30
Bioconjugate Chem. 2000, 11, 30−38
Phospholipid-Model Membrane Interactions with Branched Polypeptide Conjugates of a Hepatitis a Virus Peptide Epitope Ildiko´ B. Nagy,† Maria A. Alsina,‡ Isabel Haro,§ Francesca Reig,§ and Ferenc Hudecz*,† Research Group for Peptide Chemistry, Hungarian Academy of Science, Eo¨tvo¨s L. University, P.O. Box 32, Budapest 112, Hungary, H-1518, Physicochemical Unit, Faculty Pharmacy, Pza. Puis XII s/n 08028, Barcelona, Spain, and Department of Peptide and Protein Chemistry, Research Council, CID. CSIC. Jordi Girona 16. 08034, Barcelona, Spain. Received April 2, 1999; Revised Manuscript Received September 8, 1999
To establish correlation between structural properties (charge, composition, and conformation) and membrane penetration capability, the interaction of epitope peptide-carrier constructs with phospholipid model membranes was studied. For this we have conjugated a linear epitope peptide, 110FWRGDLVFDFQV121 (110-121), from VP3 capside protein of the Hepatitis A virus with polylysinebased branched polypeptides with different chemical characteristics. The epitope peptide elongated by one Cys residue at the N-terminal [C(110-121)] was attached to poly[Lys-(DL-Alam-Xi)] (i < 1, m ≈ 3), where x ) L (AK), Ser (SAK), or Glu (EAK) by the amide-thiol heterobifunctional reagent, 3-(2pyridyldithio)propionic acid N-hydroxy-succinimide ester. The interaction of these polymer-[C(110121)] conjugates with phospholipid monolayers and bilayers was studied using DPPC and DPPC/PG (95/5 mol/mol) mixture. Changes in the fluidity of liposomes induced by these conjugates were detected by using two fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and sodium anilino naphthalene sulfonate (ANS). The binding of conjugates to the model membranes was compared and the contribution of the polymer component to these interactions were evaluated. We found that conjugates with polyanionic/EAK-[C(110-121)] or polycationic/SAK-[C(110-121)], AK-[C(110-121)]/character were capable to form monomolecular layers at the air/water interface with structure dependent stability in the following order: EAK-[C(110-121)] > SAK-[C(110-121)] > AK-[C(110-121)]. Data obtained from penetration studies into phospholipid monolayers indicated that conjugate insertion is more pronounced for EAK-[C(110-121)] than for AK-[C(110-121)] or SAK-[C(110-121)]. Changes in the fluorescence intensity and in polarization of fluorescent probes either at the polar surface (ANS) or within the hydrophobic core (DPH) of the DPPC/PG liposomes suggested that all three conjugates interact with the outer surface of the bilayer. Marked penetration was documented by a significant increase of the transition temperature only with the polyanionic compound/EAK-[C(110-121)]. Taken together, we found that the binding/penetration of conjugates to phospholipid model membranes is dependent on the charge properties of the constructs. Considering that the orientation and number of VP3 epitope peptides attached to branched polypeptides were almost identical, we can conclude that the structural characteristics (amino acid composition, charge, and surface activity) of the carrier have a pronounced effect on the conjugate-phospholipid membrane interaction. These observations suggest that the selection of polymer carrier for epitope attachment might significantly influence the membrane activity of the conjugate and provide guidelines for adequate presentation of immunogenic peptides to the cells.
INTRODUCTION
New vistas for vaccine production arise with the application of synthetic peptide antigens, although these small molecules induce weaker immune responses than those elicited by intact proteins or viruses (1-3). To resolve this problem, multiple antigen peptide systems (4) or lipid vesicles that can elicit potent immune response against antigens entrapped or covalently linked have been introduced. The efficacy of these constructs has been clearly established for a great variety of antigens such as bacterial toxins (5, 6), parasite proteins (7), and tumor antigens (8). An alternative approach suggested by Sela (9) is the covalent attachment of * To whom correspondence should be addressed. Fax: 36 1 209 0602. Phone: 36 1 209 0555/1433. E-mail: hudecz@ szerves.chem.elte.hu. † Research Group for Peptide Chemistry. ‡ Physicochemical Unit. § Department of Peptide and Protein Chemistry.
haptens/epitopes to proteins (KLH, BSA)1 or synthetic polymers (reviewed in ref 10) as macromolecular carriers. To contribute to the rational design of synthetic macromolecular carrier, we have developed a new group of branched polypeptides based on a poly[L-Lys] backbone that contains short side chains composed of about three DL-Ala residues and one different amino acid residue (X) either at the end of the branches (XAK) or at the position next to the polylysine backbone (AXK). The general formula of these biodegradable, water-soluble compounds are poly[Lys(Xi-DL-Alam)] (XAK) or poly[Lys(DL-Alam-Xi)] 1 Abbreviations: AK, poly[Lys-(DL-Ala )]; ANS, sodium anilim no naphthalene sulfonate; AXK, poly[Lys-(DL-Alam-Xi)]; BSA, bovine serum albumin; DPH, 1,6-diphenyl-1,3,5-hexatriene; DPPC, dipalmitoyl phosphatidyl choline; EAK, poly[Lys(GluiDL-Alam)]; KLH, keyhole limpet hemocyanin; PBS, phosphatebuffered saline; PG, phosphatidyl glycerol; SAK, poly[Lys(SeriDL-Alam)]; SPDP, 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester; XAK, poly[Lys(Xi-DL-Alam)].
