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1.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639235

RESUMO

Nanomaterials have found extensive interest in the development of novel vaccines, as adjuvants and/or carriers in vaccination platforms. Conjugation of protein antigens at the particle surface by non-covalent adsorption is the most widely used approach in licensed particulate vaccines. Hence, it is essential to understand proteins' structural integrity at the material interface in order to develop safe-by-design nanovaccines. In this study, we utilized two model proteins, the wild-type allergen Bet v 1 and its hypoallergenic fold variant (BM4), to compare SiO2 nanoparticles with Alhydrogel® as particulate systems. A set of biophysical and functional assays including circular dichroism spectroscopy and proteolytic degradation was used to examine the antigens' structural integrity at the material interface. Conjugation of both biomolecules to the particulate systems decreased their proteolytic stability. However, we observed qualitative and quantitative differences in antigen processing concomitant with differences in their fold stability. These changes further led to an alteration in IgE epitope recognition. Here, we propose a toolbox of biophysical and functional in vitro assays for the suitability assessment of nanomaterials in the early stages of vaccine development. These tools will aid in safe-by-design innovations and allow fine-tuning the properties of nanoparticle candidates to shape a specific immune response.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Epitopos/imunologia , Ativação Linfocitária/imunologia , Nanopartículas/química , Dióxido de Silício/química , Vacinas/imunologia , Alérgenos/química , Humanos , Hidrogéis , Imunoglobulina E/imunologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T/imunologia
2.
ACS Catal ; 11(19): 11885-11896, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34621593

RESUMO

Protein modification by enzymatic breaking and forming of peptide bonds significantly expands the repertoire of genetically encoded protein sequences. The dual protease-ligase legumain exerts the two opposing activities within a single protein scaffold. Primarily localized to the endolysosomal system, legumain represents a key enzyme in the generation of antigenic peptides for subsequent presentation on the MHCII complex. Here we show that human legumain catalyzes the ligation and cyclization of linear peptides at near-neutral pH conditions, where legumain is intrinsically unstable. Conformational stabilization significantly enhanced legumain's ligase activity, which further benefited from engineering the prime substrate recognition sites for improved affinity. Additionally, we provide evidence that specific legumain activation states allow for differential regulation of its activities. Together these results set the basis for engineering legumain proteases and ligases with applications in biotechnology and drug development.

3.
World Allergy Organ J ; 14(3): 100516, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33717396

RESUMO

Background: Skin prick test (SPT) solutions and allergy vaccines (AVs) are crucial tools for diagnosis and therapy of allergies. It was the aim of this study to corroborate the content of products for diagnosis and treatment of dust mite allergies that are produced and sold in India. Methods: SDS-PAGE, immunoblots and high-resolution mass spectrometric analysis was performed with 16 house dust mite (HDM) SPT solutions and AVs from 3 Indian manufacturers. Authority-approved European SPT solutions and in-house extracts were used as references. Results: From the 5 Indian Dermatophagoides pteronyssinus products, none contained proteins from this source. Instead, 1 sample contained Dermatophagoides farinae and human serum proteins, 4 products contained allergens from the storage mite Suidasia medanensis, allergens from the legume Cicer arietinum (chickpea), and proteins from baker's yeast. From 4 Indian D. farinae-labeled products, 2 contained human serum proteins and a limited number of D. farinae allergens. Two contained only Suidasia, Cicer, and yeast proteins. In contrast, the European authority-approved D. pteronyssinus and D. farinae SPT solutions that were used as reference in this study, contained exclusively proteins of the respective species and covered the expected allergen spectra. The Blomia tropicalis sample contained no Blomia allergens at all, but consisted exclusively of Suidasia, Cicer, and yeast proteins. All 6 HDM samples consisted of human serum proteins and limited amounts of D. farinae allergens. Conclusions: All commercial Indian SPT solutions and AVs analyzed in this study are not suitable for dust mite allergy diagnosis and therapy, as they contain either no, or only a limited number of, HDM allergens. In addition, their use could lead to misdiagnosis since some of them contain allergens from other sources, including the storage mite Suidasia, chickpea, as well as baker's yeast. Further, their application might be harmful to patients, as some products contain large amounts of proteins of human origin. Analysis of European SPT solutions, on the other hand, confirmed their suitability for dust mite allergy diagnosis.

