Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tethered-ligand.

Thrombin is the most potent agonist of human platelets and its effects are primarily mediated through the protease-activated receptors (PARs)-1 and -4. Although PAR-1 has higher affinity for thrombin than PAR-4, both receptors contribute to thrombin-mediated actions on platelets. Recently, a potent and selective PAR-1 antagonist (vorapaxar) was approved for clinical use in selected patients. In contrast, despite the fact that several PAR-4 antagonists have been developed, few of them have been tested in clinical trials. The aim of the present study was to elucidate the molecular requirements involving the PAR-4 mechanism of activation by peptide analogues of its tethered-ligand. Eight synthetic PAR-4 tethered-ligand peptide analogues were synthesized and studied for their agonistic/antagonistic potency and selectivity toward human washed platelet aggregation, using light transmittance aggregometry. In addition, in silico studies were conducted to describe the receptor-peptide interactions that are developed following PAR-4 exposure to the above analogues. To provide a first structure-activity relationship rationale on the bioactivity profiles recorded for the studied analogues, molecular docking was applied in a homology model of PAR-4, derived using the crystal structure of PAR-1. The following peptide analogues were synthesized: AYPGKF-NH2 (1), GYPGKF-NH2 (2), Ac-AYPGKF-NH2 (3), trans-cinnamoyl-AYPGKF-NH2 (4), YPGKF-NH2 (5), Ac-YPGKF-NH2 (6), trans-cinnamoyl-YPGKF-NH2 (7), and caffeoyl-YPGKF-NH2 (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC50 value of 26.2 µM. Substitution of Ala-1 with Gly-1 resulted in peptide (2), which significantly reduces the agonistic potency of peptide (1) by 25-fold. Importantly, substitution of Ala-1 with trans-cinnamoyl-1 resulted in peptide (7), which completely abolishes the agonistic activity of peptide (1) and renders it with a potent antagonistic activity toward peptide (1)-induced platelet aggregation. All other peptides tested were inactive. Tyr-2, residue, along with its neighboring environment was a key determinant in the PAR-4 recognition mode. When the neighboring residues to Tyr-2 provided an optimum spatial ability for the ligand to enter into the binding site of the transmembrane receptor, a biological response was propagated. These results were compared with the predicted binding poses of small molecule antagonists of PAR-4, denoted as YD-3, ML-354, and BMS-986120. π-π stacking interaction with Tyr-183 appears to be critical and common for both small molecules antagonists and the peptide trans-cinnamoyl-YPGKF-NH2. Conclusively, the lipophilicity, size, and aromatic nature of the residue preceding Tyr-2 are determining factors on whether a human platelet PAR-4 tethered-ligand peptide analogue will exert an agonistic or antagonistic activity.

Design, synthesis and biological evaluation of antipicornaviral pyrrole-containing peptidomimetics.

A series of new peptidomimetics based on the tripeptide sequence Z-Leu-Phe-Gln-OH were synthesized, with ten of these including the alpha-nitrogen atom of the N-terminal amino acid incorporated into the pyrrole cycle. The synthesized compounds were tested for antiviral activity by agar-diffusion plaque inhibition test against Coxsackievirus B1 replication in FL cell. Four of the products were observed to possess an antiviral activity, which was proven to be significant for one product. N-terminal pyrrole moiety and C-terminal free carboxyl function are available in all active compounds. On the other hand, their corresponding -OBzl and -Obu t esters are inactive.

Highly constrained cyclic (S,S) -CXaaC- peptides as inhibitors of fibrinogen binding to platelets.

The Arg-Gly-Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin alpha(IIb)beta3. Binding of fibrinogen (Fg) to activated alpha(IIb)beta3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) -CXaaC- containing peptides incorporating the (S,S) -CDC- and (S,S) -CRC- motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50-values ranged from 1.7 to 570 microm). The peptides inhibited Fg binding with IC50-values up to 30-fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)-induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR-NH2 (peptide 11) and (S,S) PRCDCK-NH2 (peptide 10) did not inhibit PAC-1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR-NH(2) (peptide 11), one of the more active peptides, inhibited ADP-induced P-selectin exposure. By contrast, peptide (S,S) Ac-RWDCRC-NH2, incorporating the inverse (S,S) -DCRC- sequence (peptide 16), failed to inhibit P-selectin exposure whereas at the same concentration, it effectively inhibited PAC-1 and Fg binding. It is concluded that peptides containing the (S,S) -CDC- as well the (S,S) -CRC- sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor alpha(IIb)beta3, in its activated form. Furthermore, the (S,S) -RCDC- sequence can be used as a scaffold for developing potent non-RGD-like Fg-binding inhibitors.

