RESUMO
The carbohydrate specificities of Dioclea grandiflora lectins DGL-I1 and DGL-II, and Galactia lindenii lectin II (GLL-II) were explored by use of remodeled glycoproteins as well as by the lectin hemagglutinating activity against erythrocytes from various species with different glycomic profiles. The three lectins exhibited differences in glycan binding specificity but also showed overlapping recognition of some glycotopes (i.e. Tα glycotope for the three lectins; IIß glycotope for DGL-II and GLL-II lectins); in many cases the interaction with distinct glycotopes was influenced by the structural context, i.e., by the neighbouring sugar residues. Our data complement and expand the existing knowledge about the binding specificity of these three Diocleae lectins, and taken together with results of previous studies, allow us to suggest a functional map of the carbohydrate recognition which illustrate the impact of modification of basic glycotopes enhancing, permiting, or inhibiting their recognition by each lectin.
Assuntos
Dioclea/química , Lectinas de Plantas/imunologia , Especificidade de Anticorpos , Epitopos/química , Epitopos/imunologia , Hemaglutinação , Humanos , Lectinas de Plantas/química , Polissacarídeos/química , Polissacarídeos/imunologiaRESUMO
BACKGROUND: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. RESULTS: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing ß-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. CONCLUSIONS: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.
Assuntos
Mastócitos/imunologia , Lectinas de Plantas/imunologia , Proteínas Recombinantes/imunologia , Animais , Artocarpus/imunologia , Degranulação Celular , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Histamina/metabolismo , Imunoglobulina E/metabolismo , Imunomodulação , Interleucina-4/metabolismo , Manose/metabolismo , NF-kappa B/metabolismo , Lectinas de Plantas/isolamento & purificação , Ligação Proteica , Ratos , Proteínas Recombinantes/isolamento & purificação , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Latex allergy is a health problem that mainly affects medical environments, causing anaphylactic shocks in extreme cases. Sensitization and reactions to this material is closely linked to the use of latex gloves. The objective of this study was to purify two of the major allergens from latex surgical gloves to study the biochemical and structural changes that could be generated during the product manufacture and to compare their IgE recognition with the non-processed allergens. Glycosylated allergen Hev b 2 (ß-1,3-glucanase) and Hev b 6.02 (hevein) were purified from glove extracts using affinity (Concanavalin A) and reversed-phase chromatographies, respectively. ELISA experiments were performed with both proteins and sera from allergic patients to assess the IgE recognition, which was heterogeneous. Crystallographic methods were used to obtain the 3D structure of Hev b 6.02 from surgical gloves, which did not show evident modification when compared with the protein from the natural non-processed form. Despite having the same crystallographic structure, the IgE from some patients showed different recognition when the glove and the natural allergen were used in ELISA. Furthermore, using electrophoretic techniques, we identified three forms of Hev b 2: one corresponding to the complete polypeptide chain with posttranslational modifications, and two glycosylated fragments. The mixture of these three forms showed stronger recognition by IgE from latex-allergic patients than the pure non-processed allergen. In conclusion, IgE from subjects sensitized to latex products showed different recognition between the allergens obtained from a natural source and the processed material, even when the structure was maintained. This demonstrates the importance of using processed allergens in further investigations of diagnosis, prevalence, product allergenicity, and therapies.
