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C Sakarellos

Publications and source records attributed to C Sakarellos.

At least 37 records · Page 2Linked to original sources
PubMed1998

A major Sm epitope anchored to sequential oligopeptide carriers is a suitable antigenic substrate to detect anti-Sm antibodies.

A sensitive, highly reproducible, solid-phase enzyme immunoassay (ELISA), was developed in order to investigate whether the synthetic heptapeptide PPGMRPP-a major epitope of the Sm autoantigen-anchored in five copies to a sequential oligopeptide carrier (SOC), [(PPGMRPP)5-SOC5] is a suitable antigenic substrate to identify anti-Sm/antibodies. Sera with different autoantibody specificities [45 anti-Sm, 40 anti-U1RNP, 40 anti-Ro (SSA)/La(SSB) positive, 21 Antinuclear antibody positive, but negative for antibodies to extractable nuclear antigens (ANA + /ENA - ) and 75 normal human sera, ANA negative] and 75 sera from patients with rheumatoid arthritis (RA) were tested for anti-(PPGMRPP)5-(SOC)5 reactivity in order to evaluate the specificity and sensitivity of the method to detect anti-Sm antibodies. RNA immunoprecipitation assays for the detection of anti-Sm and anti-U1RNP antibodies and counter immunoelectrophoresis (CIE) for the detection of anti-Ro(SSA) and anti-La(SSB) antibodies were used as reference techniques. The sensitivity of the method was 98% and the specificity was 68% for the determination of anti-Sm antibodies, while for the determination of anti-Sm and/or anti-U1RNP reactivity (antibodies to snRNPs) the corresponding values were 82% and 86%, respectively. In a comparison of the above assay with an ELISA, using Sm/U1RNP purified complex as immobilized antigen it was shown that the sensitivity of the anti-Sm/U1RNP ELISA in detecting anti-snRNPs was 74%; in addition sera with anti-Sm antibodies gave higher binding in the anti-(PPGMRPP)5-(SOC)5 ELISA compared with anti-Sm/U1RNP ELISA. Intra- and inter-assay precision was measured on four sera with reactivities extending into a wide range of absorbance values showed that the intra-assay coefficient of variation (CV%) ranged from 2.7 to 6 and the inter-assay CV% ranged from 9 to 14.5. These results indicate that the PPGMRPP peptide anchored to a pentameric SOC as a carrier is a suitable antigen for detecting anti-Sm antibodies and that the above ELISA is a rapid, reproducible and valuable screening method to test anti-Sm/U1RNP reactivities.

Antibodies, Antinuclear↗
PubMed1998

Solution structures of the fibronectin-like Leishmania gp63 SRYD-containing sequence in the free and antibody-bound states--transferred NOE and molecular dynamics studies.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

Amino Acid Sequence↗
PubMed1998

The value of synthetic linear epitope analogues of La/SSB for the detection of autoantibodies to La/SSB; specificity, sensitivity and comparison of methods.

In a previous study it was shown that La/SSB contains four linear epitopes, p147-154, p291-302, p301-318 and p349-364. The aim of the present study was to investigate the value of the synthetic epitope analogues of the La/SSB autoantigen for the detection of antibodies to La/SSB, in comparison with recombinant La and fragments of this protein. A total of 122 sera with anti-La/SSB activity, from patients with primary Sjögren's syndrome (pSS) or systemic lupus erythematosus (SLE), were tested in various peptide-based assays. In addition, 62 sera from pSS or SLE patients with other autoantibody specificities and 95 sera from healthy individuals were used as controls. The autoantibody specificity was identified by counter immunoelectrophoresis and immunoblot. The peptide-based ELISA assays presented sensitivities ranging from 78% to 88-8% and specificities from 69% to 94-3%. Dot blot assays exhibited sensitivities ranging from 93-6% to 97%, but remarkably lower specificities from 56% to 88%. The most sensitive and specific peptide 349GSGKGKVQFQGKKTKF364 was synthesized and attached on a tetramer sequential oligopeptide carrier SOC4 and used for immunoassay development. Assays based on the recombinant native La protein, the La-C terminal (215 aa), and the N-terminal of La with a mutation at base pair 640 (nine adenines instead of eight) were also developed and compared with the SOC4 peptide-based assay. Of anti-La-positive sera, 88.1% were reactive with both the synthetic peptide SOC4-(349-364aa) and the recombinant La protein. Eighty-three percent of sera were reactive with the La N-terminus and 67.8% of sera were reactive with the La C-terminus. Using sera that were anti-Ro-positive but anti-La-negative, 37% were reactive with the recombinant protein, 26% with the La N-terminus, 33% with the La C-terminus and only 11 % with the synthetic peptide. Our results suggest that the synthetic peptide epitopes exhibit high sensitivity and specificity for the detection of anti-La/ SSB antibodies in ELISA and dot blot techniques. The peptide SOC4-(349-364aa) has the same sensitivity for the detection of anti-La/SSB antibodies as the recombinant protein.

Amino Acid Sequence↗
PubMed1998

Structural, molecular and immunological properties of linear B-cell epitopes of Ro60KD autoantigen.

Antibodies to Ro60KD protein are found with high frequency in sera from patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS). Two major epitopes of the Ro60KD antigen, the TKYKQRNGWSHKDLLRSHLKP (169-190) and the ELYKEKALSVETEKLLKYLEAV (211-232), were synthesized and their antigenic and structural properties were studied. Using a large panel of SLE and pSS patients' sera, it was found that the anti-Ro60KD reactivity of both Ro60KD epitopes is rather limited (approximately 45%), although they retain their original disease specificity. The epitope p.169-190 possessed sequence similarity with the peptide RPDAEYWNSQKDLLEQKRGR, shared in the beta-chain of different HLA-DR molecules, among them the HLA-DR3 (which is associated with anti-Ro/Sjögren's syndrome A (SSA) response in patients with SLE). The antigenicity of the HLA-DR3 RPDAEYWNSQKDLLEQKRGR peptide was found to be similar to the 169-190 homologous Ro60KD epitope, recognized mainly by SLE sera. Structural studies showed that the 211-232 Ro60KD epitope exhibits pronounced helical characteristics, while the 169-190 epitope and the HLA-DR3 homologous peptide possess a somewhat lower percentage of alpha-helix. A beta-folded structure was identified in the latter two peptides. Although the diagnostic value of the reported Ro60KD epitopes seems to be rather limited, correlations with other ribonucleoprotein epitopes (La/Sjögren's syndrome B, Ro52KD) may prove complementary to each other and valuable in clinical use. The ordered structure of the HLA-DR3 homologous peptide, exposed to the autoantibody binding, may offer an initiative in further investigation of the role of the HLA haplotypes, associated with the anti-Ro/SSA response, in the autoimmune stimulus.

Alanine↗
PubMed1998

Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor.

The neuromuscular junction nicotinic acetylcholine receptor (AChR), a pentameric membrane glycoprotein, is the autoantigen involved in the autoimmune disease myasthenia gravis (MG). In animals immunized with intact AChR and in human MG, the anti-AChR antibody response is polyclonal. However, a small extracellular region of the AChR alpha-subunit, the main immunogenic region (MIR), seems to be a major target for anti-AChR antibodies. A major loop containing overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) lies within residues alpha 67-76 at the extreme synaptic end of each alpha-subunit: however, anti-MIR mAbs are functionally and structurally quite heterogeneous. Anti-MIR mAbs do not affect channel gating, but are very effective in the passive transfer of MG to animals; in contrast, their Fab or Fv fragments protect the AChR from the pathogenic effects of the intact antibodies. Antibodies against the cytoplasmic region of the AChR can be elicited by immunization with denatured AChR and the precise epitopes of many such mAbs have been identified; however, it is unlikely that such antibodies are present in significant amounts in human MG. Antibodies to other extracellular epitopes on all AChR subunits are present in both experimental and human MG; these include antibodies to the acetylcholine-binding site which affect AChR function in various ways and also induce acute experimental MG. Finally, anti-AChR antibodies cross-reactive with non-AChR antigens exist, suggesting that MG may result from molecular mimicry. Despite extensive studies, many gaps remain in our understanding of the antigenic structure of the AChR; especially in relation to human MG. A thorough understanding of the antigenic structure of the AChR is required for an in-depth understanding, and for possible specific immunotherapy, of MG.

Amino Acid Sequence↗
PubMed1997

Fine specificity of autoantibodies to La/SSB: epitope mapping, and characterization.

The B cell epitope mapping of La/SSB was performed using 20mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145-164, 289-308, 301-320 and 349-368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes: 147HKAFKGSI154, 291NGNLQLRNKEVT302, 301VTWEVLEGEVEKEALKKI318 and 349GSGKGKVQFQGKKTKF364. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope 147HKAFKGSI154 presented 83.3% similarity with the 139HKGFKGVD146 region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope 147HKAFKGSI154 to 100% to epitope 349GSGKGKVQGKKTKF364. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope 147HKAFKGSI154 presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.

Adult↗
PubMed1996

Antigenicity and conformational analysis of the Zn(2+)-binding sites of two Zn(2+)-metalloproteases: Leishmania gp63 and mammalian endopeptidase-24.11.

The antigenic properties of the Zn(2+)-binding region of two Zn(2+)-metalloproteases, Leishmania surface protease gp63 and mammalian endopeptidase-24.11 (E-24.11), possessing in their active site the characteristic amino acid sequence HEXXH, were investigated by using oligoclonal antibodies raised against two synthetic peptides, V1VTHEMAHALG11 (pepgp63) and V1IGHEITHGFD11 (pepE-24.11), containing the respective Zn(2+)-binding sites of the cognate protein. The affinity-purified antibodies, tested on synthetic peptides modelled from the active sites of ten different Zn(2+)-metalloproteases, showed high selectivity for their respective peptides. However, cross-reactivity was revealed when the antibodies were tested against the gp63 and E-24.11 molecules. A panel of synthetic peptide analogues and peptides of various size was synthesized and used for the fine antigenic characterization of pepgp63 and pepE-24.11. The shortest peptides capable of significant antibody binding were the pentapeptides V1VTHE5 and E5ITHG9 for pepgp63 and pepE-24.11 respectively. His4 and Glu5 were found to be indispensable for anti-pepgp63 binding to pepgp63, whereas in the case of pepE-24.11, Glu5 and His8 were found to be critical. The conformational characteristics of the two peptides correlate well with the observed differences in their antigenicity. 1H-NMR studies showed that pepgp63 adopts a folded structure whereas pepE-24.11 takes up a rather flexible conformation. Moreover, the antigenically critical His4 of pepgp63 contributes to the structural stabilization of the peptide. Similarly, the antigenically critical His8 of pepE-24.11 is involved in partial structural stabilization of its C-terminal region. The generated antibodies may be useful tools for identifying and classifying proteins possessing similar Zn(2+)-binding motifs and/or environments.

Amino Acid Sequence↗
PubMed1996

Compared structures of the free nicotinic acetylcholine receptor main immunogenic region (MIR) decapeptide and the antibody-bound [A76]MIR analogue: a molecular dynamics simulation from two-dimensional NMR data.

Monoclonal antibodies against the main immunogenic region (MIR) of the muscle acetylcholine receptor (AChR) are capable of inducing experimental myasthenia gravis (MG) in animals. The epitope of these antibodies has been localized between residues 67 and 76 of the AChR alpha-subunit. The conformation in solution of the Torpedo californica MIR peptide and of its [A76] MIR analogue have been analyzed using molecular modeling based on nmr interproton distances and J-derived phi dihedral angles. Molecular dynamics simulations including dimethyl-sulfoxide as explicit solvent have been carried out on the free MIR peptide. Calculation of the structure of the [A76]MIR analogue bound to an anti-MIR monoclonal antibody have been performed in the presence of water molecules. A tightly folded structure appears for both peptides with alpha beta-folded N-terminal N68-P-A-D71 sequence of type I in the free state and type III in the mAb6-bound state. The C-terminal sequence is folded in two different ways according to the result in the free and bound state of the peptides: two overlapping beta/beta or beta/alpha turns result in a short helical sequence in the free MIR peptide, whereas the bound analogue is folded by uncommon hydrogen bond closing an 11-membered cycle. This structural evolution is essentially the result of the reorientation of the hydrophobic side chains that are probably directly involved in peptide--antibody recognition.

Animals↗
PubMed1996

Isomerization of the Xaa-Pro peptide bond induced by ionic interactions of arginine.

Inclusion of Arg or Pro residues in proteins and peptides has been proved to play an essential role in biochemical functions through ionic interactions, conformational transitions, and formation of turns as well. In this study we present the conformational properties of the Ac-Arg-Ala-Pro (1), Ac-Arg-Ala-Pro-NH2 (2), Ac-Arg-Pro-Asp-NH2 (3), and Ac-Arg-Pro-Asp (4) tripeptides, using 1H-nmr spectroscopy and molecular dynamics. These peptides were modeled with the aim of studying the role of the Arg-guanidinium to carboxylate ionic interactions on the Xaa-Pro peptide bond isomerization. It was found with 1 and 4 that arginine preferentially interacts with the C-terminal carboxylate group, even though the beta-carboxylate is also accessible. This tendency of the Arg moiety was found to induce the cis disposition of the Ala-Pro peptide bond in 1. It was also confirmed that the Arg...Asp side chain-side chain ionic interaction in 3 plays a key role in backbone folding and structural stabilization through a type I beta-turn. The nmr pattern for 3 showed a remarkable similarity with that for various Arg-Tyr-Asp containing peptides, a sequence that is crucial for the adhesion properties of the Leishmania gp63 glycoprotein.

Amino Acid Sequence↗
PubMed1996

Construction and application of a new class of sequential oligopeptide carriers (SOCn) for multiple anchoring of antigenic peptides--application to the acetylcholine receptor (AChR) main immunogenic region.

A new class of sequential oligopeptide carriers (SOCn), namely (Lys-Aib-Gly)n (n = 2-7), for anchoring antigenic peptides, is presented. These SOCn have been designed in order to assume a determined structural motif, exhibiting defined spatial orientations of the Lys-N epsilon H2 anchoring groups. The NMR study showed that SOCn adopt a rigid conformation with some regularity, initiated from the C-terminus of the carrier, while molecular dynamics simulation confirmed the occurrence of a distorted 3(10)-helix. It was also demonstrated, by 1HNMR, that all the antigenic peptides bound to the SOCn retain their original, folded active, structure and that probably they do not interact to each other. It is concluded that the beneficial structural elements of the SOCn impose a favorable disposition of the anchored peptides so that potent antigens with maximum molecular recognition are generated.

Antibodies, Monoclonal↗
PubMed1996

Epitope mapping of the Ro/SSA60KD autoantigen reveals disease-specific antibody-binding profiles.

Anti-Ro60KD autoantibodies are commonly found in sera from patients with primary Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). In order to identify the epitopes of this autoantigen, 22-mer, synthetic peptides overlapping by eight residues, and covering the entire sequence of the Ro60KD autoantigen were prepared. Three groups of sera were evaluated according to their autoantibody specificites. The first group consisted of monospecific anti Ro60KD sera from four patients with SLE and one with SS, the second one was composed of anti-Ro60KD+anti-La(SSB)-positive sera from four patients with SS and the third group included three normal sera and one anti Ro52KD serum. It was found that sera from SLE patients interact with a common antigenic site spanning the sequence TKYKQRNGWSHKDLLRSHLKP (169-190) of the Ro60KD protein. On the other hand, sera from SS patients recognise the ELYKEKALSVETEKLLKYLEAV (211-232) region of this autoantigen. Determination of the minimal required peptide length for optimal antibody recognition showed that the defined epitopes can be shortened to the NGWSHKDLLR (175-184) and KALSVETEKLLKYLEAV (216-232) sequences respectively. Inhibition experiments using the Ro60KD antigen and soluble peptides corresponding to the 175-184 and 216-232 segments further confirmed the specific antibody binding. These results, although only a small number of sera were used, indicate that the Ro60KD autoantigen, which is not characterized by disease specificity, contains two discrete epitopes specifically recognized from SLE and SS patient sera. Finally, the sequence similarity of the NGWSHKDLLR (175-184) epitope with some of the HLA haplotypes, associated with anti-Ro response, deserves to be noted.

Amino Acid Sequence↗
PubMed1996

Immunoreactivity and conformation of the P-P-G-M-R-P-P repetitive epitope of the Sm autoantigen.

Anti-Sm antibodies are usually considered highly specific for systemic lupus erythematosus (SLE), while anti-U1 RNP antibodies are found in high titers in patients with mixed connective tissue disease (MCTD). The sequence P1-P-G-M-R-P-P7, present in three copies in the Sm (U1-U6 RNA-protein complex) autoantigen, is an important functional domain of the antigenic determinants. The immunoreactivity of this proline-rich repetitive epitope was investigated by testing sera with various autoantibody specificities for reactivity against this epitope, as well as its conformational properties by means of 1D and 2D 1HNMR spectroscopy. It was found that the P-P-G-M-R-P-P epitope is recognized mainly by anti-U1RNP and/or anti-Sm positive sera, but also by anti-Ro(SSA) (hY1RNA-protein complex) and anti-La(SSB) (hY1RNA-protein complex) positive sera, although these sera are negative for anti-U1RNP and anti-Sm. Conformational analysis of the proline-rich epitope in DMSO-d6 solution obtained from lyophilized aqueous solution at pH 5 showed the presence of at least three conformers. The main conformer A (62%) is stabilized by an ionic interaction between the guanidinium and the C-terminal carboxylate groups, and the Pro6-Pro7 peptide bond adopts the cis form. A type II beta-turn is also present in the N-terminal sequence (Pro1-Pro-Gly-Met4-) of this conformer. Conformer B (21%) is also stabilized by a similar ionic interaction, as in conformer A, while the NMR data indicate the absence of a folded structure in the N-terminal tetrapeptide of this conformer. Conformer C (17%) adopts a completely extended structure. The multiple conformers of the P-P-G-M-R-P-P may offer some explanation for the reactivity of sera with various autoantibody specificities against this epitope.

Amino Acid Sequence↗
PubMed1995

Conformational properties of the Arg-Leu-Gly tripeptide--DMSO--water clusters with the combined use of molecular dynamics and energy minimization studies.

The Arg-Leu-Gly tripeptide is the repeating fragment of sequential arginine-rich polypeptides capable of interacting with DNA. The conformational influence of solvent molecules (DMSO/H2O) were investigated with the combined use of molecular dynamics and energy minimization. It was found that water molecules greatly contribute to the peptide structure by solvating all its hydrophylic sites even in the presence of DMSO excess, whereas one water molecule links the ammonium and carboxylic ends of the Arg-Leu-Gly. The persistence of residual water, which was confirmed by varying the computer simulation parameters, indicates that pretreatment of peptide segments in aqueous solutions should greatly affect their conformational properties in organic media. A satisfactory agreement between experimental data (1H-NMR and IR spectroscopy) and the presented computational study deserves also to be noted.

Amino Acid Sequence↗
PubMed1994

Analytical and semi-preparative separation of diastereomeric lipid amino acid conjugates.

A series of biologically active peptides and their conjugates with lipidic amino acids were investigated by systematic change of the mobile phase composition using traditional octadecylsilica stationary phase and the newly developed Supelcosil LC-ABZ column. The mobile phases contained various concentrations of methanol and acetonitrile combined with 0.1% trifluoroacetic acid (TFA). Better peak shapes and higher resolution of the isomers could be observed when the mobile phase contained 0.1% TFA. More symmetrical peaks and much higher S values (slope of the log k' vs organic phase concentration plots) were obtained on the special reversed-phase column developed for anionic, basic or zwitterionic compounds. The optimum separation conditions were scaled up to a semi-preparative reversed-phase column (15 mm i.d.) to collect mg quantities of isomers for further studies.

