AIvAmIcA CHIMICA ACT4

ELSEVIER Analytica Chimica Acta 303 (1995) 65-72

Surface and polarization fluorescence studies an RGD sequence containing a Hepatitis A

phospholipids

on the interaction of virus peptide with

J.A. Perez a, I. Haro a, I. Martin b, M.A. Alsina b2 * , F. Reig a a Peptides Department, CID, CSIC, Barcelona, Spain

b Physicochemical Unit, Faculty of Pharmacy, Auinguda Joan XXIII, S/N, 08028 Barcelona, Spain

Received 6 June 1994

Abstract

The synthesis of a VP3(110-121)~HAV peptide was carried out by solid phase methodology. A physicochemical study on the interaction of this peptide with phospholipids involving lipid mono- and bilayers is described. The surface activity of the peptide is indicative of a medium hydrophobic character suggesting the potential ability of this peptide to associate with lipids. Moreover, the presence of the peptide in the incubation media of dipalmitoylphosphatidylcholine (DPPC) and DPPC/DPPG liposomes saturated with anilinonaphthalene sulphonate CANS) or diphenylhexatriene (DPH) show a strong influence in the bilayers fluidity determined by polarization fluorescence. The presence of lipids in the gel state has a strong influence on the polarity of a Trp environment, inducing a blue shift and fluorescence intensity increases. No interaction was

detected when measurements were carried out at temperatures above T,.

Keywords: Fluorimetry; Liposomes; Peptides; Hepatitis A

1. Introduction

Hepatitis A virus (HAV) infection is widespread

throughout the world. The disease is caused by an RNA virus belonging to the hepatovirus genus of the

Picornaviridae family which is transmitted by the faecal-oral route and by person to person contact.

In developing countries the infection has a high incidence [ 11. In some mediterranean countries I-IA is

* Corresponding author.

an endemic disease and a large proportion of the

population acquires immunity through asymptomatic infection during childhood.

Until now the only way that protection could be

achieved was by passive immunization with immune serum globulins. However, since 1992 a highly im-

munogenic formalin inactivated vaccine is commer-

cially available [2]. The use of peptide fragments, enclosing epitopes,

to generate antibodies able to recognize and neutral-

ize viral particles can be a good alternative to con- ventional vaccines. Immunization experiments with

0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0003-2670(94)00570-2

66 JA. P&ez et al. /Analytica Chimica Acta 303 (1995) 65-72

peptide chimeras have shown that the immunogenic- ity of these constructs and the specificity of the antibody response induced are dependent on the relative positions of B or T cell epitopes, that is its surface exposure [3]. This aspect is specially impor- tant if liposomes are to be used as immunoadjuvants [4], because the presence of lipids can modify the conformation of the peptide and in consequence the way it is presented and processed by antigen present- ing cells. For this reason it is interesting to study interactions between lipids and peptides in order to determine how peptides are associated with lipids and which are the changes in the physicochemical properties of lipids or peptides induced by the pres- ence of both types of molecules.

This paper describes how the presence of an HAV-VP3 peptide modifies the microviscosity of lipid bilayers and the peptide conformational changes induced by the presence of lipid bilayers.

2. Experimental

2.1. Chemicals

Ncu-Fluorenylmethoxycarbonyl amino acids and Kieselguhr supported poly(dimethylacrylamide) resin with 4-hydroxymethylphenoxy acetic acid as the ‘handle’ were obtained from Novabiochem.

Dimethylformamide (DMF) (Milligen) was freshly distilled from ninhydrin and phosphorus pentoxide at reduced pressure. Washing solvents such as iso- propyl alcohol, acetic acid and diethyl ether were obtained from Merck (p.a.1. Trifluoroacetic acid (TFA) was supplied by LKB (Ultrosyn Chemicals) and scavengers such as anisole, m-cresol, thioanisole and ethanedithiol were from Merck. Coupling reagents Bop (benzotriazole-l-yl-oxy-tris_(dimethyl- amino)-phosphonium hexafluorophosphate) and py- Bop (benzotriazole-l-yl-oxy-tris-pyrrolidinophos- phonium hexafluorophosphate) were obtained from Fluka and Novabiochem, respectively.

