The effect of the branched chain polypeptide carrier on biodistribution of covalently attached B-cell epitope peptide (APDTRPAPG) derived from mucin 1 glycoprotein

In order to establish structure–function relationship for the design of a new group of oligopeptide antigen–macromolecule conjugate, multiple copies of mucin-1 B-cell epitope peptide, APDTRPAPG were conjugated with branched chain polymeric polypeptides possessing poly[L-Lys] backbone. By the synthesis, radiolabelling (125I) and in vivo treatment of BALB/c mice with epitope conjugates containing XiK/XAK type carrier, where X = Glu (EiK or EAK) or Leu (LAK), the influence of the polypeptide structure on the blood clearance profile and on tissue distribution profile concerning the epitope delivery to relevant organs (e.g. immunocompetent or involved in excretion) were investigated. We observed significant differences in the blood clearance profiles for the conjugates, the respective polypeptide carriers and free epitope peptide. All conjugates, regardless of their charge properties exhibited longer presence in the circulation than the free oligopeptide. Tissue distribution data also showed that the structural properties (e.g. amino acid composition, charge) of the carrier polypeptide have marked influence on the tissue accumulation of the epitope peptide conjugates. In contrast to conjugates with linear (K) or branched chain (LAK) polycationic polymers exhibiting rapid blood clearance and high spleen/liver uptake, amphoteric epitope peptide conjugates with different branches, but similar charge properties (EiK or EAK) had extended blood survival and generally lower tissue accumulation. The results on this systematic investigation suggest that further studies on the immune response induced by these epitope conjugates would be needed to provide correlation between biodistribution properties (presence in the blood, level of tissue accumulation) and the capacity of these conjugates to elicit antibody production

Investigation of Neutral Losses and the Citrulline Effect for Modified H4 N-Terminal Pentapeptides

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The mapping of linear B-cell epitope regions in desmoglein 1 and 3 proteins : Recognition of immobilized peptides by pemphigus patients’ serum autoantibodies

Desmosomal transmembrane glycoproteins desmoglein 1and desmoglein 3 are targets of life-threatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus foliaceus(PF). In these diseases pemphigus autoantibodies are produced against Dsg1 and Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of desmosomal cell-cell adhesion, and clinically, to the presence of blisters and erosions. Identification, characterization and detailed analysis of the binding sites of autoantibodies have an outstanding importance in understanding theimmunopathology of the disease and also in the design of novel diagnostics. Here we describe the localization of the B-cell epitope regions of Dsg1 and Dsg3 proteins extracellular parts recognized by IgG-type serum autoantibodies of patients with PV andPF. In our study overlapping pentadecapeptides were synthesized on hydroxypropylmethacrylate pins based on the results of in silicopredictions. To detect the interaction between theserum autoantibodies and the immobilized synthetic peptides, modified ELISA (Enzyme Linked Immunosorbent Assay) was performed with pin-attached peptides testing the serum samples of ten patients and four healthy donors. We identified five possible epitope regions (aa86-110, aa196-220, aa226-250, aa326-340, and aa486-520) within the Dsg1 protein sequence and four possible epitope regions (aa64-78, aa330-344, aa375-399, aa446-460) within the Dsg3 protein sequence using these methods. Our data showed that serum autoantibodies of patients, previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear epitope regions of both Dsg1 and Dsg3 proteins using pin-bound overlapping peptides in modified ELISAs

Humán és bakteriális hősokkfehérjék komplementaktiváló képességének összehasonlító vizsgálata = Study on the complement activating ability of human and bacterial heat-shock proteins

A kutatási periódusban 4 célkitűzésben vizsgáltuk humán és bakteriális hősokkfehérjék komplementaktiváló képességét. A komplementaktiváció legfontosabb regulátorainak, a Hsp60/65 ellens antitestek vonatkozásában azt kaptuk, hogy jelentős reguláló tényező az IL-6 promóter -174-es polimorfizmusa. Kimutattuk továbbá, hogy az anti-Hsp60 autoantitestek a természetes autoantitest repertoárba tartoznak, valamint epitóp szinten is elkülöníthetők a az anti-Hsp65 antitestektől. Eredményeink jelentőségét az adja, hogy eddig csak korlátozott ismeretanyaggal rendelkeztünk az anti-Hsp autoantitestek összefüggéseire és regulációjára vonatkozóan. A pályázat támogatásával az erre vonatkozó részletes immunológiai ismeretanyag gazdagodott. A Hsp70 komplementaktiváló képességének in vitro vizsgálatát megnehezítette a rekombináns fehérjék endotoxin szennyezettsége, ami mellett nem tudtuk megítélni a komplementaktiváció pontos mechanizmusát. A Hsp70 további vizsgálatát in vivo, klinikai beteganyagon valósítottuk meg, ezek a megfigyeléseink lehetővé teszik egy későbbi, in vivo komplementaktivációra vonatkozó munkahipotézis kialakítását. Eredményeinknek gyakorlati vonatkozását az adja, hogy a hősokkfehérjék immunológiai szerepe egyre több klinikai állapotban nyer bizonyítást, és az ezzel kapcsolatos részletes szabályozó folyamatok jellemzése hozzájárul az adott kórállapotok pontosabb megértéséhez. | The complement (C) activating ability of human and bacterial heat shock proteins was investigated along four objectives in this project. Investigating the main regulator of Hsp60/Hsp65 induced C activation, i.e. anti-Hsp antibodies, the primary regualting role of the IL-6 -174 promoter polymorphism was shown. Furthermore, evidences were obtained about the natural autoantibody properties of anti-Hsp60 IgM antibodies. These antibodies can also be distinguished from anti-Hsp65 antibodies based on their epitope specificities. There were only limited data available until now on the regulation and associations of anti-Hsp autoantibodies. With the support of the current research grant detailed immunological knowledge has been obtained on the above topic. The in vitro investigations on the Hsp70 induced C activation was hampered by the endotoxin contamination of the recombinant Hsp70 preparations. Therefore, our subsequent in vivo investigation on Hsp70 was done in clinical studies. The results of these provide base for follow-up in vivo research to determine the exact mechanism of Hsp70 induced C acitvaiton in vivo. The physiological relvance of our results is related to the fact of recent observations about the roles of heat shock proteins in different human diseases. Understanding the precise immunological mechanisms behind these associations will help in the future to appreciate the clinical consequences of Hsps in more depth