Assuntos
Antígenos de Plantas , Peptídeos Catiônicos Antimicrobianos , Luvas Cirúrgicas/efeitos adversos , Imunoglobulina E , Hipersensibilidade ao Látex/imunologia , Látex , Lectinas de Plantas , Proteínas de Plantas , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Antígenos de Plantas/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Cristalografia por Raios X , Feminino , Hevea/química , Humanos , Imunoglobulina E/química , Imunoglobulina E/imunologia , Látex/efeitos adversos , Látex/química , Masculino , Lectinas de Plantas/química , Lectinas de Plantas/imunologia , Lectinas de Plantas/isolamento & purificação , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/isolamento & purificação , Processamento de Proteína Pós-Traducional/imunologia , Relação Estrutura-AtividadeRESUMO
O-glycosidically-linked glycans have been involved in development, maturation, homing, and immune regulation in T cells. Previous reports indicate that Amaranthus leucocarpus lectin (ALL), specific for glycans containing galactose-N-acetylgalactosamine and N-acetylgalactosamine, recognizes human naïve CD27(+)CD25(+)CD4(+) T cells. Our aim was to evaluate the phenotype of CD4(+) T cells recognized by ALL in peripheral blood mononuclear cells obtained from healthy volunteers. CD4(+) T cells were isolated by negative selection using magnetic beads-labeled monoclonal antibodies; the expression of T regulatory cell phenotypic markers was assessed on ALL-recognized cells. In addition, IL-4, IL-10, IFN-γ, and TGF-ß intracellular production in ALL (+) cells was also evaluated. The analyses of phenotypic markers and intracellular cytokines were performed through flow cytometry. ALL-recognized CD4(+) T cells were mainly CD45RA(+), CCR7(+) cells. Although 52 ± 10% CD25(+)Foxp3(+) cells were positive to ALL, only 34 ± 4% of ALL (+) cells corresponded to CD25(+)Foxp3(-) cells. Intracellular cytokines in freshly obtained ALL (+)CD4(+) T cells exhibited 8% of IL-4, 15% of IL-10, 2% of IFN-γ, and 15% of TGF-ß, whereas ALL (-)CD4(+) T cells depicted 1% of IL-4, 2% of IL-10, <1% of IFN-γ, and 6% of TGF-ß. Our results show that galactose-N-acetylgalactosamine and N-galactosamine-bearing CD4(+) T cells expressed phenotypic markers of NnTreg cells.
Assuntos
Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Lectinas de Plantas/imunologia , Lectinas de Plantas/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígeno CTLA-4/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Glicosilação , Humanos , Imunofenotipagem , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Fenótipo , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Advances in the glycobiology and immunology fields have provided many insights into the role of carbohydrate-protein interactions in the immune system. We aim to present a comprehensive review of the effects that some plant lectins exert as immunomodulatory agents, showing that they are able to positively modify the immune response to certain pathological conditions, such as cancer and infections. The present review comprises four main themes: (1) an overview of plant lectins that exert immunomodulatory effects and the mechanisms accounting for these activities; (2) general characteristics of the immunomodulatory lectin ArtinM from the seeds of Artocarpus heterophyllus; (3) activation of innate immunity cells by ArtinM and consequent induction of Th1 immunity; (4) resistance conferred by ArtinM administration in infections with intracellular pathogens, such as Leishmania (Leishmania) major, Leishmania (Leishmania) amazonensis, and Paracoccidioides brasiliensis. We believe that this review will be a valuable resource for more studies in this relatively neglected area of research, which has the potential to reveal carbohydrate targets for novel prophylactic and therapeutic strategies.
Assuntos
Fatores Imunológicos/farmacologia , Lectinas de Plantas/farmacologia , Animais , Artocarpus/química , Artocarpus/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/química , Fatores Imunológicos/imunologia , Lectinas de Plantas/química , Lectinas de Plantas/imunologia , Células Th1/efeitos dos fármacos , Células Th1/imunologiaRESUMO
The activities of dectin-1 and mannose receptors on phagocytosis of Candida albicans and the production of TNF-α by macrophages from mice pretreated for 3 days with extract of Artocarpus intergrifolia seeds (jack extract), Artin M or jacalin were studied. Macrophages from these mice were coincubated with C. albicans CR15 (yeast), in the presence of mannose (50mM) plus mannan (100 µg) or laminarin (1mg). Phagocytosis was significantly enhanced to 52% in macrophages from mice pretreated intraperitoneally for 3 days with jack extract (500 µg/250 µl PBS). Reduction in phagocytosis from 52% to 34% (P<0.05) occurred in the presence of mannose receptor inhibitors and from 52% to 16% (P<0.01) in the presence of dectin-1 inhibitor laminarin, whereas only 20% of control macrophages phagocytosed blastoconidia. Similar results were verified for pretreatment of mice with Artin M (2.5 µg/250 µl PBS), but not for jacalin (25 µg/250 µl PBS). Macrophages from mice pretreated 3 days previously with jack extract or Artin M and then coincubated for 2h with C. albicans presented a significant increase in TNF-α production, correlating with significantly less transition of yeast to filamentous forms compared to pretreatment with jacalin. These results suggest that Artin M, but not jacalin present in jack extract significantly increased TNF-α production and the activity of mannose and dectin-1 receptors.