Disodium hydrogenphosphate dodecahydrate, monosodium dihydrogenphosphate dihydrate and sodium chloride were from Fluka (analytical grade).

Dipalmitoylphospatidylcholine (DPPC), dipalmi- toylphosphatidylglycerol (DPPG), anilinonaphtha- lene sulphonate CANS) and diphenylhexatriene (DPH)

were from Sigma. CF was from Eastman Kodak, purified as described in [5]. Quantitative phosphate analysis was carried out following the method of Marshall and Steward.

2.2. Methods

Solid phase peptide synthesis The synthesis of the peptide has been accom-

plished by the continuous-flow Fmoc-polyamide solid-phase method. A functionalised poly(dimethyl- acrylamide) resin supported in macroporous Kiesel- guhr was used. The first amino acid of the sequence was esterified to the resin-bound linkage by Castro’s reagents Bop and pyBop and the following amino acids were assembled straightforwardly in a continu- ous flow synthesizer Biolynx 4170 LKB. The syn- thetic process was carried out in a stepwise manner using largely standard procedures and will be de- scribed elsewhere.

Physicochemical studies Surface activity was determined using a Lang-

muir-Blodgett balance working with a cylindrical PTFE cuvette of 70 ml capacity (13 cm diam., depth 2 cm). The peptide solution was injected into the bulk of the aqueous subphase and surface pressure increases were detected by a platinum plate half dipped and connected to an electrobalance.

Membrane fluidity changes versus temperature were determined for DPPC small unilamellar vesi- cles (SUV) liposomes prepared from 20 mg/ml dispersions submitted to ultrasound treatment. The diameter of the vesicles as determined by quasi-elas- tic light scattering was 110 mn. Liposomes saturated with ANS or DPH were mixed with the peptide and polarization fluorescence determined as a function of temperature.

Intrinsic fluorescence measurements were carried out following the description in [6]. Starting from a peptide solution of 1 X lop6 M, small volumes of liposomes were added and the Trp fluorescence recorded. Absorbance at 280 and 340 nm was also recorded in order to use these values to introduce some corrections for inner filter effects. Fluorescence intensity data recorded were corrected for dilution, light scattering and inner filter effects, applying equation: F = F, . lo(Aex+Aem)P

JA. Pt%ez et al. /Analytica Chimica Acta 303 (1995) 65-72 61

where F, was the peptide fluorescence corrected for the light scattering effect of the liposomes. The peptide concentration ranged from 1.4 X 10m6 to 1.26 X 10e6 M, and measurements were performed in acetate buffer at pH 7.4. Additions of liposomes or buffer were made from concentrated stock solu- tions. The goodness of this approach was checked with N-acetyltryptophanamide and N-acetyltyro- sinamide. Neither molecule showed an interaction with liposomes.

Carboxyfluorescein leakage was calculated as la- tency changes in CF-loaded liposomes after incuba- tion with the peptide. SW were prepared from DPPC dry films after hydration with a 100 mM CF solution followed by ultrasound sonication. Non-en- trapped CF was removed from liposomes by gel filtration on Sephadex G50 and dialysis. The latency of initial preparations was higher than 90%.

Compression isotherms. Monolayers of DPPC and DPPG/DPPC were spread on phosphate-buffered saline (PBS) subphases, and surface pressure in- creases, after compression, recorded using a Lang- muir balance, as described elsewhere [7]. Films were spread from chloroform solutions and at least 10 min were allowed for solvent evaporation. Films were compressed continuously at a rate of 4.2 cm/min. Changes in the compressibility rate did not alter the shape of the isotherms. All the samples were run at least three times in the direction of increasing pres- sure with freshly prepared films. The accuracy of the system under the conditions in which the bulk of the reported measurements were made was 0.5 mN m-l for surface pressure.

