Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity.

Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limpet haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long-chain fatty acid like palmitic acid by a thioester or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin-releasing hormone). For chemical synthesis of thioesters, we established conditions for solution and solid-phase synthesis. In both phases, Cys(SBut) could only be deprotected efficiently using phosphines, and S-acylation was accomplished using standard coupling at pH 5. We speculate that, in vivo, the presence of an appropriate fatty acid chain, chemically linked through a labile thioester bond, greatly enhances immunogenicity, because it represents a favourable substrate for cleavage by cellular thioesterases in cells of the immune system.

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. I. Model studies using cyclic and linear peptides.

The high molecular weight (HMW) proteins from wheat contain a repetitive domain that forms 60-80% of their sequence. The consensus peptides PGQGQQ and GYYPTSPQQ form more than 90% of the domain; both are predicted to adopt beta-turn structure. This paper describes the structural characterization of these consensus peptides and forms the basis for the structural characterization of the repetitive HMW domain, described in the companion paper. The cyclic peptides cyclo-[PGQGQQPGQGQQ] (peptide 1), cyclo-[GYYPTSPQQGA] (peptide 2), and cyclo-[PGQGQQGYYPTSPQQ] (peptide 3) were prepared using a novel synthesis route. In addition, the linear peptides (PGQGQQ)n (n = 1, 3, 5) were prepared. CD, FTIR, and NMR data demonstrated a type II beta-turn structure at QPGQ in the cyclic peptide 1 that was also observed in the linear peptides 9PGQGQQ)n. A type I beta-turn was observed at YPTS and SPQQ in peptides 2 and 3, with additional beta-turns of either type I or II at GAGY (peptide 2) and QQGY (peptide 3). The proline in YPTS showed considerable cis/trans isomerization, with up to 50% of the population in the cis-conformation; the other prolines were more than 90% in the trans conformation. The conversion from trans to cis destroys the type I beta-turn at YPTS, but leads to an increase in turn character at SPQQ and GAGY (peptide 2) or QQGY (peptide 3).

Synthesis of peptide p-nitroanilides mimicking fibrinogen- and hirudin-binding to thrombin. Design of slow reacting thrombin substrates.

The design and synthesis of 20 peptide p-nitroanilides is described. The nitroanilides are used as thrombin substrates that share uncommon properties. These substrates are tested for their applicability to measure thrombin generation in activated plasma. This technique requires substrates that must slowly but selectively be hydrolyzed by thrombin. To ensure selectivity, thrombin's natural substrate and its most potent inhibitor were used as lead compounds. Eighteen peptide p-nitroanilides were synthesized using human fibrinogen A alpha [7-16] decapeptide as lead structure since this fragment constitutes the minimal sequence which binds to thrombin with high affinity. Two other chromogenic substrates were designed using 5-amino-2-nitrobenzoic acid as the chromophore. A peptide giving subsite interactions with the fibrinogen recognition exosite was coupled to the carboxylic function. As the peptide segment, the carboxyl terminal part of hirudin (residues 50-65) was chosen to ensure highly specific thrombin recognition. From the 20 nitroanilides synthesized, we indeed obtained a number of compounds which can be used as substrate in the thrombin generation assay.

The solution structure of the synthetic circular peptide CGVSRQGKPYC. NMR studies of the folding of a synthetic model for the DNA-binding loop of the ssDNA-binding protein encoded by gene V of phage M13.

The cyclic disulfide peptide CGVSRQGKPYC was prepared to obtain a constrained analogue of residues 17-27 of the DNA-binding loop of the gene-V-encoded sDNA-binding protein of filamentous bacteriophage M13. Amino acid sequences very similar to that of the beta-loop have been found in various phage-encoded ssDNA-binding proteins, and it has been proposed that such a loop may occur as a common motif in this class of proteins. The conformation, in aqueous solution, of the synthetic gene-V-protein binding-loop analogue has been investigated by means of two-dimensional-1H-NMR techniques. Subsequent structure calculations show that the molecule forms a beta-loop that includes a turn formed by three residues. This structure, very unusually for a cyclic disulfide peptide, is highly similar to that of the analogous part of the binding loop of the native protein. Comparison with experiments on other cyclic disulfide peptides indicates that the formation, of the beta-sheet (beta-hairpin) secondary structure is essentially governed by the amino acid composition of the 11-residue sequence. The disulfide bridge in the 11-residue sequence is essential for conformational stability, as indicated by the finding that the open peptide analogue that encompasses residues Ser17-Ser27 does not adopt a detectable secondary structure in water. The bridge replaces the role of the loop formed by residues 49-58 in the protein, which act as a scaffold to hold the N-terminal and C-terminal ends of the DNA-binding loop together.