5.
Int J Mol Sci ; 21(24)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371317

RESUMO

To analyze the impact of Ascaris lumbricoides infection on the pathogenesis and diagnosis of allergic diseases, new allergens should be identified. We report the identification of a new Ascaris lumbricoides allergen, Asc l 5. The aim of this study was to evaluate the physicochemical and immunological features of the Asc l 5 allergen. We constructed an A. lumbricoides cDNA library and Asc l 5 was identified by immunoscreening. After purification, rAsc l 5 was physicochemically characterized. Evaluation of its allergenic activity included determination of Immunoglobulin E (IgE) binding frequency (in two populations: 254 children and 298 all-age subjects), CD203c based-basophil activation tests (BAT) and a passive cutaneous anaphylaxis (PCA) mouse model. We found by amino acid sequence analysis that Asc l 5 belongs to the SXP/RAL-2 protein family of nematodes. rAsc l 5 is a monomeric protein with an alpha-helical folding. IgE sensitization to rAsc l 5 was around 52% in general population; positive BAT rate was 60%. rAsc l 5 induced specific IgE production in mice and a positive PCA reaction. These results show that Asc l 5 has structural and immunological characteristics to be considered as a new allergen from A. lumbricoides.


Assuntos
Alérgenos/imunologia , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Ascaris lumbricoides/imunologia , Asma/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Asma/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Imunoglobulina E/sangue , Lactente , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
6.
Int J Biol Macromol ; 164: 1545-1553, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32735921

RESUMO

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately ß-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 µM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.


Assuntos
Venenos de Crotalídeos/metabolismo , Crotalus/metabolismo , Inibidores de Fosfolipase A2/metabolismo , Fosfolipases A2/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Estrutura Secundária de Proteína , Proteômica/métodos
7.
Gut Microbes ; 12(1): 1770017, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-32584649

RESUMO

Mechanisms of host-pathogen interactions resulting in immunopathological responses upon human Campylobacter jejuni infection are not completely understood, but the recent availability of murine infection models mimicking key features of campylobacteriosis helps solving this dilemma. During a screen for proteases expressed by C. jejuni, we identified a peptidase of the M24 family as a potential novel virulence factor, which was named PepP. The gene is strongly conserved in various Campylobacter species. A constructed deletion mutant ΔpepP of C. jejuni strain 81-176 grew as efficiently compared to isogenic wild-type (WT) or pepP complemented bacteria. To shed light on the potential role of this protease in mediating immunopathological responses in the mammalian host, we perorally challenged microbiota-depleted IL-10-/- mice with these strains. All strains stably colonized the murine gastrointestinal tract with comparably high loads. Remarkably, pepP deficiency was associated with less severe induced malaise, with less distinct apoptotic and innate immune cell responses, but also with more pronounced proliferative/regenerative epithelial cell responses in the large intestine at d6post-infection. Furthermore, pro-inflammatory mediators were lower in the colon, ileum, and mesenteric lymph nodes of mice that had been challenged with the ΔpepP mutant compared to the WT or pepP complemented strains. This also held true for extra-intestinal organs including liver, kidneys, and lungs, and, strikingly, to systemic compartments. Taken together, protease PepP is a novel virulence determinant involved in mediating campylobacteriosis. The finding that apoptosis in the colon is significantly diminished in mice infected with the pepP mutant highlights the epithelial layer as the first and main target of PepP in the intestine.


Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter jejuni , Microbioma Gastrointestinal/fisiologia , Serina Endopeptidases/genética , Animais , Apoptose/fisiologia , Campylobacter jejuni/genética , Campylobacter jejuni/imunologia , Campylobacter jejuni/patogenicidade , Feminino , Doenças Transmitidas por Alimentos/microbiologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Serina Endopeptidases/metabolismo , Fatores de Virulência/genética
8.
Allergy ; 75(11): 2909-2919, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32436591

RESUMO

BACKGROUND: Tropomyosins are highly conserved proteins, an attribute that forms the molecular basis for their IgE antibody cross-reactivity. Despite sequence similarities, their allergenicity varies greatly between ingested and inhaled invertebrate sources. In this study, we investigated the relationship between the structural stability of different tropomyosins, their endolysosomal degradation patterns, and T-cell reactivity. METHODS: We investigated the differences between four tropomyosins-the major shrimp allergen Pen m 1 and the minor allergens Der p 10 (dust mite), Bla g 7 (cockroach), and Ani s 3 (fish parasite)-in terms of IgE binding, structural stability, endolysosomal degradation and subsequent peptide generation, and T-cell cross-reactivity in a BALB/c murine model. RESULTS: Tropomyosins displayed different melting temperatures, which did not correlate with amino acid sequence similarities. Endolysosomal degradation experiments demonstrated differential proteolytic digestion, as a function of thermal stability, generating different peptide repertoires. Pen m 1 (Tm 42°C) and Der p 10 (Tm 44°C) elicited similar patterns of endolysosomal degradation, but not Bla g 7 (Tm 63°C) or Ani s 3 (Tm 33°C). Pen m 1-specific T-cell clones, with specificity for regions highly conserved in all four tropomyosins, proliferated weakly to Der p 10, but did not proliferate to Bla g 7 and Ani s 3, indicating lack of T-cell epitope cross-reactivity. CONCLUSIONS: Tropomyosin T-cell cross-reactivity, unlike IgE cross-reactivity, is dependent on structural stability rather than amino acid sequence similarity. These findings contribute to our understanding of cross-sensitization among different invertebrates and design of suitable T-cell peptide-based immunotherapies for shrimp and related allergies.