The output of the pressure pickup (Sartorius mi- crobalance) was calibrated by recording the well- known isotherm of stearic acid. This isotherm is characterised by a sharp phase transition (25 mN m-l).

The PTFE trough (495 cm’/330 ml) was regu- larly cleaned with hot chromic acid; before each experiment it was washed with ethanol and rinsed with doubly distilled water.

Monolayer stability was assessed by compressing monolayers to a pressure of 25 mN m - ‘, stopping the barrier and observing the pressure decay. No pressure changes were observed after 30 min. All measurements were made at 21°C.

3. Results

3.1. Peptide synthesis

There are several methods to select potential epi- topes in a protein sequence. The most important are based on hydrophilicity. Assuming that the more hydrophilic the sequence, the most exposed will be the fragment to water media and consequently more able to elicit antibodies. For these reasons, the pro- gram Epiplot based on the semiempirical method of Chou and Fasmann (1978) was applied to the whole sequence of VP3 protein and the hydrophilic@ pro- file generated is given in Fig. 1. Although the se- lected fragment did not correspond to the highest point, its synthesis was undertaken having in mind that the sequence RGD included in it could favour its interaction (adhesion) to cells involved in the recog- nition process.

Moreover, conformational parameters calculated for this peptide applying the same programme showed a preference for P-sheet structures over an a-helix and random coil.

The synthesis has been accomplished by the con- tinuous-flow Fmoc-polyamide solid-phase method and will be published elsewhere. Briefly, a function- alised poly(dimethylacrylamide) resin supported in macroporous Kieselguhr was used. The first amino acid of the sequence was esterified to the resin bound linkage (4-hydroxymethylphenoxy acetic acid) by Castro’s reagent, Bop. This esterification step was carried out twice in order to achieve anchoring yields of 98%.

Hishcst pe*: rdamkn ( 29 - 26 )

1 * v~~[IIo-I~]: FWRGDLVFDFQV

D

B I I I 0

f $ -4

t-*t , ,~ , J 28 (8 68 80 188 120 149 168 188 288 220 248

Hopp 8 Woods index Au. I -0.176

SrnUmcE wslrmt

Fig. 1. Hydrophilicity profile of VF’3 protein and conformation prediction according to the Elem predict programm (Chou and Fasmann, 1978).

68 JA. P&ez et al. /Analytica Chimica Acta 303 f199.5) 65-72

The following amino acids were assembled straightforwardly in a continuous-flow synthesizer. A Fmoc/But scheme of protection was used. The side-chain of Asp was blocked with tert.-butyl ester and the guanidino function of Arg was protected with 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr) or pentamethylchromansulphonyl (Pmc), while Trp was used either without protection at the indole ring nitrogen or protected with t-Boc. Five-fold mo- lar excesses of activated Fmoc amino acids were used throughout the synthesis. Amino acids were coupled as their pentafluorophenyl esters in the pres- ence of an equivalent of 1-hydroxybenzotriazole ex- cept FmocTrp(Boc)-OH, which was coupled using the PyBop reagent.

Acylation was based on counter-ion distribution monitoring (CDM). The CDM system makes use of an anionic dye which is displaced from the growing peptide matrix as free amino groups react with the incoming amino acid. The concentration of the dye rises as the reaction proceeds and this is measured at 600 nm in the recycling liquid. Once the reaction is complete the computer (Olivetti M-240 PC) proceeds to the next step in the cycle. Nevertheless, aliquots of peptide resin were removed during the course of the synthesis and subsequent Kaiser’s tests were carried out, the yield of each coupling being at least 95%.

Deprotection steps were also monitored spec- trophotometrically at 304 nm. Typical acylation and deprotection traces were obtained which also indi- cated satisfactory progress in the assembly.