Multiple anchoring of myelopeptides on sequential oligopeptide carriers (SOC(n)): synthesis, conformation and studies in human leukemia cells.

Myelopeptides, MP-6 (Val-Asp-Pro-Pro) and MP-4 (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), induce metabolic changes in human leukemia cells, HL-60, characteristic of the differentiation process, which should be regarded as a promising therapeutic approach in cancer and related diseases. With the aim to optimize the differentiation effect of MPs, they were coupled to the Lys-N(epsilon)H(2) groups of a sequential oligopeptide carrier Ac-(Lys-Aib-Gly)(4), SOC(4), and the constructs obtained were studied. The rigid 3(10) secondary structure of the carrier is preserved even after linkage of the MPs, which also maintain their initial conformations without interacting either with each other or with the carrier, as demonstrated by (1)H nuclear magnetic resonance (NMR) spectroscopy. It is concluded that the carrier accommodates the presentation of MPs, thus improving their differentiation effect on human leukemia cells.

A diepitopic sequential oligopeptide carrier (SOCn) as mimic of the sm autoantigen: synthesis, conformation and biological assays.

Anti-Sm (Sm: U1-U6 RNA-protein complex) antibodies are usually considered highly specific for systemic lupus erythematosus (SLE), while anti-U1RNP (U1RNP: U1RNA-protein complex) are thought of as diagnostic criteria for the mixed connective tissue disease (MCTD). However, both antibody specificities coexist in SLE and MCTD, in varying percentages. Although the anti-Sm/anti-U1RNP immunological cross-reactivity has been initially attributed to a common motif, PPXY(Z)PP (where X, Y, Z are various amino acids), found in the Sm, U1-A and U1-C autoantigens, it appears that the conformational features of the Sm epitopes also play an important role in the immunoreactivity. The PPGMRPP and PPGIRGP main epitopes of the Sm antigen were coupled in duplicate to the tetrameric Ac-(Lys-Aib-Gly)4-OH, SOC4, carrier to form the [(PPGMRPP)2, (PPGIRGP)2]-SOC4 construct as a mimic of the native Sm. It was found that: (i) the 3(10) helical structure of SOC4 allows the epitopes to adopt an exposed orientation, similar to their free forms, that facilitates their recognition from the anti-Sm antibodies, and (ii) the U1-RNP cross-reactivity is minimized.

The position of the LysN epsilon H2-grafted antigens along the sequential oligopeptide carrier, Ac-(Aib-Lys-Aib-Gly)n (SOCn-II), influences the antibody recognition: application to the Sm main autoimmune epitope.

A sequential oligopeptide carrier of antigenic peptides is presented, incorporating two Aib residues in each repetitive moiety: Ac-(Aib-Lys-Aib-Gly)(n) (SOC(n) -II; n = 2-4). The conformational study, by (1)H-nmr, CD, and Fourier transform ir spectroscopy, indicated that the SOC(n) -II carrier displays a pronounced 3(10)-helix, compared to the Ac-(Lys-Aib-Gly)(n) (SOC(n) -I) carrier of the same approximately backbone length, previously reported. One of the dominant autoimmune epitopes of the Sm and U1RNP cellular components, the PPGMRPP sequence, was coupled to the Lys-N(epsilon)H(2) groups of the SOC(n) -II carrier and used as antigenic substrate for detecting anti-Sm/U1RNP autoantibodies in ELISA assays. Anti-Sm antibodies are highly specific for systemic lupus erythematosus, while anti-U1RNP are specific for mixed connective tissue disease. The anti-(PPGMRPP)(5)-SOC(n) -II ELISA was compared with the anti-(PPGMRPP)(n) -SOC(n) -I ELISA, provided that both antigenic substrates possess the same amount of the epitope replicates. The significance of the lysine positions along the oligopeptide backbone of the carrier for a favorable antibody recognition of the anchored antigens is also examined.

The third-dimensional structure of the complex between an Fv antibody fragment and an analogue of the main immunogenic region of the acetylcholine receptor: a combined two-dimensional NMR, homology, and molecular modeling approach.

Binding of autoantibodies to the acetylcholine receptor (AChR) plays a major role in the autoimmune disease Myasthenia gravis (MG). In this paper, we propose a structure model of a putative immunocomplex that gives rise to the reduction of functional AChR molecules during the course of MG. The model complex consists of the [G(70), Nle(76)] decapeptide analogue of the main immunogenic region (MIR), representing the major antigenic epitope of AChR, and the single chain Fv fragment of monoclonal antibody 198, a potent MG autoantibody. The structure of the complexed decapeptide antigen [G(70), Nle(76)]MIR was determined using two-dimensional nmr, whereas the antibody structure was derived by means of homology modeling. The final complex was constructed using calculational docking and molecular dynamics. We termed this approach "directed modeling," since the known peptide structure directs the prestructured antibody binding site to its final conformation. The independently derived structures of the peptide antigen and antibody binding site already showed a high degree of surface complementarity after the initial docking calculation, during which the peptide was conformationally restrained. The docking routine was a soft algorithm, applying a combination of Monte Carlo simulation and energy minimization. The observed shape complementarity in the docking process suggested that the structure assessments already led to anti-idiotypic conformations of peptide antigen and antibody fragment. Refinement of the complex by dynamic simulation yielded improved surface adaptation by small rearrangements within antibody and antigen. The complex presented herein was analyzed in terms of antibody-antigen interactions, properties of contacting surfaces, and segmental mobility. The structural requirements for AChR complexation by autoantibodies were explored and compared with experimental data from alanine scans of the MIR peptides. The analysis revealed that the N-terminal loop of the peptide structure, which is indispensable for antibody recognition, aligns three hydrophobic groups in a favorable arrangement leading to the burial of 40% of the peptide surface in the binding cleft upon complexation. These data should be valuable in the rational design of an Fv mutant with much improved affinity for the MIR and AChR to be used in therapeutic approaches in MG.

Arg side-chain-backbone interactions evidenced in model peptides by 17O-NMR spectroscopy.

The guanidinium group of arginine possesses a variety of biochemical functions, either by participating in direct interactions in recognition processes, or by stabilizing secondary structures. Three model compounds, selectively (17)O enriched, Ac-Arg-Ala-[(17)O]Pro-NH(2) (1), Piv-Arg-Pro-[(17)O]Gly-NH(2) (2) (C-terminal segment of the luteinizing hormone releasing hormone), and Piv-Nle-Pro-[(17)O]Gly-NH(2) (3), were prepared and studied by (17)O-nmr spectroscopy. A direct hydrogen-bonded interaction between the Arg side chain and the carbonyl main chain carboxy-terminus was found, thus confirming the tendency of Arg to participate in proton-acceptor functions.

No evidence of epitope spreading after immunization with the major Sm epitope P-P-G-M-R-P-P anchored to sequential oligopeptide carriers (SOCs).

