Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.129
Filtrar
1.
J Sci Food Agric ; 100(1): 308-314, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31525267

RESUMO

BACKGROUND: Peanut is among the most common of food allergies, and one of its allergens is Ara h 2. A previous study revealed that this allergen was recognized by serum immunoglobulin E (IgE) in over 90% of a peanut-allergic patient population. Enzymatic cross-linking is a popular processing method used to tailor food functionality, such as antigenicity. RESULT: The cross-linking reactions of Ara h 2 were catalyzed by polyphenol oxidase (PPO), and the relevant reaction sites were identified using mass spectrometry and StavroX software. Two pairs of intramolecular cross-linking peptides and two intermolecular cross-linking peptides were found. Intramolecular cross-linking was speculated to occur between ARG131 (amino acids 116-131) and TYR65 (amino acids 63-80) and between TYR60 (amino acids 56-62) and ARG92 (amino acids 92-102); the intermolecular cross-linking sites were ARG31 with TYR84 or TYR89 and TYR65 or TYR72 with ARG92 or ARG102 . Three out of four cross-linking peptides were found in α-helices, and destruction of this secondary structure resulted in a loose tertiary structure. Although seven linear allergen epitopes were involved in cross-linking, the IgE binding capacity of protein changed slightly, while its sensitization potential decreased in mouse model. CONCLUSION: Exploring the structural change of Ara h 2 after cross-linking is beneficial in further understanding the influence of structure on sensitization. This result indicated the future possibility of precision processing on structure of proteins to improve their properties. © 2019 Society of Chemical Industry.


Assuntos
Albuminas 2S de Plantas/química , Albuminas 2S de Plantas/imunologia , Catecol Oxidase/química , Sequência de Aminoácidos , Arachis/química , Arachis/imunologia , Biocatálise , Epitopos/química , Epitopos/imunologia , Manipulação de Alimentos , Humanos , Imunoglobulina E/imunologia , Hipersensibilidade a Amendoim/imunologia , Estrutura Secundária de Proteína
2.
Food Chem ; 309: 125603, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31707198

RESUMO

Exopalaemon modestus (EM) is a shrimp delicacy that could cause food allergy, the major allergen of EM is Exo m 1. The amino acid (AA) sequence, IgE-binding epitopes and allergenic peptides in gastrointestinal (GI) digests of Exo m 1, and their effects on basophil function were investigated. Exo m 1 has an AA-sequence of high similarity with other shrimp tropomyosins, while not 100% matching. The IgE-binding epitopes of Exo m 1 are epitope 1 (43-59, VHNLQKRMQQLENDLDS), epitope 2 (85-105, VAALNRRIQLLEEDLERSEER), epitope 3 (131-164, ENRSLSDEERMDALENQLKEARFLAEEADRKYDE), epitope 4 (187-201, ESKIVELEEELRVVG) and epitope 5 (243-280, ERSVQKLQKEVDRLEDELVNEKEKYKSITDELDQTFSE). Among the thirty-three peptides of Exo m 1 identified in GI digests, two were highly recognized by IgE, twenty-four moderately or weakly bound IgE, and seven had no IgE-reactivities. These IgE-binding epitopes and GI digestion induced-allergenic peptides could activate basophil degranulation, and CD63 and CD203c expression, they could be potential peptide-based immunotherapy for shrimp allergic individuals.


Assuntos
Alérgenos/imunologia , Epitopos/metabolismo , Palaemonidae/imunologia , Hipersensibilidade a Frutos do Mar/imunologia , Tropomiosina/imunologia , Adulto , Alérgenos/química , Alérgenos/metabolismo , Animais , Basófilos/imunologia , Criança , Pré-Escolar , Mapeamento de Epitopos , Epitopos/química , Feminino , Água Doce , Humanos , Imunoglobulina E/metabolismo , Lactente , Masculino , Peptídeos/química , Peptídeos/imunologia , Homologia de Sequência de Aminoácidos , Tropomiosina/química , Tropomiosina/metabolismo
3.
Comput Biol Chem ; 83: 107157, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31751887