3.2. Monolayer binding properties

At micromolar concentrations VP3(110-121) peptide was highly surface active, lowering the sur- face tension of the air/water interface by 21.5 mN m-l at the saturating peptide concentration of 3.7 PM. The process was very fast; after 5 min, pressure increases represented 80% of the final values achieved. Pressure increases represented as a func- tion of the peptide concentration in the subphase are given in Fig. 2. The surface excess (I’) determined by applying the equation

F = - l/RT- AH/A In c (mol/cm’) (2)

0 0 1 2 3 4 6

Pepfw COnCMMUOn (pW)

* pmraum Incmau + ama/molecule

Fig. 2. Increase of surface pressure ( A) and area per molecule

(0) of aqueous VP3(110-121) solutions versus the peptide bulk

concentration.

was used to calculate area per molecule values applying Eq. 3:

A = l/AN (3)

where R is 8.31 X lo7 mM K-’ mol-‘, T = 294 K, AlI surface pressure increase, c is molar concen- tration and N Avogadro’s number.

This peptide had important surface activity com- pared with a sequence of similar length of HAV proteins [B].

The ability of VP3(110-121) peptide to insert into phospholipid monolayers was determined by injecting a fixed concentration of peptide (2.73 X

10m6 M) in the aqueous phase beneath phospholipid monolayers composed of DPPC and DPPC/DPPG (1:l). These studies were carried out at two initial surface pressures: 5 and 20 mN m-‘, working at constant area and measuring the pressure increases after 1 h. The results obtained are given in Fig. 3.

According to the electrical charge of VP3(110- 121) peptide ( - 1) at neutral pH (7.41, a low interac- tion with DPPG monolayers would be expected. Nevertheless, the values given in Fig. 3 show no strong differences between both phospholipids thus suggesting that the electric charge of the monolayer does not affect this type of interaction or, more likely, that the strong basicity or arginine can com- pensate both carboxyls.

As usual, the initial surface pressure of the mono- layer has a negative effect on insertion of the pep-

J.A. P&ez et al. /Analytica Chimica Acta 303 (1995) 65-72 69

tides. This is a common trend for molecules with medium or strong surface activity and has already been discussed [9]. Moreover, the more condensed the monolayer the more difficult for the peptide molecules to insert between phospholipid molecules [91.

3.3. Mixed monolayers

To study the miscibility of VP3(110-121) peptide with DPPC and DPPC/DPPG (l:l), the compres- sion isotherms for mixed monolayers of both compo- nents were measured in PBS and are given in Fig. 4a and b. The peptide is able to form stable monolayers with a collapse pressure around 30 mN m-‘. As usual for this type of peptides, after spreading, monolayers show an initial surface pressure of l-2 mN m-l and, on compression, molecules are in a liquid expanded state until 3-4 mN m-r; above this value and until collapse, its shape corresponds to a liquid condensed film. At collapse the area per molecule of this peptide is 0.8 mn2. This value is in agreement with the tendency to form P-sheets, pre- dicted by the Epiplot programme.

Mixtures of VP3(110-121) with DPPC or DPPC/DPPG gave monolayers with characteristics intermediate to those of pure components. According to the additivity rule the area per molecule must show a linear relationship with molar composition if

01 I 1 I I I

0 5 10 15 20 25

Inltlal s~rta~o preuure (mN/m)

Fig. 3. Increase of surface pressure induced after injection of a 2.73 /AM VP3(110-121) solution under DPPC (A) and DPPC- DPPG (0) monolayers spread at 5 and 20 mN m-‘.