Tailoring Uptake Efficacy of HSV-1 gD Tailoring Uptake Efficacy of Hsv-1 GD Derived Carrier Peptides

Regions of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) were chosen to design carrier peptides based on the known tertiary structure of the virus entry receptor complexes. These complexes consist of the following: HSV-1 gD–nectin-1 and HSV-1 gD–herpesvirus entry mediator (HVEM). Three sets of peptides were synthesised with sequences covering the (i) N-terminal HVEM- and nectin-1 binding region -5–42, (ii) the 181–216 medium region containing nectin-1 binding sequences and (iii) the C-terminal nectin-1 binding region 214–255. The carrier candidates were prepared with acetylated and 5(6)-carboxyfluorescein labelled N-termini. The peptides were chemically characterised and their conformational features in solution were also determined. In vitro internalisation profile and intracellular localisation were evaluated on SH-SY5Y neuroblastoma cells. Peptide originated from the C-terminal region 224–247 of the HSV-1 gD showed remarkable internalisation compared to the other peptides with low to moderate entry. Electronic circular dichroism secondary structure studies of the peptides revealed that the most effectively internalised peptides exhibit high helical propensity at increasing TFE concentrations. We proved that oligopeptides derived from the nectin-1 binding region are promising candidates—with possibility of Lys237Arg and/or Trp241Phe substitutions—for side-reaction free conjugation of bioactive compounds—drugs or gene therapy agents—as cargos

Autoimmun betegségekben szerepet játszó új, diagnosztikus epitópok, valamint Fc receptorhoz kötődő és effektor funkciót kiváltó IgG Fc peptidkimérák azonosítása, szintézise és funkcionális jellemzése = Identification, synthesis and functional characterisation of new, diagnostic epitopes in autoimmune diseases, and of IgG Fc peptide chimeras binding to Fc receptor and triggering effector function

Autoimmun hólyagos bőrbetegségekre jellemző B- és T-sejt epitóppeptideket szintetizáltunk szilárdfázisú peptidszintézissel, Fmoc/tBu stratégiával. A peptideket tisztítottuk, azonosítottuk. Autoantigén B-sejt epitópok azonosítására módosított ELISA módszerrel tűhegyen korábban szintetizált átlapoló desmoglein 1 és 3 peptideken hat pemphigus foliaceusban (PF), egy pemphigus vulgarisban (PV) szenvedő beteg és egészséges donor szérumát vizsgáltuk. Megállapítottuk, hogy az egészséges kontrollhoz képest a betegszérumok desmoglein specifikus autoellenanyag-tartalma általában kicsit magasabb, ezen belül öt olyan epitóprégiót találtunk, amelyeket hét beteg közül legalább öt szérum-ellenanyagai szignifikánsan erősebben ismertek fel. Megkezdtük ezen epitóprégiók szintézisét egyedi peptidek formájában, Cys-nel meghosszabbítva, későbbi, makromolekulás hordozóhoz vagy funkcionalizált ELISA lemezhez való konjugálás céljából. Desmoglein 3 autoantigén T-sejt epitóp vizsgálata pemphigus vulgarisban Két PV-ben szenvedő és két egészséges donor perifériás vér monomorfonukleáris sejtjeit (PBMC) izoláltuk, majd egy desmoglein 3 T-sejt epitóppeptid rövidített, átlapoló változataival inkubáltuk. A sejtek felülúszóiból szendvics ELISÁ-val mutattuk ki a termelődött interferon-gammát. A betegek PBMC-i szignifikánsan nagyobb mértékben termeltek IFN-gamma citokint, mint az egészséges donorok sejtjei. Leghatékonyabban a T-sejt epitóppeptid középső szakasza stimulálta a sejteket. | B- and T-cell epitope peptides characteristic for autoimmune bullous skin diseases were prepared by solid phase synthesis, Fmoc/tBu strategy. The peptides were purified and characterised. To determine autoantigenic B-cell epitopes, modified ELISA was performed on formerly synthesised pin-bound desmoglein 1 and 3 peptides with the sera of six pemphigus foliaceus (PF), a pemphigus vulgaris (PV) patients and healthy donor. The serum autoantibody binding to the peptides was generally somewhat higher in case of the patients, and we could identify five epitope regions which were recognised by at least 5 out of 7 patients significantly more strongly by patients’ sera than by those of the control. We have started the synthesis of the selected regions as individual peptides, elongated by Cys, to facilitate future conjugation to ELISA plates or macromolecular carriers. To study a desmoglein 3 autoantigen T-cell epitope in PV, we have isolated the PBMC of two PV patients and two healthy donors. The cells were incubated with truncated overlapping derivatives of T-cell epitope peptide. Interferon gamma cytokine produced by the cells was determined from the supernatants by sandwich ELISA. PBMC of patients produce

Mapping the tandem mass spectrometric characteristics of citrulline containing peptides

RATIONALE Protein citrullination (deimination) is a post-translational modification of proteins converting arginine(s) to citrulline(s). “Overcitrullination” could be associated with severe pathological conditions. Mass spectrometric analysis of modified proteins is hindered by several problems. A comprehensive study of fragmentation of deiminated peptides is not yet available. In this paper we have made an attempt to describe the characteristics of these processes, based on the studies of epitope model oligopeptides derived from clinically relevant proteins. METHODS Solution of purified model peptides containing either one or two citrulline residues as well as their native variants were injected directly to the electrospray source of a high accuracy and resolution quadrupole-time of flight instrument and were analysed by tandem mass spectrometry using low-energy collision induced dissociation. RESULTS Loss of isocyanic acid from citrulline residues is a preferred fragmentation route for deiminated peptides, which yields ornithine residues in the sequence. However, simultaneous detection of both the isocyanic acid loss and sequence fragments is often compromised. A preferential cleavage site was observed between citrulline and any other following amino acids yielding intensive complementary b and y type ions. Also, citrulline positioned at the C-termini displays a preferential cleavage N-terminal to this residue yielding characteristic y1 ions. These phenomena are described here for the first time and are referred to as the “citrulline effect”. CONCLUSIONS We found that the citrulline effect is very pronounced and could be used as a complementary tool for the confirmation of modification sites in addition to losses of isocyanic acids from the protonated molecules or from fragment ions. Low collision energy applied to peptide ions having partially mobile protons reveal the site of modification by generating specific and intensive fragments of the sequence. On the other hand, fragmenting parent ions with mobile protons usually allow full sequence coverage, although citrulline-specific fragments may exhibit lower intensities compared to other fragments

Analysis of Linear Antibody Epitopes on Factor H and CFHR1 Using Sera of Patients with Autoimmune Atypical Hemolytic Uremic Syndrome

Introduction: In autoimmune atypical hemolytic uremic syndrome (aHUS), the complement regulator factor H (FH) is blocked by FH autoantibodies, while 90% of the patients carry a homozygous deletion of its homolog complement FH-related protein 1 (CFHR1). The functional consequence of FH-blockade is widely established; however, the molecular basis of autoantibody binding and the role of CFHR1 deficiency in disease pathogenesis are still unknown. We performed epitope mapping of FH to provide structural insight in the autoantibody recruitment on FH and potentially CFHR1. Methods: Eight anti-FH positive aHUS patients were enrolled in this study. With overlapping synthetic FH and CFHR1 peptides, we located the amino acids (aa) involved in binding of acute and convalescence stage autoantibodies. We confirmed the location of the mapped epitopes using recombinant FH domains 19-20 that carried single-aa substitutions at the suspected antibody binding sites in three of our patients. Location of the linear epitopes and the introduced point mutations was visualized using crystal structures of the corresponding domains of FH and CFHR1. Results: We identified three linear epitopes on FH (aa1157-1171; aa1177-1191; and aa1207-1226) and one on CFHR1 (aa276-290) that are recognized both in the acute and convalescence stages of aHUS. We observed a similar extent of autoantibody binding to the aHUS-specific epitope aa1177-1191 on FH and aa276-290 on CFHR1, despite seven of our patients being deficient for CFHR1. Epitope mapping with the domain constructs validated the location of the linear epitopes on FH with a distinct autoantibody binding motif within aa1183-1198 in line with published observations. Summary: According to the results, the linear epitopes we identified are located close to each other on the crystal structure of FH domains 19-20. This tertiary configuration contains the amino acids reported to be involved in C3b and sialic acid binding on the regulator, which may explain the functional deficiency of FH in the presence of auto antibodies. The data we provide identify the exact structures involved in autoantibody recruitment on FH and confirm the presence of an autoantibody binding epitope on CFHR1.Peer reviewe