RESUMO

Leishmaniosis, caused by intracellular parasites of the genus Leishmania, has become a serious public health problem around the world, and for which there are currently extensive limitations. In this work, a theoretical model was proposed for the development of a multi-epitope vaccine. The protein GP63 of the parasite was selected for epitopes prediction, due to its important biological role for the infection process and abundance. IEDB tools were used to determine epitopes B and T in Leishmania braziliensis; besides, other conserved epitopes in three species were selected. To improve immunogenicity, 50S ribosomal protein L7 / L12 (ID: P9WHE3) was used as a domain of adjuvant in the assembly process. The folding arrangement of the vaccine was obtained through homologous modeling multi-template with MODELLER v9.21, and a Ramachandran plot analysis was done. Furthermore, physicochemical properties were described with the ProtParam tool and secondary structure prediction combining GOR-IV and SOPMA tools. Finally, a molecular dynamics simulation (50 ns) was performed to establish flexibility and conformational changes. The analysis of the results indicates high conservancy in the epitopes predicted among the four species. Moreover, Ramachandran plot, physicochemical parameters, and secondary structure prediction suggest a stable conformation of the vaccine, after a minimum conformational change that was evaluated with the free energy landscape. The conformational change does not drive any substantial change for epitope exposition on the surface. The vaccine proposed could be tested experimentally to guide new approaches in the development of pan-vaccines; vaccines with regions conserved in multiple species.


Assuntos
Leishmania/imunologia , Metaloendopeptidases/imunologia , Simulação de Dinâmica Molecular , Vacinas/imunologia , Epitopos/química , Epitopos/imunologia , Metaloendopeptidases/química , Conformação Proteica , Especificidade da Espécie
4.
J Agric Food Chem ; 67(46): 12918-12926, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31668066

RESUMO

The triosephosphate isomerase (TIM), Scy p 8, is a crab allergen and shows cross-reactivity in the shellfish. Here, recombinant Scy p 8 was expressed, and its crystal structure was determined at a resolution of 1.8 Å. The three-dimensional structure of Scy p 8 is primarily composed of a (ß/α)8-barrel motif prototype. Additionally, Scy p 8 showed cross-reactivity with high sequential and secondary structural identity among TIMs from shellfish species. The site-directed mutagenesis of critical amino acids of conformational epitopes was carried out, and the mutants of Trp 168 and Lys 237 to Ala reduced immunoglobulin E (IgE)-binding activity by approximately 30%, compared with wild-type TIM in an inhibition ELISA; however, it still induced basophil activation despite the interpatient variability between patients. These results can help to provide an accurate template for the analysis of the IgE binding and establish meaningful relationships between structure and allergenicity.


Assuntos
Braquiúros/enzimologia , Epitopos/química , Triose-Fosfato Isomerase/imunologia , Sequência de Aminoácidos , Animais , Braquiúros/química , Braquiúros/genética , Braquiúros/imunologia , Reações Cruzadas , Cristalização , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoglobulina E/imunologia , Conformação Molecular , Conformação Proteica , Alinhamento de Sequência , Frutos do Mar/análise , Hipersensibilidade a Frutos do Mar/imunologia , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genética
5.
Mol Immunol ; 116: 199-207, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31731097

RESUMO

A 38 kDa ß-1,3-glucanase allergen from Cryptomeria japonica pollen (CJP38) was recombinantly produced in E. coli and purified to homogeneity with the use of Ni-affinity resin. CJP38 hydrolyzed ß-1,3-glucans such as CM-curdlan and laminarioligosaccharides in an endo-splitting manner. The optimum pH and temperature for ß-1,3-glucanase activity were approximately 4.5 and 50 °C, respectively. The enzyme was stable at 30-60 °C and pH 4.0-10.5. Furthermore, CJP38 catalyzed a transglycosylation reaction to yield reaction products with a molecular weight higher than those of the starting laminarioligosaccharide substrates. The three-dimensional structure of CJP38 was determined using X-ray crystallography at 1.5 Å resolution. CJP38 exhibited the typical (ß/α)8 TIM-barrel motif, similar to allergenic ß-1,3-glucanases from banana (Mus a 5) and rubber tree latex (Hev b 2). Amino acid sequence alignment of these proteins indicated that the two-consensus IgE epitopes identified on the molecular surfaces of Mus a 5 and Hev b 2 were highly conserved in CJP38. Their conformations and surface locations were quite similar for these proteins. Sequence and structural conservation of these regions suggest that CJP38 is a candidate allergen responsible for the pollen-latex-fruit syndrome relating to Japanese cedar pollinosis.


Assuntos
Alérgenos/química , Antígenos de Plantas/química , Cryptomeria/química , Pólen/química , Alérgenos/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/imunologia , Reações Cruzadas/imunologia , Cryptomeria/imunologia , Cristalografia por Raios X/métodos , Epitopos/química , Epitopos/imunologia , Escherichia coli/imunologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina E/química , Imunoglobulina E/imunologia , Látex/química , Látex/imunologia , Musa/química , Musa/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Temperatura Ambiente
6.
Nat Commun ; 10(1): 4403, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562305

RESUMO

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.


