Novel anoplin-based (lipo)-peptide models show potent antimicrobial activity.

The subject of this study is the synthesis and biological evaluation of anoplin-based (Gly-Leu-Leu3 -Lys-Arg5 -Ile-Lys-Thr8 -Leu-Leu-NH2 )-designed (lipo)-peptides, aiming at the development of new antibiotic substances. The design of synthetic compounds based on natural bioactive molecules is an optimistic strategy for the development of new pharmaceutics. Antimicrobial peptides (AMPs) and (lipo)-peptides are two classes of promising compounds, with characteristics that allow them to express their activity by differentiated mechanisms of action. On this basis, anoplin, a natural AMP, was used as a scaffold to design five peptides and seven lipopeptide analogs of them. Substitutions were made on residues Leu3 and Arg5 of the interphase and on Thr8 of the polar phase, as well as N-terminus conjunctions with octanoic and decanoic acid. The outcome of the biological evaluation revealed that some analogs might have substantial clinical potential. Specifically, Ano 1-F, Ano 3-F, Ano 4-C10 , and Ano 5-F are strongly active against Gram-negative bacteria at minimum inhibitory concentration (MIC) values of 3 µg/ml, while Ano 4-F is active against Gram-positive bacteria at 1 µg/ml. Ano 2-C10 , C10 -Gly-Leu-Lys3 -Lys-Ile5 -Ile-Lys-Lys8 -Leu-Leu-NH2 , is the most promising compound (MIC = 0.5 µg/ml) for the development of new pharmaceutics. The conformational features of the synthetic peptides were investigated by circular dichroism spectroscopy, and their physicochemical parameters were calculated. Our study shows that appropriate substitutions in the anoplin sequence in combination with Nα -acylation may lead to new effective AMPs.

Cationic amphipathic peptide analogs of cathelicidin LL-37 as a probe in the development of antimicrobial/anticancer agents.

Cathelicidin LL-37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL-37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13-amino acid cathelicidin 17-29 amide segment F17 KRIV21 QR23 IK25 DF27 LR-NH2 were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R23 and K25 in the hydrophilic side, V21 and F27 in the hydrophobic side of the interphase, and F17 that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17-29 fragment to investigate how they affect activity. Substitution of the amino-terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac-FKRIVQRIL25 DFLR-NH2 resulted in significant cytotoxicity against A549 cancer cells with an IC50 value 3.90 µg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac-FKRIVQI23 IKK26 FLR-NH2 , which incorporates the IIKK motif and the peptides FKRIVQL23 L24 KK26 L27 LR-NH2 and Ac-FKRIVQL23 L24 KK26 L27 LR-NH2 , which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram-negative bacteria (MIC 3-7.5 µg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC50 12.9-9.8 µg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17-29 segment, may lead to the preparation of effective biological compounds.

Synthesis and biological activity of lipophilic analogs of the cationic antimicrobial active peptide anoplin.

Anoplin is a short natural cationic antimicrobial peptide which is derived from the venom sac of the solitary wasp, Anoplius samariensis. Due to its short sequence G1 LLKR5 IKT8 LL-NH2 , it is ideal for research tests. In this study, novel analogs of anoplin were prepared and examined for their antimicrobial, hemolytic activity, and proteolytic stability. Specific substitutions were introduced in amino acids Gly1 , Arg5 , and Thr8 and lipophilic groups with different lengths in the N-terminus in order to investigate how these modifications affect their antimicrobial activity. These cationic analogs exhibited higher antimicrobial activity than the native peptide; they are also nontoxic at their minimum inhibitory concentration (MIC) values and resistant to enzymatic degradation. The substituted peptide GLLKF5 IKK8 LL-NH2 exhibited high activity against Gram-negative bacterium Zymomonas mobilis (MIC = 7 µg/ml), and the insertion of octanoic, decanoic, and dodecanoic acid residues in its N-terminus increased the antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC = 5 µg/ml). The conformational characteristics of the peptide analogs were studied by circular dichroism. Structure activity studies revealed that the substitution of specific amino acids and the incorporation of lipophilic groups enhanced the amphipathic α-helical conformation inducing better antimicrobial effects. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Collagen and keratin polypeptide models for assessing the natural and artificial protein decay of organic materials.

