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1.
Artigo em Inglês | MEDLINE | ID: mdl-34427974

RESUMO

Within the last decade, the application range of aptamers in biochemistry and medicine has expanded rapidly. More than just a replacement for antibodies, these intrinsically structured RNA- or DNA-oligonucleotides show great potential for utilization in diagnostics, specific drug delivery, and treatment of certain medical conditions. However, what is analyzed less frequently is the process of aptamer identification known as systematic evolution of ligands by exponential enrichment (SELEX) and the functional mechanisms that lie at its core. SELEX involves numerous singular processes, each of which contributes to the success or failure of aptamer generation. In this review, critical steps during aptamer selection are discussed in-depth, and specific problems are presented along with potential solutions. The discussed aspects include the size and molecule type of the selected target, the nature and stringency of the selection process, the amplification step with its possible PCR bias, the efficient regeneration of RNA or single-stranded DNA, and the different sequencing procedures and screening assays currently available. Finally, useful quality control steps and their role within SELEX are presented. By understanding the mechanisms through which aptamer selection is influenced, the design of more efficient SELEX procedures leading to a higher success rate in aptamer identification is enabled.

2.
Clin Transl Allergy ; 11(1): e12004, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33900048

RESUMO

AIM: Molecular sensitization profile analyses of allergic individuals to the house dust mites (HDM) Blomia tropicalis and Dermatophagoides pteronyssinus from Brazil and Austria, in the attempt to comprehend the individual contribution of the molecular components in the diagnosis of HDM allergy. METHODOLOGY: These analyses were made using a new in vitro multiplex allergen assay which allows simultaneous measurement of specific IgE against the whole allergen extract as well its components. RESULTS AND CONCLUSION: The data showed that in Brazil the inclusion of the molecular components Blo t 5 and/or Blo t 21 major allergens and Blo t 2 can increase the sensitivity and specificity of the assay for the diagnosis of allergy to B. tropicalis, using matrix-based methodologies. Also we highlighted, for the first time, the importance of Blo t 2 analysis for a sensitive diagnosis, since some individuals were sensitized only to this molecular component. Regarding the sensitization profile of individuals sensitized to D. pteronyssinus, we point out the importance of analyzing the molecular components Der p23 and Der p 7, in addition to Der p 1 and Der p 2 for an accurate diagnosis based on matrices.

3.
Allergy ; 76(8): 2383-2394, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33655520

RESUMO

Until recently, glycan epitopes have not been documented by the WHO/IUIS Allergen Nomenclature Sub-Committee. This was in part due to scarce or incomplete information on these oligosaccharides, but also due to the widely held opinion that IgE to these epitopes had little or no relevance to allergic symptoms. Most IgE-binding glycans recognized up to 2008 were considered to be "classical" cross-reactive carbohydrate determinants (CCD) that occur in insects, some helminths and throughout the plant kingdom. Since 2008, the prevailing opinion on lack of clinical relevance of IgE-binding glycans has been subject to a reevaluation. This was because IgE specific for the mammalian disaccharide galactose-alpha-1,3-galactose (alpha-gal) was identified as a cause of delayed anaphylaxis to mammalian meat in the United States, an observation that has been confirmed by allergists in many parts of the world. Several experimental studies have shown that oligosaccharides with one or more terminal alpha-gal epitopes can be attached as a hapten to many different mammalian proteins or lipids. The classical CCDs also behave like haptens since they can be expressed on proteins from multiple species. This is the explanation for extensive in vitro cross-reactivity related to CCDs. Because of these developments, the Allergen Nomenclature Sub-Committee recently decided to include glycans as potentially allergenic epitopes in an adjunct section of its website (www.allergen.org). In this article, the features of the main glycan groups known to be involved in IgE recognition are revisited, and their characteristic structural, functional, and clinical features are discussed.


Assuntos
Alérgenos , Imunoglobulina E , Animais , Carboidratos , Reações Cruzadas , Epitopos , Humanos
4.
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.

6.
Allergy ; 76(1): 210-222, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32621318

RESUMO

BACKGROUND: Allergen-specific immunotherapy via the skin targets a tissue rich in antigen-presenting cells, but can be associated with local and systemic side effects. Allergen-polysaccharide neoglycogonjugates increase immunization efficacy by targeting and activating dendritic cells via C-type lectin receptors and reduce side effects. OBJECTIVE: We investigated the immunogenicity, allergenicity, and therapeutic efficacy of laminarin-ovalbumin neoglycoconjugates (LamOVA). METHODS: The biological activity of LamOVA was characterized in vitro using bone marrow-derived dendritic cells. Immunogenicity and therapeutic efficacy were analyzed in BALB/c mice. Epicutaneous immunotherapy (EPIT) was performed using fractional infrared laser ablation to generate micropores in the skin, and the effects of LamOVA on blocking IgG, IgE, cellular composition of BAL, lung, and spleen, lung function, and T-cell polarization were assessed. RESULTS: Conjugation of laminarin to ovalbumin reduced its IgE binding capacity fivefold and increased its immunogenicity threefold in terms of IgG generation. EPIT with LamOVA induced significantly higher IgG levels than OVA, matching the levels induced by s.c. injection of OVA/alum (SCIT). EPIT was equally effective as SCIT in terms of blocking IgG induction and suppression of lung inflammation and airway hyperresponsiveness, but SCIT was associated with higher levels of therapy-induced IgE and TH2 cytokines. EPIT with LamOVA induced significantly lower local skin reactions during therapy compared to unconjugated OVA. CONCLUSION: Conjugation of ovalbumin to laminarin increased its immunogenicity while at the same time reducing local side effects. LamOVA EPIT via laser-generated micropores is safe and equally effective compared to SCIT with alum, without the need for adjuvant.


Assuntos
Asma , Pneumonia , beta-Glucanas , Alérgenos , Animais , Asma/terapia , Lasers , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina
7.
Clin Transl Allergy ; 10(1): 50, 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33292509

RESUMO

BACKGROUND: Artemisia weed pollen allergy is important in the northern hemisphere. While over 350 species of this genus have been recorded, there has been no full investigation into whether different species may affect the allergen diagnosis and treatment. This study aimed to evaluate the variations in amino acid sequences and the content of major allergens, and how these affect specific IgE binding capacity in representative Artemisia species. METHODS: Six representative Artemisia species from China and Artemisia vulgaris from Europe were used to determine allergen amino acid sequences by transcriptome, gene sequencing and mass spectrometry of the purified allergen component proteins. Sandwich ELISAs were developed and applied for Art v 1, Art v 2 and Art v 3 allergen quantification in different species. Aqueous pollen extracts and purified allergen components were used to assess IgE binding by ELISA and ImmunoCAP with mugwort allergic patient serum pools and individual sera from five areas in China. RESULTS: The Art v 1 and Art v 2 homologous allergen sequences in the seven Artemisia species were highly conserved. Art v 3 type allergens in A. annua and A. sieversiana were more divergent compared to A. argyi and A. vulgaris. The allergen content of Art v 1 group in the seven extracts ranged from 3.4% to 7.1%, that of Art v 2 from 1.0% to 3.6%, and Art v 3 from 0.3% to 10.5%. The highest IgE binding potency for most Chinese Artemisia allergy patients was with A. annua pollen extract, followed by A. vulgaris and A. argyi, with A. sieversiana significantly lower. Natural Art v 1-3 isoallergens from different species have almost equivalent IgE binding capacity in Artemisia allergic patients from China. CONCLUSION AND CLINICAL RELEVANCE: There was high sequence similarity but different content of the three group allergens from different Artemisia species. Choice of Artemisia annua and A. argyi pollen source for diagnosis and immunotherapy is recommended in China.

8.
PLoS One ; 15(11): e0241560, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33151990

RESUMO

The monoclonal anti-CD20 IgG1 antibody rituximab is used as a first-line treatment for B cell lymphoma. Like all therapeutic antibodies, it is a complex protein for which both safety and efficacy heavily depend on the integrity of its three-dimensional structure. Aptamers, short oligonucleotides with a distinct fold, can be used to detect minor modifications or structural variations of a molecule or protein. To detect antibody molecules in a fold state occurring prior to protein precipitation, we generated DNA aptamers that were selected for extensively heat-treated rituximab. Using the magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX), we obtained six DNA aptamer sequences (40-mers) specific for 80°C heat-treated rituximab. In silico fold prediction and circular dichroism analysis revealed a G-quadruplex structure for one aptamer, while all others exhibited a B-DNA helix. Binding affinities ranging from 8.8-86.7 nM were determined by an enzyme-linked apta-sorbent assay (ELASA). Aptamers additionally detected structural changes in rituximab treated for 5 min at 70°C, although with lower binding activity. Notably, none of the aptamers recognized rituximab in its native state nor did they detect the antibody after it was exposed to lower temperatures or different physical stressors. Aptamers also reacted with the therapeutic antibody adalimumab incubated at 80°C suggesting similar aptamer binding motifs located on extensively heat-treated IgG1 antibodies. Within this work, we obtained the first aptamer panel, which is specific for an antibody fold state specifically present prior to protein aggregation. This study demonstrates the potential of aptamer selection for specific stress-based protein variants, which has potential impact for quality control of biopharmaceuticals.


Assuntos
Anticorpos/imunologia , Aptâmeros de Nucleotídeos/metabolismo , Temperatura Alta , Rituximab/farmacologia , Aptâmeros de Nucleotídeos/química , Dicroísmo Circular , Simulação por Computador , Humanos , Conformação de Ácido Nucleico , Técnica de Seleção de Aptâmeros
9.
J Biol Chem ; 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037072

RESUMO

Identification of antibody binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome that is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort is frequently involved in this cross-reactivity, but no antibody binding epitopes have been determined so far.  To reveal human IgE binding regions of Art v 3, we produced three murine high-affinity monoclonal antibodies (mAbs), which showed 70-90% coverage of the allergenic epitopes from mugwort pollen allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4, as well as a novel Art v 3-specific epitope at the C-terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP-allergy and facilitates design of therapeutics.

10.
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
11.
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
12.
Int J Mol Sci ; 21(7)2020 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-32260156

RESUMO

Plant cell wall proteins play major roles during plant development and in response to environmental cues. A bioinformatic search for functional domains has allowed identifying the PAC domain (Proline-rich, Arabinogalactan proteins, conserved Cysteines) in several proteins (PDPs) identified in cell wall proteomes. This domain is assumed to interact with pectic polysaccharides and O-glycans and to contribute to non-covalent molecular scaffolds facilitating the remodeling of polysaccharidic networks during rapid cell expansion. In this work, the characteristics of the PAC domain are described in detail, including six conserved Cys residues, their spacing, and the predicted secondary structures. Modeling has been performed based on the crystal structure of a Plantago lanceolata PAC domain. The presence of ß-sheets is assumed to ensure the correct folding of the PAC domain as a ß-barrel with loop regions. We show that PDPs are present in early divergent organisms from the green lineage and in all land plants. PAC domains are associated with other types of domains: Histidine-rich, extensin, Proline-rich, or yet uncharacterized. The earliest divergent organisms having PDPs are Bryophytes. Like the complexity of the cell walls, the number and complexity of PDPs steadily increase during the evolution of the green lineage. The association of PAC domains with other domains suggests a neo-functionalization and different types of interactions with cell wall polymers.


Assuntos
Parede Celular/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Biologia Computacional/métodos , Sequência Conservada , Cisteína/metabolismo , Bases de Dados de Proteínas , Evolução Molecular , Modelos Moleculares , Mucoproteínas/metabolismo , Filogenia , Prolina/metabolismo , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína
15.
J Biol Chem ; 295(51): 17398-17410, 2020 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-33453986

RESUMO

Identification of antibody-binding epitopes is crucial to understand immunological mechanisms. It is of particular interest for allergenic proteins with high cross-reactivity as observed in the lipid transfer protein (LTP) syndrome, which is characterized by severe allergic reactions. Art v 3, a pollen LTP from mugwort, is frequently involved in this cross-reactivity, but no antibody-binding epitopes have been determined so far. To reveal human IgE-binding regions of Art v 3, we produced three murine high-affinity mAbs, which showed 70-90% coverage of the allergenic epitopes from mugwort pollen-allergic patients. As reliable methods to determine structural epitopes with tightly interacting intact antibodies under native conditions are lacking, we developed a straightforward NMR approach termed hydrogen/deuterium exchange memory (HDXMEM). It relies on the slow exchange between the invisible antigen-mAb complex and the free 15N-labeled antigen whose 1H-15N correlations are detected. Due to a memory effect, changes of NH protection during antibody binding are measured. Differences in H/D exchange rates and analyses of mAb reactivity to homologous LTPs revealed three structural epitopes: two partially cross-reactive regions around α-helices 2 and 4 as well as a novel Art v 3-specific epitope at the C terminus. Protein variants with exchanged epitope residues confirmed the antibody-binding sites and revealed strongly reduced IgE reactivity. Using the novel HDXMEM for NMR epitope mapping allowed identification of the first structural epitopes of an allergenic pollen LTP. This knowledge enables improved cross-reactivity prediction for patients suffering from LTP allergy and facilitates design of therapeutics.