Assuntos
Epitopos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/metabolismo , Células 3T3 , Animais , Células COS , Epitopos/química , Epitopos/genética , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Camundongos , Microscopia de Fluorescência , Mutação , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética
7.
Org Biomol Chem ; 17(39): 8878-8901, 2019 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-31513223

RESUMO

Melioidosis and glanders, respectively caused by the Gram-negative bacteria Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), are considered as urgent public health issues in developing countries and potential bioterrorism agents. Bp and Bm lipopolysaccharides (LPS) have been identified as attractive vaccine candidates for the development of prophylactic measures against melioidosis and glanders. Bp and Bm express structurally similar LPSs wherein the O-antigen (OAg) portion consists of a heteropolymer whose repeating unit is a disaccharide composed of d-glucose and 6-deoxy-l-talose residues, the latter being diversely acetylated and methylated. Herein we report the synthesis of two tetrasaccharides mimicking the main substitution epitopes of Bp and Bm LPS OAgs. The assembly of the tetrasaccharides was achieved using a sequential glycosylation strategy while relying on the late-stage epimerization of the inner rhamnose into a 6-deoxy-l-talose residue. We show that these synthetic compounds strongly react with culture-confirmed Thai melioidosis patient serum and closely mimic the antigenicity of native Bp OAg. Our results suggest that these tetrasaccharides could be suitable candidates for the development of vaccines and/or diagnostic tools against melioidosis and glanders.


Assuntos
Burkholderia mallei/imunologia , Burkholderia pseudomallei/imunologia , Epitopos/química , Melioidose/sangue , Melioidose/imunologia , Antígenos O/imunologia , Oligossacarídeos/química , Oligossacarídeos/imunologia , Burkholderia mallei/química , Burkholderia pseudomallei/química , Epitopos/sangue , Epitopos/imunologia , Humanos , Antígenos O/química , Oligossacarídeos/sangue , Tailândia
8.
PLoS Pathog ; 15(9): e1008026, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527908

RESUMO

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage.


Assuntos
Vacinas contra a AIDS/imunologia , /imunologia , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/imunologia , HIV-1/genética , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/química , Vacinas contra a AIDS/genética , Substituição de Aminoácidos , Afinidade de Anticorpos , Sítios de Ligação , Antígenos CD4/metabolismo , Desenho de Drogas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Células HEK293 , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Engenharia de Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
9.
ACS Appl Mater Interfaces ; 11(35): 32431-32440, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31393695

RESUMO

Molecularly imprinted polymers were commonly used for drug delivery. However, single-template molecularly imprinted polymers often fail to achieve both drug delivery and precise targeting. To address this issue, a dual-template molecularly imprinted polymer nanoparticle used for targeted diagnosis and drug delivery for pancreatic cancer BxPC-3 cells (FH-MIPNPs) was prepared. In the FH-MIPNPs, the 71-80 peptide of human fibroblast growth-factor-inducible 14 modified with glucose (Glu-FH) and bleomycin (BLM) were used as templates simultaneously, so that the FH-MIPNPs could load BLM and bind to the BxPC-3 cells, which overexpress human fibroblast growth-factor-inducible 14 (FN14). Targeted imaging experiments in vitro show that the FH-MIPNPs could specifically target BxPC-3 cells and that there is no targeting effect on cells without expression of FN14. In vivo antitumor experiment results demonstrated that the FH-MIPNP-loaded BLM (FH-MIPNPs/BLM) could inhibit the growth of xenografts tumor of BxPC-3 (tumor volume increased to 1.05×), which shows that FH-MIPNPs/BLM had obvious targeted therapeutic effect compared to the other three control groups of BLM, FH-NIPNPs/BLM, and physiological saline (tumor volume increased to 1.5×, 1.6×, and 2.4×, respectively). What is more, FH-MIPNPs have low biotoxicity through toxicity experiments in vitro and in vivo, which is favorable toward making molecularly imprinted polymers an effective platform for tumor-targeted imaging and therapy.


Assuntos
Sistemas de Liberação de Medicamentos , Epitopos , Nanopartículas , Imagem Óptica , Neoplasias Pancreáticas , Peptídeos , Receptor de TWEAK/química , Animais , Linhagem Celular Tumoral , Epitopos/química , Epitopos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Impressão Molecular , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Peptídeos/química , Peptídeos/farmacologia
10.
J Agric Food Chem ; 67(37): 10458-10469, 2019 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-31469568

RESUMO

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.