Among the materials constituting the natural and cultural heritage, organic materials of proteinaceous origin as bone (collagen), parchment and woolen textiles (keratin) are the most susceptible to damage and decay because of their exposure to air pollution, inappropriate values of ambient temperature, humidity and light. Aiming at contributing to the development of a reliable and reproducible immunoassay for the evaluation of collagen and keratin decay, three polypeptide models of these proteins were designed, synthesized and studied. Polypeptide [Pro-Ser(OBzl)-Gly]n incorporates the typical motif Pro-X-Gly of collagen; polypeptide [Pro-Cys(Acm)-Gly]n is a model of the C-terminal domain of type I keratin, corresponding to the repeating unit Pro-Cys-X of keratin, while polypeptide Ac-YRSGGGFGYRSGGGFGYRS-ßAla-NH2 encloses the characteristic repeating sequence GGGFGYRS of the N-terminal part of Type II keratin. These polypeptides may be considered as simplified models that mimic fragments of collagen and keratin resulting from artificial and natural ageing or decay. It is concluded that high recognition of anti-polypeptide antibodies, produced after immunizations, by the bone, parchment and textile samples is indicative of high deterioration, while high anti-collagen or anti-keratin recognition is indicative of low deterioration. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Palmitoylated peptide, being derived from the carboxyl-terminal sequence of the integrin αIIb cytoplasmic domain, inhibits talin binding to αIIbß3.

The αIIb cytoplasmic domain of platelet integrin αIIbß3 contains an unorganized acidic membrane-distal (1000)LEEDDEEGE(1008) 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 (989)KVGFFKR(995). 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-(1000)LEEDDE(1005) (pal-K-1000-1005) and palmitoyl-K-(1005)EEGE(1008) (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 αIIbß3 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 αIIbß3.

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 (SOC(4) ), to formulate human and avian receptor mimics, respectively. SOC(4) , formed by the tripeptide unit Lys-Aib-Gly, adopts a rigid helicoids-type conformation, which enables the conjugation of biomolecules to the Lys-N(ε) H(2) groups. By doing so, it preserves their initial conformations and functionalities of the epitopes. We report that SOC(4) -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. SOC(4) -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 SOC(4) -glyco-conjugates immobilized onto nitrocellulose membrane. It is concluded that Ac-SOC(4) [(Ac)(2) ,(3'SL-Aoa)(2) ]-NH(2) 5 and Ac-SOC(4) [(Ac)(2) ,(6'SL-Aoa)(2) ]-NH(2) 6 mimic the HA receptors. These findings could be useful for easy screening of binding and inhibition assays of virus-receptor interactions.

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 (T(1) , T(3) , T(7) and T(8) )] and four cytotoxic lymphocyte [CTL (T(2) , T(4) , T(5) and T(6) )] epitopes were coupled in two copies each to an artificial carrier, SOC(4) , which was formed by the repeating tripeptide Lys-Aib-Gly. The helical conformation of the SOC(4) -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 SOC(4) -conjugates.

HLA-DQ7 beta(1) and beta(2) derived peptides as immunomodulators.

Modulation of protein-protein interactions involved in the immune system by using small molecular mimics of the contact interfaces may lead to the blockage of the autoimmune response and the development of drugs for immunotherapy. The nonpolymorphic beta-regions, exposed to the microenvironment, of the modeled HLA-DQ7, which is genetically linked to autoimmune diseases, were determined. Peptides 132-141 and 58-67, located at the beta(1) and beta(2) domains of HLA-DQ7, respectively, were tested for their involvement in the interactions with CD4(+) T lymphocytes. Linear, cyclic, and dimeric analogs that mimic the exposed surfaces of HLA-DQ7 were designed and synthesized. Their immunosuppressory activities, found in the secondary, humoral immune response to sheep erythrocytes (SRBC) in mice in vitro, ranged from 11% to 53%. The significance of the total charge of the peptides, the pattern of the hydrogen bonding, and the presence of secondary structure were investigated in relation to the immunomodulatory effect of the peptides. Two dimeric analogs of the HLA-DQ7 58-67 fragment, consisting of the two monomers covalently linked by a polyethylene glycol (PEG) spacer, able to mimic the superdimers, were also synthesized and studied. As the 58-67 segment is located at the beta(1) region of HLA-DQ7, close to the major histocompatibility complex (MHC) groove, one may assume that the 58-67 peptide could accommodate the association between T-cell receptor (TCR) and human leukocyte antigen (HLA) by activating a co-stimulatory molecule of the TCR/HLA interaction. This hypothesis is supported by the confocal laser image of the fluorescein-labeled 58-67 peptide and by the fact that it is an immunostimulator at low concentration.

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

Platelet integrin alpha(IIb)beta(3) contains an acidic membrane distal motif, 1000LEEDDEEGE1008, in the cytoplasmic domain of the alpha(IIb) 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 microM). 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 alpha(IIb), 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 microM). 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 alpha(IIb) with the acidic terminal 1000LEEDDEEGE1008 motif may be an important structural factor in platelet signaling, leading to platelet activation and aggregation.

