Eugenia Panou-Pomonis

The clinical relevance of antibodies to ribosomal-P common epitope in two targeted systemic lupus erythematosus populations: a large cohort of consecutive patients and patients with active central nervous system disease

OBJECTIVES To develop an enzyme linked immunosorbent assay (ELISA) using as substrate a synthetic 22-aminoacid peptide, corresponding to the ribosomal P0, P1 and P2 common epitope. To study the specificity and sensitivity of the method and evaluate the frequency and clinical associations of anti-P antibodies in two groups of systemic lupus erythematosus (SLE) patients: (a) unselected SLE patients and (b) SLE patients with central nervous system (CNS) involvement. PATIENTS AND METHODS The C-terminal 22 aminoacid peptide of the ribosomal P proteins (Lys-Lys-Glu-Glu-Lys-Lys-Glu-Glu-Lys-Ser-Glu-Glu-Glu-Asp-Glu-Asp-Met-Gly-Phe-Gly-Leu-Phe-Asp) was synthesised according to Merrifields solid phase procedure. Purification of the peptide was performed by preparative high performance liquid chromatography and confirmed by amino acid analysis. Using this peptide, in a concentration 5 μg/ml, an ELISA was developed. The presence of anti-P antibodies was evaluated by western blot using purified ribosomal proteins from rat liver. Sera from 178 consecutive patients with SLE and 28 patients with SLE and CNS manifestations were tested. Sera from 58 patients with rheumatoid arthritis and 57 patients with primary Sjögrens syndrome were used as controls. The cut off point of the assay was defined using 124 normal sera. RESULTS The specificity of the assay was evaluated by homologous inhibition. Pretreatment of positive sera with soluble 22mer peptide of the ribosomal P proteins resulted in 88% inhibition. The concordance between the peptide assay and western blot was found to be 83%. Thirty three of 178 (18.6%) of the unselected SLE patients had antibodies to P-protein common epitope. Their presence was associated with more active disease (European Consensus Lupus Activity Measurement, ECLAM scoring system) (p<0.001), higher levels of anti-ds DNA antibodies (p<0.05) and lower levels of the C4 component of complement (p<0.01). Eleven of 28 (39.3%) patients with SLE and active CNS involvement had antibodies to P-protein. The overall prevalence of anti-P antibodies in active CNS disease patients was statistically significantly higher, as compared with unselected SLE patients (χ2=6.04, p<0.05). These antibodies were found in a high proportion of patients without anticardiolipin antibodies (52.4%) and they were associated with diffuse CNS involvement (psychiatric disorders (71%) and epilepsy (75%)). CONCLUSIONS A synthetic analogue of the common epitope of ribosomal P-proteins can be use as an antigen for the detection of anti-P antibodies. These antibodies are associated with active SLE and CNS involvement particularly in patients without anticardiolipin antibodies.

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.

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.

Zinc Ion Dependent B-Cell Epitope, Associated with Primary Sjogren's Syndrome, Resides within the Putative Zinc Finger Domain of Ro60kD Autoantigen: Physical and Immunologic Properties

The Ro/La ribonucleoprotein (RNP) complex is composed of the proteins Ro60kD, Ro52kD, and La48kD that are in association with one small cytoplasmic RNA (YRNA). Specific protein-RNA and protein-protein interactions are thought to occur through the RNP and zinc-finger secondary structure elements of the Ro60kD protein. The aim of our study was to investigate the antigenic properties of the zinc finger domain of the Ro60KD autoantigen and its contribution to the formation of Ro/La RNP complex. It was found that the peptide VSLVCEKLCNEKLLKKARIHPFHILIA (Zif-1), which corresponds to the natural sequence of the zinc finger domain (301-327), and the peptide C(Acm)NEKLLKKARIC(Acm), analogous to the intermediate loop 310-319 (Zif-3) of the same domain of Ro60KD, are recognized by the majority of anti-Ro/SSA and anti-La/SSB positive sera (82.6% and 77.1%, respectively) in the absence of zinc ions. The same sera failed to react with Zif-1 peptide in the presence of Zn2+. In contrast, the addition of zinc ions was necessary for the binding of Zif-1 to recombinant Ro52KD as shown by direct binding experiments of the recombinant protein with synthetic peptides. Our data suggest the zinc finger domain of Ro60kD contains a B-cell epitope with high specificity for primary Sjogrens syndrome. Furthermore, depending on the presence of zinc ions, the zinc finger domain of the Ro60KD protein can exist in two different conformational states favoring either an interaction with the Ro52KD protein or binding with autoantibodies.

