RESUMO
Background: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.
Assuntos
Equinococose/diagnóstico , Echinococcus granulosus/imunologia , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Lipoproteínas/química , Lipoproteínas/imunologia , Sequência de Aminoácidos , Animais , Equinococose/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas de Helminto/síntese química , Humanos , Lipoproteínas/síntese química , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Anhydrobiotic (i.e., life without water) organisms are known to produce group 3 late embryogenesis abundant (G3LEA) proteins during adaptation to severely water-deficient conditions. Their primary amino acid sequences are composed largely of loosely conserved 11-mer repeat units. However, little information has been obtained for the structural and functional roles of these repeat units. In this study, we first explore the consensus sequences of the 11-mer repeat units for several native G3LEA proteins originating from anhydrobiotic organisms among insects (Polypedilum vanderplanki), nematodes, and plants. Next, we synthesize four kinds of model peptides (LEA models), each of which consists of four or two repeats of the 11-mer consensus sequences for each of the three organisms. The structural and thermodynamic properties of the LEA models were examined in solution, in dehydrated and rehydrated states, and furthermore in the presence of trehalose, since a great quantity of this sugar is known to be produced in the dried cells of most anhydrobiotic organisms. The results of Fourier transform infrared (FTIR) spectroscopic measurements indicate that all of the LEA models transform from random coils to alpha-helical coiled coils on dehydration and return to random coils again on rehydration, both with and without trehalose. In contrast, such structural changes were never observed for a control peptide with a randomized amino acid sequence. Furthermore, our differential scanning calorimetry (DSC) measurements provide the first evidence that the above 11-mer motif-containing peptides themselves vitrify with a high glass transition temperature (>100 degrees C) and a low enthalpy relaxation rate. In addition, they play a role in reinforcing the glassy matrix of the coexisting trehalose. On the basis of these results, we discuss the underlying mechanism of G3LEA proteins as desiccation stress protectants.
Assuntos
Adaptação Fisiológica , Desidratação , Vidro/química , Proteínas de Helminto/síntese química , Proteínas de Insetos/síntese química , Biossíntese Peptídica/fisiologia , Proteínas de Plantas/síntese química , Motivos de Aminoácidos , Animais , Brassica napus/embriologia , Proteínas de Caenorhabditis elegans/síntese química , Sequência Consenso , Dípteros/embriologia , Gossypium/embriologia , Estrutura Secundária de Proteína , Sequências Repetitivas de Aminoácidos , Trealose/síntese química , Tylenchoidea/embriologiaRESUMO
Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14-18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113-122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83.3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis.
Assuntos
Antígenos de Helmintos/sangue , Cisteína Endopeptidases/sangue , Doenças Endêmicas , Proteínas de Helminto/sangue , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/diagnóstico , Esquistossomose mansoni/epidemiologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Cisteína Endopeptidases/síntese química , Cisteína Endopeptidases/imunologia , Proteínas de Helminto/síntese química , Proteínas de Helminto/imunologia , Humanos , Imunoensaio , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Coelhos , Schistosoma mansoni/imunologia , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/sangue , Esquistossomose mansoni/imunologia , Sensibilidade e Especificidade , Especificidade da Espécie , Venezuela/epidemiologiaRESUMO
Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin ß-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of ß-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.
Assuntos
Proliferação de Células/efeitos dos fármacos , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Opisthorchis/química , Cicatrização/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Feminino , Proteínas de Helminto/síntese química , Proteínas de Helminto/uso terapêutico , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/síntese química , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Progranulinas , Alinhamento de SequênciaRESUMO
The assay of choice for serological detection of cysticercosis in humans and pigs is the enzyme-linked immunoelectrotransfer blot (EITB), a Western blot assay that relies on the use of seven lentil-lectin-purified glycoproteins (LLGPs) derived from Taenia solium metacestodes. The EITB is has a sensitivity of 98% and a specificity of 100% in detecting cysticercosis, yet scarcity of native source material and the labor-intensive process of metacestode purification hinder its practicality. These limitations have necessitated the reproduction of the EITB antigens in synthetic forms. Four chemically synthesized LLGP antigens, TS14, TS18var1, TSRS1, and TSRS2var1, were assayed individually by enzyme-linked immunosorbent assay (ELISA) and Western blot for immunoreactivity against a large cohort of sera from clinically defined neurocysticercosis patients. The sensitivity and specificity of all four of these antigens using the ELISA format were well below the standards set by the LLGP EITB, whereas results of the Western blot format closely mirrored those of the LLGP EITB.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos , Proteínas de Helminto/síntese química , Neurocisticercose/diagnóstico , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/imunologia , Western Blotting/métodos , Ensaio de Imunoadsorção Enzimática , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Humanos , Dados de Sequência Molecular , Sensibilidade e Especificidade , Testes SorológicosRESUMO
Cysticercosis caused by Taenia solium is a very common disease in developing countries that seriously affects human health. Diagnosis can only be confirmed with the aid of computerized tomography or nuclear magnetic resonance (NMR) creating obvious difficulties for epidemiological studies. Reliable immunoassays employing cerebrospinal fluid (CSF) have been developed, based on the use of cysticercal antigens. However, the reliance on parasite material is restrictive. Herein, we report the advances in the design of a diagnostic kit based on immunodominant synthetic peptides, targeting four candidate epitopes KETc1, KETc12, 410 and 413 which were identified from three different clones (KETc1, 12 and 4) selected from a cDNA library of Taenia crassiceps. CSF antibodies against T. solium cysticercal antigens (TCA) as well as the four peptides were determined by enzyme-linked immunoabsorbent assays (ELISA) using two panels of CSF from patients with confirmed neurocysticercosis and other neurological diseases. In the first CSF panel which included patients with high level of antibodies against TCA, KETc12 exhibited almost the same sensitivity (87.5%) as TCA (93.7%) and 100% specificity. In the second panel of 110 CSF collected at random, two peptides (KETc1 and KETc12) exhibited sensitivities of 40 and 36% respectively, and were 100% specific.
Assuntos
Anticorpos Anti-Helmínticos/análise , Antígenos de Helmintos/imunologia , Cisticercose/diagnóstico , Cysticercus/imunologia , Ensaio de Imunoadsorção Enzimática , Proteínas de Helminto/imunologia , Neurocisticercose/diagnóstico , Peptídeos/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/líquido cefalorraquidiano , Cisticercose/imunologia , Epitopos de Linfócito B/imunologia , Proteínas de Helminto/síntese química , Proteínas de Helminto/genética , Humanos , Epitopos Imunodominantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neurocisticercose/imunologia , Peptídeos/síntese química , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
We are attempting to design a simpler assay based on synthetic or recombinant antigens to replace the labor-intensive enzyme-linked immunoelectrotransfer blot (EITB-C), which is currently used to diagnose Taenia solium cysticercosis. From the lentil lectin-bound fraction of cyst glycoproteins (the LLGP fraction used in the EITB-C), we previously identified and purified 2 related polypeptides of 14- and 18-kDa that demonstrated diagnostic usefulness. Using degenerate oligonucleotide primers corresponding to amino acid sequences of these polypeptides and a cDNA library prepared from T. solium cysticerci, we amplified cDNA clones that represent the 14- and 18-kDa polypeptides. These clones share sequence homology at the nucleotide and amino acid levels. Synthetic polypeptides that represented the full-length, mature proteins (sTS14 and sTS18) were assessed for serologic potential using an ELISA. sTS14, but not sTS18, demonstrated utility as a diagnostic antigen. sTS14 was recognized by antibodies in a majority of the sera from patients with cysticercosis and none of the sera from persons with other helminth infections or uninfected human sera. Furthermore, polyclonal antibodies to sTS14 reacted with 6 discrete proteins present in the LLGP cyst fraction, suggesting that TS14 is a subunit of other previously described antigens used for diagnosing cysticercosis.