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 Sjogren's 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, SOC(n), 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.

Chirality and helix stability of polyglutamic acid enantiomers.

In this work the chirality and the relative thermal stability of ordered micellar aggregates of poly-L- and poly-D-glutamic acids with the cationic surfactant C14TAB is examined. The complexed mesophases poly-L-Glu/C14TAB and poly-d-Glu/C14TAB were characterized by circular dichroism (CD) in the temperature range 10-70 degrees C for their chirality and thermal stability as well as by X-ray diffraction (XRD) for the micellar ordered structure. Low angle XRD analysis showed that both micellar aggregates poly-L-Glu/C14TAB and poly-D-Glu/C14TAB are hexagonally packed in a MCM-41 fashion with an intermicellar distance identical and equal to 3.55+/-0.10 nm. The CD spectra indicated that both complexes poly-L-Glu/C14TAB and poly-D-Glu/C14TAB possess a mainly alpha-helix structure and are exact mirror images to each other. The same mirror images and a mainly alpha-helix configuration were also observed by CD for the free poly-l- and poly-d-glutamic acids at room temperature. As the temperature increases from 10 up to 70 degrees C the alpha-helix of the poly-l-glutamic acid is gradually transformed to a mixture containing increased amounts of the 3(10)-helix while the alpha-helix structure of the poly-d-glutamic acid is constantly degenerated. In contrast the alpha-helices of the corresponding complexes poly-L-Glu/C14TAB and poly-d-Glu/C14TAB are degenerated upon heating without appreciable increase of the 3(10)-helices as an intermediate configuration. This difference in helix conservation is attributed to increase protection of the l-enantiomers, compared to d-enantiomers, which might be related to the survival of l-aminoacids in the living world.

A palmitoyl-tailed sequential oligopeptide carrier for engineering immunogenic conjugates.

The main guideline in designing effective immunogens as vaccine candidates capable of eliciting potent and specific immune responses is to combine B/T cell epitopes and adjuvants as immunostimulators on the same carrier that links the major histocompatibility complex with T cell receptors. Aiming at contributing to the development of carriers for human usage a helicoid type sequential oligopeptide carrier, SOC(n)-II, formed by the repeating tetrapeptide unit (Aib-Lys-Aib-Gly)(n), n=2-7, elongated from the amino-terminus by the palmitoyl group, known for its adjuvanticity, is now presented. The main B cell epitope, PPGMRPP, of the Sm autoantigen against which the majority of antibodies in patients with systemic lupus erythematosus is directed, was coupled to the Lys-N(epsilon)H(2) groups of the carrier in four copies and the resulting conjugate Palm-SOC(4)-II-Sm(4) was subjected to animal immunizations without utilizing any adjuvant. The induced immune response was comparable with that produced when Ac-SOC(4)-II-Sm(4) was administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. High titers of anti-Palm-SOC(4)-II-Sm(4) antibodies were generated, which recognize the priming immunogenic conjugate, as well as reconstituted Sm mimics but not the carrier alone. It is concluded that Palm-SOC(n)-II carrier is a valuable tool for engineering immunogens eliciting enhanced and specific humoral immune responses.

Collagen models as a probe in the decay of works of art: synthesis, conformation and immunological studies.

Proteinaceous substances such as collagen, casein and albumin have been widely used as binding media in a variety of works of art. Damages of these 'sensitive' materials, mainly caused of the influence of the environment, are responsible for the overall decay of works of art, and their identification is essential to understand ancient technologies, determine the extent of deterioration and help in future restoration and preservation processes. The most commonly used techniques for the identification of proteinaceous binding media are staining techniques, chromatography, spectrometry and immunological methods, although for the latter no systematic studies have been carried out until now. Aiming at contributing to the development of a reliable and reproducible immunoassay for the evaluation of the collagen-based decay of works of art, sequential polypeptides (Pro-X-Gly)n where X represents amino acid residues Val, Lys, Glu and (Hyp-Val-Gly)n were prepared as models of collagen fragments derived from artificially and naturally aged animal collagens. Conformational studies of the polypeptides by CD revealed the occurrence of polyproline II-like structures comparable with those of collagen. Polypeptides and collagen I were administered to animals, and the induced antibodies were used for the immunochemical detection and differentiation of collagen and collagen fragments. The combined application of (i) anti-collagen antibodies, which strongly interact with native collagen, but poorly recognized by artificially aged collagen and (ii) anti-polypeptide antibodies, which do not associate with native collagen, but are strongly recognized by collagen fragments in naturally or artificially aged collagen, is a valuable tool in determining the extent of decay in works of art.

Artificial carriers: a strategy for constructing antigenic/immunogenic conjugates.