Assuntos
Artocarpus/química , Candida albicans/imunologia , Lectinas Tipo C/metabolismo , Macrófagos/efeitos dos fármacos , Lectinas de Ligação a Manose/metabolismo , Fagocitose/efeitos dos fármacos , Lectinas de Plantas/farmacologia , Receptores de Superfície Celular/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Animais , Glucanos , Macrófagos/imunologia , Macrófagos/metabolismo , Mananas/metabolismo , Manose/metabolismo , Receptor de Manose , Camundongos , Lectinas de Plantas/química , Lectinas de Plantas/imunologia , Polissacarídeos/metabolismo , Sementes/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Mast cells are inflammatory cells that respond to signals of innate and adaptive immunity with immediate and delayed release of mediators. ArtinM, a lectin from Artocarpus integrifolia with immunomodulatory activities, is able to induce mast cell activation, but the mechanisms remain unknown. This study sought to further investigate the effects of the lectin on mast cells. We showed that ArtinM binds to mast cells, possibly to the high affinity receptor for immunoglobulin E (IgE) - FcεRI - and/or to IgE bound to FcεRI. Binding of the lectin resulted in protein tyrosine phosphorylation and release of the pre- and newly-formed mediators, ß-hexosaminidase and LTB(4) by mast cells, activities that were potentiated in the presence of IgE. ArtinM also induced the activation of the transcription factors NFκB and NFAT, resulting in expression of some of their target genes such as IL-4 and TNF-α. In view of the established significance of mast cells in many immunological and inflammatory reactions, a better understanding of the mechanisms involved in mast cell activation by ArtinM is crucial to the pharmacological application of the lectin.
Assuntos
Artocarpus/química , Mastócitos/imunologia , Lectinas de Plantas/imunologia , Receptores de IgE/imunologia , Animais , Linhagem Celular , Expressão Gênica , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , NF-kappa B/agonistas , Fatores de Transcrição NFATC/agonistas , Fosforilação , Lectinas de Plantas/metabolismo , Lectinas de Plantas/farmacologia , Ratos , Tirosina/metabolismoRESUMO
ArtinM and Jacalin (JAC) are lectins from the jackfruit (Artocarpus integrifolia) that have important role in modulation of immune responses to pathogens. Neospora caninum is an Apicomplexa parasite that causes neuromuscular disease in dogs and reproductive disorders in cattle, with economic impact on the livestock industry. Hence, we evaluated the adjuvant effect of ArtinM and JAC in immunization of mice against neosporosis. Six C57BL/6 mouse groups were subcutaneously immunized three times at 2-week intervals with Neospora lysate antigen (NLA) associated with lectins (NLA+ArtinM and NLA+JAC), NLA, ArtinM and JAC alone, and PBS (infection control). Animals were challenged with lethal dose of Nc-1 isolate and evaluated for morbidity, mortality, specific antibody response, cytokine production by spleen cells, brain parasite burden and inflammation. Our results demonstrated that ArtinM was able to increase NLA immunogenicity, inducing the highest levels of specific total IgG and IgG2a/IgG1 ratio, ex vivo Th1 cytokine production, increased survival, the lowest brain parasite burden, along with the highest inflammation scores. In contrast, NLA+JAC immunized group showed intermediate survival, the highest brain parasite burden and the lowest inflammation scores. In conclusion, ArtinM presents stronger immunostimulatory and adjuvant effect than Jacalin in immunization of mice against neosporosis, by inducing a protective Th1-biased pro-inflammatory immune response and higher protection after parasite challenge.