0.6 1.2 1.6

Atea (nrnz melee-1 )

55

a 50

DPPC -, 40

1 - 35

t I 30

! 25

6 ii!

f

20

b

DPPC-DPPG

.2 1.6 Area (nm2molec-‘j

Fig. 4. Compression isotherms of DPPC/W3(110-121) and DPPC-DPPG (l:l)/VP3(110-121) mixed monolayers of diier- ent molar composition spread on subphases containing PBS at pH 7.4. (a) DPPC/peptide and (b) DPPC-DPPG/peptide: O:l, Ir; 0.2:0.8, *; 0.4~0.6, r; 0.6~0.4, W; O&0.2, open star; l:O, 0 (0 for DPPC-DPPG).

miscibility is ideal or if there is a lack of interaction. Nevertheless, in the present case, Fig. 4 suggests that the presence of small amounts of PL has a condens- ing effect on the monolayer. This behaviour is in- dicative of an increase in the stability of mixed monolayers because the excess free energy of mixing associated with the decrease in the area per molecule has a negative value. Thermodynamic calculations showed that the excess energy of mixing and the interaction energy are very low.

3.4. Effect of VP3(110-121) in the bilayer microvis- cosity

Fluorescence anisotropy was performed by using two different probes located in different regions of the lipid bilayer. This technique is very sensitive and the micromolar concentrations of fluorescent probe required do not noticeably perturb the bilayer proper-

70 J.A. P&ez et al. /Analytica

0,s r a

Chimica Acta 303 (I 995) 65-72

0,s b

084 -

I O-3 -

E c

f

092 -

091 -

0.4 -

s 0,3

0 3

a L 0,2 -

091 -

0 ’ ’ ’ ’ ’ I ’ 0 ’ ’ ’ ’ ’ ’ ’ J

lb 20 26 30 35 40 45 50 65 60 16 20 26 30 35 40 45 50 55 60

Tamparature T*mpwaturm

+ Reference * VP3 (110-121) - Rderence * VP3(110-121)

Fig. 5. Changes in polarization of DPH liposomes after incubation with VP3(110-121) measured at different temperatures. (a) DPPC, (b) DPPC-DPPG.

ties of liposomes. DPH is a rigid rod-shaped probe whose wobbling motion during its excited lifetime is highly dependent on the degree of ordering of the surrounding hydrocarbon chains. The presence of a molecule added to the PL probe system can intro- duce changes in the packing and motion of alkyl chains that can be determined by polarization fluo-

16 20 26 30 36 40 45 50 55 60

w7lp.r.tur.

+ Reference * VP3(110-121)

rescence measurements. This and liposomes com- posed of DPPC and DPPC/DPPG saturated with DPH were incubated with VP3(110-121) at a PL/peptide molar ratio x/y, and the polarization fluorescence of DPH was recorded as a function of temperature. The results are given in Fig. 5a and b.

One can appreciate that the presence of

_I 15 20 26 30 36 40 45 50 65 60

Tempwature

-Reference *VP3(110-121)

Fig. 6. Polarization temperature dependence of ANS-saturated liposomes in the presence of VP3(110-121): (a) DPPC, (b) DPPC-DPPG.

JA. P&ez et al, /Analytica Chimica Acta 303 (1995) 65-72 71

VP3(110-121) has no effect on the thermotropic transition of DPPC, T, being the same for both reference and sample. Nevertheless, the peptide has a rigidifying effect at temperatures above and below T,. In contrast, when liposomes where composed of DPPC/DPPG the peptide has a rigidifying effect only at temperatures < T,. This behaviour has already been described for other peptide/lipid sys- tems, and seems to be due to the presence of defect sites [lO,ll] on the bilayers that allow peptide molecules to insert easily and decrease the existing small motion.

To study the interfacial region of phospholipid bilayers, anilinonaphthalene sulfonate CANS) was chosen. As stated before, vesicles saturated previ- ously with this marker were incubated with VP3 (110-121) and the polarization fluorescence at dif- ferent temperatures was measured. Fig. 6 shows these values for DPPC and DPPC/CPPG liposomes.

FIFO 6, I

I

1.0 1.2 1.4

DPPC/vP3 x lo+

FIFO

6

bj

0’ I 0 .2 .4 .6 .8 1.0 1.2 1.4 1.6 1.8 2.0

DPPC-DPPG/VP3 X lo+

(-20% +5ooc 1

Fig. 7. Changes in fluorescence intensity (F/F,) of VF%llO- 121) at 340 nm versus lipid/peptide molar ratio. (a) DPPC, (b) DPPC-DPPG.

A max (nm) 370, I

a

350

340 ’ I

0 .2 .4 .6 .6 1.0 1.2 1.4 1.6 1.8 2.0

DPPC/vPJ x 1o-3

I”2OOC f509C 1

.amax (nm) 370,

340 I

02 4 6 8 1 .o 1.2 1.4 1.6 1.8 2.0

DPPC-DPPGiVP3 X lo-$

Fig. 8. Chauges iu wavelength of the emission maximum (A,,). Excitation at 280 nm. Peptide concentration: 1.4 X 10e6. (a) DPPC, (b) DPPC-DPPG.

In both cases the presence of the peptide has a rigidifying effect over the range of temperatures assayed.

One can appreciate that polarization is higher for DPPC liposomes than for DPPC/DPPG, especially at temperatures < T,. This larger mobility of DPPC/DPPG polar heads was to be expected be- cause packing is optimum when the molecular species are equal, as is the case for the palmitoyl alkyl chains.

3.5. Permeability studies

To complete this study the interaction of CF- loaded liposomes with VP3 (110-121) was deter- mined by measuring latency changes for 2 h at 21’C. After this period less than 2% of marker had been released from the liposomes thus indicating that pep- tide molecules are unable to insert deeply in bilayers.

72 J.A. P&z et at. /Analytica Chimica Acta 303 (1995) 65-72

The interaction of VP3 with DPPC and DPPC- DPPG was also investigated by measuring the fluo- rescence quantum yield and the wavelength of the emission maximum in the presence of different con- centrations of lipid vesicles, both above and below T, (20°C and SO”C, respectively). Titration curves are given in Figs. 7 and 8. The wavelength of maximum fluorescence emission ( Amax) of the pep- tide in acetate buffer was 356.7 nm, close to the value of 360 nm generally accepted for the trypto- phan ring in a polar medium. This number is indica- tive of a highly polar environment of the tryptophan residue in the peptide. Nevertheless, the fluorescence properties of the peptide were markedly altered upon addition of DPPC or DPPC-DPPG vesicles in a gel state; there was also a clear blue shift with both populations of liposomes.

These two results, increase in fluorescence and blue shift, are indicative of a strong interaction of peptide with lipids. The maximum wavelength shift was 12-14 nm at a lipid to peptide ratio of 400:1, and an increase in intensity of 5, thus indicating a truly hydrophobic enviroment for the tryptophan residue. Comparable large shifts have also been de- scribed for small peptides [12].

The most striking feature of these curves is that the fluorescence of the peptide did not change with the liposomes in a fluid state, showing on the con- trary a strong interaction with phospholipids in gel state. This preferential interaction, although not ex- pected, has also been described for pentagastrin-re- lated peptides and glucagon [13]. It has been sug- gested that peptides interact preferentially with de- fect sites in the gel phase; the lipid molecules at such sites are isoenergetic and this energy will be lower than that of the other molecules away from the defects, thus acting as the peptide binding sites in the bilayer. Besides, as a certain number of molecules become immobilized by interaction with the peptide, the entropy loss will be smaller in the lamellar gel state than in the lipid crystalline state, thus favouring interaction below T,.

No strong differences are produced by the pres- ence of negative lipids in the bilayer, in agreement with the results obtained in monolayer studies.

All these results indicate that this peptide interacts mainly with lipid layers in the gel state and is able to insert into mono- and bilayers efficiently. The pres- ence of lipids has a certain influence on the peptide conformation, giving rise to structures in which Trp is surrounded by a non-polar medium.

Acknowledgements

This work was supported by research grant BIO- 92-0982~CO2-02 from CICYT, Spain.

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111

121

131 [41 El

[61

171

Bl

t91

WI

ml

WI

[131

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