The rational design of artificial carriers for anchoring multiple copies of B and/or T cell epitopes, built-in vaccine adjuvants and "promiscuous" T cell epitopes for the construction of conjugates as antigenic substrates or potent immunogens has been the stimulus of intensive efforts nowadays. The unambiguous composition, the reliability and the versatility of the production of reconstituted antigens or immunogens has found a great number of biochemical applications in developing immunoassays of high sensitivity, specificity and reproducibility and in generating site-specific antibodies for usage as human vaccine candidates. In this review are summarized different types of artificial carriers currently used as dendrimers bearing branching segments, multimeric core matrices and templates with built-in folding devices. Emphasis is given to the construction and application of a helicoid-type Sequential Oligopeptide Carrier (SOCn) developed in our laboratory. The beneficial structural elements of SOCn induce a favorable arrangement of the conjugated peptides, which also retain their initial "active" conformation, so that potent antigens and immunogens are generated.

Phosphorylated and nonphosphorylated epitopes of the La/SSB autoantigen: comparison of their antigenic and conformational characteristics.

La/SSB phosphoprotein is the target antigen of autoantibodies in sera of patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Among other structural and function motifs, four phosphorylation sites are encompassed in the primary sequence of La/SSB. Two of them (Thr-362 and Ser-366) are located within GSGKGKVQFQGKKTKFASDD (346-368) and one (Thr-302) within VTWEVLEGEVEKEALKKI (301-318), which are main B-cell epitopes of La/SSB. With the aim to investigate how phosphorylation, one of the most common posttranslational protein modifications, affects the antigenic and conformational characteristics of the La/SSB epitopes, we synthesized and studied the phosphorylated epitopes La/SSB(346-368)-P, La/SSB(359-368)-P, and La/SSB(301-318)-P with respect to their nonphosphorylated counterparts. Anti-La/SSB positive sera from SS and SLE patients are better recognized by the phosphorylated epitopes compared to their nonphosphorylated counterparts. Conformational analysis by (1)H nuclear magnetic resonance spectroscopy and molecular dynamics showed that the phosphorylated epitopes adopt different structural characteristics from those of the corresponding nonphosphorylated epitopes. It is concluded that phosphorylation can create neoepitopes with altered functions, compared to the nonphosphorylated epitopes, which might be seen from the immune system as "foreign."

Innovative, multifunctional sequential oligopeptide carriers Soc(n)-I and SOC(n)-II: functions-technology-perspectives.

An innovative type of multifunctional helicoid artificial carriers, formed by the repetitive Lys-Aib-Gly (SOC(n)-I) or by the Aib-Lys-Aib-Gly (SOC(n)-II), with structural rigidity and regularity were successfully applied in our laboratory for anchoring antigenic/immunogenic peptides. The carriers, designed to display a predetermined 3D structure, adopt the 3(10) helical conformation, while the attached peptides preserve their original "active" conformation. The constructed conjugates were used as substrates in solid phase immunoassays, as well as for generating potent and specific immune responses.

A peptide carrier with a built-in vaccine adjuvant: construction of immunogenic conjugates.

A multifunctional carrier combining B/T cell epitopes (i), a built-in vaccine adjuvant (ii), and a universal T cell epitope (iii) for the construction of potent and specific immunogenic conjugates is presented. The IL-1beta(163-171) fragment known to reproduce the immunostimulatory and adjuvant effects of the whole IL-1beta without possessing any of the pro-inflammatory properties of IL-1beta was covalently anchored to the N-terminus of the Sequential Oligopeptide Carrier, SOC(n), formed by the repeating tripeptide unit Lys-Aib-Gly. A promiscuous T cell epitope derived from the tetanus toxin, TT(593-599), was also positioned in the carboxy terminus of SOC(n) as a universal immunogen to provide broad immunogenicity. Selected B/T cell epitopes from the Sm and La/SSB autoantigens, against which is directed the humoral autoimmunity in patients with systemic lupus erythematosus and Sjögren's Syndrome, respectively, were coupled to the Lys-N(epsilon)H2 groups of the carrier, and the formulated constructs were administered in animals following the conventional immunization protocol of complete/incomplete Freund's adjuvant. The induced immune responses were compared with that produced when the Sm- and La/SSB-reconstituted immunogenic conjugates were injected alone. High titers of specific antibodies recognizing the priming construct, as well as the cognate autoantigen, were obtained when administered alone without the assistance of Freund's adjuvant. It is concluded that our approach provides the conceptual and experimental framework for the development of multifunctional immunogenic conjugates eliciting enhanced, specific, and prolonged humoral response for usage as human vaccine candidates.

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 Sjogren's 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.