Assuntos
Adjuvantes Imunológicos/farmacologia , Coccidiose/prevenção & controle , Lectinas de Ligação a Manose/farmacologia , Neospora/patogenicidade , Lectinas de Plantas/farmacologia , Animais , Anticorpos Antiprotozoários/sangue , Anticorpos Antiprotozoários/imunologia , Artocarpus/química , Encéfalo/parasitologia , Coccidiose/imunologia , Citocinas/imunologia , Feminino , Imunização , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Carga Parasitária , Lectinas de Plantas/imunologia , Baço/citologia , Baço/imunologiaRESUMO
OBJECTIVE: To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography) from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. METHOD: Fifty-six cerebrospinal fluid (CSF) samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders) and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons) were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA). RESULTS: The VF ELISA showed 100% sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9% and 97.1%, with the CSF samples and 95.5% and 100% with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. CONCLUSION: Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.
Assuntos
Anticorpos Anti-Helmínticos/líquido cefalorraquidiano , Antígenos de Helmintos , Imunoglobulina G/líquido cefalorraquidiano , Neurocisticercose/diagnóstico , Taenia solium/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Estudos de Casos e Controles , Cromatografia de Afinidade , Líquido Cístico/imunologia , Cysticercus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas/imunologia , Humanos , Imunoglobulina G/sangue , Neurocisticercose/imunologia , Lectinas de Plantas/imunologia , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography) from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. METHOD: Fifty-six cerebrospinal fluid (CSF) samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders) and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons) were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA). RESULTS: The VF ELISA showed 100 percent sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9 percent and 97.1 percent, with the CSF samples and 95.5 percent and 100 percent with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. CONCLUSION: Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.
OBJETIVO: Avaliar o desempenho de duas preparações antigênicas (líquido vesicular - LV e uma fração glicoprotéica, fração LL a-Gp, purificada do extrato total dos parasitas por cromatografia de afinidade com lentil lectina) de cisticercos de Taenia solium para o imunodiagnóstico da neurocisticercose. MÉTODO: Cinquenta e seis amostras de líquido cefalorraquidiano (LCR) (22 de pacientes com neurocisticercose e 34 de pacientes com outras doenças neurológicas) e 57 amostras de soro (22 de pacientes com neurocisticercose, 18 de pacientes com outras infecções e 17 de pessoas presumivelmente sadias) foram analisadas quanto à presença de anticorpos IgG anti-cisticercos com uma reação imunoenzimática (ELISA). RESULTADOS: A reação ELISA LV apresentou 100 por cento de sensibilidade e especificidade em amostras de LCR e soro, enquanto a sensibilidade e a especificidade da reação ELISA LLa-Gp em amostras de LCR e soro foram de 90,9 por cento e 97,1 por cento e 95,5 por cento e 100 por cento, respectivamente. Não foram encontradas diferenças significativas na sensibilidade e especificidade das duas preparações antigênicas utilizadas, tanto para amostras de LCR como para amostras de soro. CONCLUSÃO: Considerando a complexidade e o alto custo de obtenção da fração LLa-Gp, o LV pode ser mais adequado para a pesquisa de anticorpos específicos por ELISA em amostras de LCR e soro de pacientes com neurocisticercose.
Assuntos
Animais , Humanos , Anticorpos Anti-Helmínticos/líquido cefalorraquidiano , Antígenos de Helmintos , Imunoglobulina G/líquido cefalorraquidiano , Neurocisticercose/diagnóstico , Taenia solium/imunologia , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Estudos de Casos e Controles , Cromatografia de Afinidade , Líquido Cístico/imunologia , Cysticercus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas/imunologia , Imunoglobulina G/sangue , Neurocisticercose/imunologia , Lectinas de Plantas/imunologia , Sensibilidade e EspecificidadeRESUMO
Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.
Assuntos
Lectinas de Ligação a Manose/química , Phaseolus/metabolismo , Lectinas de Plantas/química , Sementes/metabolismo , Sequência de Aminoácidos , Aminobutiratos/química , Animais , Sítios de Ligação , Cálcio/química , Carragenina , Simulação por Computador , Cristalografia por Raios X , Edema/induzido quimicamente , Edema/imunologia , Hemaglutinação , Membro Posterior , Ligação de Hidrogênio , Masculino , Manganês/química , Lectinas de Ligação a Manose/antagonistas & inibidores , Lectinas de Ligação a Manose/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Monossacarídeos/farmacologia , Lectinas de Plantas/antagonistas & inibidores , Lectinas de Plantas/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Alinhamento de Sequência , Análise de Sequência de Proteína , Trissacarídeos/químicaRESUMO
Cramoll 1,4 is a lectin with specific glucose/mannose binding, which is extracted from seeds of Cratylia mollis Mart. Many assays have shown the cytokine expression activity and anti-inflammatory profile of this lectin. The aim of this study was to evaluate the immunostimulatory response, in vitro, of splenocytes in mice previously inoculated, in vivo, with C. mollis (Cramoll 1,4) and Canavalia ensiformis (Con A) lectins. Results demonstrated higher proliferation indexes induced by Cramoll 1,4 than Con A lectin in relation to all experimental groups. Cramoll 1,4 and Con A also induced high levels of IL-2, IL-6, IFN-γ and nitric oxide production. Moreover, Cramoll 1,4 did not induce apoptosis and stimulated a significant number of cells in the S phase of the cell cycle. Results showed that Cramoll 1,4 lectin induces proliferative response and suggested that this lectin can be used as a mitogenic agent in immunostimulatory assays.
Assuntos
Interferon gama/imunologia , Interleucina-2/imunologia , Interleucina-6/imunologia , Mitose , Lectinas de Plantas/imunologia , Baço/citologia , Baço/imunologia , Animais , Proliferação de Células , Células Cultivadas , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-6/biossíntese , Linfócitos/citologia , Linfócitos/imunologia , Masculino , Camundongos , Óxido Nítrico/biossínteseRESUMO
The tumor-associated structure N-acetyl-galactosamine-O-Ser/Thr (Tn antigen), which is overexpressed in various tumor cell types, notably of the breast, ovary and colon, is an interesting determinant that is useful for cancer diagnosis and follow-up. The aim of this research was to study different assay strategies in order to determine the most sensitive system for further application in epitope characterization and binding assessment. The tetrameric isolectin obtained from Vicia villosa seeds (VVLB(4)) shows high affinity for the tumor-associated structure. A monoclonal antibody against VVLB(4), MabVV(34), was generated, and the interaction between MabVV(34) and VVLB(4) was studied by means of binding and inhibition assays. Several synthetic peptides (10 amino acid sequences) designed from the amino acid sequence of VVLB(4) and obtained from trypsin digestion were tested to determine which amino acids were involved in the interaction between MabVV(34) and VVLB(4). The further unraveling of this epitope was investigated by inhibition using designed synthetic peptides as well as mixtures mimicking variable density effect. Under the experimental circumstances, MabVV(34) was able to inhibit the binding of VVLB(4) to Tn. Two of the four peptide sequences assayed showed better inhibition properties. Finally, mixtures containing these selected sequences allowed the evaluation of binding and inhibition as a function of Tn density. We conclude that the present study facilitates the further development of a specific Tn marker and may contribute to the development of Tn-like radiolabelled peptides or Tn-specific radiolabelled fragments providing a highly selective tool for cancer diagnosis and treatment. This strategy may contribute to characterize the new generation of radiopharmaceuticals for diagnosis and therapy based on biomolecules like antibodies, fragments or peptides, whose application is directly guided by their specific molecular recognition.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Ligação Competitiva , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Antígenos Glicosídicos Associados a Tumores/metabolismo , Sítios de Ligação , Epitopos/imunologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Lectinas de Plantas/química , Lectinas de Plantas/imunologia , Radioimunoensaio , ViciaRESUMO
Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.
Assuntos
Alérgenos/química , Peptídeos Catiônicos Antimicrobianos/química , Mapeamento de Epitopos , Epitopos de Linfócito B/química , Hipersensibilidade ao Látex/imunologia , Lectinas de Plantas/química , Alérgenos/imunologia , Sequência de Aminoácidos , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Epitopos de Linfócito B/imunologia , Humanos , Imunoglobulina E/sangue , Dados de Sequência Molecular , Lectinas de Plantas/imunologia , Conformação Proteica , Alinhamento de SequênciaRESUMO
The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.