Assuntos
Alérgenos , Tropomiosina , Animais , Reações Cruzadas , Imunoglobulina E , Camundongos , Linfócitos T
9.
Clin Exp Allergy ; 50(7): 835-847, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32314444

RESUMO

INTRODUCTION: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens. AIM: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies caused by B tropicalis. METHODS: Using in silico design of B tropicalis hybrid proteins, we chose two hybrid proteins for heterologous expression. Wild-type Blo t 5/Blo t 21 hybrid molecule and a hypoallergenic version, termed BTH1 and BTH2, respectively, were purified by ion exchange and size exclusion chromatography and characterized by physicochemical, as well as in vitro and in vivo immunological, experiments. RESULTS: BTH1, BTH2 and the parental allergens were purified to homogeneity and characterized in detail. BTH2 displayed the lowest IgE reactivity that induced basophil degranulation using sera from allergic rhinitis and asthmatic patients. BTH2 essentially presented the same endolysosomal degradation pattern as the shortened rBlo t 5 and showed a higher resistance towards degradation than the full-length Blo t 5. In vivo immunization of mice with BTH2 led to the production of IgG antibodies that competed with human IgE for allergen binding. Stimulation of splenocytes from BTH2-immunized mice produced higher levels of IL-10 and decreased secretion of IL-4 and IL-5. In addition, BTH2 stimulated T-cell proliferation in PBMCs isolated from allergic patients, with secretion of higher levels of IL-10 and lower levels of IL-5 and IL-13, when compared to parental allergens. CONCLUSIONS AND CLINICAL RELEVANCE: BTH2 is a promising hybrid vaccine candidate for immunotherapy of Blomia allergy. However, further pre-clinical studies addressing its efficacy and safety are needed.


Assuntos
Alérgenos , Proteínas de Artrópodes , Hipersensibilidade , Ácaros , Vacinas , Alérgenos/genética , Alérgenos/imunologia , Alérgenos/farmacologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/farmacologia , Citocinas , Feminino , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Masculino , Camundongos Endogâmicos BALB C , Ácaros/genética , Ácaros/imunologia , Vacinas/genética , Vacinas/imunologia , Vacinas/farmacologia
11.
Clin Exp Allergy ; 50(3): 401-405, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31880850

RESUMO

BACKGROUND: Small, basic peanut proteins are often poorly extracted in pH-neutral buffers that are optimal for the extraction of peanut storage proteins such as Ara h 1. As a result, such proteins are easily missed as potential allergens. OBJECTIVE: To analyse the allergenic composition of the basic peanut protein (BPP) fraction. METHODS: A peanut extract prepared at pH 4 was fractionated by physicochemical procedures. Chemical analysis was performed by SDS-PAGE and mass spectrometry. Because immunoblotting was found to be inefficient for most of these small basic proteins, IgE-binding activity was measured by coupling the fractions to CNBr-activated Sepharose, followed by incubation with sera from 55 Dutch peanut-allergic children and 125 I-labelled anti-IgE. RESULTS: Most IgE reactivity of the BPP fraction was due to the 5-7 kDa amino-terminal fragment of Ara h 1. This finding was confirmed by the use of the fragment in recombinant form, to which 25/55 of the sera was IgE-positive. CONCLUSION: The amino-terminal fragment of Ara h 1, a member of a family of small anti-microbial proteins, is an allergen independent of the carboxy-terminal fragment of Ara h 1.