Assuntos
Alérgenos/imunologia , Proteínas de Transporte/imunologia , Reações Cruzadas , Epitopos/química , Imunoglobulina E/imunologia , Espectroscopia de Ressonância Magnética/métodos , Antígenos de Plantas/imunologia , Deutério/química , Hidrogênio/química , Pólen/imunologia , Conformação Proteica
16.
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.

17.
Front Immunol ; 10: 2600, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31798576

RESUMO

The World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee was established in 1986 by leading allergists to standardize names given to proteins that cause IgE-mediated reactions in humans. The Sub-Committee's objective is to assign unique names to allergens based on a critical analysis of confidentially submitted biochemical and clinical data from researchers, often prior to publication to preserve consistency. The Sub-Committee maintains and revises the database as the understanding of allergens evolves. This report summarizes recent developments that led to updates in classification of cockroach group 1 and 5 allergens to animal as well as environmental and occupational allergens. Interestingly, routes, doses, and frequency of exposure often affects allergenicity as does the biochemical properties of the proteins and similarity to self and other proteins. Information required by the Sub-Committee now is more extensive than previously as technology has improved. Identification of new allergens requires identification of the amino acid sequence and physical characteristics of the protein as well as demonstration of IgE binding from subjects verified by described clinical histories, proof of the presence of the protein in relevant exposure substances, and demonstration of biological activity (skin prick tests, activation of basophils, or mast cells). Names are assigned based on taxonomy with the abbreviation of genus and species and assignment of a number, which reflects the priority of discovery, but more often now, the relationships with homologous proteins in related species.


Assuntos
Alérgenos/classificação , Terminologia como Assunto , Alérgenos/química , Alérgenos/imunologia , Animais , Humanos
18.
Mol Immunol ; 116: 140-150, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31654938

RESUMO

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are important allergens in fruits, pollen, vegetables, nuts and latex. Due to their compact structure, LTPs are highly resistant to heat treatment. Here, Art v 3 from mugwort pollen and Pru p 3 from peach were used as model allergens to in-depth investigate structural and immunological properties upon thermal treatment at different buffer conditions. METHODS: Recombinant Art v 3 and Pru p 3 were purified from E. coli and incubated at 95 °C up to 120 min using sodium phosphate buffer pH 3.4 or 7.3. Physicochemical properties of allergens were analyzed in circular dichroism spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, size exclusion chromatography, and mass spectrometry. The crystal structure of Art v 3.0201 was determined to 1.9 Šresolution. IgG and IgE binding was investigated in ELISA using murine and LTP allergic patients' sera. RESULTS: Highly pure and homogenous recombinant allergens were obtained from bacterial production. The crystal structure of Art v 3.0201 revealed an antiparallel four helix bundle with a C-terminal extension mediating an asymmetric, transient dimer interface and differently sized cavities. Both allergens showed high thermal stability at acidic conditions. In contrast, extensive heat treatment in neutral buffer induced irreversible structural changes due to lanthionine-based cysteine rearrangement. This fostered loss of the typical α-helical structure, increased molecular size and abrogation of IgG and IgE binding epitopes. Pru p 3 lost its structural integrity at shorter heat stress duration than Art v 3, which did however only partially affect the molecule's IgE binding epitopes. CONCLUSION: During thermal treatment, susceptibility to structural changes of the LTP-fold is highly dependent on the surrounding environment but also on intrinsic features of individual LTPs. This is a crucial fact to consider when processing LTP-containing food or food products as this will directly influence their allergenic potential.


Assuntos
Alanina/análogos & derivados , Antígenos de Plantas/metabolismo , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Proteínas de Plantas/metabolismo , Sulfetos/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Artemisia/metabolismo , Reações Cruzadas/fisiologia , Epitopos/metabolismo , Escherichia coli/metabolismo , Hipersensibilidade Alimentar/metabolismo , Humanos , Imunoglobulina E/metabolismo , Imunoglobulina G/metabolismo , Camundongos , Pólen/metabolismo , Prunus/metabolismo
19.
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
20.
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
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