The PPGMRPP repetitive epitope of the Sm autoantigen: Antigenic specificity induced by conformational changes. Application of the Sequential Oligopeptide Carriers (SOCs)

The PPGMRPP sequence, found in several copies in the Sm and U1RNP autoantigens, is the main target of anti-Sm and anti-U1RNP antibodies in systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) patients sera. It is also recognized, to a lower extent, by anti-Ro/SSA and anti-La/SSB specificities. The PPGMRPP-NH2 peptide amide and the PPGMRPP peptide, which is bound to a pentameric sequential oligopeptide carrier (SOC5), were examined by1H-NMR spectroscopy and ELISA assays, using sera from patients with autimmune rheumatic diseases. Among the three main conformers found for the free PPGMRPP, the extended one was also identified for PPGMRPP-NH2 and (PPGMRPP)5-SOC5. This can be attributed to the absence of ionic interactions between the Arg-guanidinium and the carboxylate group in the amide and SOC5-bound forms of the peptide. Immunoassays using sera from various specificities showed an enhanced anti-Sm and anti-U1RNP recognition of PPGMRPP-NH2 and (PPGMRPP)5-SOC5, and lowering of the anti-Ro/SSA and anti-La/SSB reactivity. The presence of multiple conformers of free PPGMRPP may explain the unexpected cross-reactivity to the anti-Ro/La positive sera, while the prevalence of the extended conformation in PPGMRPP-NH2 and (PPGMRPP)5-SOC5 is mainly responsible for the enhanced recognition from the anti-Sm and anti-U1RNP autoantibodies. it is concluded that the antigenic specificity of PPGMRPP-NH2 and (PPGMRPP)5-SOC5 is mainly induced by conformational changes resulting from the conversion of the C-terminal carboxylate group to the amide form.

Sialic acid and sialyl-lactose glyco-conjugates: design, synthesis and binding assays to lectins and swine influenza H1N1 virus.

The terminal parts of the influenza hemagglutinin (HA) receptors α2,6‐ and α2,3‐sialyllactoses were conjugated to an artificial carrier, named sequential oligopeptide carrier (SOC4), to formulate human and avian receptor mimics, respectively. SOC4, formed by the tripeptide unit Lys‐Aib‐Gly, adopts a rigid helicoids‐type conformation, which enables the conjugation of biomolecules to the Lys‐NεH2 groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC4‐glyco‐conjugate bearing two copies of the α2,6‐sialyllactose is specifically recognized by the biotinylated Sambucus nigra (elderberry) bark lectin, which binds preferentially to sialic acid in an α2,6‐linkage. SOC4‐glyco‐conjugate bearing two copies of the α2,3‐sialyllactose was not recognized by the biotinylated Maackia amurensis lectin, despite its well‐known α2,3‐sialyl bond specificity. However, preliminary immune blot assays showed that H1N1 virus binds to both the SOC4‐glyco‐conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac‐SOC4[(Ac)2,(3′SL‐Aoa)2]‐NH2 5 and Ac‐SOC4[(Ac)2,(6′SL‐Aoa)2]‐NH2 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus–receptor interactions. Copyright

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.

A complementary La/SSB epitope anchored to Sequential Oligopeptide Carrier regulates the anti-La/SSB response in immunized animals.

Complementary peptide epitopes, derived from complementary RNA sequences, have been used for suppressing the autoimmune response in experimental autoimmune diseases as myasthenia gravis, allergic neuritis and allergic encephalomyelitis. Aiming at contributing to the development of a tool that could regulate the autoantibody production against La/SSB, which is the main target of autoantibodies in Sjogrens syndrome (SS) and systemic lupus erythematosus (SLE), the complementary epitope, cpep349–364, of the minor T/major B cell epitope of La/SSB, pep349–364, was utilized for the induction of neutralizing anti‐cpep349–364 antibodies in rabbit immunizations. Complementary peptides were coupled to an artificial carrier, developed in our laboratory, in order to enhance the complementary potency of cpep349–364 and its counterpart. This carrier, named Sequential Oligopeptide Carrier, SOCn, formed by the repeating tripeptide Lys‐Aib‐Gly, adopts helical conformation, which allows the anchored peptide epitopes to preserve their initial reactivity such as molecular recognition, antigenicity/immunogenicity. Our study provides proof of evidence of specific interactions between idiotypic (Id)/anti‐idiotypic (anti‐Id) antibodies generated in immunized animals by the sense epitope (conjugate I) of La/SSB and its complementary counterpart (conjugate II). It was also demonstrated that the Id/anti‐Id association is specifically disrupted by adding either the sense epitope (conjugate I) or its complementary counterpart (conjugate II). A mutual neutralization of Id/anti‐Id antibodies was observed in vivo, which implies that generation of anti‐Id antibodies by immunization with the complementary La/SSB epitope could scavenge the anti‐La/SSB response. Copyright

Palmitoylated peptide, being derived from the carboxyl-terminal sequence of the integrin αIIb cytoplasmic domain, inhibits talin binding to αIIbβ3

Abstract The αIIb cytoplasmic domain of platelet integrin αIIbβ3 contains an unorganized acidic membrane-distal 1000LEEDDEEGE1008 region. We have shown that a platelet permeable peptide corresponding to the above region the palmitoyl-K-LEEDDEEGE (pal-K-1000–1008) inhibits platelet aggregation induced by thrombin or by pal-K-989–995, a palmitoylated peptide corresponding to the membrane-proximal αIIb cytoplasmic domain 989KVGFFKR995. We now tested the anti-aggregatory activity of (i) a lipid-modified scrambled acidic peptide (pal-K-GDDEELEEE), (ii) two smaller peptides derived from the acidic amino sequence: palmitoyl-K-1000LEEDDE1005 (pal-K-1000–1005) and palmitoyl-K-1005EEGE1008 (pal-K-1005–1008) and (iii) lipid-modified palmitoyl-acidic peptides with alanine (Ala) substitution at residues 1001, 1003, 1004 and 1005 and one peptide with a double Ala substitution at residues 1001 and 1004 of the 1000–1008 sequence. All the peptides tested showed an inhibitory activity, however, the palmitoylated peptide with the natural and the whole acidic sequence, being the most active. Our results suggest that the whole acidic sequence, rather than some specific amino acids, contributes to the aggregation inhibitory activity. The inhibitory peptide, pal-K-1000–1008, inhibited the association of talin with αIIbβ3 in thrombin-activated platelets, as demonstrated by co-immunoprecipitation experiments, while the scrambled peptide was inefficient. We suggest that, by interacting with αIIb cytoplasmic domain, pal-K-1000–1008 has an anti-aggregatory inhibitory activity due to a specific inhibition of talin binding to αIIbβ3.

Influenza virus H5N1 hemagglutinin (HA) T-cell epitope conjugates: design, synthesis and immunogenicity.

The influenza virus, major surface glycoprotein hemagglutinin (HA) is one of the principal targets for the development of protective immunity. Aiming at contributing to the development of a vaccine that remains the first choice for prophylactic intervention, a reconstituted model of HA, mimicking its antigenic properties was designed, synthesized and tested in mice for the induction of protective immunity. Four helper T lymphocyte [HTL (T1, T3, T7 and T8)] and four cytotoxic lymphocyte [CTL (T2, T4, T5 and T6)] epitopes were coupled in two copies each to an artificial carrier, SOC4, which was formed by the repeating tripeptide Lys‐Aib‐Gly. The helical conformation of the SOC4‐conjugates preserves the initial topology of the attached epitopes, which is critical for their immunogenic properties. Survival of immunized animals, ranged from 30 to 50%, points out the induction of protective immunity by using the SOC4‐conjugates. Copyright