RESUMO
Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N-linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to ß1-6 branched N-glycans' characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.
Assuntos
Lectinas/química , Neoplasias/metabolismo , Phaseolus/química , Polissacarídeos/química , Proteínas Recombinantes/química , Cristalografia por Raios X , Glicosilação , Humanos , Lectinas/biossíntese , Ligação Proteica , Proteínas Recombinantes/biossínteseRESUMO
We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 µM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans.
Assuntos
Proteínas de Algas/química , Fucose/química , Lectinas/química , Polissacarídeos/química , Rodófitas/química , Proteínas de Algas/biossíntese , Proteínas de Algas/isolamento & purificação , Animais , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Eritrócitos/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Lectinas/biossíntese , Lectinas/isolamento & purificação , Dados de Sequência Molecular , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por SubstratoRESUMO
Spermadhesins, a family of secretory proteins from the male genital tract of ungulate species, belong to the group of animal lectins. Spermadhesins have a prominent role in different aspects of fertilisation, such as spermatozoid capacitation, acrosomal stabilisation, sperm-oviduct interaction and during sperm-oocyte fusion. Proteins (spermadhesins) in buck seminal plasma were described. In the present study, bodhesin Bdh-2 cDNA present in buck seminal plasma was subcloned with the expression plasmid pTrcHis TOPO used to transform Escherichia coli Top10 One shot cells. The recombinant clones were selected by growth in 50 µg mL⻹ ampicillin-containing LB broth and polymerase chain reaction amplification. Recombinant rBdh-2His6 synthesis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by immunoblotting using monoclonal anti-His antibody. Production of rBdh-2 using low temperatures was not satisfactory. Greater production of rBdh-2 occurred with 1.5mM isopropyl ßd-thiogalactoside after 2h of induction. The method used to purify rBdh-2 was affinity chromatography on a His-Trap column following ion-exchange chromatography on a DEAE-Sephacel column. The secondary structure of the rBdh-2His6 was evaluated by spectral profile circular dichroism (CD). The prevalence of secondary structures like ß-sheets, with fewer unfolded structures and α-helices, was confirmed. The structure of rBdh-2His6 remained stable up to 35°C. However, significant structural changes were observed at temperatures higher than 40 °C related to a distortion of the CD spectrum.
Assuntos
Cabras/metabolismo , Sêmen/metabolismo , Proteínas de Plasma Seminal/biossíntese , Proteínas de Plasma Seminal/química , Animais , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Dicroísmo Circular , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Isopropiltiogalactosídeo/farmacologia , Lectinas/biossíntese , Lectinas/química , Lectinas/genética , Lectinas/isolamento & purificação , Masculino , Biossíntese de Proteínas/efeitos dos fármacos , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas de Plasma Seminal/genética , Proteínas de Plasma Seminal/isolamento & purificação , TemperaturaRESUMO
Elimination of the helminth parasite Nippostrongylus brasiliensis from infected mice is mediated by IL-4 or IL-13 and dependent on the IL-4Ralpha chain and the transcription factor Stat6 in non-hematopoietic cells. However, it is not clear which Stat6-dependent effector molecules mediate worm expulsion. We identified intelectin-1 and -2 as Stat6-dependent genes that are induced during infection. Intelectins can bind galactofuranose, a sugar present only in microorganisms and might therefore serve as microbial pattern element. To analyze whether constitutive expression of intelectin-1 or -2 leads to accelerated pathogen clearance, transgenic mice were generated which express high levels of these genes selectively in the lung. Infection with N. brasiliensis or Mycobacterium tuberculosis did not result in accelerated pathogen clearance in transgenic as compared to wild-type mice. Further, no significant modulation of the immune response in lung or lymph nodes was observed. Thus, under these conditions, intelectins did not enhance pathogen clearance.
Assuntos
Citocinas/genética , Lectinas/genética , Nippostrongylus/genética , Fator de Transcrição STAT6/fisiologia , Infecções por Strongylida/genética , Animais , Citocinas/biossíntese , Citocinas/imunologia , Citometria de Fluxo , Proteínas Ligadas por GPI , Expressão Gênica , Mucosa Intestinal/metabolismo , Lectinas/biossíntese , Lectinas/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Nippostrongylus/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Strongylida/imunologia , Infecções por Strongylida/metabolismoRESUMO
A soluble beta-galactoside lectin purified from Bufo arenarum ovary agglutinated homologous neuraminidase-treated spermatozoa. Microscopic observations of sperm clusters showed that spermatozoa agglutinated in a random way, but the head-to-head type of sperm agglutination was the most common (94-98%). The lectin activity was specifically inhibited by D-galactose and its derivatives, thio-digalactoside being the most active saccharide inhibitor.