10.1021/bc9900385 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/02/1999
Phospholipid−Model Membrane Interactions
(AXK), where i < 1, m ≈ 3, and X represents an optically active residue (11-14). Considering the structural [e.g., solution conformation (16)] and functional characteristics [e.g., in vitro cytotoxicity (15), immunoreactivity (14, 17), and biodistribution (18)] of these polypeptides, various bioconjugates were prepared. In these constructs, a B-cell epitope from mucin-1 glycoprotein (19), peptide fragment from Herpes simplex virus glycoprotein D1 (17), and T cell epitopes of 16 and 38 kDa proteins of Mycobacterium tuberculosis (20, 21) were attached to branched polypeptides. In the interaction between conjugates and cellular/ subcellular membranes, the polymeric polypeptide component might have an important role. Therefore, the aim of present study was to investigate the influence of branched polymeric polypeptide in conjugates on phospholipid mono- and bilayers used as biomembrane models. As first step, we have analyzed the interaction of some branched polypeptides with simple mono- and bilayer membranes composed of DPPC or DPPC/PG mixture (22). We found that all three polymers studied were capable of inserting into lipid monolayers, but this interaction was more pronounced for the polycationic poly[Lys(Seri-DL-Alam)] (SAK) and poly[Lys(DL-Alam)] than for the amphoteric poly[Lys(Glui-DL-Alam)] (EAK). Using fluorescent reporter molecules, we have also demonstrated that even the most marked penetration of polycationic polypeptide (SAK) did not result in alteration of the ordered state of the alkyl chains of liposomal bilayers composed of DPPC/PG. For the present experiments, we have prepared conjugates with a linear epitope peptide, 110FWRGDLVFDFQV121 (110-121) from VP3 capside protein of the Hepatitis A virus (23) with known phospholipid interaction characteristics (24). We have previously shown that branched polypeptide conjugates of the elongated and Abu-substituted 20-mer peptide, 101LASI(Abu)QMF(Abu)FWRGDLVFDFQV121 influence the stability of mono- and bilayers composed of DPPC (25). To get insight about the carrier effect on membrane activity a new set of conjugates with AK, SAK, or EAK were prepared and tested on DPPC/PG phospholipid model membranes. These conjugates possess similar relative molecular mass and number of epitope (110-121) copies, but the hydrophobicity and electrical charge of the carrier are different (22). In this paper, we report the preparation of branched polypeptide conjugates of the 12-mer peptide elongated by one cysteine residue at the N-terminal C110FWRGDLVFDFQV121 [C(110-121)]. For the disulfide bond formation between the polymer (AK, SAK, and EAK) and the epitope peptide the amide-thiol heterobifunctional coupling reagent, 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester (SPDP) (19, 26) was used. The interaction of these polymer-[C(110-121)] conjugates dissolved in aqueous solution with phospholipid monolayers and bilayers was studied using DPPC and DPPC/ PG (95/5 mol/mol) mixture mimicking cell membranes containing the smallest percentage of negatively charged lipids (27). Changes in the fluidity of liposomes induced by these conjugates were detected by using two fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and sodium anilino naphthalene sulfonate (ANS). The binding of conjugates to the model membranes was compared and the contribution of the polymer component to these interactions was evaluated.
Bioconjugate Chem., Vol. 11, No. 1, 2000 31 EXPERIMENTAL SECTION
Synthesis of Polypeptide-[C(110-121)] Conjugates. Experimental details of the synthetic procedures for branched chain polypeptides were described previously (12, 13). Briefly, the poly[L-Lys] backbone was prepared by the polymerization of NR-carboxy-N-benzyloxycarbonyl-lysine anhydride under conditions that allowed a number average degree of polymerization approximately 60. After cleavage of benzyloxycarbonyl protecting groups, DL-alanine oligomers were grafted to the -amino groups of polylysine by polymerization of N-carboxy-DL-alanine anhydride to produce poly[Lys-(DLAlam)] (AK). The suitably protected Glu/Ser residue was coupled to R-amino groups of branch terminal Ala of AK. The amide bond was formed by HOBt-catalyzed active ester method (28). After the removal of protecting groups from Glu (EAK) and Ser (SAK) residues by HBr/AcOH, samples were dialyzed against distilled water and freezedried. Peptide C(110-121) was synthesized by Fmoc/OtBu strategy on solid support (24) and after HPLC purification was coupled to the N-terminal amino acid residue of the AK, SAK, and EAK using SPDP (26). Conjugation was performed as described earlier with mucin peptides (19). Briefly, polymeric polypeptides (AK, SAK, or EAK) (215 µmol) were dissolved in 7.5 mL of 0.1 M phosphate buffer (pH 7.4, adjusted with 0.1 M NaOH). A total of 20 mg (64.0 µmol) of SPDP dissolved in 2 mL of absolute methanol was added dropwise to the solution. The reaction mixture was stirred for 30 min at room temperature. After dialysis against distilled water for 24 h, the modified polymers were isolated by freeze-drying. To determine the average