
phosphatidylcho-line orcholesterol atthe concentrations tested.
Results shown in
Fig.3 suggestthat removalofcholesterol from cells
byapo A-I or
phospholipid-rich particlesthat actas cholesterol acceptors.
To furthertest forthis
37pAto stimulate cellular cholesterol and
promotedefflux of[3H]cholesterol from the
mem-branes of fibroblasts (0 kLM
37pA, Fig.4A). In contrast, the vesicles had no measurable effect on effiux of [3H
(Fig.4 B) nor on
followingthe 6-hincubations. In thepresenceof
37pAhad no additional
abilityto stimulate [3H]cholesterol effiux
(comparedto vesicles alone) atconcentrations up to 2
peptide,however, was still
inhibitingcholes-terolesterificationwhenvesicleswereincludedin the medium,
bytwo-to threefold.This concentrationshiftmay havebeencaused
re-sults supportthe conceptthatefflux of
depends largelyon the presenceof extracellular
phos-pholipid particles,whereas both effiux of
phospholipidsand removalof ACAT-accessible cholesterol oc-cur
possibly involvingthe direct inter-action of
amphipathichelices with cell-surface
bindingsites. The different mechanisms for removal of[3H]cholesterol and ACAT-accessible cholesterol suggest that these sterols reside indifferentcellular
bindingof HDL to
putativecell-surface receptors is
requiredto promote removal ofexcesscholesterol fromtheACAT substrate
pools,and that intact
bythe same mechanism, we
bindingsitesonthe surfaceofcholesterol-loaded fibroblasts. Forthese studies, we
12 A Figure4.Effect of37pAon
cho-10 lesterol efflux(A),
phosphati-8 dylcholineefflux (B),and
cho-lesterol esterification (C)in the
vesi-4 cles. Parallel cultures of choles-2 terol-loaded human skin
fibro-________________ blastswere labeled with either B 0.2,.sCilml [3H]cholesterolor
~ride~~~~~ for 18 hasdescribed in
06 Methods. The cellswerethen in-3Jcubated for 6 hat370(2with the
2 indicated concentrations of
sym--E 2.0 C bols)of 100
,ig/mlof phospha-tidylcholinevesicles added to e1.5
~~~~~~tion,theoneincubations.setof dishesAfter incuba-was
E0. pulsed with 14C-oleate to
cholesterolesterificationas 0 0 0 1 2 3 4 described forFig. 1.
37pAdia and celllayerswereanalyzed
[3H]cholesterol,totalcounts were13,848± 1,469and 14,114±135cpm/
-Figoure5.Interaction of synthetic amphipathic helical peptides with cell-surface HDL bind-ing sites. Cholesterol-loaded fibroblasts were chilledoniceand incu-bated with ice-cold se-rum-free medium con-taining 1 mg/ml BSA and the indicated con-centrations of 37pA, 18A, 18R, apoA-I,or HDL.After 3 h, dishes were rinsed three times with PBS/BSA, 1 ml of serum-freemedium con-taining5
"MI-la-beled HDLwasaddedtoeachdish, and incubationswerecontinued for 1honice. Cellswerethen washed five times with PBS/BSA and twice
withPBS and dissolved in 0.1 M NaOH, and aliquotsweretaken for
125I and protein determinations. Resultsaremeansof duplicate dishes (3.8% coefficient of variation) representingatleastthreeseparate
exper-iments. In the absence of competitors, cells bound 33-3ng12"I-labeled
HDL/mg cell protein (n= 6).
usedasequential competitive binding protocoltoassayfor the
abilityof differentpeptidestoblockhigh-affinityHDLbinding. Cellswerefirstexposed at0°C toincreasingconcentrations of unlabeled competitor and, after removal of unbound competitor,
were then incubated at0°C with
'"I-HDLat a concentration nearitshigh-affinity Kd. Because high-affinityHDLbindingis
essentially irreversible at0°C (26, 27), moleculesthat bindto these sites with similar kinetics as HDLshould remain bound
duringthesecond 0°C incubationandblock high-affinity bind-ingof'251I-HDL.Itwasnecessarytouse asequential competition
assayrather thana moreconventional simultaneous incubation
tominimize the interaction ofpeptidesand
1"I-HDLinsolution, whichmay produce artifacts due to the displacement of
'"I-apolipoproteinsfrom HDLparticles (10, 23).Asimilar
sequen-tialcompetition assayhas been used previouslytoidentify
re-ceptorligandsinplasmathat interact with otherplasma compo-nents(22).
