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C Sakarellos

Publications and source records attributed to C Sakarellos.

At least 55 records · Page 3Linked to original sources
PubMed1993

Conformational requirements for molecular recognition of acetylcholine receptor main immunogenic region (MIR) analogues by monoclonal anti-MIR antibody: a two-dimensional nuclear magnetic resonance and molecular dynamics approach.

The conformational properties of two [D-A70, A76] and [Aib70, A76] analogues of the alpha 67-76 Torpedo acetylcholine receptor fragment, with low binding capacity for the anti main immunogenic region (MIR) antibodies, were studied in DMSO by two-dimensional nmr techniques and molecular dynamics simulations. The results were compared to the free and bound conformations of the [A76] analogue, which has twice more affinity for the anti-MIR monoclonal antibody 6 (mAb6), than the natural Torpedo sequence. It appeared that a single substitution of the A70, at a crucial position, by the D-A70 or Aib70, could modify completely the conformational behavior of the peptide and reduced its recognition by the anti-MIR antibody. The WNPADY rigid structure at the N-terminal part was essential for antibody recognition. The adjacent more flexible C-terminal sequence (GGIK) gives additional stability to the monoclonal antibody-peptide complex probably due to an adequate orientation of the peptide side chains in the complex, by setting them in close contact with the antibody.

Amino Acid Sequence↗
PubMed1993

Calreticulin synthetic peptide analogues: anti-peptide antibodies in autoimmune rheumatic diseases.

Autoantibodies in sera from patients with systemic lupus erythematosus (SLE) and onchocerciasis recognize calreticulin (CaR), a calcium-binding protein, as antigen. In this study we present the immunological properties of two synthetic peptides prepared to correspond to the 1-24 and 7-24 amino acid sequence of CaR. In contrast to information previously reported for the recombinant protein, the CaR-peptide analogues appeared immunoreactive to anti-Ro/SSA autoimmune sera. Human sera from patients with SLE, Sjögren's syndrome (SS), rheumatoid arthritis (RA), as well as mixed connective tissue disease (MCTD), demonstrated a positive autoimmune response (binding of antibodies), to the CaR-peptide analogues. These findings suggest that anti-calreticulin autoantibodies are not restricted to any disease specificity.

Amino Acid Sequence↗
PubMed1992

Hydration of the Gly2 and Gly3 peptide oxygens of [Leu5]-enkephalin in aqueous solution as revealed by the combined use of 17O-NMR and Fourier-transform infrared spectroscopy.

Solvent-induced and temperature-induced 17O chemical shifts of [17O-Gly2, Leu5]-enkephalin and [17O-Gly3, Leu5]-enkephalin and solvent-induced spectral modifications of the amide-I' stretching vibrations of [1-13C-Gly2, Leu5]-enkephalin and [1-13C-Gly2, Leu5]-enkephalin are reported and correlated with the spectroscopic characteristics of model amides. It is demonstrated that both Gly2 and Gly3 peptide oxygens are motionally equivalent and form solvation species which are essentially monohydrated in aqueous solution, contrary to several simple amides and model peptides in which water largely forms dihydrates. It is shown that the combined use of 17O-NMR and Fourier transform infrared is a unique methodology for studying the hydration state of specific peptide oxygens in peptide hormones.

Deuterium↗
PubMed1992

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.

Amino Acid Sequence↗
PubMed1992

17O NMR and FT-IR study of the ionization state of peptides in aprotic solvents. Application to Leu-enkephalin.

The ionization state of Leu-enkephalin in DMSO and MeCN/DMSO (4/1) solution was studied by the combined use of 17O NMR and FT-IR spectroscopy. After lyophilization of an aqueous solution at nearly neutral pH, Leu-enkephalin essentially exists in the uncharged state in MeCN/DMSO (4/1) solution. In pure DMSO, only 40% of the Leu-enkephalin molecules are in the zwitterionic state under the same conditions.

Amino Acid Sequence↗
PubMed1991

Two-dimensional 1H-NMR study of antigen-antibody interactions: binding of synthetic decapeptides to an anti-acetylcholine receptor monoclonal antibody.

Two-dimensional NMR experiments [correlated spectroscopy (COSY) and two-dimensional transferred nuclear Overhauser enhancement spectroscopy (TR-NOESY)] have been applied to study the interactions of a monoclonal antibody (mAb) directed to the main immunogenic region (MIR) of the acetylcholine receptor (AChR), and four synthetic decapeptides from the MIR. The decapeptides were the Torpedo AChR alpha 67-76 fragment (W67-N68-P69-A70-D71-Y72-G73-+ ++G74-I75-K76) and its three [A69], [A73], and [A76] analogues. The results led to the following conclusions: (1) the magnitude of the TR-NOE cross peaks does not depend only on the structuration of the peptide in the bound state, but also on restrictions of the mobility, i.e., on the correlation time tau c, which can be different for every residue; (2) the binding capacity of the synthetic peptides to mAbs measured by radioimmunoassay is directly correlated to the NOE magnitude; and (3) the combined interpretation of the COSY and TR-NOESY experiments gives a qualitative information about the nature and the overall conformation of the sequence which is in contact with the mAb binding site.