Design and synthesis of thrombin substrates with modified kinetic parameters.

For the continuous registration of thrombin formation in plasma (1), selective thrombin substrates are required, that show moderate binding affinities (high Km) and low turnover numbers (low kcat). Previously we have used SQ68 (CH3O-CO-CH2-CO-Aib-Arg-pNA) for this purpose. In order to find more substrates suitable for this application, we synthesized a series of 25 peptide p-nitroanilides. As lead structures SQ68 and S2238 (H-D-Phe-Pip-Arg-pNA) were used. By introduction of specific structure modifications we tried to alter the kinetic data in the required direction. The modifications were designed on basis of existing knowledge on the structure of the thrombin active-site and its surroundings. We indeed obtained a number of substrates with the kinetic constants in the desired range.

Protected peptide disulfides by oxidative detachment from a support.

A new and efficient procedure for the preparation of protected cyclized and protected symmetrical dimeric peptide disulfides is described. A thiol is immobilized onto a solid phase through coupling of the thiol function with a resin-linked trityl group. Following conventional peptide assembly using the Fmoc-strategy, detachment is performed by oxidation with iodine in a suitable organic solvent. When N,N-dimethylformamide is used as the solvent, and the peptide chain contains an acetamidomethylthio function, located N-terminally in a N alpha-(9-fluorenylmethyloxycarbonyl), or N alpha-tert-butyloxycarbonyl cysteinyl residue, or occurring in the chain, then the corresponding fully protected cyclic peptide disulfide will be obtained in high yield and purity. In other solvents (e.g. dioxane or chloroform-methanol 1:1, v/v), the iodine-mediated oxidation gave not only the cyclic product, but also substantial amounts of the parallel symmetrical dimeric peptide retaining Cys(Acm) at the two identical N-termini.

Exploring the DNA binding domain of gene V protein encoded by bacteriophage M13 with the aid of spin-labeled oligonucleotides in combination with 1H-NMR.

The DNA binding domain of the single-stranded DNA binding protein gene V protein encoded by the bacteriophage M13 was studied by means of 1H nuclear magnetic resonance, through use of a spin-labeled deoxytrinucleotide. The paramagnetic relaxation effects observed in the 1H-NMR spectrum of M13 GVP upon binding of the spin-labeled ligand were made manifest by means of 2D difference spectroscopy. In this way, a vast data reduction was accomplished which enabled us to check and extend the analysis of the 2D spectra carried out previously as well as to probe the DNA binding domain and its surroundings. The DNA binding domain is principally situated on two beta-loops. The major loop of the two is the so-called DNA binding loop (residues 16-28) of the protein where the residues which constitute one side of the beta-ladder (in particular, residues Ser20, Tyr26, and Leu28) are closest to the DNA spin-label. The other loop is part of the so-called dyad domain of the protein (residues 68-78), and mainly its residues at the tip are affected by the spin-label (in particular, Phe73). In addition, a part of the so-called complex domain of the protein (residues 44-51) which runs contiguous to the DNA binding loop is in close vicinity to the DNA. The NMR data imply that the DNA binding domain is divided over two monomeric units of the GVP dimer in which the DNA binding loop and the tip of the dyad loop are part of opposite monomers. The view of the GVP-ssDNA binding interaction which emerges from our data differs from previous molecular modeling proposals which were based on the GVP crystal structure (Brayer & McPherson, 1984; Hutchinson et al., 1990). These models implicate the involvement of one or two tyrosines (Tyr34, Tyr41) of the complex loop of the protein to participate in complex formation with ssDNA. In the NMR studies with the spin-labeled oligonucleotides, no indication of such interactions has been found. Other differences between the models and our NMR data are related to the structural differences found when solution and crystal structures are compared.

Exploration of the single-stranded DNA-binding domains of the gene V proteins encoded by the filamentous bacteriophages IKe and M13 by means of spin-labeled oligonucleotide and lanthanide-chelate complexes.

Scrutiny of NOE data available for the protein encoded by gene V of the filamentous phage IKe (IKe GVP), resulted in the elucidation of a beta-sheet structure which is partly five stranded. The DNA-binding domain of IKe GVP was investigated using a spin-labeled deoxytrinucleotide. The paramagnetic-relaxation effects observed in the 1H-NMR spectrum of IKe GVP, upon binding of this DNA fragment, could be visualized using two-dimensional difference spectroscopy. In this way, the residues present in the DNA-binding domain of IKe GVP can be located in the structure of the protein. They exhibit a high degree of identity with residues in the gene V protein encoded by the distantly related phage M13 (M13 GVP), for which similar spectral perturbations are induced by such a spin-labeled oligonucleotide. Binding studies with negatively charged lanthanide-1,4,7,10-tetraazacyclodecanetrayl-1,4,7-10- tetrakis(methylene)tetrakisphosphonic acid (DOTP) complexes, showed that these complexes bind to IKe and M13 GVP at two spatially remote sites whose affinities have different pH dependencies. Above pH 7, there is one high-affinity binding site for Gd(DOTP)5-/M13 GVP monomer, which coincides with the single-stranded DNA-binding domain as mapped with the aid of spin-labeled oligonucleotide fragments. The results show that single-stranded DNA binds to conserved (phosphate binding) electropositive clusters at the surface of M13 and IKe GVP. These positive patches are interspersed with conserved or conservatively replaced hydrophobic residues. At pH 5, a second Gd(DOTP)(5-)-binding site becomes apparent. The corresponding pattern of spectral perturbations indicates the accommodation of patches of conserved, or conservatively replaced, hydrophobic residues in the cores of the M13 and IKe dimers.

Preparation and characterization of monoclonal antibodies against ornithine decarboxylase.

In order to develop a method for the immunocytochemical detection of ornithine decarboxylase (ODC), EC 4.1.1.17, we have prepared and characterized monoclonal antibodies (MAbs) against ODC. The primary structure of rat ODC (Rattus Norvegicus) was used for the selection of an epitope by computer calculations. The epitope (P16), a hexadecapeptide representing ODC-(345-360), was synthesized by means of solid phase peptide synthesis and coupled to a carrier protein. A bovine serum albumin conjugate of the P16 peptide was used as the immunogen for the production of MAbs in mice. Hybridoma clones were screened and the specificity of the monoclonal antibodies was tested in an ELISA utilizing a thyroglobulin conjugate of the hexadecapeptide. Two hybridoma cell lines were developed, i.e., MP16-2 and MP16-3. The epitope specificity of the MAbs produced by these cell lines was characterized in an ELISA using a set of small peptides representing parts of the P16 hexadecapeptide chain. MP16-2 recognized the ODC-(355-360) portion whereas MP16-3 reacted with the ODC-(345-350) part of the hexadecapeptide. Further studies showed that both MAbs also recognized native ODC but not the inhibited (i.e., ODC labelled with 3H-DFMO) enzyme indicating that the selected epitope was associated with the active site of ODC or a locus in its direct vicinity.

Binding of ethylenediamine to phosphatidylserine is inhibitory to Na+/K(+)-ATPase.

Covalent linkage of ethylenediamine with the Na+/K(+)-ATPase complex from rabbit kidney outer medulla by the use of the water-soluble carbodiimide, N-ethyl,N'-(3-dimethylaminopropyl)carbodiimide, resulted in a 73% reaction with phosphatidylserine and only 27% with carboxylic groups in the proteic component of the enzyme. Condensation products from the reaction between phosphatidylserine and ethylenediamine, N-(O-phosphatidylseryl)ethylenediamine, N,N'-bis(O-phosphatidylseryl)ethylenediamine and its intermediary product O-phosphatidyl-[N,N'-bis(seryl)]ethylenediamine, were synthesised. Symmetrically substituted ethylenediamine was the most likely condensation product of ethylenediamine with endogenous phosphatidylserine. The synthesised lipids were incorporated in proteoliposomes containing Na+/K(+)-ATPase and only the addition of the phospholipid phosphatidylcholine. The ratio of phospholipid to protein was 52 (w/w). These proteoliposomes were perforated by the addition of 0.5% cholate and both the Na(+)-dependent phosphorylation level and its dependence on Na+, Mg2+ and ATP were measured. Phosphatidylcholine alone increased the half-maximal activation concentration for Na+ ([Na+]0.5) from 0.2 to 1-2 mM, for Mg2+ from 0.1 to 0.8 microM and for ATP from 0.02 to 0.3 microM. The Ki for K+ (in the absence of Na+) was unaffected: 12.8 microM vs. 12.5 microM in the non-reconstituted system. Replacing 10 mol% of phosphatidylcholine by phosphatidylethanolamine: or phosphatidylserine had no significant effect on [Na+]0.5: 1.1 and 0.7 mM, respectively. Replacing 5 mol% phosphatidylcholine by the bis(phosphatidylseryl) substituent of ethylenediamine further increased [Na+]0.5 to 13.7 mM, while half-maximal activation concentrations for Mg2+ and ATP were unaltered. The mono-phosphatidylseryl derivatives of ethylenediamine, each 5 mol%, also increased [Na+]0.5, but to a lesser extent (3.2-3.8 mM). In addition to their competitive effects, the phosphatidylseryl-substituted ethylenediamine compounds exerted a slowly-increasing non-competitive inhibition, not only in phosphorylation, but also in overall ATPase activity, which was reduced, although not abolished, by exogenous protein (bovine serum albumin). A detergent-like action in the usual sense is unlikely since liposomes containing these lipids remained intact. These studies prove that phospholipids are not only required for optimal activity of this transport enzyme, but in excess or in compositions deviating from the normal, may also be inhibitory.

Enhancement of solubility by temporary dimethoxybenzyl-substitution of peptide bonds. Towards the synthesis of defined oligomers of alanine and of lysyl-glutamyl-glycine.

The synthesis of the model compound Aloc-Ala-Ala-Dma-Ala-Ala-OMe has been described as an illustration of the fact that a large group reversibly alkylating the amido group of an oligomer can disturb the regularity of a peptide backbone, oppose its aggregation and thus enhance its solubility greatly, affording synthons for further oligomerization. Application of such a group not only affects the solubility, but alters also the properties of the intermediates. The concomitant change in reactivity may run to such an extent that N-alkylation of oligomers has to be abandoned (this was encountered in the attempted synthesis of Lys-Glu-Dmg). Consequently, the solubility of the growing protected peptide chain will become progressively less and in the mentioned example the oligomerization had to be terminated at the dodecapeptide level, indicating the severe need for reversible "structure-breaking" functions.

The sequence A alpha-(154-159) of fibrinogen is capable of accelerating the t-PA catalysed activation of plasminogen.

The rate of activation of plasminogen by tissue-type plasminogen activator (t-PA) is greatly increased by fibrin, but much less by fibrinogen. Fibrin(ogen) fragments such as the fibrin(ogen) cyanogen bromide fragment FCB-2 and FCB-5, and a synthetic peptide with the sequence of fibrinogen A alpha-(148-160), a constituent of FCB-2, also have rate-enhancing properties. In order to find a possibly smaller, still stimulating site within A alpha-(148-160) we synthesized successive linear amino-terminally acylated hexapeptides [i.e. A alpha-(148-153), A alpha-(149-154)'d, .... A alpha-(155-160)] from the sequence A alpha-(148-160). The only hexapeptide within the sequence A alpha-(148-160) capable of enhancing the rate of plasminogen-to-plasmin conversion by t-PA appears to be the amino-terminally acylated peptide comprising the sequence A alpha-(154-159). This peptide enhances the plasminogen activation rate six-fold; half-maximal activation rate is reached at a peptide concentration of 56 microM.

The sequence gamma-(312-324) is a fibrin-specific epitope.

Fibrin accelerates the activation of plasminogen catalyzed by tissue-type plasminogen activator much stronger than fibrinogen. Detailed studies showed that (part of) this rate-enhancing effect of fibrin is brought about by two sites in the fibrin molecule: one in A alpha-(148-160) and one in the gamma-chain stretch 311-379 (also known as FCB-5). During the fibrinogen-to-fibrin conversion, A alpha-(148-160) appears to become accessible, because a monoclonal antibody against synthetic A alpha-(148-160) reacts with fibrin, but not with fibrinogen. Because a similar situation may exist for (at least parts of) FCB-5, we have prepared a monoclonal antibody against a part (ie, gamma-[312-324]) of FCB-5, and found that this is fibrin-specific and does not bind fibrinogen. We conclude that gamma-(312-324) is hidden in fibrinogen and is exposed by the formation of fibrin.

Use of Mpc-amino acids in solid phase peptide synthesis leads to improved coupling efficiencies.

The Mpc-group has a somewhat better stability than the Fmoc-group, resists catalytic hydrogenolysis, is highly stable in acidic media and its elimination product does not polymerize spontaneously. In a direct comparison of coupling efficiencies obtained in solid phase peptide syntheses using Mpc- or Fmoc-amino acids it is shown that the use of Mpc-amino acids leads to better coupling efficiencies and, consequently, a more homogeneous peptide. An improved synthesis of Mpc-ONSu and of Mpc-amino acid derivatives is presented.

Preparation and characterization of polyclonal and monoclonal antibodies to polyamines.

In order to develop an immunocytochemical method suitable for the study of the cellular localization and intracellular distribution of polyamines we have prepared and characterized antibodies to polyamines. Artificial immunogens were prepared by coupling putrescine, spermidine and spermine to a carrier protein. Immunogens containing bovine serum albumin as a carrier protein were used to immunize rabbits (polyclonal antibodies) and mice (for the production of Mabs). The specificity of the antibodies was tested in an ELISA system utilizing antigens synthesized from thyroglobulin and one of the polyamines. Polyclonal antibodies to putrescine, spermidine and spermine were obtained. However, these antibodies showed a variable degree of cross-reactivity to the polyamines not used for immunization. Two hybridoma cell lines were developed. The first, MPut88, selectively produces a Mab to putrescine, the second, MSpm/d88 produces a Mab which recognizes spermine and spermidine but does not react with putrescine.

Structural requirements of fibrinogen A alpha-(148-160) for the enhancement of the rate of plasminogen activation by tPA.

Fibrin, not fibrinogen, enhances the rate of tPA catalysed plasminogen activation. In earlier studies we have shown that a site involved in this rate enhancement is located in a tridecapeptide, i.e. fibrinogen A alpha-(148-160). This sequence comprises a special charge distribution in which a stretch with alternating neutral and acidic amino acids is embraced by basic amino acids. In this study we found that the disruption of charge distribution as caused by replacing valine 152 by other (charged and/or polar) amino acids leads to loss of rate-enhancing capacity. Also lysine at position A alpha-157 was replaced by lysine derivatives and other amino acids. We found that the side chain of the amino acid at position A alpha-157 must contain no (as in glycine) or one carbon atom without substitution (alanine). When the side chain contains two or more carbon atoms, there should also be a polar group in the side chain. We also synthesized a series of hexapeptides covering the sequence of A alpha-(148-160), and found that only A alpha-(154-159) is stimulatory, notwithstanding the fact that the peptides A alpha-(152-157), A alpha-(153-158) and A alpha-(155-160) also contain lysine A alpha-157. We conclude that the shortest peptide with stimulation activity is A alpha-(154-159); that the charge distribution in A alpha-(148-160) is important; that it is not lysine A alpha-157 per se that is crucial, but rather the properties and orientation of the side chain of A alpha-157.

The sequence A alpha-(148-160) in fibrin, but not in fibrinogen, is accessible to monoclonal antibodies.

Fibrin, but not fibrinogen, accelerates the activation of plasminogen catalyzed by tissue-type plaminogen activator. Previous work showed that essential information for this accelerating capacity of fibrin resides in the sequence corresponding to residues 148-160 of the A alpha chain of fibrinogen [A alpha-(148-160)]. Our working hypothesis, based on those findings, is that A alpha-(148-160) is buried in fibrinogen and becomes accessible to proteins such as plasminogen and/or tissue-type plasminogen activator when fibrinogen is converted to fibrin. To test this hypothesis we have raised a monoclonal antibody against synthetic A alpha-(148-160) and found that this antibody reacts with fibrin and not with fibrinogen. This finding shows that A alpha-(148-160) becomes accessible when fibrinogen is converted to fibrin and that A alpha-(148-160) is a fibrin-specific neoantigenic determinant.

Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates.

Peptide-carrier conjugates are widely used to raise antipeptide antibodies. In a model system using angiotensin and tetanus toxoid as the peptide and the carrier protein respectively, four cross-linking reagents were employed to study their effect on the immunogenicity of the conjugates. Optimization of the conjugation method for these heterobifunctional reagents, all succinimidyl active esters, resulted in well-defined conjugates of predictable composition. ELISA assays were performed to compare the antigenicity and the immunogenicity of the conjugates. The antipeptide antibody titres were of the order of 2 X 10(4)-2 X 10(5). The anti-carrier antibody titres were high, in spite of the modification of the protein. Three of the four coupling reagents used for conjugation were of the 'maleimide' type: succinimidyl 6-(N-maleimido)-n-hexanoate (MHS), succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) and succinimidyl m-maleimidobenzoate (MBS). One coupling reagent contained an activated disulphide: succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The constrained linkers originating from SMCC and MBS induced very high linker-specific antibody levels. The more flexible non-aromatic linkers originating from MHS and SPDP showed almost no reactivity. For this reason and since the thioether linkage is more stable than the disulphide bond, we recommend MHS as the crosslinking reagent of choice.

Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe. II. Characterization of the DNA-binding wing with the aid of spin-labelled oligonucleotides.

The DNA-binding domain of the single-stranded DNA-binding protein IKe GVP was studied by means of 1H nuclear magnetic resonance, through use of oligonucleotides of two and three adenyl residues in length, that were spin-labelled at their 3' and/or 5' termini. These spin-labelled ligands were found to cause line broadening of specific protein resonances when bound to the protein, although they were present in small quantities, i.e. of the order of 0.04 molar equivalent and less. The line broadening of protein resonances was made manifest by means of difference one and two-dimensional spectroscopy. Difference one-dimensional experiments revealed line broadening of the same protein resonances upon binding of either 3' or 5' spin-labelled oligonucleotides. Evidence in favour of the existence of a fixed 5' to 3' orientation in the binding of oligonucleotides to the protein surface was therefore not obtained from the spin-labelled oligonucleotide binding studies. Residue-specific assignments of broadened resonances could not, or could only sparsely, be derived from the difference one-dimensional spectra, because of the tremendous overlap in the aliphatic region of the spectrum. In contrast, such assignments were easily obtained from the difference two-dimensional spectra, which were recorded by means of both total correlated spectroscopy and nuclear Overhauser effect spectroscopy. Difference signals were detected for 15 spin systems; ten out of these were assigned to the residues I29, Y27, S20, G18, R16, T28, K22, Q21, V19 and S17 in the amino acid sequence of IKe GVP; the other five spin systems could be assigned to a phenylalanyl residue, an arginyl or lysyl residue, an aspartic acid or asparagyl residue, a glycyl residue and a glutamic acid or glutamyl residue. From the evaluation of the relative difference signals, it was concluded that the direct surroundings of the spin-label group of the labelled oligonucleotide in the bound state is composed of the first five residues in the former group of residues and the five residues in the latter group.(ABSTRACT TRUNCATED AT 400 WORDS)