Results using the sequential competitive binding assay
showed that neither 18Anor18R could prevent thesubsequent bindingof '25I-HDL (Fig.5A).In contrast,apoA-Iand37pA effectivelyblocked sitesrecognized by '251-HDL(Fig. 5,Aand B).These sites werealso blockedby37aA(datanotshown). The concentrations for half-maximal inhibition of
1"I-HDLbinding by HDL, apo A-I, and 37pA were 3 X 10-8 M, 1
x 10-8M,and 5X 10-8M,respectively.Theseresults indicate that high-affinity HDL binding sites on cholesterol-loaded
fi-broblasts also interact withapoA-I, 37pA, and37aA, butnot
with 18A and 18R.
We also examined direct binding of iodinated 37pA and 18Ato cholesterol-loaded fibroblasts (Fig. 6). Both peptides boundextensivelytocells,but with different saturation kinetics. Over a concentration range of 0.01 to 2.0 ,iM, 18Abinding
wasnearlylinear. Athigherconcentrations(not shown), some
saturability was evident, but thesehigh-capacity binding sites hadarelatively low-affinity (apparent Kdof10-5 M). In
con-trast, computeranalysis of
"25I-37pAbindingindicated that the concentration curvebest fita single saturablebinding
compo-nent with a Kd of 2 x
component.Thus,37pA appears to interact with saturable cell-surfacebinding sites that haveamuchhigher affinity than those for 18A.
We characterized thespecificity of the 37pA binding sites by competitive binding studies. To assess competition by HDL, weused the sequential competitive binding assay to avoid po-tential interactions between iodinated 37pA and lipoprotein par-ticles. At a concentration of iodinated 37pA near its Kd for high-affinity binding (10-7 M), pretreatment of cholesterol-loaded fibroblasts with unlabeled37pA and 37aA blocked 40% of the iodinated 37pA binding in a concentration-dependent and satu-rable manner (Fig. 7). Pretreatment with 18A had no effect on 37pA binding, even at concentrations of 18A that exhibited twofold greater total cellularbinding than 37pA (Fig. 6). In contrast, pretreatment of cells with apo A-I or HDL blocked approximately 20% of the 37pA binding sites (half that blocked by 37pA). These results suggest that 37pA is interacting with multiple high-affinity binding sitesoncholesterol-loaded fibro-blasts,none of which interactwith 18Awith thesame kinetic properties. A subset of these sites, however, appears to bind both HDL and apo A-I, consistent with results showing that 37pA interacts with thehigh-affinity HDL binding sites.
Resultspresented in this report demonstrate that the synthetic peptides 37pA and 37aA, composed of two 18-mer class A amphipathic helical repeats (6-11), promoted cholesterol ef-flux andinhibited cellular cholesterol esterification in both fi-broblasts andmacrophages. These effectsweresimilartothose mediated by lipid-free apo A-I and HDL, except that HDL had a greatercapacity topromote efflux ofplasma membrane
cholesterol, probably dueto exchange of cholesterol between
0.0 0.5 1.0 1.5 2.0
Figure6.Cell-surfacebindingofiodinated18A and37pAto cholesterol-loadedfibroblasts.Cholesterol-loaded humanskinfibroblastswere incu-batedwithincreasingconcentration of iodinatedpeptidefor 3 hat40C. Then,cellswerewashed,andradioactivitywasdeterminedasdescribed inFig.5. Thespecificactivities of"SI-labeled 18Aand "MI-labeled
37pAwere810 and2,100cpm/pmol,respectively.Each value represents themeanofduplicatedishes(5.5% coefficient ofvariation).
1702 A.J.Mendez, G.M.Anantharamaiah, J.P. Segrest, and J.F. Oram
0 50 100 150 2C
0 2 4 6 8 10
cholesterol.Conversely, addition of37pAtomediumcontaining phospholipid vesicles increased cellular phosphatidylcholine ef-flux severalfold anddepletedoverhalf of the ACAT-accessible cholesterol when there was no further increase in cholesterol efflux from the
One explanation for these findings is that, of these three lipidtransport processes,only phospholipid efflux anddepletion
ofACAT-accessible cholesterolinvolve interactions of amphi-pathic helices withcell-surface binding sites. Promotion of cho-lesterolefflux from the plasma membranemaybeasecondary o
37pAresponsetoformation of membrane-derived
phospholipidcom-V 37aA plexesgenerated byreversible cell-surfacebinding of peptides * 18A orlipid-freeapolipoproteins. Thesenewly formedparticlesmay o apoA-I act as sterol acceptors and enhance passive diffusion of choles-U
HDL3terolfrom the cell surface (1, 2). This two-steplipidtransport 0.0 0.2 0.4 0.8 0.8 1.0 1.2 has been postulated for promotion ofcholesterol efflux from cells by various lipid-free apolipoproteins (25, 28-31). It is
jIM ProteinorPeptide likely, however, that removal of plasma membrane phospholipid InteractionofHDL, apo A-I, andsynthetic peptideswith cell is significant only for those apolipoproteins that are lipid-defi-7pA binding sites. Cholesterol-loaded fibroblasts were chilled cient. In contrast, the interaction ofapolipoproteins with cells d incubatedwith ice-cold serum-freemediumcontaining1 depletes ACAT-accessible cholesterol even when the apolipo-SAand the indicated concentrations of37pA, 37aA, 18A, apo proteins are associated with HDL phospholipid. This process DL. After3h,dishes were rinsed threetimes withPBS/BSA, may be initiated by the release of intracellular signals which ,rum-free medium containing 0.5
jig/ml(0.11 M) "NI-labeled activate a cholesterolexcretory pathway (3, 4).
sadded to eachdish,and incubationswere continued for 1 h Competitive binding studies showed that apo A-I, 37pA, ellsweretreated andradioactivitydetermined as described in and 37aA blocked theinteraction of HDL with its high-affinity tal cellbindingof'"I-labeled37pA(specific activityof2,100
bindingsites on cholesterol-loaded fibroblasts, while 18Ahad 1) in the absenceofcompetitorwas 64pmol/mgcell
protein,no effecton HDL binding. These results strongly suggest that le representsthemeanOf duplicate dishes(3.5%/ coefficient
)n).the interaction of HDL with its cell-surface
bindingsite is medi-ated by two or more tandem repeats of class A amphipathic helices withinHDLapolipoproteins. Thus, peptides containing two of these amphipathic helicescan mimic apo A-I in both roteinparticle and cells. Additionally, 37pA and 37aA stimulating lipid transport from cells and in binding to high-)moteefflux of cellular [3H]phosphatidylcholine, simi- affinity HDL binding sites. The higher efficacy of the dimeric effects of apo A-I, while HDL hadalimitedcapacity peptides comparedtothemonomers cannotbeexplained byan
pholipidefflux. In contrast tothepeptide dimers, pep- extension of the polar and nonpolar faces along the helical axis, taining only a single 18-meramphipathic helix (18A for linkage of the two 18-mer helices bya single amino acid
8A-NH2)had littleor noabilitytostimulate cholesterol causes a350 rotation of these faces. Moreover, linkage by
pro-;pholipid efflux and to deplete cells of substrate for line compared to alanine causes a "kink" between the axes, olesterification by ACAT.Thus, although thesemono- yet 37pA and 37aA were equally active. It is most likely that ebeen showntointeract withlipids and have biological cooperativity between two or more amphipathic helices is re-8-15), theywerealmostcompletely inactive in mim- quiredfor optimum binding to high-affinity sites on the cell ko A-Ior HDLinstimulating lipid transport from cells. surface, as has been described previously for the interaction of sureof fibroblaststoapoA-I or37pA increased efflux HDL apolipoproteins with cells (28, 32). This property may plasmamembrane ofboth cholesterol and phosphati- confer specificity for lipid-poor subclasses of HDL particles ieinparallel to depletion of ACAT-accessible choles- which have morehelices exposed for cellular interactions (7,
plyingthat these three lipid transport processes are 33, 34).
by thesame cellularinteractions. Several other lines Studies withiodinated peptides revealed that both 18A and of
evidence, however,suggest that theseprocessesareoccurring
by independentmechanisms.First,removal of cholesterol from either the
requiresimultaneous release ofplasmamembrane
showingthatHDLstimulatedefflux of bothsources of cholesteroltothesameextent as 37pAand apo A-I but had
abilityto promote efflux of cellular
phospholipids. Second,efflux of radiolabeled choles-terol from the
plasmamembranecanbedissociated from both
phospholipidefflux anddepletionof ACAT-accessible choles-terol. For
example,addition of phospholipid vesicles to the medium increasedcholesterol efflux from theplasmamembrane
severalfold within 6
h,but there was no significant effecton
37pA bind extensively to the surface of cholesterol-loaded fi-broblasts,butonly 37pA bindstosaturable, high-affinitysites. High-affinity binding of 37pAwasnotblockedby 1 8A andwas
only partially blocked by HDL and apo A-I, suggesting that noneof these37pA binding sites interactswith 18A and onlya
subsetinteracts withHDLapolipoproteins. Therefore,although both apoA-Iand 37pA arerecognized by HDL binding sites,
toadditional cell-surface sites that do notbind 18A, apo A-I,
Previous studiesshowed thatmodifying HDLsoas to
de-crease its interaction with cellular high-affinity binding sites
also reduces its ability to remove excess cellular cholesterol
(4, 5, 35-37), suggestingthat thesetwoprocesses areclosely
It2 V- 20
Figure7. surface 3
onicean mg/mlB A-I,orH]
1ml ofse 37pAwas onice. Cc Fig.5. To cpmlpmol Each valu of variatic
thelipopi could prc lartothe forphosi tidescon and Ac-l and phos cholester mershavt
ickingap Expor from the dylcholin
-coupled. The findings that both high-affinity binding to cells and removal of cellular cholesterol requiretwo or moretandem amphipathic helices further support this concept. The current study, however, does not provide direct evidence thatHDL,apo A-I, and dimeric peptides are removing cellular lipids through their interactions with a common binding site. The interactions ofamphipathic helices with the plasma membrane are complex and appearto involve multiple binding sites, and the relative contribution of each of these sites to lipid transport processes is unknown. In addition, comparisons ofsaturation curves re-vealed that the apparent half-maximal concentrations for inhibi-tion of HDL binding were approximately fivefold lower than those for removing cholesterol. However, this may reflect some of the special conditions of these assays, particularly the differ-ent temperatures used (370C vs. 0C). The kinetics of high-affinityHDLbinding is highly temperature sensitive, in that the dissociation rateis much faster at 370C than at 0C (26, 27). Moreover, the active conformation of lipid-free amphipathic peptides in solution may vary with temperature. Despite these differences between assays,therelativeefficacy of the peptides was similar for each process. The half-maximal molar concen-trations for stimulation of cholesterol efflux and inhibition of HDLbinding were approximately fivefold higher for 37pA than for apo A-I. This may be relatedto thefact that apoA-Ihas a molecularmass sixtimes that of 37pA and contains four times the number of amphipathic helices. These and other peptide analogues will be useful tools for testing the hypothesis that HDLapolipoproteins stimulate clearance of cellular cholesterol through the cooperative interaction of their amphipathic helices withcell-surface binding sites or receptors.
Numerouspopulationstudies have shownaninverse corre-lation between plasmaHDLlevels and riskfor coronary heart disease, suggesting that HDL protects against atherosclerosis. This may be related to the ability of HDL apolipoproteins to stimulate clearance of cholesterol from peripheral cells,
particu-larly those of the artery wall. The finding thatsynthetic peptides
with structural features ofHDLapolipoproteins canmimic the biological activity of HDL raises the possibility that specific
agonists could bedeveloped forthe cellularcholesterol excre-tory pathway. Theseagonistsmay havetherapeuticbenefits for theprevention of coronaryheartdisease.
Wewould liketoacknowledge Rosario Bowen for technical support, and Julie Kirk and PatBurnsforassistance inpreparationof themanuscript. This study was supported by National Institutes of Health grants HL-31194,HL-18645,and HL-34343.
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