degree of 2-pyridyl-disulfide group incorporation, 400 µL of (SSP)polymer solution was reacted with 200 µL of DL-dithiotreitol (5 mg/mL PBS), and after 10 min, the absorbance of the released pyridine2-thione was measured at λ ) 343 nm using ) 8080 M-1 cm-1 (29). In the second step of conjugation, 10 mg of (SSP)polymer samples (containing 5 µmol protected thiol groups) were dissolved in 1 mL in 0.1 M phosphate buffer (pH 7.4) and mixed with 10 µmol of peptide C(110121) dissolved in the same buffer (1 mL). The reaction mixture was stirred for 30 min at room temperature. Conjugate samples were purified by dialysis and isolated by freeze-drying, and the substitution degree was determined by amino acid analysis. Abbreviations for Amino Acids and Their Derivatives. Abbreviations for amino acids and their derivatives follow the revised recommendation of the IUPAC-IUB Committee on Biochemical Nomenclature, entitled Nomenclature and Symbolism for Amino Acids and Peptides (recommendations of 1983). Nomenclature of branched polypeptides is used in accordance with the recommended nomenclature of graft polymers (30). For the sake of brevity, codes of branched polypeptides were constructed by us using the one-letter symbols of amino acids. The abbreviations used in this paper are the following: AK, poly[Lys(DL-Alam)]; XAK, poly[Lys(Xi-DL-Alam)]; X ) Ser (SAK) or Glu (EAK). All amino acids are of L configuration unless otherwise stated. The symbols used for the conjugates are AK-[C(110-121)], SAK-[C(110-121)]. and EAK-[C(110-121)]. Chemicals. 3-(2-Pyridyldithio)propionic acid N-hydroxy-succinimide ester (SPDP), 1,6-diphenyl-1,3,5hexatriene (DPH), sodium anilino naphthalene sulfonate (ANS), dipalmitoyl phosphatidyl choline (DPPC), and phosphatidyl glycerol (PG) of bovine origins were ob-
32 Bioconjugate Chem., Vol. 11, No. 1, 2000
tained from Merck (Barcelona, Spain). All solvents as well as dithiotreitol were from Reanal (Budapest, Hungary). MATERIALS AND METHODS
Surface Activity Measurement. Hydrophobic character of conjugate samples was analyzed by Wilhelmy plate method as reported earlier (31). All measurements were carried out in a cylindrical Teflon cuvette with capacity of 70 mL (type A). All conjugates were dissolved in 0.1 M sodium acetate buffer (pH 7.4) and were injected under the air-water interface at different final bulk concentration [(0.2-1.8) × 10-8 M]. Pressure increase was recorded as a function of time up to 60 min as detailed in ref 32. Compression Isotherms. Surface pressure vs area (Π-A) isotherms were recorded in the same device using a rectangular Teflon cuvette of 28.5 cm × 17.5 cm (type B). Conjugate samples dissolved in 0.1 M sodium acetate buffer (pH 7.4) were either spread on or injected under the air-water interface. After 10 min, the monolayer was compressed by a Teflon barrier at a speed of 4.2 cm/min. The stability of all conjugate monolayers was assessed by compression till 10 mN/m and after stopping the barrier, changes in pressure were recorded for 1 h. Experiments were carried out in triplicate at 22 °C using 0.1 M sodium phosphate buffer (pH 7.4) as subphase. The reproducibility of the measurements was in the range (0.02 nm2/molecule. Penetration of Polymer-[C(110-121)] Conjugates into Phospholipid Monolayers. For the insertion experiments, phospholipid solutions of DPPC or DPPC/PG (95/5 mol/mol) mixture were spread on the aqueous phase to form a monolayer with a given initial surface pressure in the type A cuvette. Once the monolayer at 5, 10, or 20 mN/m was established, conjugate solutions were injected in the subphase and pressure increases were recorded as a function of time. In addition, phospholipid monolayers were formed on aqueous subphase in the presence or absence of conjugate in type B cuvette, and after 10 min of stabilization period, compression isotherms were recorded. Preparation of Liposomes. Liposomes were prepared using mixture of DPPC and PG (95/5, mol/mol) as follows. Phospholipids dissolved in chloroform were mixed in a round-bottomed flask and solvent was eliminated by rotary evaporation from the clear solution at 55 °C in a vacuum. The liposome preparation was dried in high vacuum for 2 h. Lipid film was hydrated with 2 mL of 0.1 M sodium acetate buffer (pH 7.4), and multilamellar vesicles thus formed were submitted to sonication in an ultrasound bath until the solution was only slightly turbid. Size of liposomes measured in Malvern Autosizer and as described earlier (33) was lower than 80 nm in diameter. Fluorescence Studies. Fluorescence experiments were performed by detecting changes in fluorescence intensity and polarization of ANS and DPH probes located in the bilayers. For this, a PE-LS50 spectrofluorometer provided with four cuvettes and thermostated bath was used. Liposomes (small unilamellar vesicles) described above were incubated with different concentrations of fluorescent probes in the dark at 55 °C for 60 min. Fluorescence intensity was measured to determine the saturation. Once the optimal phospholipid/probe ratio was selected [ANS/phospholipid ) 1/26, DPH/phospholipid ) 1/240 (mol/mol)], liposomes were mixed with either conjugate solution or 0.1 M sodium acetate (pH
Nagy et al.
7.4) and fluorescence intensity as well as polarization were recorded. Temperature was increased step by step, leaving enough time between lectures to allow the system to equilibrate. The suspension was continuously stirred, and the temperature inside the cuvettes monitored before and after the scan. Experiments were carried out in duplicate. Excitation (λex) and emission (λem) wavelengths were 380 and 480 nm for ANS and 365 and 425 nm for DPH, respectively. The degree of fluorescence polarization was calculated according to Shinitzky and Barenholz (34),
P ) (I| - I⊥*G)/(I| + I⊥*G) where I| and I⊥ are the intensities measured with its polarization plane parallel (|) and perpendicular (⊥) to that of the exciting beam. G is a factor used to correct polarization of the instrument and is given by the ratio of vertically to horizontally polarized emission components when the excitation light is polarized in the horizontal direction. RESULTS AND DISCUSSION
Synthesis and Structure of Branched Polypeptide-[C(110-121)] Conjugates. Polymeric branched polypeptides (DPn ) 61) with polycationic (AK, SAK) or amphoteric (EAK) charge properties (13, 14) were conjugated with a 12-mer synthetic peptide representing a B-cell epitope of VP3 capside protein of Hepatitis A virus. The schematic structure of branched polymer-peptide conjugates is depicted in Figure 1. To achieve uniform orientation in conjugates, disulfide bridge was introduced between the branches of the polymer and the peptide epitope. For this the native epitope sequence, 110FWRGDLVFDFQV121 was elongated at the N-terminal by incorporation of a Cys residue to provide free SH group for conjugation. At the same time, protected thiol function was formed at the end of the branches of polymers using an amine-thiol type heterobifunctional cross-linking reagent, SPDP (19, 26). The polymer-dithio-propionate derivatives prepared (Scheme 1) were purified, isolated, and characterized by UV spectroscopy. After reduction of an aliquot samples, the amount of pyridine-2-thione coupled to the R-amino groups of the branches was determined. The average degree of substitution calculated was between 27 and 37%. In the second step of the synthesis, disulfide bond was formed by thiolysis using free thiol group of peptide C(110-121) (Scheme 1). The characteristics of conjugates are summarized in Table 1. These data obtained from two independent methods (UV-chromophore content and amino acid composition determination) indicate that the average incorporation of C(110-121) was in the range 27-37%. Peptide C(110-121) bears net negative charge under physiological pH. Therefore, its coupling to the polymers changes the net electrical charge of the polymers. In case of conjugates with polycations (AK and SAK), 27-37% of the positively charged amino groups was replaced by the epitope peptide. However, these conjugates still had pronounced polycationic character (Figure 1.). In contrast, the amphoteric polymer, EAK, after reaction with C(110-121) had a 31% increase in negatively charged groups. It is due to the blocking of approximately 1/3 of the amino groups of glutamic acid residues of the branches by C(110-121) (Figure 1.). Consequently, this compound can be considered as a polyanionic conjugate in which the ratio of positive and negative charges is 2-3. Surface Characteristics of Polypeptide-[C(110121)] Conjugates. The properties of branched polypep-
Phospholipid−Model Membrane Interactions
Bioconjugate Chem., Vol. 11, No. 1, 2000 33
Figure 1. Simplified structure of the branched polypeptide-[C(110-121)] conjugates. (A) AK-[C(110-121)], (B) SAK-[C(110121)], and (C) EAK-[C(110-121)]. Scheme 1. Schematic Presentation of the Synthesis of AK-[C(110-121)] Conjugatea
(A) Introduction of the 2-piridyl disulfide groups into a-amino group of the branches. (B) Formation of disulfide bond between the modified branches and [C(110-121)].
tide-epitope conjugates in absence of phospholipids at the air-water interface was studied first. Conjugate solutions at different concentrations were injected to the subphase, and surface pressure vs time curves were recorded. In all three cases, we have observed a certain period of time needed for the conjugate incorporation to the interface. This induction time decreased with sample concentration (data not shown). The kinetics of the process for the conjugates was similar (data not shown). Similar findings were reported for proteins (35, 36) as
well as for the free polymeric-branched polypeptides used for conjugate preparation (22). This phenomenon could be related to the presence of an ordered/packed structure whose adsorption to the air-water interface required time to reach the equilibrium. Surface pressure values achieved after 60 min are depicted as a function of conjugate concentration in Figure 2. The pattern of these curves indicates that the maximum pressures achieved were dependent on the macromolecular component of the conjugate. A tendency
34 Bioconjugate Chem., Vol. 11, No. 1, 2000
Nagy et al.
Table 1. Characteristics of Branched Polypeptide-[C(110-121)] Conjugates amino acid compositionb polypeptide
codea
Lys
Ala (m)
Ser/Glu (i)
conjugate codec
DSd
Mwe ((5%)
poly[Lys(DL-Alam) poly[Lys(Seri-DL-Alam) poly[Lys(Glui-DL-Alam)
AK SAK EAK
1.00 1.00 1.00
4.20 4.20 4.20
0.75 0.95
AK-[C(110-121)] SAK-[C(110-121)] EAK-[C(110-121)]
0.27 0.37 0.31
51 500 57 000 59 000
a Code of branched polypeptides based on one letter symbol of amino acids. b Determined by amino acid analysis after hydrolysis in 6 M HCl at 105 °C for 24 h as described in ref 22. c Code of polymer-peptide conjugates based on one letter symbol of amino acids. d Average degree of substitution determined from the amino acid analysis after hydrolysis of the conjugate samples in 6 M HCl at 105 °C for 24 h. e Calculated from the number average degree of polymerization of polylysine (DP ) 61) and from the amino acid composition of conjugates. n
Figure 2. Effect of concentration of branched polypeptide[C(110-121)] conjugate on the increase of surface pressure. Conjugate samples were dissolved in 0.1 M sodium acetate buffer, (pH 7.4) and injected in the subphase and surface pressure values were determined after 60 min: AK-[C(110121)] (2), SAK-[C(110-121)] ([), and EAK-[C(110-121)] (O).
of surface pressure to increase with concentration was also revealed, up to 0.7 × 10-8 M for AK-[C(110-121)], but at higher concentration (1.4 × 10-8 M) for SAK[C(110-121)] and EAK -[C(110-121)]. Above these values, there was a soft decrease in surface pressure that could be indicative of formation of aggregates. Similar data were reported for poly(ethyleneglycol)s (37). According to the maximum pressure values achieved, EAK[C(110-121)] conjugate incorporated to the air-water interface most efficiently (22 mN/m). Formation and stability of monolayers were also investigated by the analysis of surface pressure/area isotherms of conjugates. The monolayers were prepared in two ways. Conjugate solution was either spread on or injected under the aqueous surface. After 10 min, the monolayer was compressed by a movable barrier. The stability of monolayers was determined by compressing them up to 5 mN/m and leaving the system in equilibrium for 1 h. The unchanged surface pressure indicated that monolayers were stable under these conditions (data not shown). The pattern of compression isotherms indicated that all three conjugates formed monomolecular layer independently from the method used (Figure 3). In the case of conjugates with AK and SAK, the pressure vs area curves obtained after spreading samples on the surface displayed a similar pattern (Figure 3, panels A and B). Monolayers were formed in liquid expanded state at low compression pressures and after phase change around 9 mN/m isotherms showed a typical profile of condensed state without collapse. In contrast, no phase change was observed with EAK-[C(110-121)] conjugate compression isotherms (Figure 3C). The monolayer was almost in liquid state during the whole compression process, and no collapse occurred at the end of compression. The monomolecular layer of this conjugate was more
Figure 3. Compression isotherms of monomolecular layers formed by branched polypeptide conjugates AK-[C(110-121)] (A), SAK-[C(110-121)] (B), and EAK-[C(110-121)] (C) spread on the surface (black curve) or injected into (gray curve) the 0.1 M phosphate buffer subphase, (pH 7.4).
expanded than those of AK and SAK. These data are in agreement with the characteristics described in surface activity measurements (Figure 2) where EAK-[C(110121)] was the most active compound. These differences can be explained by the altered charge properties of the conjugates. Repulsion forces between negatively charged groups characteristic for EAK-[C(110-121)] seems to be more pronounced than that of positively charged groups which predominate the net charge of AK- or SAK-based conjugates. Maximum surface pressure values obtained for EAK-[C(110-121)] conjugate [31.8 mN/m (spreading on the surface) and 29.1 mN/m (injecting under the surface)] indicate that these values are interestingly in the range of those described for hydrophobic proteins (38). The area/amino acid residue values at the end of compression and maximal pressures achieved are summarized in Table 2.
Phospholipid−Model Membrane Interactions
Bioconjugate Chem., Vol. 11, No. 1, 2000 35
Table 2. Area/Amino Acid Residue Values Calculated and Maximum Pressure (ΠMax) Values Obtained from the Compression Isotherms of the Polypeptide-[C(110-121)] Conjugates Spread on or Injected under the Air-Water Interface spread
a
injected
conjugate
area/residuea (nm2/m)
Πmax (mN/m)
area/residuea (nm2/m)
Πmax (mN/m)
AK-[C(110-121)] SAK-[C(110-121)] EAK-[C(110-121)]
0.031 ( 0.000 32 0.020 ( 0.000 32 0.024 ( 0.000 32
22.8 23.0 31.8
0.057 ( 0.000 32 0.028 ( 0.000 32 0.030 ( 0.000 32
22.2 17.6 29.1
The area/residue values were calculated from compression isotherms recorded at 10 mN/m.
Figure 4. Effect of initial surface pressure on penetration of branched polypeptide-[C(110-121)] conjugates into DPPC (open symbol) and DPPC/PG (95/5, mol/mol) (filled symbol) monolayers. AK-[C(110-121)] (4,2), SAK-[C(110-121)] (],[) and EAK-[C(110-121)] (O,b).
The method of conjugate sample application had some effect on the properties of monolayers. Films formed after injection to the subphase were qualitatively the same, but maximal pressures achieved were slightly lower and area/amino acid residue values higher than those of calculated for monolayers obtained after spreading of conjugate samples. As monolayers were stable in both cases, these differences can be interpreted by different conformation adopted by conjugates at the air-water interface. Structures formed after spreading were probably more stable/packed than those adopted after conjugate injection into the subphase. Conjugation of the 12-mer epitope peptide increased the hydrophobicity of all polymeric polypeptides (22). This effect was dependent on the polymer structure. The most marked increase was observed for EAK-[C(110-121)] (almost 4-fold), whereas the surface activity of AK[C(110-121)] and of SAK-[C(110-121)] was elevated by about 50% (22). Interaction of Polypeptide-[C(110-121)] Conjugates with Phospholipid Monolayers. The ability of conjugates to insert in phospholipid monolayers was evaluated using two phospholipid compositions: DPPC and DPPC/PG (95/5, mol/mol). Aqueous solutions of the conjugates were injected into the phosphate buffer subphase under monolayers spread at different initial surface pressures (5, 10, and 20 mN/m). Results summarized in Figure 4. indicate that EAK-[C(110-121)] induced the highest surface pressure increase both for DPPC and for DPPC/PG (95/5, mol/mol) monolayers. No significant differences were observed between the two conjugates possessing polycationic carrier (AK or SAK). These findings are in agreement with results obtained in surface activity measurements and compression isotherms at the air-water interface. Stronger interaction of EAK-[C(110121)] with these model membranes initiates an expansion/deformation of the phospholipid monolayer. This
Figure 5. Compression isotherms of monolayers formed by DPPC (A) or DPPC/PG (95/5, mol/mol) (B) spread on 0.1 M phosphate buffer subphase, (pH 7.4) (s). Containing AK[C(110-121)] (- - -), SAK-[C(110-121)] (- - -), or EAK[C(110-121)] (‚‚‚) in the subphase.
results in an elevated surface pressure recorded at all initial surface pressure values studied. The interactions of all three conjugates with phospholipid monolayers were also investigated in a dynamic model spreading either DPPC or DPPC/PG (95/5, mol/ mol) on the air-water interface of the conjugate solutions (Figure 5). Pattern of the compression isotherms of DPPC monolayers (Figure 5A) indicated an expansion due to the presence of conjugates in the subphase at low and medium surface pressures, but at 20 mN/m, curves were indistinguishable. This value was in the range of the collapse pressures for conjugates, suggesting that epitopepolypeptide conjugates are squeezed out from the films under this condition. In addition, conjugates had also expanding effect on monolayers composed of DPPC and PG (95/5, mol/mol) all over the compression pressures (Figure 5B). This is indicative for a more pronounced interaction compared with neutral monolayers. This finding also suggests that,
36 Bioconjugate Chem., Vol. 11, No. 1, 2000
though an exclusion of conjugates is evident at high pressures, fractions incorporated initially in the lipid monolayers still remain there during the whole compression process. A similar behavior has been described for multiple antigenic peptides (39). Data summarized in Figure 5 show that all three conjugates inserted strongly in charged monolayers. Conjugate EAK-[C(110-121)] proved to be the most active compound; however, differences recorded among conjugates were small. Taken together, these results document that conjugation of C(110-121) peptide epitope increased the penetration of all three polymers. The most prominent effect was observed in case of EAK-[C(110-121)] conjugate vs EAK polymer where the surface pressure increased to almost 4-fold (22). Interaction of Polypeptide-[C(110-121)] Conjugates with Phospholipid Bilayers. The interaction of these conjugates with lipid bilayers was further studied using liposomes with DPPC/PG (95/5 mol/mol) phospholipid composition. Bilayers were saturated either with negatively charged ANS or with DPH possessing hydrophobic character. Altered fluorescent properties of the probes (fluorescence intensity and polarization) were recorded and related to the degree of ordered structure of the bilayers. The former compound incorporated into the surface of liposomes was used to analyze the effect of conjugates on the outer part, while the latter one was applied to monitor changes in the hydrophobic core of bilayers. The presence of conjugate SAK-[C(110-121)] or AK[C(110-121)] increased the fluorescence of the samples containing ANS. In contrast, conjugate EAK-[C(110121)] reduced this parameter (data not shown). Fluorescence polarization values obtained with ANS/liposomes are shown in Figure 6. These data indicate that all conjugates gave values higher than the reference thus reflecting the rigidification of the bilayer at the outer polar heads level. The intensity of fluorescence of ANS in conjugates’ solution in the absence of liposomes was slightly higher than in water (data not shown). This might indicate the presence of hydrophobic component for ANS provided by conjugate aggregates. It should be noted that these intensity values were low; therefore, the corresponding polarization was not reliable. Consequently, the polarization changes detected in the presence of liposomes are by far reflecting the interaction between liposomes polar heads and conjugates. The interaction of epitope conjugates with the internal part of the bilayer was studied on liposomes saturated with DPH. Fluorescence polarization changes determined as a function of the temperature are summarized in Figure 7. The transition temperature was also determined (Figure 7, insert). In all three cases, conjugates induce a clear rigidification of bilayers mainly at temperatures above the phase transition (Tc ) 40.5 °C), where bilayer is more fluid and insertion is favored. These data demonstrate that the presence of conjugates resulted in an increased temperature range of transition reflecting the reduction in the cooperativity of the process. Conjugates with polycationic polymers, SAK[C(110-121)] or AK-[C(110-121)] slightly reduced the transition temperature (Tc ) 39.5 °C). In contrast, this value was shifted by about 2 °C in case of EAK-[C(110121)] (Tc ) 43.5 °C). These findings are in agreement with those described earlier in the sense that EAK-[C(110121)] conjugate has more pronounced effect on phospholipid monolayers than SAK-[C(110-121)] or AK-[C(110121)]. As the PG content of the liposomes was relatively low, it seems that differences in this behavior were more
Nagy et al.
Figure 6. Polarization of ANS/DPPC/PG liposome preparations in the presence or absence of branched polypeptide-[C(110121)] conjugates: AK-[C-VP3(110-121)] (2), SAK-[C-VP3(110-121)] ([), and EAK-[C-VP3(110-121)] (O), or with 0.1 M sodium acetate buffer, (pH 7.4) (4).
Figure 7. Polarization and its first derivative (insert) of DPH/ DPPC/PG (95/5, mol/mol) liposome preparation in the presence or absence of branched polypeptide-[C(110-121)] conjugates: AK-[C(110-121)] (- - -), SAK-[C(110-121)] (- ‚ -), EAK-[C(110121)] (s). Sodium acetate buffer, 0.1 M (pH 7.4) (‚‚‚).
likely due to the higher charge density of EAK-[C(110121)] conjugate versus the SAK or AK analogues. Considering that free polymers interact only with the outer surface of the phospholipid bilayer and did not result in alteration of the ordered state of the alkyl chains (22), it can be assumed that the presence of peptide C(110-121) in the conjugates has marked effect on the stabilization of both the outer and the inner regions of the bilayer. CONCLUSIONS
Taken together, these data indicate that branched polypeptide-[C(110-121)] conjugates interact with lipid mono- and bilayers in fluid state, inserting the peptide containing side chains into phospholipid membranes. We found that the binding/penetration of conjugates to phospholipid model membranes composed of dipalmitoylphosphatidyl-choline (DPPC) and phosphatidylglycerol (PG) is dependent on the charge properties of the constructs. Considering that the orientation and number of VP3 epitope peptides attached to branched polypeptide carrier were almost identical, we can conclude that the characteristics (amino acid composition, charge, and surface activity) of the carrier have a pronounced effect on the conjugate-phospholipid membrane interaction.
Phospholipid−Model Membrane Interactions
These observations suggest that the selection of polymer carrier for epitope attachment might significantly influence the membrane activity of the conjugate and provide guidelines for adequate way to present immunogenic peptides to the cells in order to induce biological response. ACKNOWLEDGMENT
This work was financed by a grants from HungarianSpanish Intergovernmental Program (E-13/1997), CICYT SAF-93-0063 and the Hungarian Research Fund (OTKA) no. T 014964 and T 03838. LITERATURE CITED (1) Emini, E. A., Hughes, J. V., Perlow, D. S., and Boger, J. (1985) Induction of Hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J. Virol. 55, 836-839. (2) Wheeler, C. M., Robertson, B. H., Van Nest, G., Dina, G., Bradley, D. W., and Fields, H. A. (1986) Structure of the hepatitis A virion: peptide mapping of the capsid region. J. Virol. 58, 307-313. (3) Neurath, A. R., Kent, S. B. H., and Strick, S. (1984) Antibodies to hepatitis B surface antigen (HBsAg) elicited by immunization with synthetic peptide covalently linked to liposomes. J. Gen. Virol. 65, 1009-1014. (4) Tam, J. P. (1988) Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85, 5409-5413. (5) Allison, A. C., and Gregoriadis, G. (1974) Liposomes as immunological adjuvants. Nature 252, 252-253. (6) Davis, D., and Gregoriadis, G. (1989) Primary immune response to liposome tetanus toxoid in mice: the effect of mediators. Immunology 68, 277-282. (7) Hui, G. S., Chang, S. P., Gibson, H., Hashimoto, A., Hashiro, C., Barr, P. J., and Kotani, S. (1991) Influence of adjuvants on the antibody specificity to the Plasomdium falciparum major merozoite surface protein, gp195. J. Immunol. 147, 3935-3941. (8) Ullrich, S. E., and Fidler, I. J. (1992) Liposomes containing muramyl tripeptide phosphatidylethanolamine (MTP-PE) are excellent adjuvants for induction of an immune response to protein and tumour antigens. J. Leukocyte Biol. 52, 489-494. (9) Sela, M., and Arnon, R. (1980) In New Developments with Human and Veterinary Vaccines (A. Mizrahi, I. Hertman, M. A. Klingberg, and A. Kohn, Eds.) pp 315-323, Liss, New York. (10) Hudecz, F., and To´th, G. K. (1994) In Synthetic peptide constructs to increase the immunogenity of B cell epitopes. Synthetic peptides in the search for B and T cell epitopes (E. Rajnavo¨lgyi, Ed.) pp 97-119, R. G. Landres Company, Austin. (11) Hudecz, F., and Szekerke, M. (1980) Investigation of drugprotein interactions and the drug-carrier concept by the use of branched polypeptides as model systems. Synthesis and characterization of the model peptides. Collect. Czech. Chem. Commun. 45, 933-940. (12) Hudecz, F., and Szekerke, M. (1985) Synthesis of new branched polypeptides with poly(lysine) backbone. Collect. Czech. Chem. Commun. 50, 103-113. (13) Mezo¨, G., Kajta´r, J., Nagy, I., Majer, Zs., Szekerke, M., and Hudecz, F. (1997) Carrier design: Synthesis and conformational studies of poly[L-lysine] based branched polypeptides with hydroxyl groups. Biopolymers 42, 719-730. (14) Hudecz, F. (1995) Design of synthetic branched-chain polypeptides as carriers for bioactive molecules. Anti-Cancer Drugs 6, 171-193. (15) Hudecz, F., Gaa´l, D., Kurucz, I., La´nyi, S., Kova´cs, A. L., Mezo¨, G., Rajnavo¨lgyi, E Ä ., and Szekerke, M. (1992) Carrier design: cytotoxicity and immunogenicity of synthetic branched polypeptides with poly(L-Lys) backbone. J. Control. Relat. 19, 231-243. (16) Hudecz, F., Kutassi-Kova´cs, S., Mezo¨, G., and Szekerke, M. (1989) Biodegradability of synthetic branched polypeptide with poly(L-lysine) backbone. Biol. Chem. Hoppe-Seyler 370, 1019-1026. (17) Hudecz, F., Hilbert, A Ä ., Mezo¨, G., Mucsi, I., Kajta´r, J., Bo¨sze, Sz., Kurucz, I., and Rajnavo¨lgyi, E Ä . (1993) Epitope
Bioconjugate Chem., Vol. 11, No. 1, 2000 37 mapping of the 237-284 region of HSV glycoprotein D by synthetic branched polypeptide-carrier conjugates. Pept. Res. 6, 263-271. (18) Clegg, J. A., Hudecz, F., Mezo¨, G., Pimm, M. V., Szekerke, M., and Baldwin, R. W. (1990) Carrier design: biodistribution of branched polypeptides with poly-(L-lysine) backbone. Bioconjugate Chem. 2, 425-430. (19) Hudecz, F., and Price, M. R. (1992) Monoclonal antibody binding to peptide epitopes conjugated to synthetic branched polypeptide carriers. Influence of the carrier upon antibody recognition. J. Immunol. Methods. 147, 201-210. (20) Hudecz, F., Bogda´n, K., Vordermeier, H. M., Jurcevic, S., and Ivanyi, J. (1997) In Modulation of T cell specific immune responses against 16 kDa protein of M. tuberculosis by alteration of flanks and/or conjugation to synthetic branched polymeric carrier. Peptides 1996, Proc. 24th European Peptide Symposium (R. Ramage and R. Epton, Eds.) pp 115-118, Mayflower Scientific, Birmingham. (21) Wilkinson, K. A., Vordermeier, H., Wilkinson, R., Ivanyi, J., and Hudecz, F. (1998) Synthesis and in vitro T-cell immunogenicity of conjugates with dual specificity: attachment of epitope peptides of 16 kDa and 38 kDa proteins from Mycobacterium tuberculosis to branched polypeptide. Bioconjugate Chem. 9, 539-547. (22) Nagy, I. B., Alsina, M. A., Haro, I., Reig, F., and Hudecz, F. (1998) Surface and polarisation fluorescence study of branched polymeric polypeptides. Biopolymers 46, 169-179. (23) Perez, J. A., Gonzales-Dankart, J. F., Reig, F., Pinto´, R. M., Bosch, A., and Haro, I. (1995) Solid-phase synthesis and immunogenicity of a VP3 peptide from Hepatitis A virus. Biomed. Pept., Proteins Nucleic Acids 1, 93-100. (24) Perez, J. A., Haro, I., Martı´n, I., Alsina, M. A., and Reig, F. (1995) Surface and polarization fluorescence studies on the interaction of an RGD sequence containing a Hepatitis A virus peptide with phospholipids. Anal. Chim. Acta 303, 65-72. (25) Garcı´a, M., Nagy, I. B., Alsina, M. A., Mezo¨, G., Reig, F., Hudecz, F., and Haro, I. (1998) Analysis of the interaction with biomembrane models of the HAV-VP3(101-121) sequence conjugated to synthetic branched chain polypeptide carriers with a poly[L-Lysine] backbone. Langmuir 14, 1861-1869. (26) Carlsson, J., Drevin, H., and Axen, R. (1978) Protein thiolation and reversible protein-protein conjugation. Biochem. J. 173, 723-737. (27) Bretscher, M. (1973) Membrane structure: some general principles. Science 181, 622-629. (28) Mezo¨, G., Votavova´, H., Hudecz, F., Kajta´r, J., Sponar, J., and Szekerke, M. (1988) Conformation of branched polypeptides: The influence of DL-alanine oligomer spacers in the side chains. Collect. Czech. Chem. Commun. 53, 2843-2853. (29) Stuchbury, T., Shipton, M., Norris, R., Paul, J., Malthouse, G., and Brocklehurst, K. (1975) A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem. J. 151, 417432. (30) IUPAC-IUB Joint Commission on Biochemical Nomenclature and Symbolism for amino acids and peptides, Recommendation 1983 (1984) Eur. J. Biochem. 138, 9-37. (31) Verger, R., and De Haas, G. H. (1973) Enzyme reactions in a membrane model. 1. A new technique to study enzyme reactions in monolayers. Chem. Phys. Lipids 10, 127-136. (32) Rubio´, N., Pujol, M., Munoz, M., Alsina, M. A., Haro, I., and Reig, F. (1997) Synthesis and physicochemical characterisation of Laminin related peptides. Supramol. Sci. 4, 449453. (33) Cajal, Y., Alsina, M. A., Rabanal, F., and Reig, F. (1996) A fluorescence and CD study on the interaction of synthetic lipophilic Hepatitis B virus preS(120-145) peptide analogues with phospholipid vesicles. Biopolymers 38, 607-618. (34) Shinitzky, N., and Barenholz, Y. (1978) Fluorescence parameters of lipid regions determined by fluorescence polarisation. Biochim. Biophys. Acta 515, 367-394. (35) Ardite, E., Egea, M. A., Haro, I., Reig, F., and Alsina, M. A. (1994) Conformational changes and physicochemical properties of transferrin upon derivatization with cholesterol. Anal. Chim. Acta 290, 75-85.
38 Bioconjugate Chem., Vol. 11, No. 1, 2000 (36) Egea, M. A., Garcia, M. L., Alsina, M. A., and Reig, F. (1994) Coating of liposomes with transferrin: Physicochemical study of the transferrin-lipid system. J. Pharm. Sci. 83, 169-173. (37) Winterhalter, M., Bu¨rner, H., Marzinka, S., Benz, R., and Kasianowicz, J. J. (1995) Interaction of Poly(etylene-glycols) with air-water interfaces and lipid monolayers: investigations on surface pressure and surface potential. Biophys. J. 69, 1372-1381.
Nagy et al. (38) Nag, K., Perez-Gil, J., Cruz, A., and Keough, K. M. W. (1996) Fluorescently labeled pulmonary surfactant protein C in spread phospholipid monolayers. Biophys. J. 71, 246-256. (39) Haro, I., Busquets, M. A., Ortiz, A., Reig, F., and Alsina, M. A. (1995) Analysis of the perturbation of phospholipid model membranes by a multiple antigenic peptide. Anal. Chim. Acta 303, 57-64.
BC9900385
