Biomedical subjects
Publications and source records attributed to E Giralt.
Electrospray ionization mass spectrometry has been used to investigate simple multicharged [1(4)]heterophanes containing heterocyclic betaine subunits as building blocks. The formation of stable noncovalent polymolecular self-assembled structures in the gas phase is reported.
A foot-and-mouth disease virus (FMDV) field variant, isolate C-S30 (also named C(1)-Barcelona), is known to contain four changes within the main antigenic site A (GH loop of capsid protein VP1, residues 136-150), at least one of which (Leu147-->Val) involves a highly conserved position, critical for antibody recognition in the reference strain C-S8c1. However, immunoenzymatic analysis of FMDV C-S30 showed it was recognised by 4C4, a monoclonal antibody that specifically targets site A. This remarkable behaviour has led us to analyse the individual and combined contributions of the four mutations to the antigenicity of C-S30, by surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA) studies of pentadecapeptides displaying all possible combinations of the four replacements. Analysis of this family of C-S30-derived analogues shows a certain level of antibody recognition by SPR. In addition, SPR data suggest that kinetic rate constants provide an indirect measure, on the one hand, of paratope accessibility (association rate constant) and, on the other hand, of peptide fitness to the same paratope (dissociation rate constant).
The effects of supernatant from the bacterial strain Serratia marcescens 2170 (CS-2170) on the viability of different haematopoietic cancer cell lines (Jurkat, NSO, HL-60 and Ramos) and nonmalignant cells (NIH-3T3 and MDCK) was studied. We examined whether this cytotoxic effect was due to apoptosis, and we purified the molecule responsible for this effect and determined its chemical structure. Using an MTT assay we showed a rapid (4 h) decrease in the number of viable cells. This cytotoxic effect was due to apoptosis, according to the fragmentation pattern of DNA, Hoechst 33342 staining and FACS analysis of the phosphatidylserine externalization. This apoptosis was blocked by using the caspase inhibitor Z-VAD.fmk, indicating the involvement of caspases. Prodigiosin is a red pigment produced by various bacteria including S. marcescens. Using mutants of S. marcescens (OF, WF and 933) that do not synthesize prodigiosin, we further showed that prodigiosin is involved in this apoptosis. This evidence was corroborated by spectroscopic analysis of prodigiosin isolated from S. marcescens. These results indicate that prodigiosin, an immunosuppressor, induces apoptosis in haematopoietic cancer cells with no marked toxicity in nonmalignant cells, raising the possibility of its therapeutic use as an antineoplastic drug.
DKP formation is a serious side reaction during the solid-phase synthesis of peptide acids containing either Pro or Gly at the C-terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt-Cl-resin with a limited incorporation of the C-terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC-ESMS is a useful method for analysing complex samples, such as those formed when C-terminal Pro peptides are prepared by non-optimized solid-phase strategies.
Kaliotoxin (KTX), a potassium channel blocker found in the venom of the scorpion Androctonous Mauretanicus is a 38 residue polypeptide with a well defined structure consisting of a alpha-helix and a three strand antiparallel beta-sheet interconnected by three disulfide bonds. Although the 3D structure has been determined by NMR, there is a number of features, mainly concerning the conformation and flexibility of the side chains, but also the long range order in the peptide and its fluctuations, that may have escaped the experimental study. These questions are now being addressed using molecular dynamics (MD) simulations. Accordingly, the present work reports the analysis of a 430 ps molecular dynamics trajectory of the polypeptide soaked with 4700 TIP3 water molecules inside a 56 A box. MD calculations were performed with periodic boundary conditions. Analysis of the conformational space sampled by each of the residues along the trajectory, suggests a special behavior of Pro17 and Lys19 both located on the helix. Furthermore, analysis of the relative movements of the secondary structure elements indicates that the alpha-helix and beta-sheets fluctuate in a correlated motion, preserving the tertiary structure of the polypeptide along the trajectory. Finally, analysis of the charge distribution was also examined. The direction of the dipole moment, computed from the center of masses appears to be an interesting feature of the structure probably related to the biological function of the molecule.
