Vassilios Tsikaris

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

Summary: 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 a‐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 α67–76 at the extreme synaptic end of each a‐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 cytoplas‐mic 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 noti‐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.

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 Sjögrens 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.

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.

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.

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 α‐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 ϕ dihedral angles. Molecular dynamics simulations including dimethylsulfoxide 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 a β‐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 β / β or β / α turns result in a short helical sequence in the free MIR peptide, whereas the bound analogue is folded by an 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.

A three-residue cyclic scaffold of non-RGD containing peptide analogues as platelet aggregation inhibitors: Design, synthesis, and structure–function relationships

Antagonists of fibrinogen at the GPIIb/IIIa receptor, which is the most abundant membrane protein on the platelet surface, are under active investigation as potential antithrombotics. The critical interaction between GPIIb/IIIa and fibrinogen can be inhibited by either linear or cyclic RGDS-containing peptides, which have been proved as lead compounds in the design of platelet aggregation inhibitors. In this study we present the design and construction of a new class of cyclic (S,S) non-RGD containing peptide sequences, using two Cys as a structural scaffold for the development of antiaggregatory agents. The (S,S)-CDC- sequence was incorporated as a conformational constraint, in molecules bearing at least one positive charge with the general formula (S,S)XCDCZ, where X = Ac-Arg, Pro-Arg, Pro-Ser-Lys, and Pro-Ser-Arg, and Z = -NH(2) and Arg-NH(2). Investigation of the structure-function relationships was performed on the basis of (a) the local conformation induced by the (S,S)-CDC motif, (b) the distance of the positively (R-C(zeta) or K-N(zeta)) and negatively (D-C(gamma)) charged centers, (c) the presence of a second positive or negative charge on the molecule, and (d) the orientation of the basic and acidic side chains defined by the pseudo dihedral angle (Pdo), which is formed by the R-C(zeta), R-C(alpha), D-C(alpha), and D-C(gamma) atoms in the case of (S,S)-RCDC and by the K-N(zeta), K-C(alpha), D-C(alpha), and D-C(gamma) atoms in the case of (S,S)-KCDC.

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.

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H‐Asp110‐Lys‐Ile‐Asp‐Trp‐Arg‐Glu‐Ser‐Gly‐Tyr‐Val‐Thr‐Glu‐Val123‐OH (Fas14p) sequence of F. gigantica cathepsin L‐cysteine proteinase. This sequence was synthesized in three different forms: as Nα acetylated (Ac‐Asp110‐Lys‐Ile‐Asp‐Trp‐Arg‐Glu‐Ser‐Gly‐Tyr‐Val‐Thr‐Glu‐Val123‐OH, FasAc14p), bearing at the amino‐terminus an Nα acetylated cystein (Ac‐Cys‐Asp110‐Lys‐Ile‐Asp‐Trp‐Arg‐Glu‐Ser‐Gly‐Tyr‐Val‐Thr‐Glu‐Val123‐OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac‐[Lys‐Aib‐Gly]4‐OH (Ac‐SOC4) through an amide bond formed between Val123 carboxylic group of the epitope and the lysine Nϵ groups of the carrier (Ac‐[Lys(Fas14p)‐Aib‐Gly]4‐OH). Ac‐[Lys(Fas14p)‐Aib‐Gly]4‐OH failed to readily discriminate between naïve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite‐free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola‐infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin‐4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.

A palmitoylated peptide, derived from the acidic carboxyl-terminal segment of the integrin αIIb cytoplasmic domain, inhibits platelet activation

Platelet integrin αIIbβ3 contains an acidic membrane distal motif, 1000LEEDDEEGE1008, in the cytoplasmic domain of the αIIb subunit. We showed that a lipid-modified peptide corresponding to the above region, palmitoyl-K-LEEDDEEGE (pal-K-1000-1008), is platelet permeable and has inhibited platelet aggregation induced by 0.4 U/ml of thrombin (IC50 = 164 µM). Moreover the peptide inhibited both Fibrinogen and PAC-1, binding to activated platelets. The non palmitoylated analog was inactive. A modified, scrambled acidic peptide (palmitoyl-K-GDDEELEEE), showed significant lower inhibitory activity than pal-K-1000-1008. A palmitoylated peptide corresponding to the membrane proximal cytoplasmic domain of αIIb, 989KGVFFKR995 (pal-989-995), is known to specifically induce platelet aggregation. Pal-K-1000-1008 was an inhibitor of human washed platelet aggregation induced by pal-K-989-995 (IC50 = 15 µM). Moreover, pal-K-1000-1008 inhibited phosphorylation of ERK and FAK, two protein kinases involved in platelet activation and aggregation. Our results favour the assumption that the interaction of the membrane proximal sequence 989KGVFFKR995 of the cytoplasmic domain of αIIb with the acidic terminal 1000LEEDDEEGE1008 motif may be an important structural factor in platelet signaling, leading to platelet activation and aggregation.

Side reactions in the SPPS of Cys-containing peptides

Alkylation of sensitive amino acids during synthesis of biologically important peptides is a common and well-documented problem in Fmoc-based strategy. Herein, we probed for the first time an unexpected S-alkylation of Cys-containing peptides that occur during the final TFA cleavage of peptides from the Wang solid support. Through a battery of approaches (NMR, UV and LC–MS) the formed by-product was assigned as the alkylation of the cysteine sulfydryl group by the p-hydroxyl benzyl group derived from the acidic Wang linker decomposition. Factors affecting this side reaction were monitored and a protocol that minimizes the presence of the by-product is reported.