Assuntos
Antígenos de Helmintos , Clonagem Molecular , Cisticercose/diagnóstico , Cysticercus/genética , Cysticercus/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Sequência de Bases , Cisticercose/parasitologia , Cysticercus/crescimento & desenvolvimento , Proteínas de Helminto/síntese química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/genética , Peptídeos/imunologia , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNARESUMO
Sheep fasciolosis is a devastating burden for the livestock industry. We herein report on immunodiagnosis of fasciolosis, and significant protection of sheep against challenge infection with Fasciola gigantica following immunization with a peptide based on the H-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH (Fas14p) sequence of F. gigantica cathepsin L-cysteine proteinase. This sequence was synthesized in three different forms: as N(alpha) acetylated (Ac-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAc14p), bearing at the amino-terminus an N(alpha) acetylated cystein (Ac-Cys-Asp(110)-Lys-Ile-Asp-Trp-Arg-Glu-Ser-Gly-Tyr-Val-Thr-Glu-Val(123)-OH, FasAcCys14p), and conjugated to sequential oligopeptide carrier Ac-[Lys-Aib-Gly](4)-OH (Ac-SOC(4)) through an amide bond formed between Val(123) carboxylic group of the epitope and the lysine N(epsilon) groups of the carrier (Ac-[Lys(Fas14p)-Aib-Gly](4)-OH). Ac-[Lys(Fas14p)-Aib-Gly](4)-OH failed to readily discriminate between naïve and infected sheep. In contrast, the free peptides reproducibly differentiated between parasite-free sheep, sheep infected with parasites other than Fasciola, and experimentally Fasciola-infected sheep. The data together indicated that the peptides might be of considerable use for discriminating between early and late, and low and high burden, sheep infection with F. gigantica. FasAc14p was chosen to determine whether a peptide based on a critical enzymatic site of cathepsin L proteinase may induce protection against challenge infection. Sheep immunization with FasAc14p peptide induced significant expression of interleukin-4 mRNA, and humoral antibodies that bound to molecule(s) on the intact surface membrane of newly excysted juvenile worms, and mediated their attrition. The immune responses were associated with significant (P < 0.02) decrease of 23.1% in worm recovery, but with no decrease in the size or maturation of worms recovered.
Assuntos
Catepsinas/síntese química , Catepsinas/imunologia , Cisteína Endopeptidases/síntese química , Cisteína Endopeptidases/imunologia , Fasciolíase/prevenção & controle , Proteínas de Helminto/síntese química , Proteínas de Helminto/imunologia , Testes Imunológicos , Peptídeos/síntese química , Sequência de Aminoácidos , Animais , Catepsinas/química , Cromatografia Líquida de Alta Pressão , Cisteína Endopeptidases/química , Fasciolíase/veterinária , Proteínas de Helminto/química , Testes Imunológicos/veterinária , Peptídeos/química , Peptídeos/isolamento & purificação , Ovinos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The chemical synthesis of peptides may simplify the production of molecules for diagnosis of Schistosoma mansoni. Seventeen polymeric, 20-amino acids long, peptides comprising the entire Sm31 molecule of the adult worm, were synthesized under the t-boc strategy and their immunogenicity was evaluated. Of these, 10 peptides were immunogenic in rabbits. The peptides containing the sequence Gly74-Ser93 (peptide IMT-172) and the sequence Val154-Ala173 (peptide IMT-180) were responsible for the recognition of the Sm31 molecule by Western blot. This was confirmed by the specific inhibition of recognition of each molecule with the homologous peptide. Additionally, antibodies against these peptides strongly fixed to the adult worm gut. The present results, together with the strong immunogenicity shown for the adult worm 31 kDa antigen, establish the basis for the development of an immunodiagnostic method using synthetic peptides.