Amino Acid Sequence↗
PubMed1991

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies.

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.

Adult↗
PubMed1991

Changes in the c.d. spectra of calf thymus DNA induced by sequential polypeptides in aqueous solutions. Part I.

Interactions of calf thymus DNA with sequential polypeptides were studied using c.d. spectroscopy in aqueous solutions. It was found that DNA structural alterations induced by sequential polypeptides (L-Arg-X-Gly)n (where X = L-Val, Leu, Ile, Nva, Nle) are modulated by the nature of the X residue. Thus, the polypeptide (L-Arg-L-Nva-Gly)n induced the 10.2B-DNA form, whereas the polypeptides (L-Arg-L-Ile-Gly)n having one methyl group less on the X residue side chain, did not provide any significant modification to the structure of DNA. The effect of ionic strength from 0.14 M NaCl (physiological value) to zero was also analysed on the basis of the observed c.d. changes and the degree of complexation in the DNA-polypeptides was estimated.

Amino Acid Sequence↗
PubMed1991

Changes in the c.d. spectra of calf thymus DNA induced by sequential polypeptides in trifluoroethanol solutions. Part II.

Interactions between calf thymus DNA and (L-Arg-X-Gly)n sequential polypeptides (where X = L-Ala, Val, Leu, Ile, Nva, Nle) in trifluoroethanol: water (40:60) solutions in the salt range of 0.12-0.5 M NaCl, were studied using c.d. spectroscopy. It was found that DNA tertiary structure (psi form) is modulated by the nature of the polypeptides (variation of X residue). The effect of the secondary structure of polypeptides on the formation of psi-DNA was also analysed. Unordered polypeptides destabilized psi aggregates, while helical polypeptides favoured DNA tertiary structure. A loss of tertiary structure was observed in the presence of the (L-Arg-L-Val-Gly)n, which can be attributed to the ability of valine to suppress psi-type DNA.

Amino Acid Sequence↗
PubMed1991

The main immunogenic region of the acetylcholine receptor. Structure and role in myasthenia gravis.

Auto-antibodies to the nicotine acetylcholine receptor (AChR) cause the disease myasthenia gravis (MG). Animals immunized with AChR or receiving anti-AChR antibodies acquire MG symptoms. The majority of the monoclonal antibodies (mAbs) raised in rats against intact AChR bind to a region on the extracellular side of the AChR's alpha-subunit, the main immunogenic region (MIR). The major loop of the overlapping epitopes for several anti-MIR mAbs has been localised between residues 67-76 of the alpha-subunit. Anti-MIR mAbs are very potent in accelerating AChR degradation (antigenic modulation) in muscle cell cultures and transferring experimental MG in animals. Fab fragments of single anti-MIR mAbs when bound to the AChR inhibit two-thirds of the MG patients' antibodies from binding and from inducing antigenic modulation of the AChR. This suggest that the majority of the human MG antibodies are also directed against the MIR. It has however to be verified by direct experiments.

Animals↗
PubMed1990

Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible.

Amino Acid Sequence↗
PubMed1990

17O- and 14N-NMR studies of Leu-enkephalin and enkephalin-related fragments in aqueous solution.

17O- and 14N-nmr chemical shifts and line widths of the carboxyl and amino terminal groups of Leu-enkephalin--Tyr-Gly-Gly-Phe-[17O]Leu-Oh--and enkephalin-related fragments--[17O]Leu-OH, Phe-[17O]Leu-OH, Gly-Phe-[17O]Leu-OH, and Gly-Gly-Phe-[17O]Leu-OH--were measured in aqueous solution over the entire H pH range. Enrichment in 17O was achieved by saponification of the corresponding O-methyl esters. Ionization constants and titration shifts were obtained by nonlinear least-squares fits to one-proton titration curves. [17O]Leu-OH exhibits a profound pH-dependent solvation change on deprotonation of the carboxyl group, as shown by 17O- and 14N-nmr line widths. In contrast, the peptides studied do not exhibit pH-dependent conformational (solvation) changes on deprotonation of the carboxyl group, and a head-to-tail intramolecular association between the ionic terminal groups should be excluded. It is shown that the peptides do not exhibit isotropic overall molecular motion and that segmental motion rather than fast internal motion influences the effective correlation times at the sites of the carboxyl oxygens and the amino nitrogen.

Amino Acid Sequence↗
PubMed1990

Interaction of the peptide CF3-Leu-Ala-NH-C6H4-CF3 (TFLA) with porcine pancreatic elastase. X-ray studies at 1.8 A.

The peptide trifluoroacetyl-Leu-Ala-(p-trifluoromethylanilide), is a reversible inhibitor of pancreatic porcine elastase and is characterized by a Km of 2.5 x 10(-8) M. Co-crystals of the 1:1 complex were obtained in an acetate buffer + dimethylformamide solution at pH 5.7. Diffraction data were recorded on films at the LURE synchrotron facility. The inhibitor was localized on difference Fourier maps, and the refinement of the structure was performed by simulated annealing (XPLOR). The current agreement factor is R = 19% (for 13224 observed structure factors and 1.8 A effective resolution). The RMS deviations from ideality of bond distances and angles are 0.02 A and 2 degrees, respectively. The inhibitor molecule was found in the active site, bent around the side chain of Phe-215 in a geometry that resembles the previously reported structure of the CF3-Lys-Ala complex at 2.5 A, in a parallel beta-sheet association with the loop 214-216. The analysis of the close contacts (less than 3.5 A) indicates that the trifluoromethylamide bond interacts with the active site and not the Leu-Ala or Ala-anilide bonds. The two fluorinated groups of the inhibitor exhibit different specificities: the trifluoroacetyl group (N terminus) is tightly stacked between the two chain loops 191-195 and 213-215, while the trifluoromethylanilide (C terminus) shows less specificity and only a single contact.

Animals↗
PubMed1990

Local interactions in peptides. 1H-1H, 13C-H coupling constants and calculations for the conformational analysis of N-acetyl-N'-methylamides of aliphatic amino acids.

We report the results of a joint NMR and theoretical investigation devoted to the conformational properties of N-acetyl-N'-methylamides of aliphatic amino acids with side chains of increasing bulkiness: Gly, Ala, Leu, Ile, and tert.Leu. In this series, determination of the coupling constants 3JHNC alpha H together with the coupling constants 3JC'NC alpha H (thanks to specific carbon-13 labeling of the N-acetyl carbonyl group) led to the derivation of alternative A, B, and C parameters in a Karplus-type relation expressing the dependence of 3JC'NC alpha H upon the phi dihedral angle. The value of the latter is found to increase regularly following the increase of the side-chain bulkiness. The theoretical conformational analysis is performed by applying the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. The conformational energy maps illustrate the progressive distortion of the backbone conformation incurred in the series Gly to tert.Leu. Theoretical values computed for 3JHNC alpha H and 3JC'NC alpha H are found to be in a good quantitative agreement with the experimental ones.

Alanine↗
PubMed1989

Investigation of the active center of rat pancreatic elastase.

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.

Anilides↗
PubMed1989

17O-NMR studies of the conformational and dynamic properties of enkephalins in aqueous and organic solutions using selectively labeled analogues.

The synthesis of Leu-enkephalin selectively 17O-enriched in Gly2 and Gly3 is reported. The 17O-nmr chemical shifts of [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in H2O are almost identical and independent of the pH. Since hydrogen bonding is the dominant factor governing the chemical shifts of the peptide oxygen, it can be concluded that the hydration state of both oxygens is identical and independent of the pH. The 17O chemical shifts of the [17O-Leu5]-enkephalin terminal carboxyl group at pH approximately 1.9 and 5.6 are very different in H2O but very similar in CH3CN/DMSO (4:1) solution. This suggests that the protonation state of the carboxyl group at both pH values in CH3CN/DMSO solution is the same and consequently that Leu-enkephalin exists in the neutral form at pH approximately 5.6. In this organic mixed solvent system both Gly2 and Gly3 oxygen resonances exhibit a significant shift to high frequency by the same extent (delta delta approximately 30 ppm). It is concluded that both peptide oxygens are not hydrogen bonded to an appreciable extent and that no specific 2----5 hydrogen bonding exists to an appreciable extent. This conclusion is in agreement with the energy of activation for molecular rotation, as determined from T1 measurements, which was found to be almost identical for both [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in CH3CN/DMSO (4:1) mixed solvent.

Enkephalins↗
PubMed1989

A crystal molecular conformation of leucine-enkephalin related to the morphine molecule.

Leucine-enkephalin (Try1-Gly2-Gly3-Phe4-Leu5) has been crystallized as a trihydrate from water solution. X-ray diffraction reveals a tightly folded molecular conformation with two fused beta III- (Gly2-Gly3) and beta I- (Gly3-Phe4) turns. The Tyr1 and Phe4 aromatic rings have a close orthogonal arrangement analogous to the tyramine and cyclohexenyl rings in morphine. This suggests that the conformation found in the trihydrate crystal structure could be required for recognition by mu-receptor sites.

Enkephalin, Leucine↗