Assuntos
Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Imunoglobulina E/imunologia , Proteínas de Membrana/imunologia , Proteínas de Plantas/imunologia , Proteínas Citotóxicas Formadoras de Poros/imunologia , Antígenos de Plantas/genética , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Plantas/genética , Proteínas Citotóxicas Formadoras de Poros/genética
12.
Geroscience ; 42(1): 19-38, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31676965

RESUMO

Originally Lipid droplets (LDs) were considered as being droplets for lipid storage only. Increasing evidence, however, demonstrates that LDs fulfill a pleiotropy of additional functions. Among them is the modulation of protein as well as lipid homeostasis. Under unfavorable pro-oxidative conditions, proteins can form aggregates which may exceed the overall proteolytic capacity of the proteasome. After stress termination LDs can adjust and support the removal of these aggregates. Additionally, LDs interact with mitochondria, specifically take over certain proteins and thus prevent apoptosis. LDs, which are loaded with these harmful proteins, are subsequently eliminated via lipophagy. Recently it was demonstrated that this autophagic process is a modulator of longevity. LDs do not only eliminate potentially dangerous proteins, but they are also able to prevent lipotoxicity by storing specific lipids. In the present study we used the model organism Saccharomyces cerevisiae to compare the proteome as well as lipidome of mitochondria and LDs under different conditions: replicative aging, stress and apoptosis. In this context we found an accumulation of proteins at LDs, supporting the role of LDs in proteostasis. Additionally, the composition of main lipid classes such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols, phosphatidylglycerols, triacylglycerols, ceramides, phosphatidic acids and ergosterol of LDs and mitochondria changed during stress conditions and aging.


Assuntos
Gotículas Lipídicas , Saccharomyces cerevisiae , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Mitocôndrias/metabolismo , Proteostase
13.
Int J Biol Macromol, v. 164, p. 1545-1553, dez. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3419

RESUMO

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately β-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 μM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.

14.
Int Arch Allergy Immunol ; 180(3): 159-172, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31563904

RESUMO

BACKGROUND: The dawn of the "omics" technologies has changed allergy research, increasing the knowledge and identification of new allergens. However, these studies have been almost restricted to Dermatophagoides spp. Although Blomia tropicalis has long been established as a clinically important source of allergens, a thorough proteomic characterization is still lacking for this dust mite. OBJECTIVE: To increase knowledge of B. tropicalis allergens through proteomic analysis. METHODS: Eleven in-bred lineages of B. tropicalis were obtained from 11 unique different pregnant females. Their somatic extracts were analyzed and compared with a commercially available extract by liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Considerable differences in the protein expression profiles were found among the breeds, and most of them displayed higher expression levels of major allergens than the commercially available extract. Blo t 2 was the most prominent allergenic protein in the analyzed extracts. Six identified allergens and 14 isoforms have not yet been recognized by IUIS. Conversely, 3 previously recognized B. tropicalis allergens were not found. CONCLUSIONS: The clear impact of inbreeding on allergen content shown by our study leads us to conclude that the quantification and/or identification of allergens from in-bred lines should be routinely considered for mite cultivation in order to select breeds with higher amounts of major allergens. In this sense, LC-MS/MS may be a useful method to achieve this quality control for research and commercial purposes.


Assuntos
Alérgenos/imunologia , Extratos Celulares/imunologia , Hipersensibilidade/imunologia , Feromônios/imunologia , Sarcoptidae/imunologia , Alérgenos/isolamento & purificação , Animais , Animais Endogâmicos , Variação Biológica da População , Extratos Celulares/química , Cromatografia Líquida , Feminino , Humanos , Feromônios/isolamento & purificação , Gravidez , Especificidade da Espécie , Espectrometria de Massas em Tandem , Transcriptoma
15.
Medicina (Kaunas) ; 55(8)2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434264

RESUMO

Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin-proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.


Assuntos
Alérgenos/imunologia , Artemisia/imunologia , Proteínas de Plantas/imunologia , Defensinas/análise , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Imunoglobulina E , Extratos Vegetais/imunologia , Prolina/análise
16.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 6): 419-427, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31204688

RESUMO

Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of this parasite. Here, the expression, purification and crystallization of this enzyme are reported. The cruzain crystals diffracted to 1.2 Šresolution, yielding two novel cruzain structures: apocruzain and cruzain bound to the reversible covalent inhibitor S-methyl thiomethanesulfonate. Mass-spectrometric experiments confirmed the presence of a methylthiol group attached to the catalytic cysteine. Comparison of these structures with previously published structures indicates the rigidity of the cruzain structure. These results provide further structural information about the enzyme and may help in new in silico studies to identify or optimize novel prototypes of cruzain inhibitors.


Assuntos
Apoproteínas/química , Apoproteínas/metabolismo , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Desenho de Fármacos , Metanossulfonato de Metila/análogos & derivados , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/química , Metanossulfonato de Metila/química , Metanossulfonato de Metila/metabolismo , Modelos Moleculares , Conformação Proteica
17.
Allergy ; 74(12): 2382-2393, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31230350

RESUMO

BACKGROUND: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. METHODS: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs. RESULTS: We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. CONCLUSION: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Endossomos/enzimologia , Peptídeo Hidrolases/metabolismo , Basófilos/imunologia , Basófilos/metabolismo , Betula/imunologia , Degranulação Celular/imunologia , Ativação Enzimática , Humanos , Imunoglobulina E/imunologia , Ligantes , Pólen/imunologia , Ligação Proteica , Proteínas Recombinantes
18.
Mol Nutr Food Res ; 63(18): e1900336, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31207117

RESUMO

SCOPE: Allergies to lipid transfer proteins involve severe adverse reactions; thus, effective and sustainable therapies are desired. Previous attempts disrupting disulfide bonds failed to maintain immunogenicity; thus, the aim is to design novel hypoallergenic Pru p 3 variants and evaluate the applicability for treatment of peach allergy. METHODS AND RESULTS: Pru p 3 proline variant (PV) designed using in silico mutagenesis, cysteine variant (CV), and wild-type Pru p 3 (WT) are purified from Escherichia coli. Variants display homogenous and stable protein conformations with an altered secondary structure in circular dichroism. PV shows enhanced long-term storage capacities compared to CV similar to the highly stable WT. Using sera of 33 peach allergic patients, IgE-binding activity is reduced by 97% (PV) and 71% (CV) compared to WT. Both molecules show strong hypoallergenicity in Pru p 3 ImmunoCAP cross-inhibition and histamine release assays. Immunogenicity of PV is demonstrated with a phosphate-based adjuvant formulation in a mouse model. CONCLUSIONS: An in silico approach is used to generate a PV without targeting disulfide bonds, T cell epitopes, or previously reported IgE epitopes of Pru p 3. PV is strongly hypoallergenic while structurally stable and immunogenic, thus representing a promising candidate for peach allergen immunotherapy.


Assuntos
Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas Recombinantes/imunologia , Adolescente , Adulto , Animais , Antígenos de Plantas/genética , Criança , Modelos Animais de Doenças , Feminino , Humanos , Imunização , Imunoglobulina E/sangue , Imunoglobulina E/metabolismo , Camundongos Endogâmicos BALB C , Proteínas de Plantas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relação Estrutura-Atividade , Adulto Jovem
19.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096561

RESUMO

Pollinosis is sub-diagnosed and rarely studied in tropical countries. Cashew tree pollen has been reported as an allergen source although the knowledge of its immunoglobulin E (IgE)-reactive molecules is lacking. Therefore, this work aimed to identify IgE-reactive molecules and provide a proteomic profile of this pollen. From the 830 proteins identified by shotgun analysis, 163 were annotated to gene ontology, and a list of 39 proteins filtered for high confidence was submitted to the Allfam database where nine were assigned to allergenic families. Thus, 12 patients from the northeast of Brazil with persistent allergic rhinitis and aggravation of symptoms during cashew flowering season were selected. Using a 2D-based approach, we identified 20 IgE-reactive proteins, four already recognized as allergens, including a homolog of the birch isoflavone-reductase (Bet v 6). IgE-reactivity against the extract in native form was confirmed for five patients in ELISA, with three being positive for Bet v 6. Herein, we present a group of patients with rhinitis exposed to cashew tree pollen with the first description of IgE-binding proteins and a proteomic profile of the whole pollen. Cashew tree pollen is considered an important trigger of rhinitis symptoms in clinical practice in the northeast of Brazil, and the elucidation of its allergenic molecules can improve the diagnostics and treatment for allergic patients.


Assuntos
Alérgenos/imunologia , Anacardium/química , Imunoglobulina E/imunologia , Pólen/efeitos adversos , Pólen/química , Rinite Alérgica Sazonal/induzido quimicamente , Adolescente , Adulto , Idoso , Alérgenos/química , Animais , Antígenos de Plantas/efeitos adversos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Betula/metabolismo , Brasil , Proteínas de Transporte/análise , Proteínas de Transporte/imunologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Dermatophagoides farinae , Dermatophagoides pteronyssinus , Feminino , Humanos , Masculino , Proteínas de Plantas/efeitos adversos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Pólen/genética , Proteômica , Rinite Alérgica Sazonal/imunologia , Testes Cutâneos
20.
Int J Mol Sci ; 20(8)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-31013713

RESUMO

Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the ß-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.


Assuntos
Leishmania infantum/genética , Leishmania infantum/metabolismo , Leishmaniose Visceral/parasitologia , Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Técnicas de Visualização da Superfície Celular , Citocinas/metabolismo , Humanos , Leishmania infantum/efeitos dos fármacos , Leishmania infantum/imunologia , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/tratamento farmacológico , Modelos Moleculares , Conformação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Nanomedicina Teranóstica , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologia
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