Assuntos
Alérgenos/imunologia , Proteínas de Artrópodes/imunologia , Braquiúros/genética , Clonagem Molecular , Epitopos/imunologia , Cadeias Leves de Miosina/imunologia , Alérgenos/química , Alérgenos/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Braquiúros/química , Braquiúros/imunologia , Degranulação Celular , Epitopos/química , Epitopos/genética , Humanos , Mastócitos/imunologia , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Fases de Leitura Aberta , Alinhamento de Sequência
11.
Mol Immunol ; 114: 189-195, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31376732

RESUMO

The presence in cypress pollen of an important allergen, belonging to the gibberellin-regulated protein (GRP) family, has been suggested for many years. However, it has never been isolated and sometimes the homologous peach allergen, Pru p 7, has been used as a surrogate to perform immunological investigations. The aim of this study has been the isolation and molecular characterization of the GRP contained in the Cupressus sempervirens pollen. This protein, named Cypmaclein, has been purified from the natural source using conventional biochemical methods consisting in different chromatographic separations. Cypmaclein has been identified by direct protein sequencing of the N-terminal region and of internal fragments of the molecule. In SDS-PAGE, its apparent molecular mass is slightly higher than that of Pru p 7. Nevertheless, the mass spectrometry experiments reveal that the exact molecular mass of Cypmaclein (6821.88 Da) is very close to that of Pru p 7 (6909.90 Da). Two regions of Cypmaclein have been sequenced providing 50% of its primary structure. A high overall sequence identity of Cypmaclein with all the analyzed GRP has been observed, although in the N-terminal region the high identity is limited to the homolog of Cryptomeria japonica. In circular dichroism experiments Cypmaclein produced a spectrum overlapping that of Pru p 7. However, the comparative analysis of Cypmaclein, Pru p 7 and Pun g 7 IgE reactivity revealed a behavior that was not completely overlapping, thus suggesting that the IgE epitopes are only partially shared. In single point highest inhibition achievable assays performed with the FABER test, Cypmaclein efficiently competed with the allergenic peach and pomegranate GRP in the binding of specific IgE of patients sensitized to Pru p 7. In conclusion, the natural cypress pollen GRP has been isolated for the first time, its structural features have been investigated and its cross-reactivity with Pru p 7 and Pun g 7 has been demonstrated. This protein is now available for further investigations aimed at understanding its clinical relevance in the allergy to cypress pollen. In addition, the prevalence of sensitization directly to Cypmaclein, and not limited to the homologs, can be defined.


Assuntos
Cupressus/química , Cupressus/imunologia , Giberelinas/química , Giberelinas/imunologia , Imunoglobulina E/imunologia , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Criança , Reações Cruzadas/imunologia , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Masculino , Pólen/química , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto Jovem
12.
BMC Bioinformatics ; 20(1): 387, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296178

RESUMO

BACKGROUND: Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits in replacing a protein which is difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitopes predicted by bioinformatics tools are commonly used and these sequential epitopes are used as is in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools have been proposed so far to predict peptide sequences: Superficial and PEPOP 2.0. PEPOP 2.0 can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. RESULTS: We have developed an improved version of PEPOP (PEPOP 2.0) dedicated to answer to experimentalists' need for a tool able to handle proteins and to turn them into peptides. The PEPOP 2.0 web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP 2.0 proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature. CONCLUSION: Through the presentation of its user-friendly, well-structured new web site conceived in close proximity to experimentalists, we report original methods that show how PEPOP 2.0 can assist biologists in dealing with discontinuous epitopes.


Assuntos
Biologia Computacional/métodos , Epitopos/metabolismo , Software , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Epitopos/química , Soros Imunes , Internet , Camundongos , Peptídeos/sangue , Peptídeos/química , Peptídeos/imunologia , Domínios Proteicos , Proteínas/química
13.
Molecules ; 24(14)2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31319525

RESUMO

The functionalization of VHHs enables their application in almost every aspect of biomedical inquiry. Amino modification remains a common strategy for protein functionalization, though is considered to be inferior to site-specific methods and cause protein property changes. In this paper, four anti-ß2M VHHs were selected and modified on the amino group by NHS-Fluo. The impacts of amino modification on these VHHs were drastically different, and among all th examples, the modified NB-1 maintained the original stability, bioactivity and homogeneity of unmodified NB-1. Specific recognition of VHHs targeting ß2M detected by fluorescence imaging explored the possible applications of VHHs. Via this study, we successfully functionalized the anti-ß2M VHHs through amino modification and the results are able to instruct the simple and fast functionalization of VHHs in biomedical researches.


Assuntos
Epitopos/química , Anticorpos de Domínio Único/isolamento & purificação , Microglobulina beta-2/química , Calmodulina/química , Calmodulina/imunologia , Epitopos/imunologia , Humanos , Imagem Óptica , Estabilidade Proteica , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/imunologia , Microglobulina beta-2/imunologia
14.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269680

RESUMO

Much of the experimental data, especially in life sciences, is considered to be useless if it demonstrates a large standard deviation from the mean value. The Renaissance distribution, as presented in this study, allows one to extract true values from such statistical data with large noise. To obtain proof of the Renaissance distribution, high-throughput synthesis of deep substitutions for a target amino acid sequence was performed, and the known epitope was identified in assay with human serum antibodies. In addition, the Renaissance distribution was shown to approach the epitope affinity maturation by the deep alanine substitution. The Renaissance distribution may have an impact in the development of novel specific drugs.


Assuntos
Alanina/genética , Substituição de Aminoácidos , Peptídeos/genética , Alanina/química , Sequência de Aminoácidos , Epitopos/química , Epitopos/genética , Humanos , Modelos Moleculares , Peptídeos/química , Análise Serial de Proteínas , Distribuições Estatísticas , Processos Estocásticos
15.
MAbs ; 11(6): 1036-1052, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257988

RESUMO

Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies. Abbreviations: ADCC: antibody-dependent cellular cytotoxicity; ADCP: antibody-dependent cellular phagocytosis; CFSE: carboxyfluorescein succinimidyl ester; Fab: fragment antigen binding; Fc: fragment crystallizable; FcγR: Fcγ receptor; Ig: immunoglobulin; IND: investigational new drug; MDM⊘: monocyte-derived macrophage; NOD: non-obese diabetic; scFv: single chain fragment variable; SCID: severe combined immunodeficiency; SIRP: signal-regulatory protein.


Assuntos
Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos de Diferenciação , Receptores Imunológicos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos de Diferenciação/química , Antígenos de Diferenciação/imunologia , Antígeno CD47/imunologia , Galinhas , Cristalografia por Raios X , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunoterapia , Masculino , Neoplasias/imunologia , Neoplasias/terapia , Domínios Proteicos , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/química , Receptores Imunológicos/imunologia
16.
Virol J ; 16(1): 75, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159841

RESUMO

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Parvovirus Suíno/imunologia , Animais , Antígenos Virais/química , Mapeamento de Epitopos , Epitopos/química , Masculino , Testes de Neutralização , Peptídeos/genética , Peptídeos/imunologia , Suínos
17.
Artigo em Inglês | MEDLINE | ID: mdl-31176265

RESUMO

Herein an approach to prepare molecularly imprinted polymer nanoparticles (nanoMIPs) with specific binding affinity for antibodies is reported. The process relied on the covalent immobilization of the template (whole immunoglobulin G (IgG), Fc domain of human IgG and peptide epitope) onto the surface of a solid support, polymerization and affinity separation of nanoMIPs. The binding between nanoMIPs and their corresponding templates was analyzed and evaluated as being in sub-nanomolar and nano-molar range. The nanoMIPs prepared for Fc domain and epitope demonstrated a specific recognition of both human and goat IgGs, therefore they could be considered as a synthetic analogue of protein A and benefit from its intrinsic stability, short time and low cost of preparation.


Assuntos
Epitopos/química , Imunoglobulina G/química , Animais , Cabras , Humanos , Impressão Molecular , Nanopartículas/química , Polimerização , Polímeros/síntese química , Polímeros/química , Domínios Proteicos
18.
Food Chem ; 297: 124986, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253255

RESUMO

The microwave heating of wheat kernels, flour, and gluten, has attracted attention lately because it has been claimed to abolish gluten toxicity for celiac patients. Nevertheless, contradictory results have been reported regarding the effect on gluten celiac-immunotoxicity. In order to better understand the effect of the microwave treatment on gluten structure, conformation, functionality and celiac-immunotoxicity, a central composite design with two factors, power level, and treatment time, was used to investigate a possible quadratic and interaction effects between both factors. Extractable gliadins content was affected by the power and time in a linear and quadratic fashion; extractable glutenins were not affected. Gluten secondary structure was affected by the microwave treatment and related to the polymer's disaggregation phenomenon observed. In fact, the microwave treatment increased the amount of potentially toxic epitopes released after peptic and tryptic digestion, showing inefficiency as a treatment to detoxify the gluten for celiac disease patients.


