Bioconjugate Chem. 2006, 17, 1116−1124
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Amino-Terminal Dimerization of Peptides on the Solid Support. Synthesis and Biological Activity of the Immunosuppressive HLA-DR Fragments Linked by Poly(ethylene glycol)s Monika Biernat,† Piotr Stefanowicz,† Michał Zimecki,‡ and Zbigniew Szewczuk*,† Faculty of Chemistry, University of Wrocław, Wrocław, Poland, and Institute of Immunology and Experimental Therapy, PAN, Wrocław, Poland. Received December 22, 2005; Revised Manuscript Received July 27, 2006
The nonapeptide fragment of the HLA-DR molecule, located in the exposed loop of the β chain (164-172) and having the sequence VPRSGEVYT, suppresses the immune response. On the basis of the three-dimensional structure of the HLA-DR superdimer, we designed new dimeric analogs in which the VPRSGEVYT peptides are linked through their N-termini by poly(ethylene glycol) linkers of different lengths and are able to mimic the dimeric nature of the immunosuppressive fragments of HLA class II molecules. The analogs were synthesized using standard solid-phase peptide synthesis protocols. The dimerization was achieved by cross-linking the N-terminal positions of the peptides, attached to an MBHA resin, with R,ω-bis(acetic acid) poly(ethylene glycol), activated by esterification with pentafluorophenol. Our results demonstrate that the amino-terminal dimerization of the peptide results in enhanced immunosuppressive activity and that the potency of the conjugates depends on the length of the poly(ethylene glycol) linker. MS/MS analysis of the obtained dimeric peptides is also presented.
INTRODUCTION MHC1
class II molecules present antigenic peptides to helper T cells and thereby alert the immune system to infectious attack (reviewed by Pieters (1), Hiltbold and Roche (2)). MHC class II molecules are heterodimers with a molecular mass of 60 kDa consisting of noncovalently associated polymorphic R and β subunits. The class II molecules are highly polymorphic proteins. The presence of several isotypes (DP, DQ, and DR) of human MHC (HLA) class II molecules further increases their diversity. Our previous studies showed that fragments located in the β164-172 loop of HLA-DQ suppress the humoral and cellular immune responses (3, 4) and inhibit some integrins (5). The fragments contain the RGD sequence, known to be important in several proteins in mediating cell adhesion interactions. The sequence is located in an exposed loop of the HLA-DQ molecule and may therefore be involved in interactions with other proteins. The immunosuppressive region is situated in close spatial proximity to the site identified as interacting with the T-cell coreceptor CD4 (6). The pentateptide fragment of the loop, Arg-Gly-Asp-Val-Tyr-NH2, is the shortest immunosuppressive sequence found, although its potency is relatively low (7). The corresponding fragments of HLA-DP and HLADR show immunological properties similar to the HLA-DQ fragment (8). Particularly, the nonapeptide fragment of HLADR (Val-Pro-Arg-Ser-Gly-Glu-Val-Tyr-Thr) is a strong suppressor of the immune response, although the fragment does * Correspondence to Z. Szewczuk, Wydział Chemii, Uniwersytet Wrocławski, ul. F. Joliot-Curie 14, 50-383 Wrocław, Poland. Fax: +48-71-3282348. Tel.: +48-71-3757212. E-mail: szewczuk@ wchuwr.chem.uni.wroc.pl. † University of Wrocław. ‡ Institute of Immunology and Experimental Therapy. 1 Abbreviations and symbols are in accordance with recommendations of the European Peptide Society (J. Peptide Sci. 5, 465-471, 1999). AFC, antibody-forming cells; HLA, human leukocyte antigen; MHC, major histocompatibility complex; PEG-Pfp2, poly(ethyleneglycol)bis(carboxymethyl)ether activated by esterification with pentafluorophenol; SRBC, sheep red blood cells.
not contain the RGD sequence. We also designed and synthesized constrained (by cyclization) analogs of the fragment to mimic the immunosuppressive loop of HLA class II molecules and found that some of the investigated peptides suppress immune responses more strongly than their linear counterparts (9, 10). On the basis of our results, we suggested that the loop may serve as a functional epitope on the HLA class II surface for intermolecular binding and that a possible mechanism of biological action of the synthesized peptides is connected with specific interference with the adhesion of HLA class II molecules to their coreceptors, e.g., some specific integrins and/ or the T-cell coreceptor CD4 (11). Recently, we pointed out some topological correspondence between the β-164-172 loop of HLA-DQ and the 50-59 loop of ubiquitin, suggesting that the proteins may interact with similar proteins, although the ubiquitin fragment contains the retro-RGD sequence. We showed that the linear and cyclic fragments of ubiquitin, designed to mimic the loop, may significantly inhibit both humoral and cellular immune responses (12). The low toxicity of such compounds compared with classic suppressors such as cyclosporine A (CsA) may lead to potential application of the peptides in therapy. A combined use of these peptides with conventional immunosuppressors may have even better therapeutic perspectives. The three-dimensional structure of the HLA class II molecule has been determined by X-ray crystallography (13). The crystal structure shows that the Rβ heterodimer can itself dimerize to form a four-chain (Rβ)2 superdimer of 120 kDa. There is also evidence for the existence of the 120 kDa (Rβ)2 complex of the class II molecules in animal B cells (14). It has been suggested that MHC class II molecules may interact with the T-cell receptor (TCR) and CD4+ as an (Rβ)2 superdimer, potentially ensuring more stable and stimulatory interactions than can be provided by the simple Rβ heterodimer alone (15, 16). Evidence has also been presented that the formation or rearrangement of a T-cell receptor dimer is necessary and sufficient for initiation of T-cell signaling (17). Self-association of the CD4 coreceptor may also affect T-cell activation (18). Therefore, substances that are able to modulate the receptors’
10.1021/bc050360h CCC: $33.50 © 2006 American Chemical Society Published on Web 08/22/2006
N-Terminal Dimerization of Immunosuppressive HLA Fragment
dimerization may control such a process and are potential immunomodulators. Dimeric ligands are known candidates for mediating dimerization of these types of receptors (19, 20). It was previously found (21) that peptides that consist of two tandemly repeated epitopes joined by a flexible linker have an increased affinity for class II molecules and are more potent in inducing proliferation of T-cell clones than monomeric epitopes. These results support a role of the MHC class II dimer of heterodimers in amplifying the proliferative response of T cells to antigen by dint of the superdimers having a higher affinity for CD4 than the nominal class II Rβ heterodimers. Recently, we synthesized a series of dimeric analogs of the immunosuppressive fragments of HLA class II molecules. The dimeric analogs consist of two VPRSGEVYT sequences joined through their C-termini by oligoglycine spacers of different length connected to the lysine residue (VPRSGEVYTGn)2K-NH2 (n ) 4, 5, and 6). Our results demonstrate that the immunosuppressive activities of the resulting dimeric analogs are significantly higher than those of their monomeric counterparts and their potencies depend on the length of their linker (22). However, employment of the oligoglycine sequences in the dimers significantly decreased their solubility in aqueous media. This problem might be solved by replacing the linker by a more soluble one, for example, poly(ethylene glycol). Conjugation of poly(ethylene glycol) with bioactive proteins or peptides (PEGylation) increases their solubility in physiological fluids as well as their plasma half-life and resistance to proteolytic cleavage (23). In addition, the conjugate’s immunogenicity may also be decreased (24, 25). It has also been shown that the length of the PEG chains that are attached to the protein affects the protein’s biological activities (26). In this paper, we report the synthesis of dimeric peptides linked through their N-termini by poly(ethylene glycol) spacers of different length. On the basis of the three-dimensional structure of the HLA-DR superdimer, we designed new dimeric analogs able to mimic the dimeric nature of the immunosuppressive fragments of HLA class II molecules by which the VPRSGEVYT peptides were linked with a commercially available mixture of R,ω-bis(acetic acid) poly(ethylene glycol) (PEG-diacid) activated by esterification with pentafluorophenol. The dimeric peptides (peptides 1 and 2), in which the sequences VPRSGEVYT were linked with polyether linkers of defined length, were also obtained. The synthesized dimers were analyzed by ESI-MS/MS spectrometry, and their immunomodulatory properties were also tested.
MATERIALS AND METHODS Peptide Synthesis. Materials. The derivatives of amino acids for peptide synthesis and the coupling reagent (TBTU) were purchased from NovaBiochem. The side-chain-protecting groups for Fmoc-amino acids were t-butyl for Thr, Tyr, Glu, and Ser, and Pmc for Arg. The MBHA-Rink amide resin (0.55 mmol/g) was purchased from NovaBiochem. Poly(ethylene glycol) 600 diacid and poly(ethylene glycol) 250 diacid R,ω-bis(acetic acid) poly(ethylene glycol) were purchased from Aldrich. The poly(ethylene glycol) 250 diacid used for the synthesis of dimeric peptide 2 contains R,ω-bis(acetic acid) tri(ethylene glycol) as a main component. The homogeneous R,ω-bis(propionic acid) dodecae(ethylene glycol) used for the synthesis of dimeric peptide 1 was purchased from Iris Biotech GmbH (Germany). The solvents for peptide synthesis were obtained from Riedel de Hae¨n (DMF) and J.T. Baker (methanol). Preparation of Peptides. Peptides were prepared by manual solid-phase techniques using the standard Fmoc synthesis procedure. The first amino acid attached to the MBHA-Rink amide resin was Thr(But). Fmoc-protecting groups were removed in the presence of 25% piperidine in dimethylformamide
Bioconjugate Chem., Vol. 17, No. 5, 2006 1117
(DMF). Single coupling by TBTU was performed in DMF followed by acetylation with acetic anhydride at the end of each cycle. Preparation of Pentafluorophenol-PEG Diesters (PEGPfp2). The poly(ethylene glycol) 600 diacid (90 mg, 0.15 mmol (according to the average molecular weight)) and pentafluorophenol (55 mg, 0.3 mmol) components were dissolved in 20 mL of ethyl acetate at 0 °C, and DCC (62 mg, 0.3 mmol) was added. The reaction mixture was stirred for 1.5 h at 0 °C. Dicyclohexylurea was filtered off, and the filtrate was evaporated in vacuo. The resulting PEG-Pfp2 product was used for the following synthesis without further purification. Syntheses of the pentafluorophenol diesters of the individual PEG-diacids (R,ω-bis(propionic acid) dodecae(ethylene glycol) and R,ω-bis(acetic acid) tri(ethylene glycol)) were carried out with an identical procedure. Dimerization. The peptide on the resin was linked by forming an amide bond between the N-terminal amino acid residues and PEG-Pfp2 in DMF at room temperature in the presence of 1-hydroxybenzotriazol. The peptidyl resin (120 mg, 0.7 mmol) was mixed in DMF in the reaction vessel. The PEG diester (34 mg, 0.35 mmol) and HOBT (50 mg, 1.75 mmol) were added to the reaction mixture in five equal portions (one portion per day). The peptide was cleaved from the resin using Reagent K (TFA/ thioanisole/water/phenol/TIS 33:2:2:2:1, v/v) at room temperature for 4 h and extracted with trifluoroacetic acid, followed by precipitation with cold diethyl ether. Purification and Characterization of the Product. The crude product was characterized by HPLC on a 5-µm C-18 column (ODS 4.6 mm × 250 mm). Solvent systems were S1, 0.1% aqueous trifluoroacetic acid (TFA); S2, 80% acetonitrile + 0.1% TFA, linear gradient from 0-100% of S2 for 60 min, flow rate 1.0 mL/min, determined at 220 nm. The main heterogeneous reaction product was further purified and fractionated to three approximately equal fractions by preparative reversed-phase HPLC on a Vydac C-18 column (22 mm × 250 mm) using the solvent systems S1, 0.1% aqueous TFA; S2, 40% acetonitrile + 0.1% TFA, linear gradient from 50-100% of S2 for 60 min, flow rate 7.0 mL/min, determined at 220 nm. The fractions were collected, transformed into acetate forms, and lyophilized. The homogenic peptides 1 and 2 were purified by the same method. Their purity was over 98% measured by reversed-phase HPLC analyses. The obtained products possessed the correct amino acid composition (measured on a DIONEX-AAA Direct System). Their molecular weights were checked on a Finnigan MAT TSQ 700 mass spectrometer equipped with a Finnigan electrospray ionization source and Q-TOF instrument (Micromass, Manchester, U.K.). The identity of peptides 1 and 2 was confirmed using a micrOTOF-Q (Bruker, Germany) electrospray mass spectrometer on the basis of high-resolution mass measurement. Structural Analysis. MS/MS Spectra. Mass spectrometric measurements were performed on a quadruple time-of-flight instrument (Q-TOF Micromass, Manchester, U.K.) equipped with an electrospray source. Spectra were recorded using aqueous solutions of acetonitrile (50%) and formic acid (1%) at a peptide concentration of typically 5 µM (according to the average molecular weight). The potential between the spray needle and the orifice was set to 2.5 kV, and the cone voltage was 35 V. In MS/MS mode, the quadrupole was used to select the precursor ions, which were fragmented in the hexapole collision cell, generating product ions that were subsequently mass analyzed by the orthogonal reflectron TOF mass analyzer. For CID MS/MS measurements, the voltage over the hexapole collision cell varied from 25 to 70 V, and argon was used as the collision gas at a pressure of 11 kPa. MS/MS data were processed by a maximum entropy data enhancement program,
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Biernat et al.
Table 1. Effect of the Peptides on the Secondary Humoral Immune Response of Splenocytes from CBA Mice to SRBC peptide controlb fraction A fraction C control peptide 1 control peptide 2 control VPRSGEVYTc
dose (µg/mL) 1 10 100 1 10 100 1 10 100 1 10 100 1 10 100
AFC/ 106 splenocytes
(SEa
2169 2085 566 1518 1908 1396 686 1040 910 625 550 912 682 337 283 1382 1253 956 813
120 201 102 107 164 80 85 41 37 61 33 45 76 72 13 11 9 14 14
P (Student’s t-test)
suppression (%)
NS