The sequence Pro-Pro-Gly-Meth-Arg-Pro-Pro (PPGMRPP) is the major B-cell epitope of the Sm autoantigen. The aim of the present study was to evaluate the immune response against the native forms of Sm and U1RNP and immune mediated tissue injury after immunization with the sequence PPGMRPP anchored in five copies to a new type helicoid sequential oligopeptide carrier (SOC) formed by the repetitive Lys-Aib-Gly moiety, [(PPGMRPP)(5)SOC(5)]. Rabbits (n=3) were immunized with 0.5 mg of (PPGMRPP)(5)SOC(5)in complete Freud's adjuvant and boosted at days 26, 53, 99; control rabbits were immunized with the PPGMRPP alone (n=3), phosphate buffered saline (PBS) (n=1), SOC(5)alone (n=1), a peptide at aminoacid (aa) position 158-177 of myelin basic protein (MBP aa 158-147) (n=1) and three La/SSB autoantigen B-cell epitopes (n=3). Antibodies to (PPGMRPP)(5)SOC(5)were determined by enzyme linked immunosorbent assay (ELISA); precipitating anti-Sm and anti-U1RNP antibodies were detected by RNA precipitation and western blot on HeLa total cellular and nuclear extract and 12s sucrose gradient fraction of rat liver extracts. High titres of anti-(PPGMRPP)(5)SOC(5)antibodies not recognizing the native forms of Sm or U1RNP antigens were detected in the (PPGMRPP)(5)SOC(5)immunized but not in the control animals. The sera of two (PPGMRPP)(5)SOC(5)immunized but not of the control rabbits recognized a 67 kDa protein in HeLa nuclear extract, distinct from the 70 kDa U1RNP antigen. Diffuse and segmental increase of mesangeal matrix and cells, crescent formation, segmental glomerular necrosis, rarely massive subendothelial deposits occluding the lumen and C3 complement component in the mesangeal area were seen in the kidneys of one (PPGMRPP)(5)SOC(5)immunized, but not of the remaining animals. In conclusion, the immune response induced by (PPGMRPP)(5)SOC(5)was specific for the immunizing epitope but not for the native forms of Sm and U1RNP antigens, but it was associated with immune mediated kidney injury.

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.

Design, synthesis, and conformational study of biologically active photolabeled analogues of the main immunogenic region of the acetylcholine receptor.

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding to acceptor molecules, which provides important insights for mapping the bimolecular interfaces. The autoimmune disease myasthenia gravis is caused by autoantibodies against the acetylcholine receptor (AChR). The majority of the anti-AChR antibodies bind to the "main immunogenic region" (MIR) of the AChR. To identify the contact points between the complementarity determining regions of the anti-MIR antibodies that recognize the MIR contact sites of the AChR, we present here three photoreactive dodecapeptide MIR analogues containing the photolabel p-benzoyl-L-phenylalanine (Bpa) moiety, either in position 1 or 11. The structure of the produced 12-mers was analyzed using two-dimensional (1)H-NMR spectroscopy, whereas their binding to anti-MIR monoclonal antibodies (mAbs) was determined by immunochemical assays. In all cases the modifications resulted in conservation of the beta-turn conformation of the N-terminus, which has been proved essential for antibody recognition and increased anti-MIR binding relative to the MIR decapeptide.

A new helicoid-type sequential oligopeptide carrier (SOC(n)) for developing potent antigens and immunogens.

A new class of sequential oligopeptide carriers (SOC(n)) for anchoring antigenic/immunogenic peptides has been constructed. The carrier, formed by the repetitive Lys-Aib-Gly moiety, is designed to display a predetermined 3D structure, so that the attached peptides would obtain a defined spatial orientation. Conformational analysis showed that SOC(n) adopt a distorted 3(10)-helical structure, while the coupled peptides preserve their original 'active' structure. Coupling to the carrier may also result to the enhancement of one conformer of the anchored peptide. It is concluded that the structure of SOC(n) offers an optimal presentation of the attached peptides, so that potent antigens or immunogens are generated.

Peptide carriers: A helicoid-type sequential oligopeptide carrier (SOC(n)) for multiple anchoring of antigenic/immunogenic peptides.

A new peptide carrier with three-dimensional predetermined structural motif has been constructed by the repetitive Lys-Aib-Gly moiety. The sequential oligopeptide carrier (SOC(n)), (Lys-Aib-Gly)(n), adopts a distorted 3(10)-helical conformation and the Lys-N(epsilon)H(2) anchoring groups exhibit defined spatial orientations. Conformational analysis of the SOC(n) conjugates showed that the coupled peptides retain their initial "active" structure, while prevalence of one conformer was also observed. It is concluded that the beneficial structural elements of SOC(n) induce a favorable arrangement of the conjugated peptides, so that potent antigens and immunogens are generated.

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.

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.

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.

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.

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.

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.