Assuntos
Doença Celíaca/prevenção & controle , Glutens/efeitos da radiação , Micro-Ondas , Conformação Molecular/efeitos da radiação , Triticum/efeitos da radiação , Epitopos/química , Epitopos/efeitos da radiação , Epitopos/toxicidade , Farinha/análise , Gliadina/química , Gliadina/efeitos da radiação , Glutens/química , Glutens/toxicidade , Humanos , Imunotoxinas/química , Imunotoxinas/efeitos da radiação , Imunotoxinas/toxicidade , Fatores de Tempo , Triticum/química
19.
Food Funct ; 10(7): 3934-3941, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31197293

RESUMO

Shrimp tropomyosin (TM) is a glycoprotein allergen with high lysine content, however, there is little data on how deglycosylation and glycation modify the allergenicity of TM. In this work, the effects of deglycosylation (enzymatic and chemical) and glycation of TM by glucose on in vivo allergenicity and mast cell function were investigated using BALB/c mouse and RBL-2H3 mast cells. Compared with TM, after deglycosylation, the deglycosylated TMs (DGTMs) exacerbated the in vivo allergenicity and mast cell allergy response. As for the TM glycated by glucose (TM-G), glycation led to weaker allergy responses on mouse and mast cells, which might be due to glucose destroying the epitopes with glycation sites. Moreover, the glucose residues on TM-G functioned as both a resistance to prevent the accessibility of TM-G to the IgE on mast cell and masking epitopes. Therefore, deglycosylation of TM was not suitable for desensitizing TM-induced food allergy, while glycation of TM might be applied to desensitize TM allergenicity, which could provide new insight into food allergy desensitization for larger cohorts of shrimp-allergic patients.


Assuntos
Alérgenos/química , Alérgenos/imunologia , Mastócitos/imunologia , Tropomiosina/química , Tropomiosina/imunologia , Anafilaxia/sangue , Animais , Citocinas/análise , Epitopos/química , Feminino , Hipersensibilidade Alimentar/prevenção & controle , Glucose/metabolismo , Glicosilação , Histamina/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Espécies Reativas de Oxigênio/metabolismo , Alimentos Marinhos , Hipersensibilidade a Frutos do Mar/imunologia
20.
Cells ; 8(6)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31212633

RESUMO

The human leukocyte antigen HLA-B27 is a strong risk factor for Ankylosing Spondylitis (AS), an immune-mediated disorder affecting axial skeleton and sacroiliac joints. Additionally, evidence exists sustaining a strong protective role for HLA-B27 in viral infections. These two aspects could stem from common molecular mechanisms. Recently, we have found that the HLA-B*2705 presents an EBV epitope (pEBNA3A-RPPIFIRRL), lacking the canonical B27 binding motif but known as immunodominant in the HLA-B7 context of presentation. Notably, 69% of B*2705 carriers, mostly patients with AS, possess B*2705-restricted, pEBNA3A-specific CD8+ T cells. Contrarily, the non-AS-associated B*2709 allele, distinguished from the B*2705 by the single His116Asp polymorphism, is unable to display this peptide and, accordingly, B*2709 healthy subjects do not unleash specific T cell responses. Herein, we investigated whether the reactivity towards pEBNA3A could be a side effect of the recognition of the natural longer peptide (pKEBNA3A) having the classical B27 consensus (KRPPIFIRRL). The stimulation of PBMC from B*2705 positive patients with AS in parallel with both pEBNA3A and pKEBNA3A did not allow to reach an unambiguous conclusion since the differences in the magnitude of the response measured as percentage of IFNγ-producing CD8+ T cells were not statistically significant. Interestingly, computational analysis suggested a structural shift of pEBNA3A as well as of pKEBNA3A into the B27 grooves, leaving the A pocket partially unfilled. To our knowledge this is the first report of a viral peptide: HLA-B27 complex recognized by TCRs in spite of a partially empty groove. This implies a rethinking of the actual B27 immunopeptidome crucial for viral immune-surveillance and autoimmunity.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Epitopos/imunologia , Antígeno HLA-B27/genética , Herpesvirus Humano 4/metabolismo , Espondilite Anquilosante/diagnóstico , Alelos , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/química , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Plasmídeos/genética , Plasmídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/metabolismo , Espondilite Anquilosante/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA