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PLoS One ; 15(12): e0243867, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33338036


The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ß subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.

Proteínas de Bactérias/metabolismo , Periplasma/metabolismo , Proteômica , Xanthomonas/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono/metabolismo , Genes Bacterianos , Anotação de Sequência Molecular , Fosfoglucomutase/metabolismo , Reprodutibilidade dos Testes , Xanthomonas/enzimologia , Xanthomonas/genética , Xanthomonas/crescimento & desenvolvimento
Eur Biophys J ; 39(8): 1193-205, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20047048


Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-D-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.

Fabaceae/química , Lectinas de Plantas/química , Acetilgalactosamina/química , Neoplasias da Mama/química , Carboidratos/química , Adesão Celular , Linhagem Celular Tumoral , Dicroísmo Circular , Feminino , Gleiquênias , Hemaglutinação , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Lectinas de Plantas/genética , Lectinas de Plantas/isolamento & purificação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Raios Ultravioleta , Difração de Raios X
FEBS J ; 275(5): 948-59, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18215161


Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.

Abrus/química , Proteínas Inativadoras de Ribossomos Tipo 2/química , Proteínas Inativadoras de Ribossomos Tipo 2/toxicidade , Sequência de Aminoácidos , Carboidratos/química , Células HeLa , Hemaglutinação/efeitos dos fármacos , Testes de Inibição da Hemaglutinação , Humanos , Concentração Inibidora 50 , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/toxicidade , Estrutura Secundária de Proteína , Proteínas Inativadoras de Ribossomos Tipo 2/isolamento & purificação , Sementes/química , Alinhamento de Sequência
Biochim Biophys Acta ; 1770(12): 1660-6, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17920772


Most of the type 2 ribosome-inactivating proteins (RIPs) are toxins formed by an RNA-N-glycosidase A-chain polypeptide linked to a lectin B-chain by a single disulfide bond. Members of this protein class vary greatly in cytotoxity, correlating more with B-chain diversity rather than to A-chain differences. Pulchellin is a type 2 ribosome-inactivating protein toxin found in the seeds of Abrus pulchellus tenuiflorus. Recombinant pulchellin B-Chain (rPBC) has been previously produced as inclusion bodies in Escherichia coli and successfully refolded recovering biological activity. New approaches for using this kind of protein as a biotechnological tool require a better understanding of cell targeting, binding, uptake, intracellular routing and delivery. In this work, cell adhesion experiments were used to determine the interaction of rPBC with mammalian cells. Fluorescence and confocal microscopy revealed the intracellular localization and trafficking. Subcellular sorting of the native pulchellin could also be determined. The results support that the endosomal internalization pathway and the retrograde transport through the Golgi apparatus might be used by both native protein and rPBC.

Endocitose , Proteínas Inativadoras de Ribossomos Tipo 2/metabolismo , Animais , Humanos , Células K562 , Camundongos , Ligação Proteica , Proteínas Recombinantes/metabolismo
FEBS J ; 272(5): 1201-10, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15720394


Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of approximately 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 microg x kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.

Abrus/genética , Proteínas de Plantas/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/metabolismo , Abrus/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , DNA de Plantas/metabolismo , Injeções Intraperitoneais , Camundongos , Dados de Sequência Molecular , N-Glicosil Hidrolases/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/toxicidade , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/toxicidade , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/toxicidade , Saccharomyces cerevisiae/metabolismo , Sementes/química , Homologia de Sequência de Aminoácidos