Acetonitriles↗
PubMed1994

Antibodies to EYRKK vesicular stomatitis virus-related peptide account only for a minority of anti-Ro60kD antibodies.

Previous studies demonstrated a possible antigenic relation between the carboxyl terminal portion of anti-Ro60kD autoantigen and a nucleocapsid protein (N) of vesicular stomatitis virus (VSV). In order to investigate whether anti-Ro60kD autoantibodies react with the VSV homologous region of the Ro60kD protein we synthesized, according to Merrifield's method, the EYRKKMDI octapeptide (8p) sharing a common sequence with the N protein of VSV. Sera from 61 patients with autoimmune rheumatic diseases (34 systemic lupus erythematosus (SLE), 21 Sjörgren's syndrome (SS) and six rheumatoid arthritis (RA)) as well as 59 from normal blood donors were tested for the presence of anti-Ro60kD autoantibodies by ELISA and immunoblot (IB) and anti-8p antibodies by ELISA. Antibodies to 8p were found in 9/31 of anti-Ro60kD IB-positive sera, 5/30 of anti-Ro60kD-negative sera and 2/59 of normal control sera. The concordance between the anti-8p ELISA and the anti-Ro60kD IB was very poor (chi 2 = 0.71, P = 0.4) in contrast to the anti-Ro60kD ELISA and the anti-Ro IB (chi 2 = 27.6, P = 10(-7)). Subsequent affinity purification of the anti-8p antibodies from a strong positive anti-8p and anti-Ro60kD SLE serum yielded 95% depletion of the anti-8p activity and 37% reduction of the anti-Ro60kD activity. Inhibition assays with the affinity-purified anti-8p antibodies demonstrated that the octapeptide gave 94.5% inhibition of the anti-Ro60kD activity, while Ro60kD protein led to 42.3% inhibition of the anti-8p. Preincubation of the serum with the octapeptide produced 4% inhibition of anti-Ro60kD ELISA. These results indicate that the anti-8p antibodies account only for a minority of the anti-Ro60kD autoantibodies.

Amino Acid Sequence↗
PubMed1993

High-resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor. Improving the binding activity of synthetic analogues of the region.

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR) is an immunodominant area of the molecule, both in human and in experimental autoimmune myasthenia gravis. Anti-MIR monoclonal antibody (mAb) binding has been earlier localized between amino acid residues 67-76 of the AChR alpha-subunit. A thorough study of the epitope(s) for anti-MIR mAbs, by the use of a large panel of overlapping synthetic peptides and multiple peptide analogues, is now presented and offers clues for potential therapeutic applications of the obtained data. Use of all possible overlapping hexapeptides within Torpedo and human alpha 40-91 AChR and of selected peptides of various sizes, showed that the shortest peptide capable of significant antibody binding is the pentapeptide alpha 67-71. Systematic screening of peptide analogues, where each amino acid residue within alpha 67-76 and alpha 67-74 of both Torpedo and human AChRs was substituted by various amino acids, was performed. Asn68 and Asp71 were found to be indispensable for anti-MIR mAb binding, whereas Pro69 and Ala/Asp 70 were less but still significantly important. mAb binding to alpha 67-76 from various AChR species further supported the significance of these results. An additional series of selected peptide analogues was then constructed, aiming at the identification of analogues with high antigenic activity. Many analogues with either single substitutions of alpha 76 or combinations of two substitutions at alpha 73 and alpha 76 were tested. Several of these analogues (mainly His76, Arg76, Val73Ala76, His73Ala76, Val73Arg76) exhibited dramatic mAb binding enhancement. Some anti-MIR mAbs that do not bind to alpha 67-76 bound significantly to certain analogues. Such analogues could find applications in studies of therapeutic models of myasthenia gravis.

Amino Acid Sequence↗