Assuntos
Adjuvantes Imunológicos , Euphorbiaceae/química , Leishmaniose Cutânea/imunologia , Lectinas de Plantas/imunologia , Animais , Anticorpos Antiprotozoários/imunologia , Formação de Anticorpos , Antígenos de Protozoários/imunologia , Citocinas/genética , Citocinas/imunologia , Hipersensibilidade Tardia/imunologia , Imunização , Imunoglobulina G/imunologia , Látex/química , Leishmania/imunologia , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Lectinas de Plantas/isolamento & purificação , Vacinas Protozoárias/imunologia , Vacinas Protozoárias/farmacologia , Pele/patologiaRESUMO
Hevein (Hev b 6.02) is a major IgE-binding allergen in natural rubber latex and manufactured products. Both tryptophans (Trp(21) and Trp(23)) of the hevein molecule were chemically modified with BNPS-skatole (2-nitrophenylsulfenyl-3-methyl-3(')-bromoindolenine); derivatized allergen failed to significantly inhibit binding of serum IgE in ELISA assays. Similarly, skin prick tests showed that hevein-positive patients gave no response with the modified allergen. Dot blot experiments carried out with anti-hevein mono- and polyclonal antibodies confirmed the importance of Trp(21) and Trp(23) for antibody-recognition, and demonstrated the specific cross-reactivity of other molecules containing hevein-like domains. We also report the structure of Hev b 6.02 at an extended resolution (1.5A) and compare its surface properties around Trp residues with those of similar regions in other allergens. Overall our results indicate that the central part of the protein, which comprises three aromatic and other acidic and polar residues, constitutes a conformational epitope.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Mapeamento de Epitopos/métodos , Epitopos/química , Imunoglobulina E/química , Hipersensibilidade ao Látex/induzido quimicamente , Modelos Moleculares , Lectinas de Plantas/química , Triptofano/química , Alérgenos/química , Alérgenos/imunologia , Alérgenos/toxicidade , Sequência de Aminoácidos , Criança , Pré-Escolar , Simulação por Computador , Cristalografia por Raios X , Humanos , Hipersensibilidade Imediata/induzido quimicamente , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/imunologia , Lactente , Recém-Nascido , Hipersensibilidade ao Látex/imunologia , Dados de Sequência Molecular , Lectinas de Plantas/imunologia , Lectinas de Plantas/toxicidade , Conformação Proteica , Testes Cutâneos , Relação Estrutura-AtividadeRESUMO
The SALT protein is a 14.5 kDa mannose-binding lectin, originally described as preferentially expressed in rice plant roots in response to NaCl stress. Recombinant SALT lectin was produced in Escherichia coli from a cDNA clone encoding protein. After isopropyl-beta-d-thiogalactopyranoside induction, the expression level achieved was 23% of the soluble protein. The recombinant agglutinin was purified by a single-step process by dialyses against a high concentrated salt solution. After purification, hemagglutination assays of rabbit erythrocytes revealed that the recombinant SALT protein is a potent agglutinin (0.078 microg ml(-1) minimal concentration). The purified recombinant lectin was also used for comparative estimation of native protein amounts in protein extracts from rice plants by Western blot assay.
Assuntos
Oryza/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/isolamento & purificação , Animais , Eletroforese em Gel de Poliacrilamida/métodos , Eritrócitos/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Testes de Hemaglutinação/métodos , Oryza/genética , Lectinas de Plantas/biossíntese , Lectinas de Plantas/genética , Lectinas de Plantas/imunologia , Lectinas de Plantas/isolamento & purificação , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
A novel lectin from Talisia esculenta seeds (TEL) has recently been purified and characterized. In this study we investigated the proinflammatory activity of TEL in mice using both the air-pouch and peritoneal cavity as well as paw oedema models. TEL (10-40 microg) induced significant neutrophil and mononuclear cell recruitment when injected into either mouse air-pouch or peritoneal cavity. The neutrophil accumulation into the air-pouch was dose- and time-dependent with a maximal response at 16 h, returning to control levels at 72 h whereas maximal mononuclear cell accumulation was observed at 24 h after TEL injection. The same profile of neutrophil accumulation was observed when this lectin was injected into mouse peritoneal cavity, although the maximal mononuclear cell recruitment was observed 48 h after TEL injection. Additionally, TEL (12.5-200 microg/paw) caused a dose-dependent mice paw, as evaluated at 4 h after the lectin injection. D-mannose, better than D-glucose, significantly inhibited TEL-induced neutrophil migration into the peritoneal cavity or air-pouch. D-galactose had no effect on TEL-induced neutrophil migration in either cavity studied. On the other hand, D-mannose slightly inhibited the TEL-induced paw oedema, whereas neither D-glucose nor D-galactose affected this phenomenon. In conclusion, our data show that TEL induces neutrophil and mononuclear cell accumulation by a mechanism related to their specific sugar-binding properties.
Assuntos
Inflamação/induzido quimicamente , Inflamação/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Neutrófilos/imunologia , Lectinas de Plantas/toxicidade , Análise de Variância , Animais , Carboidratos/imunologia , Quimiotaxia de Leucócito/imunologia , Relação Dose-Resposta Imunológica , Edema/induzido quimicamente , Edema/imunologia , Camundongos , Cavidade Peritoneal/citologia , Lectinas de Plantas/imunologia , Sapindaceae/química , Sementes/químicaRESUMO
Ligaria cuneifolia has been used in Argentine folk medicine and is currently employed as substitute for the European mistletoe (Viscum album) as hypotensor agent. Extracts from V. album are widely used in cancer therapy and the antineoplasic effect is attributed to their cytostatic/cytotoxic and immunomodulatory actions. When studying immunomodulatory effects of L. cuneifolia extracts (Lc extracts), they inhibited proliferation of murine mitogen-activated lymphocytes, leukaemic lymphocytes (LB) and breast tumour cells (MMT). The aim of this work was to isolate and identify lectins from Lc extracts and investigate their immunobiological actions. A galactoside lectin (L-Lc) of 57 kDa was isolated. A polyclonal antiserum obtained against Lc extract recognised both L-Lc and MLI (V. album lectin), suggesting the possibility of shared epitopes. Treatment of LB tumour cells with L-Lc (0.01 and 0.1 microg/ml) produced up to 40.0+/-6.9% inhibition of cell growth, which seems partly mediated by apoptosis (apoptosis of L-Lc treated cells 58.4+/-10.3% versus non-treated cells 38.1+/-8.8%; P<0.05), analysed by acridine orange and ethidium bromide staining. Inhibitory effect on ConA stimulated splenocyte growth was non-significant, while a mitogenic effect was observed on normal murine splenocytes and MMT cells. L-Lc in non-cytotoxic concentrations (250 ng/ml) modified mRNA expression of IL-10 but neither that of TGF-beta nor of IL-2 produced by LB cells. In addition, 43.9+/-0.5% reduction in NO production by LPS-stimulated murine macrophages was found. Finally, survival rates of LB tumour-bearing mice treated or not with Lc extract or L-Lc failed to show significant differences.
Assuntos
Adjuvantes Imunológicos/farmacologia , Galactosídeos/farmacologia , Loranthaceae , Lectinas de Plantas/farmacologia , Adjuvantes Imunológicos/isolamento & purificação , Animais , Apoptose , Argentina , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Galactosídeos/imunologia , Galactosídeos/isolamento & purificação , Técnicas In Vitro , Loranthaceae/química , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias Experimentais/mortalidade , Óxido Nítrico/biossíntese , Extratos Vegetais/farmacologia , Lectinas de Plantas/imunologia , Lectinas de Plantas/isolamento & purificação , RNA Mensageiro/metabolismo , Baço/citologia , Baço/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 degrees C; however, at 55 degrees C EiLL lost more than 90% of its activity and at 60 degrees C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS.