Marilena Manea1 Ga´bor Mezo¨2 Ferenc Hudecz2,3 Michael Przybylski1 1
University of Konstanz, Department of Chemistry, Laboratory of Analytical Chemistry, 78457 Konstanz, Germany 2
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s L. University, 1518 Budapest, Hungary
Polypeptide Conjugates Comprising a -Amyloid Plaque-Specific Epitope as New Vaccine Structures Against Alzheimer’s Disease* 3
Department of Organic Chemistry, Eo¨tvo¨s L. University, 1518 Budapest, Hungary
Received 17 June 2004; accepted 4 August 2004 Published online 21 October 2004 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20160
Abstract: Immunotherapeutic approaches designed to induce a humoral immune response have recently been developed for possible vaccination to the treatment of Alzheimer’s disease (AD). Based on the identification of A(4 –10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, branched polypeptide conjugates with this epitope peptide were synthesized and characterized. In order to produce immunogenic constructs, the A(4 –10) epitope alone or together with a promiscuous T-helper cell epitope peptide (FFLLTRILTIPQSLD) were attached via thioether linkage to different branched chain polymeric polypeptides with Ser or Glu in the side chains. A single peptide containing both an A(4 –10) and T-helper cell epitope, joined by a dipeptide Cys–Acp spacer, was also attached through the thiol function to chloroacetylated poly[Lys(Seri–DL–Alax)] (SAK). Comparative binding studies of the conjugates with a monoclonal antibody against the -amyloid(1–17) peptide in mice were performed by direct ELISA. The conformational preferences of carriers and conjugates in water and in a 9:1 triflouroethanol:water mixture (v/v) was analyzed by CD spectroscopy. Experimental data showed that the chemical nature of the carrier macromolecule, and the attachment site of the
Correspondence to: Michael Przybylski; email: Michael.
[email protected] Contract grant sponsors: National Science Fund (NSF) and Deutsche Forschungsgemeinschaft (DF) Contract grant numbers: T032425 and T043576 (NSF); PR/ 175/4 and PR/175/5 (DF) * We dedicate this article to the memory of Professor Murray Goodman, who was an inspiration to us all in the field of peptide science, including the last scientific conference in Capri that he attended, at which this article was presented. Biopolymers (Peptide Science), Vol. 76, 503–511 (2004) © 2004 Wiley Periodicals, Inc.
503
504
Manea et al. epitope to the carrier, have significant effects on antibody recognition, but have no marked influence on the solution conformation of the conjugates. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 76: 503–511, 2004 Keywords: carrier branched polypeptides; -amyloid(4 –10) epitope peptide; antibody recognition; chemical ligation; peptide conjugates
INTRODUCTION Abnormal accumulation of the -amyloid peptide (A) into extra cellular toxic plaques is responsible for the neurodegeneration and resulting dementia in Alzheimer’s disease (AD) (the “amyloid cascade hypothesis” of AD pathogenesis). Therefore, A represents an important molecular target for intervention in AD, and agents that can prevent its formation and accumulation or stimulate its clearance might ultimately be of therapeutic benefit.1 The immunological approach to the treatment of AD involves either stimulating the host immune system to recognize and attack A or providing passively pre-formed antibodies. Both strategies might enhance the clearance and/or even prevent the deposition of A plaques.2 Immunization of transgenic mouse models of AD with A(1– 42) has been effective in blocking A fibrillogenesis, in disaggregating preformed fibrils, and in inhibiting A(1– 42) fibril-induced cytotoxicity. Furthermore, the reduction in A burden was accompanied by improved cognitive performance in mouse models. No adverse effects of A(1– 42) immunization were observed in these experiments.3– 6 However, a therapeutic trial of immunization with A(1– 42) in humans was discontinued due to significant toxic side effects such as meningo-encephalitic cellular inflammatory reactions.7 The use of nonfibrillar/nontoxic A homologous peptides might be a safer vaccination approach in humans. One report of immunization with a fragment of the A peptide coupled to a hexalysine oligomer ([Lys]6–A1–30) has provided preliminary evidence that a peptide conjugate could also be an effective way of triggering a specific immune response that results in reduced ADlike pathology in amyloid precursor protein (APP) transgenic mice.8 Using selective proteolytic digestion of the antigen–antibody complex (epitope excision) in combination with high resolution mass spectrometry,9 the Nterminal A(4 –10) sequence has been identified as the epitope recognized by therapeutically active antibodies raised against protofibrillar/oligomeric assemblies of A(1– 42).10 –12 The knowledge of this epitope provides the basis for the design of molecular mimics for vaccination, particularly specific immunization antigens that minimize the side effects induced