The main antigenic site (site A) of foot-and-mouth disease virus (FMDV, strain C-S8c1) may be adequately reproduced by a 15-peptide with the amino acid sequence H-YTASARGDLAHLTTT-NH(2) (A15), corresponding to the residues 136-150 of the viral protein VP1. The effect of amino acid substitutions within A15 on its antigenicity towards monoclonal antibodies (MAb) raised against antigenic site A, has been studied by means of BIAcore technology, based on surface plasmon resonance (SPR). Although these antigenicities have previously been determined from enzyme-linked immunosorbent assays (ELISA), the SPR-based technique is superior in that it allows a fast and straightforward screening of antigens while simultaneously providing kinetic data of the antigen-antibody interaction. With a view to screening fairly large libraries of individual peptides, we have inverted the typical SPR experiment by immobilizing the MAb on the sensor surface and using peptides as soluble analytes. We report the validation of this approach through the screening of 44 site A peptides, with results generally in good agreement with the relative antigenicities previously determined by competition ELISA.
A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.
The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.
A new synthesis of 9-hydroxy- and 9-(alkylamino)thiazolo[5,4-b]quinolines by cyclization of 4-(ethoxycarbonyl)-5-(arylamino)thiazoles and 5-(arylamino)-4-carbamoylthiazoles, respectively, is described. In vitro cytotoxicity of a large number of derivatives of these compounds has been tested against several cell lines. The highest activities observed are associated with the presence of a 2-[[(N,N-diethylamino)ethyl]amino] substituent at C-2 and a fluorine atom at the C-7 position of the tricyclic planar heteroaromatic framework. Three structural features seem to be essential for antitumor activities: a positive charge density at carbon C-7, a side chain at position C-2 or C-9 of the thiazoloquinoline skeleton with two basic nitrogens and a pKa value of 7.5-10 in the most basic center, and a conformational flexibility of this basic side chain. These structural requirements must be simultaneously satisfied in order to ensure a significant antitumor activity.
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A cyclic disulfide peptide corresponding to the G-H loop sequence 134-155 [replacement Tyr136 and Arg153 with Cys] of the capsid protein VP1 of foot-and-mouth disease virus (FMDV) isolate C-S8c1 was examined by proton 2D-NMR spectroscopy in water and in 25% HFIP/water. In water, NMR data supported the presence of a non-canonical turn in the central, conserved cell adhesion RGD motif and suggested the presence of a nascent helix in the C-terminal part, stabilized and slightly extended upon addition of 25% HFIP, a secondary structure stabilizing cosolvent. The formation of the C-terminal helix was evidenced by combined analysis of NOE connectivities, H alpha chemical shifts, 3JNH-H alpha coupling constants and amide temperature coefficients. Surprisingly, these global structural features of the cyclic peptide in solution show similarities to previous X-ray structure analysis of (a) a shortened linear peptide complexed with a antivirus antibody and (b) the G-H loop represented on the chemical reduced viral surface of a different serotype. Thus, even in entirely different biological environments the cyclic peptide reflect similar structural features, reinforcing the concept that this viral loop behaves as an independent structural and functional unit.
A large-scale vaccination experiment involving a total of 138 cattle was carried out to evaluate the potential of synthetic peptides as vaccines against foot-and-mouth disease. Four types of peptides representing sequences of foot-and-mouth disease virus (FMDV) C3 Argentina 85 were tested: A, which includes the G-H loop of capsid protein VP1 (site A); AT, in which a T-cell epitope has been added to site A; AC, composed of site A and the carboxy-terminal region of VP1 (site C); and ACT, in which the three previous capsid motifs are colinearly represented. Induction of neutralizing antibodies, lymphoproliferation in response to viral antigens, and protection against challenge with homologous infectious virus were examined. None of the tested peptides, at several doses and vaccination schedules, afforded protection above 40%. Protection showed limited correlation with serum neutralization activity and lymphoproliferation in response to whole virus. In 12 of 29 lesions from vaccinated cattle that were challenged with homologous virus, mutant FMDVs with amino acid substitutions at antigenic site A were identified. This finding suggests the rapid generation and selection of FMDV antigenic variants in vivo. In contrast with previous studies, this large-scale vaccination experiment with an important FMDV host reveals considerable difficulties for vaccines based on synthetic peptides to achieve the required levels of efficacy. Possible modifications of the vaccine formulations to increase protective activity are discussed.
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The crystal structure of a synthetic peptide representing the major antigenic loop of foot-and-mouth disease virus (FMDV), complexed with the Fab fragment of a neutralizing monoclonal antibody raised against the virus, has been determined at 2.8 A resolution. The peptide shows a high degree of internal structure with a nearly cyclic conformation. The conserved Arg-Gly-Asp motif, involved in the viral attachment of aphtoviruses to cells, participates directly in the interaction with several complementarity determining regions of the antibody molecule. The Arg-Gly-Asp triplet shows the same open turn conformation found in the reduced form of FMDV of another serotype and also in integrin binding proteins. The observed interactions provide a molecular interpretation of the amino acid replacements observed to occur in mutants resistant to neutralization by this antibody. The structure also suggests a number of restrictions to variation within the epitope which are imposed to keep the Arg-Gly-Asp motif in its functional conformation.
The immunodominance of a major antigenic site of foot-to-mouth disease virus (FMDV) (serotype C; clone C-S8c1) in a natural host has been evaluated by serum immunoglobulin fractionation. Nineteen sera from either convalescent or vaccinated swine were fractionated by affinity chromatography using a synthetic peptide representing antigenic site A (the G-H loop of capsid protein VP1) coupled to a Sepharose matrix. Antigen-binding and neutralizing activities of serum fractions were quantitated. On average, about 57 or 27% of the virus-neutralizing activity (and about 35 or 12% of the virus-binding activity) from convalescent or vaccinated swine, respectively, corresponded to antibodies against site A. The results provide direct evidence of the important contribution of site A, and also of additional sites unrelated to site A, in the evoking of neutralizing antibodies by FMDV in a natural host. The proportion of antibodies directed to site A varied greatly among individual swine. Some animals evoked remarkably low levels of antibodies specific for site A although they were competent to raise antibodies against other antigenic sites of FMDV. Thus, the major antigenic site of FMDV shows heterogeneous dominance in a natural host. Possible implications for evolution of viral quasispecies are discussed.
Aphthoviruses are an important group of animal pathogens. A combination of genetic and structural studies has revealed one of the main principles governing their evolution: severe limitations to variation imposed by functional and structural constraints, in conjunction with high mutation and recombination rates operating during genome replication. Evolution occurs by positive selection and random drift acting on complex quasispecies distributions. The mutant composition of a quasi-species (or mutant spectrum) is largely dictated by tolerance to nucleotide and amino acid substitutions in viral RNAs and proteins, which must remain functionally competent. We review recent evidence to support this proposal, and we suggest that similar concepts may apply to other RNA viruses as well.
The purification of a range of protected peptide segments has been carried out using modified reversed-phase chromatographic techniques in which DMF was added to the water and acetonitrile mixtures used as eluents. The purity of the recovered peptides was excellent and recoveries were high in all cases, even for longer hydrophobic segments. In several cases purifications were carried out on the hundreds of milligrams scale. For protected peptide segments containing Met, protection as the sulfoxide avoids its unwanted alkylation and oxidation, and the increased overall polarity can be useful in the purification of protected peptides incorporating this residue.
Conformationally restricted cyclic peptide mimics of the antigenic site A of foot-and-mouth disease virus serotype C-S8c1 have been designed, first by comparison to the three-dimensional structure of the O1BFS serotype, later more accurately on the basis of X-ray diffraction data from a complex between a linear peptide reproducing site A and an FMDV-derived monoclonal antibody Fab fragment. A variety of cyclization strategies have been attempted, both in solution and in the solid phase, involving disulfide, side chain lactam and head-to-tail arrangements. Preliminary immunological results have shown one of the cyclic disulfide mimics to be a better immunogen than its linear counterpart.