by antibodies recognizing other domains of the amyloid precursor protein. It has been recently shown that immunization with the complete A peptide is not necessary for efficacy, and antibodies directed against the amino-terminal of the peptide provide protection against amyloid pathology.13 On the other hand, passive administration of peptide-specific monoclonal antibodies reduced plaque burden and neuritic pathology to a similar extent to active immunization.14,15 Thus, both immunoconjugates containing defined epitopes of A and mAb against appropriate A epitopes offer alternatives to whole A immunization for the treatment of AD.13 The identification of the A(4 –10) epitope enables the preparation of specific peptide conjugates for vaccination, which is independent of the A structure, aggregation propensity, and toxicity. It is known that the administration of an epitope oligopeptide rarely elicits an appropriate immune response.16,17 Considering these findings, we designed peptide conjugates containing the A(4 –10) epitope peptide (FRHDSGY) alone or together with a peptide corresponding to the promiscuous T-helper cell epitope of a hepatitis B surface antigen (FFLLTRILTIPQSLD), and synthetic branched chain polypeptides as carriers.18,19 These carriers have already been successfully applied in conjugates with B- or T-cell epitopes derived from the herpes simplex virus (HSV) and Mycobacterium tuberculosis,20,21 respectively. Here we report on the synthesis, structural characterization, and antibody binding properties of a new set of conjugates, in which a peptide representing the A(4 –10) epitope was attached to polycationic (poly[Lys(Ser0.9–DL-Ala3.5)], SAK) or amphoteric (poly[Lys(Glu1-DL-Ala3.5)], EAK) branched chain polypeptides18,19 by introducing a thioether bond using chemical ligation.22–24 The conjugation reaction between the chloroacetylated carrier and the epitope peptide containing a Cys residue in the sequence was performed in solution at alkaline conditions. We prepared conjugates in which the A(4 –10) epitope peptide was attached through its N- or C-terminal end as well as a construct containing both A(4 –10) and a promiscuous T-helper cell epitope peptide (Figures 1 and 2). Antigenicity data were determined for all conjugates and suggest that the chemical structure of
Polypeptide Conjugates
505
FIGURE 1 Schematic structure of poly[Lys(1–Glu1–DL-Ala3.5)] conjugate (A) and of poly[Lys(3–Ser0.9–DL-Ala3.5)] conjugate with dual specificity (B).
the conjugate has a significant influence on antibody binding.
EXPERIMENTAL Synthesis Synthesis of A(4 –10)-Related Epitope Peptides: H-FRHDSGYGGGGGC-NH2 (A4 –10G5C, 1), H-CGGGGGFRHDSGY-NH2 (CG5A4 –10, 2), HFFLLTRILTIPQSLD-C-Acp-FRHDSGY-NH2 [A(4 –10)–T-cell epitope peptide, 3]. The H-FRHDSGYGGGGGC-NH2 (1) and H-CGGGGGFRHDSGYNH2 (2) epitope peptides were synthesized manually by 9-fluorenylmethoxycarbonyl/t-butyl ether (Fmoc/ tBu) strategy on Rink amide 4-methylbenzhydrylamine (MBHA) resin (NovaBiochem, La¨ufelfingen, Switzerland) with a capacity of 0.78 mmol/g. The following side-chain-protected amino acid derivatives were used: Fmoc–Tyr(tBu)–OH, Fmoc–Ser(tBu)–OH, Fmoc–Asp(OtBu)–OH, Fmoc–His(Trt)–
OH, Fmoc–Arg(Pbf)–OH, and Fmoc–Cys(Trt)–OH (from Reanal, Budapest, Hungary, and NovaBiochem, La¨ufelfingen, Switzerland). The protocol of the Fmoc solid-phase peptide synthesis was as follows: (i) dimethylformamide (DMF) washing (3 ⫻ 0.5 min), (ii) Fmoc cleavage by 2% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2% piperidine in DMF (2 ⫹ 2 ⫹5 ⫹ 10 min), (iii) DMF washing (8 ⫻ 0.5 min), (iv) coupling 3 equiv of Fmoc amino acid–N,N⬘-diisopropylcarbodiimide (DIC)–HOBt (HOBt: 1-hydroxybenzotriazole) in DMF (60 min), (v) DMF washing (3 ⫻0.5 min), (vi) dichloromethane (DCM) washing (2 ⫻ 0.5 min), and (vii) ninhydrine assay. The peptides were cleaved from the resin, for 2 h using the following cleavage mixture: 1.5 g crystalline phenole, 0.5 mL 1,2-ethandithiol (EDT), 1mL thioanisole, 1 mL deionized (d.i.) water, 20 mL triflouroacetic acid (TFA). After cleavage, the peptides were precipitated with cold diethyl ether and then filtered. The solid material was washed three times with cold ether and dissolved in
506
Manea et al.
FIGURE 2 Outline of the synthesis of A(4 –10) epitope peptide conjugates obtained by chemical ligation.
10% acetic acid before freeze-drying. Crude peptides were purified by reverse phase high performance liquid chromatography (RP-HPLC) on a semipreparative Phenomenex Jupiter C18 column. H-FFLLTRILTIPQSLD-C-Acp-FRHDSGY-NH2 [A(4 –10)–T-cell epitope peptide, 3] was prepared manually on MBHA resin (NovaBiochem, La¨ufelfingen, Switzerland) with a capacity of 1.4 mmol/g. tert-Butyloxycarbonyl/benzyl (Boc/Bzl) chemistry was applied for peptide synthesis using the following side-chain-protected amino acid derivatives: Boc– Cys(Meb)–OH, Boc–Asp(OcHex)–OH, Boc–Ser(Bzl)–OH, Boc–Thr(Bzl)–OH, Boc–Arg(Tos)–OH, Boc–His(Bom)–OH, and Boc–Tyr(BrZ)–OH (all from Reanal, Budapest, Hungary). The synthetic protocol was as follows: (i) DCM washing (3 ⫻ 0.5 min), (ii) Boc cleavage using 33% TFA in DCM (2 ⫹ 20 min), (iii) DCM washing (5 ⫻ 0.5 min), (iv) neutralization with 10% diisopropylethylamine (DIEA) in DCM (4 ⫻ 1 min), (v) DCM washing (4 ⫻ 0.5 min), (vi) coupling 3 equivalents of Boc-amino acid–N,N⬘dicyclohexylcarbodiimide (DCC)–HOBt in DCM– DMF (4:1 or 1:4, depending on the solubility of
amino acid derivatives) mixture (60 min), (vii) DMF washing (2 ⫻ 0.5 min), (viii) DCM washing (2 ⫻ 0.5 min), (ix) ninhydrine assay. Anhydrous hydrogen fluoride (HF) in the presence of p-cresol and dithiothreitol (DTT) as scavengers (HF–p-cresol–DTT ⫽ 10 mL: 1g 0.1g) was used for the removal of side-chainprotecting groups as well as for the cleavage of the peptide from the resin (1.5 h at 0°C). Crude peptide isolated from the cleavage mixture as described above was purified by RP-HPLC on a semipreparative Vydac C4 column. Purified peptides were analyzed by RP-HPLC and matrix-assisted laser desorption ionization–Fourier transform–ion cyclotron resonance (ICR) (MALDI-FT-ICR) mass spectrometry (Table I). Synthesis of Branched Polypeptides. Poly[Lys(Xi– (X ⫽ Glu, Ser) was synthesized as previously described15 with minor modification using HOBt as a catalyst in the active ester coupling method for the attachment of the glutamic acid or serine residues to the end of the side chains of the branched DL-Alam)]
Polypeptide Conjugates
507
Table I
Chemical Characterization of Peptides
Peptide
No.
Sequence
HPLC Rt (min)
[M⫹H]⫹ calculated/found***
1 2 3
H-FRHDSGYGGGGGC-NH2 H-CGGGGGFRHDSGY-NH2 H-FFLLTRILTIPQSLD-C-Acp-FRHDSGY-NH2
17.1* 17.7* 26.6**
1268.52/1268.52 1268.52/1268.51 2854.43/2854.62
A(4–10)G5C CG5A(4–10) A(4–10)-T-cell epitope
RP-HPLC columns: * Phenomex Synergy C12 column (4 m, 80 Å, 250⫻4.6 mm I.D). ** Vydac C4 column (5 m, 300 Å, 250⫻4.6 mm I.D.); eluents: 0.1% TFA/water (A), 0.1% TFA/AcN-water 80:20, V/V (B); flow rate: 1 mL/min; gradient: 0 min 0% B, 5 min 0% B, 50 min 90% B. *** Mass spectrometric analysis was performed with a Bruker APEX II FT-ICR instrument equipped with an actively shielded 7T superconducting magnet (Bruker Daltonik, Bremen, Germany).
polypeptide poly[Lys(DL-Alam)] (AK).21 To explain the procedure briefly: poly(L-Lys) (DPn ⫽ 60) was prepared by the polymerization of N␣-carboxy-N⑀(benzyloxycarbonyl)lysine anhydride. After cleavage of the protecting groups with HBr in glacial acetic acid, poly[Lys(DL-Ala3.5)] was formed by grafting of short oligomeric DL-alanine side chains onto the ⑀-amino groups of polylysine using N-carboxy-DLalanine anhydride. Attachment of suitable protected and activated glutamic acid or serine residues to AK was carried out by the active ester method using a 50% molar excess of Z–Glu(OBzl)–pentachlorophenyl ester (OPcp) or Z–Ser–OPcp and HOBt over AK in a 1:9 water:DMF (v/v) solvent mixture in the presence of 4-methyl-morpholine (at pH 7– 8). The overnight reaction at room temperature was followed by removal of the solvent in vacuum, and the residue was triturated several times with ether containing 10% DCM. The protecting groups were cleaved with HBr/ acetic acetic mixture afterwards. Finally, EAK or SAK were purified by extensive dialysis using Viking tubes (cut-off 8000 –12000 Da) followed by lyophilization. The removal of protecting groups was verified by UV spectroscopy. Amino acid analysis yielded Lys:Ala:Glu ⫽ 1:3.5:1 and Lys:Ala:Ser ⫽ 1:3.5:0.9, respectively. Synthesis of Chloroacetylated Branched Polypeptides. Poly[Lys(Glu1.0–DL-Ala3.5)] (EAK) and poly[Lys(Ser0.9–DL-Ala3.5)] (SAK) were dissolved in an appropriate d.i. water volume respectively and the solutions were diluted with 4 mL DMF. The amount of 0.5 equivalents of ClAc–pentachlorophenyl ester (ClAc–OPcp) in 5 mL DMF was added to each polymer solutions. After stirring at room temperature for 18 h, the reaction mixtures were dialyzed against d.i. water for 24 h (using Visking tubes, cutoff 8000 – 12,000). After filtration, the products were freezedried. The chlorine ion content analysis showed that
around 50% of the side chains were modified by the chloroacetyl (ClAc) groups.
Conjugation of Epitope Peptides with Chloroacetylated Poly[Lys(ClAci– Ser0.9–DL-Ala3.5)] (ClAc–SAK) and poly[Lys(ClAcj–Glu1–DL-Ala3.5)] (ClAc– EAK) Carriers Using Thioether Linkage Branched polypeptides containing ClAc groups, poly[Lys(ClAci–Ser0.9–DL-Ala3.5)] (ClAc–SAK) and poly[Lys(ClAcj–Glu1–DL-Ala3.5)] (ClAc–EAK), were dissolved in 0.1M Tris buffer, pH ⫽ 8.1, and the peptides containing the A(4 –10) epitope (1, 2, or 3) were added to the solution of chloroacetylated polypeptides from time to time in solid form. The conjugations proceeded for 24 h and were terminated by addition of an excess of L-Cys to block the unreacted ClAc groups. The solutions were dialyzed to remove unreacted epitope peptide and cysteine in Visking tubes (cutoff 8000 –12000 Da) against d.i. water for 24 h. After filtration, the products were freeze-dried and the average degree of substitution was calculated from the amino acid analysis (Table II).22,25
Reverse Phase High Performance Liquid Chromatography Analytical RP-HPLC was performed on a Knauer (H. Knauer, Bad Homburg, Germany) HPLC system using a Phenomex Synergy C12 column (250 ⫻ 4.6 mm I.D.) with 4 m silica (80 Å pore size) (Torrance, CA) or a Vydac C4 column (250 ⫻ 4.6 mm I.D.) with 5 m silica (300 Å pore size) (Hesperia, CA) as stationary phases. Linear gradient elution (0 min 0%B; 5 min 0%B; 5 0min 90%B) with eluent A (0.1% TFA in water) and eluent B [0.1% TFA in acetonitrile–water (80:20, v/v)] was used at a flow rate of 1 mL/min at ambient temperature. Peaks were detected at ⫽ 220 nm. The samples were dissolved in eluent A. The crude products were purified on a semipreparative Phenomex Jupiter C18 column (250 ⫻ 10 mm I.D.) with 10 m silica (300 Å pore size) (Torrance, CA) or a Vydac C4 column (250 ⫻ 10 mm I.D.) with 10 m silica (300 Å pore
508
Manea et al.
Table II
Characteristics of Peptide-Conjugates
Peptide/Conjugate Poly[Lys(1-Glu1-DL-Ala3.5)] Poly[Lys(2-Glu1-DL-Ala3.5)] Poly[Lys(1-Ser0.9-DL-Ala3.5)] Poly[Lys(2-Ser0.9-DL-Ala3.5)] Poly[Lys(3-Ser0.9-DL-Ala3.5)] A(4–10)-T-cell epitope
No.
Epitope/Conjugate* (mol/mol)
Mw ⫾ 5%**
Concentration (mol/L A4-10)***
4 5 6 7 8 3
50 20 23 55 5 —
70000 49100 48100 70500 40300 2854
0.02 ⫾ 0.004 0.01 ⫾ 0.001 0.15 ⫾ 0.025 0.01 ⫾ 0.003 0.07 ⫾ 0.015 0.41 ⫾ 0.005
* Molar ratio calculated from the amino acid composition. ** Calculated from the average degree of polymerization of polylysine backbone and amino acid composition of the conjugates. The average degree of polymerization of the polylysine backbone (27) is 60. *** The lowest amount of A (4 –10) epitope peptide in mol/L to obtain an OD450 ⫽ 1.0 in direct ELISA experiment.
size) (Hesperia, CA) using a flow rate of 4 mL/min, with the appropriate linear gradient.
Amino Acid Analysis The amino acid compositions of peptides, carriers, and conjugates were determined by a Beckman Model 6300 analyzer (Fullerton, CA). Prior to analysis, samples were hydrolyzed in 6M HCl in sealed and evacuated tubes at 110°C for 24 h.
MALDI–FT–ICR Mass Spectrometry Mass spectrometric analysis was performed with a Bruker APEX II FT–ICR instrument equipped with an actively shielded 7T superconducting magnet, a cylindrical infinity ICR analyzer cell, and an external Scout 100 fully automated X-Y target stage MALDI source with pulsed collision gas (Bruker Daltonik, Bremen, Germany). The pulsed nitrogen laser is operated at 337 nm. A one hundred milligram per milliliter solution of 2,5-dihydroxybenzoic acid (DHB) (Aldrich, Germany) in acetonitrile:0.1% trifluoroacetic acid in water (2:1) was used as the matrix. The amount of 0.5 L matrix solution and 0.5 L of sample solution were mixed on the stainless-steel MALDI sample target and allowed to dry. Calibration was performed with a standard peptide mixture with an m/z range of approximately 5000.26
CD Spectroscopy CD spectra were recorded on a Jasco spectropolarimeter, model J-720 (Jasco, Finland), at room temperature, in quartz cells of 0.05 cm path length, under constant nitrogen flush. The instrument was calibrated with 0.06% (w/v) ammonium-d-camphor-10-sulfonate (Katayama Chemical, Japan) in water. Double-distilled water and a 9:1 mixture (v/v) of triflouroacetic acid (TFE) (Fluka, Buchs, Switzerland) and water were used as solvents. The sample concentration was 0.5 g/L. The spectra were averages of six scans between
⫽ 190 and 260 nm. Results are expressed in terms of ellipticity after subtraction of the buffer baseline.
Enzyme-Linked Immunosorbent Assay Enzyme-linked immunosorbent assay (ELISA) studies were performed by the purified type immunoglobulin G1 (IgG1) monoclonal antibody (mAb) 6E10 (Chemicon, Temecula, CA), raised against A(1–17) at c ⫽ 1 mg/mL in phosphatebuffered saline (PBS) solution. A standard dilution of the mAb 6E10 was used in combination with 12 serial dilutions of the conjugates or peptide 3, used as coating antigens. The 96-well ELISA plates (BioRad, Hercules, CA) were coated overnight at room temperature with 100 L/well of conjugates or an A(4 –10)–T-cell epitope peptide using serial dilutions from 20 to 0.0001 M of A(4 –10) epitope concentration. The dilutions were made in coating buffer (PBS buffer containing 5 mM Na2HPO4 and 150 mM NaCl, pH ⫽ 7.5). The antigen concentrations were expressed as the A(4 –10) epitope concentration and they were as follows: 20, 6.66, 2.22, 0.74, 0.246, 0.082, 0.027, 0.0091, 0.003, 0.001, 0.0003, and 0.0001 M. After coating, the wells were washed four times with washing buffer (0.05% Tween-20 v/v in PBS, pH ⫽ 7.5), and the nonspecific adsorption sites were blocked with 5% bovine serum albumin (BSA) w/v in PBS for 2 h. The mAb 6E10 at c ⫽ 1 g/L (Chemicon, Temecula, CA) was diluted 4000 times in 5% BSA (SigmaAldrich, Steinheim, Germany) and added to each well (100 L). Thereafter, the plates were incubated at room temperature for 2 h and subsequently washed four times with washing buffer. Then 100 L of peroxidase-labeled goat antimouse IgG (Jackson Immuno Research, West Grove, PA) diluted 1000 times in 5% BSA was added to each well. After incubation for 2 h, the plates were washed three times with washing buffer and once with 0.05M sodium phosphate– citrate buffer, pH ⫽ 5. Then 100 L of o-phenylenediamine dihydrochloride (Merck, Darmstadt, Germany) in substrate buffer (phosphate– citrate) at c ⫽ 1 mg/mL with 2 L of 30% hydrogen peroxide (Merck, Darmstadt, Germany) per 10 mL of substrate buffer was added. The ab-
Polypeptide Conjugates
509
FIGURE 3 CD spectra of poly[Lys(Ser0.9–DL-Ala3.5)] (SAK) and poly[Lys(Glu1.0–DL-Ala3.5)] (EAK) branched polypeptides (a) and their conjugates 4–7 (b) in 9:1 TFE:water (v/v).
sorbance at ⫽ 4 50 nm was measured on a Wallac 1420 Victor2 ELISA Plate Counter (Perkin-Elmer, Boston, MA). The concentration of the peptide/conjugate solution, which gave an OD450 value of 1.0, was calculated (Table II).
RESULTS AND DISCUSSION Synthesis and Chemical Characterization of A(4 –10) Epitope Peptide Conjugates In this work we report on the preparation of synthetic antigens, in which the epitope peptides 1–3 were attached to branched chain polymeric polypeptides as carriers by chemical ligation (thioether bond formation) (Figure 1). The thioether bond was formed rapidly under mild alkaline conditions between a thiol group and a chloroacetylated amino group. Besides the simplicity of the synthesis, an important advantage of the thioether bond is the increased stability in vivo compared to the disulfide bridge. The A(4 –10) epitope peptides, FRHDSGYGGGGGC-NH2 (1) and H-CGGGGGFRHDSGYNH2 (2), were synthesized by solid-phase peptide synthesis according to Fmoc/tBu strategy. Cysteine as a conjugation site was attached to the N- or C-terminal of the peptides. Another peptide, containing both A(4 –10) and a promiscuous T-helper cell epitope, joined by a dipeptide Cys–Acp spacer (H-FFLLTRILTIPQSLD-C-Acp-FRHDSGY-NH2, 3) was synthesized by Boc/Bzl chemistry. Because of its pronounced hydrophobicity, the T-cell epitope peptide was synthesized with a double-coupling procedure. Crude peptides were purified by RP-HPLC and analyzed by mass spectrometry (Table I). After synthesis of poly[Lys(Ser0.9–DL-Ala3.5)] (SAK) and poly[Lys(Glu1–DL-Ala3.5)] (EAK) branched polypeptides, the chloroacetylation reaction was carried out in solution using ClAc–pentachlorophenyl ester. The
analysis of Cl-ion content showed that approximately 50% of the side chains were modified by the chloroacetylation. The conjugation reaction was carried out in solution under a slightly alkaline condition (0.1M Tris buffer, pH ⫽ 8). To avoid oxidation, the cysteinecontaining epitope peptides were added to the solution of chloroacetylated polypeptides from time to time in solid form. The conjugation reaction was terminated after 48 h by addition of an excess of cysteine to block unreacted chloroacetyl groups (Figure 2). The crude products were dialyzed against d.i. water for removal of uncoupled peptide and cysteine. The average degree of substitution was calculated from the amino acid analysis (Table II).
Conformational Preferences by CD Spectroscopy Considering the structural differences that may exist between natural epitope peptides and their conjugates with carrier molecules, various peptide conjugates were synthesized and their conformation was characterized. The secondary structure of carriers and conjugates in water and in a 9:1 TFE:water mixture (v/v) was analyzed by CD spectroscopy. TFE is known to preferentially stabilize proteins and peptides in helical conformation. In water, EAK and SAK carriers showed CD spectral features of some ordered structure (a negative maximum at 200 nm and an intensive negative shoulder at 223 nm). Their conjugation with various epitope peptides containing the A(4 –10) sequence resulted in random coil conformation, characterized by the presence of a negative minimum at 197–198 nm (data not shown). In a TFE–water solution, both carriers adopt a helical structure, as indicated by two negative maxima at 208 nm (–* transition) and at 223 nm (n–* transition) in the spectra. In case of conjugates, the CD spectra in a 9:1
510
Manea et al.
FIGURE 4 Binding of mouse antiamyloid protein (1–17) monoclonal antibody to A(4 –10) epitope peptide conjugates 4–8 and to A(4 –10)–T-cell epitope peptide, 3, as measured by direct ELISA.
TFE:water (v/v) mixture also indicated the presence of ordered structure regardless of primary structure differences (Figure 3).
Comparison of the Antigenicity of the A(4 –10) Epitope Peptide Conjugates Against an anti-A(1–17) Monoclonal Antibody The A(4 –10)–T-cell epitope peptide and the conjugates were compared for binding to an anti-A(1–17) monoclonal antibody by direct ELISA. The conjugates containing the A(4 –10) sequence alone (peptide 1 or 2), together with the T-cell epitope (peptide 3), or the free carriers were coated to ELISA plates. Subsequently, mAb and a peroxidase-conjugated second mAb and o-phenylendiamine (OPD) substrate were added, and absorbance was measured on a Wallac 1420 Victor2 ELISA Plate Counter (Table II). The carriers without the A(4 –10) epitope peptide did not show any reactivity with mAb (data not shown). The curves from the direct ELISA determination are shown in Figure 4. Based on ELISA data, the influence of the chemical nature of the carrier, the presence of the T-cell epitope peptide, and the attachment site of the A(4 –10) epitope peptide to the carrier on the antigenic properties of the conjugates could be well compared. All conjugates showed increased binding activity compared to the linear A(4 –10)–Tcell epitope peptide to the mAb 6E10. This result may
be explained by the conjugation of the A(4 –10) epitope peptide— elongated with a pentaglycine spacer at N- or C-termini—to a branched polypeptide carrier, which is superior to its attachment to a short, linear peptide, such as the T-cell epitope shown above. By comparison of the conjugation attachment site, the A(4 –10) epitope (with a five Gly spacer) coupled at the N-terminal end to polypeptide carrier (SAK or EAK) yielded increased antibody binding relative to A(4 –10) epitope coupled to the carrier at the C-terminal end. This observation is in good agreement with previous evidence of enhanced binding of this epitope by an N-terminal extension in an A precursor protein.11 The poly[Lys(1–Glu1–DL-Ala3.5)] and poly[Lys(2–Glu1–DL-Ala3.5)] conjugates exhibited equal or more pronounced antibody binding than the corresponding constructs with SAK carrier (conjugate 4 vs. conjugate 6, conjugate 5 vs. conjugate 7 in Table II). This indicates that the use of the amphoteric branched polypeptide, EAK, as carrier in combination with certain epitope configurations could be advantageous.
CONCLUSION In this study we report on the preparation, and structural and binding characterization, of synthetic antigen conjugates containing the -amyloid(4 –10) epitope, as lead structure for new types of vaccine
Polypeptide Conjugates
against Alzheimer’s disease. The attachment of small epitopes into macromolecular carriers may represent a true possibility for yielding increased antigenicity. Branched chain polymeric polypeptides based on the polylysine backbone with serine or glutamic acid in the side chains were used as carriers to which epitope peptides containing the A(4 –10) sequence were attached through a thioether bond. The conjugation via a thioether linkage between the chloroacetylated carrier and epitope peptides with Cys at the N- or Cterminus can be easily performed with high yields and appropriate molecular homogeneity. Correlating the primary structure of the conjugates with the antibody recognition data, we concluded that the chemical nature of the carrier and the attachment site of the epitope peptide to the carrier both play a significant role in optimizing antigenicity. This work was supported by grants from the Hungarian National Science Fund (OTKA nos. T032425 and T043576) and the Deutsche Forschungsgemeinschaft (PR/175/4 and PR/175/5). The authors thank Dr. Szilvia Bo¨sze and Dr. Nikolay Youhnovski for expert help with the amino acid analysis and mass spectrometry, respectively.
REFERENCES 1. Schenk, D. Nature Rev Neurosci 2002, 3, 824 –28. 2. Selkoe, D. J.; Schenk, D. Annu Rev Pharmacol Toxicol 2003, 43, 545–584. 3. Schenk, D.; Barbour, R.; Dunn, W.; Gordon, G.; Grajeda, H.; Guido, T.; Hu, K.; Huang, J.; Johnson-Wood, K.; Khan, K.; Kholodenko, D.; Lee, M.; Zhenmei, L.; Lieberburg, I.; Motter, R.; Mutter, L.; Soriano, F.; Shopp, G.; Vasquez, N.; Vandevert, C.; Walker, S.; Wogulis, M.; Yednock, T.; Games, D.; Seubert, P. Nature 1999, 400, 173–177. 4. Janus, C.; Pearson, J.; McLaurin, J.; Mathews, P. M.; Jiang, Y.; Schmidt, S. D.; Chishti, M. A.; Horne, P.; Heslin, D.; French, J.; Mount, H. T.; Nixon, R. A.; Mercken, M.; Bergeron, C.; Fraser, P. E.; St GeorgeHyslop, P.; Westaway, D. Nature 2000, 408, 979 –982. 5. Morgan, D.; Diamond, D. M., Gottschall, P. E.; Ugen, K. E., Dickey, C.; Hardy, J.; Duff, K.; Jantzen, P.; DiCarlo, G.; Wilcock, D.; Connor, K.; Hatcher, J.; Hope, C.; Gordon, M.; Arendash, G. W. Nature 2000 408, 982–985. 6. Weiner, H. L.; Lemere, C. A.; Maron, R.; Spooner, E. T.; Grenfell, T. J.; Mori, C.; Issazadeh, S.; Hancock, W. W.; Selkoe, D. J. Ann Neurol 2000, 48, 567–579. 7. Check, E. Nature 2002, 415, 462. 8. Sigurdsson, E. M.; Scholtzova H.; Mehta D. P.; Frangione B., Wisniewski T. Am J Pathol 2001, 159, 439 – 447. 9. Macht, M.; Fiedler, W.; Kurzinger, K.; Przybylski, M. Biochemistry 1996, 35, 15633– 15639.
511
10. McLaurin, J.; Cecal, R.; Kierstead, M. E.; Tian, X.; Phinney, A. L; Manea, M.; French, J. E.; Lambermon, M. H.; Darabie, A. A.; Brown, M. E., Janus, C., Chishti, M. A.; Horne, P.; Westaway, D.; Fraser, P. E.; Mount, H. T.; Przybylski, M.; St George-Hyslop, P. Nat Med 2002, 8, 1263–1269. 11. Przybylski, M.; McLaurin, J.; Cecal, R. E.; Kierstead, M. E.; Tian, X.; Janus, C.; Manea, M.; Horne, P.; Westaway, D.; Fraser, P. E.; St George-Hyslop, P. In Peptides 2002; Benedetti, E., Pedone, C., Eds.; Edizioni Zino: Napoli, Italy, 2002; pp 862– 863. 12. Cecal, R.; McLaurin, J.; Tian, X.; Manea, M.; Janus, C.; Westaway, D.; Fraser, P.; St George-Hyslop, P.; Przybylski, M. In Peptides 2002; Benedetti, E., Pedone, C., Eds.; Edizioni Zino: Napoli, Italy, 2002; pp 700 –701. 13. Bard, F.; Barbour, R.; Cannon, C.; Carretto, R.; Fox, M.; Games, D., Guido, T.; Hoenow, K.; Hu, K.; JohnosWood, K.; Khan, K.; Kholodenko, D.; Lee, C.; Lee, M.; Motter, R.; Nguyen, M.; Reed, A.; Shenk, D.; Tang, P.; Vasqzez, N.; Seubert, P.; Yednock, T. Proc Natl Acad Sci USA 2003, 100, 2023–2028. 14. Bard, F.; Cannon, C.; Barbour, R.; Burke, R-L.; Games, D.; Grajeda, H.; Guido, T.; Hu, K.; Huang, J.; JohnsonWood, K.; Khan, K.; Kholodenko, D.; Lee, M.; Lieberburg, I.; Motter R.; Nguyen, M.; Soriano, F.; Vasquez, N.; Weiss, K.; Welch, B.; Seubert, P.; Schenk, D.; Yednock, T. Nat Med 2000, 6, 916 –920. 15. DeMattos, R. B.; Bales, R. K.; Cummins, D. J.; Dodart, J. K.; Paul, S. M.; Holzman, D. M. Proc Natl Acad Sci USA 2001, 98, 8850 – 8855. 16. Purcell, A. W.; Zeng, W.; Mifsud, N. A.; Ely, L. K.; MacDonald, W. A.; Jackson, D. C. J Peptide Sci 2003, 9, 255–281. 17. Downard, M. K. J Mass Spectrom 2000, 35, 493–503. 18. Hudecz, F.; Szekerke, M. Collect Czech Chem Commun 1984, 50, 103–113. 19. Mezo¨, G.; Kajta´r, J.; Nagy, I.; Szekerke, M.; Hudecz, F. Biopolymers 1997, 42, 719 –730. 20. Hudecz, F. Biologicals 2001, 29, 197–207. 21. Mezo¨, G.; Mihala, N.; Andreu, D.; Hudecz, F. Bioconj Chem 2000, 11, 484 – 491. 22. Mezo¨, G.; de Oliveira, E.; Krikorian, D.; Feijlbrief, M.; Jakab, A.; Tsikaris, V.; Sakarellos, C.; Welling-Wester, S.; Andreu, D.; Hudecz, F. Bioconj Chem 2003, 14, 1260 –1269. 23. Robey, F.; Fields, R. L. Anal Biochem 1989, 177, 373–377. 24. Mezo¨, G.; Votavova, H.; Hudecz, F.; Kajtar, J.; Sponar, J.; Szekerke, M. Collect Czech Chem Commun 1988, 53, 2843–2853. 25. Mezo¨, G.; Manea, M.; Jakab, A.; Kapuvari, B.; Bo¨sze, S.; Schlosser, G.; Przybylski, M.; Hudecz, F. J Peptide Sci 2004, in press. 26. Damoc, E.; Youhnovski, N.; Crettaz, D.; Tissot, J. D.; Przybylski, M. Proteomics 2003, 3, 1425–1433. 27. Hudecz, F.; Kovacs, P.; Kutassi-Kovacs, S.; Kajtar, J Colloid Polym Sci 1984, 262, 208.
