RESUMEN
Lectin is a carbohydrate-binding protein that recognizes specific cells by binding to cell-surface polysaccharides. Tumor cells generally show various glycosylation patterns, making them distinguishable from non-cancerous cells. Consequently, lectin has been suggested as a good anticancer agent. Herein, the anticancer activity of Bryopsis plumosa lectins (BPL1, BPL2, and BPL3) was screened and tested against lung cancer cell lines (A549, H460, and H1299). BPL2 showed high anticancer activity compared to BPL1 and BPL3. Cell viability was dependent on BPL2 concentration and incubation time. The IC50 value for lung cancer cells was 50 µg/mL after 24 h of incubation in BPL2 containing medium; however, BPL2 (50 µg/mL) showed weak toxicity in non-cancerous cells (MRC5). BPL2 affected cancer cell growth while non-cancerous cells were less affected. Further, BPL2 (20 µg/mL) inhibited cancer cell invasion and migration (rates were Ë20%). BPL2 induced the downregulation of epithelial-to-mesenchymal transition-related genes (Zeb1, vimentin, and Twist). Co-treatment with BPL2 and gefitinib (10 µg/mL and 10 µM, respectively) showed a synergistic effect compared with monotherapy. BPL2 or gefitinib monotherapy resulted in approximately 90% and 70% cell viability, respectively, with concomitant treatment showing 40% cell viability. Overall, BPL2 can be considered a good candidate for development into an anticancer agent.
Asunto(s)
Antineoplásicos , Chlorophyta , Lectinas de Unión a Manosa , Humanos , Antineoplásicos/farmacología , Línea Celular Tumoral , Chlorophyta/química , Gefitinib/farmacología , Neoplasias Pulmonares , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/farmacologíaRESUMEN
Apoptosis, a well-known pattern of programmed cell death, occurs in multicellular organisms not only for controlling tissue homeostasis but also for getting rid of severely damaged cells in order to protect the redundant growth of abnormal cells undergoing cancerous cells. The epidermis of the human skin, composed largely of keratinocytes (KCs), is renewed continuously. Therefore, KCs apoptosis plays a critical role in the maintenance of epidermis structure and function. However, regulated cell death can be disturbed by environmental factors especially ultraviolet radiation (UV) B, leading to the formation of sunburn cells (KCs undergoing UVB-induced apoptosis) and impairing the skin integrity. In the present study, we firstly reported the potential of the natural artocarpin (NAR) to regulate UVB-induced human KCs apoptosis. The NAR showed antilipid peroxidation with an IC50 value of 18.2 ± 1.6 µg/mL, according to TBARS assay while the IC50 value of trolox, a well-known antioxidant, was 7.3 ± 0.8 µg/mL. For cell-based studies, KCs were pretreated with 3.1 µg/mL of the NAR for 24 hr and then exposed to UVB at 55 mJ/cm2. Our data indicated that the NAR pretreatment reduces UVB-induced oxidative stress by scavenging free radicals and nitric oxide and therefore prevents reactive oxygen species (ROS) and reactive nitrogen species- (RNS-) mediated apoptosis. The NAR pretreatment has been shown also to reduce the UVB-induced cyclobutane pyrimidine dimer (CPD) lesions by absorbing UVB radiation and regulating the cell cycle phase. Additionally, the NAR pretreatment was found to modulate the expression of cleaved caspases-3 and 8 that trigger different signalling cascades leading to apoptosis. Thus, these results provide a basis for the investigation of the photoprotective effect of the NAR isolated from A. altilis heartwood and suggest that it can be potentially used as an agent against UVB-induced skin damages.
Asunto(s)
Apoptosis/efectos de los fármacos , Lectinas de Unión a Manosa/química , Lectinas de Plantas/química , Protectores contra Radiación/farmacología , Rayos Ultravioleta , Antioxidantes/química , Apoptosis/efectos de la radiación , Artocarpus/química , Artocarpus/metabolismo , Caspasa 3/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Cromatografía Líquida de Alta Presión , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/farmacología , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/química , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/farmacología , Protectores contra Radiación/química , Protectores contra Radiación/aislamiento & purificación , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Plant lectins are carbohydrate-binding proteins with nonimmune origin, which can reversibly bind with carbohydrates, agglutinate cells, and precipitate polysaccharides and glycoconjugates. Plant lectins have attracted much attention for their anti-virus, anti-proliferation, and pro-apoptosis properties. Thus the exploration of new lectins has received special attention. Here we purified a mannose-binding lectin from the rhizomes of Liparis nervosa by ion exchange chromatography on DEAE-Sepharose, affinity chromatography on Mannose-Sepharose 4B, and gel filtration chromatography on Sephacryl S-100. The purified L. nervosa lectin (LNL) was identified to be a monomeric protein with a molecular mass of 13 kDa. LNL exhibited hemagglutinating activity towards rabbit erythrocytes, and its activity could be strongly inhibited by D-mannose, N-acetyl glucosamine and thyroglobulin. In vitro experiments showed that LNL exhibited a comparable anti-fungal activity against Piricularia oryzae (Cavara), Bipolaris maydis, Fusarium graminearum, and Sclerotium rolfsii, and anti-proliferation activity against tumor cells by inducing apoptosis. The full-length cDNA sequence of LNL is 715 bp in length and contains a 525 bp open reading frame (ORF) encoding a 110-residue mature protein. It was predicted to have three mannose-binding conserved motifs 'QXDXNXVXY'. The binding pattern of LNL was further revealed by homology modeling and molecular docking. We demonstrated that LNL is not only a potential therapeutic candidate against tumor but also a new anti-fungal agent.
Asunto(s)
Antifúngicos/farmacología , Antineoplásicos/farmacología , Lectinas de Unión a Manosa/farmacología , Orchidaceae/química , Lectinas de Plantas/farmacología , Secuencia de Aminoácidos , Animales , Antifúngicos/química , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Basidiomycota/efectos de los fármacos , Bipolaris/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Fusarium/efectos de los fármacos , Hemaglutinación/efectos de los fármacos , Humanos , Manosa/metabolismo , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/metabolismo , Simulación del Acoplamiento Molecular , Peso Molecular , Orchidaceae/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/metabolismo , Conejos , Homología de Secuencia de AminoácidoRESUMEN
Animal leguminous-type (L-type) lectins, including ERGIC-53 and VIP36 are responsible for intracellular transport and quality control of N-linked glycoproteins in the early secretory pathway. These lectins possess the carbohydrate recognition domain (CRD), which recognizes high-mannose-type glycans in a Ca2+-dependent manner. Here we describe the procedures involved in bacterial overproduction and purification of the CRDs of the animal L-type lectins.
Asunto(s)
Lectinas/aislamiento & purificación , Lectinas/metabolismo , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/metabolismo , Manosa/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Animales , Calcio/metabolismo , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Lectinas de Unión a Manosa/química , Proteínas de Transporte de Membrana/química , Unión Proteica , Dominios ProteicosRESUMEN
The Galanthus nivalis lectin, abbreviated as GNA, is the model protein for a large group of mannose-binding lectins. Here, we describe the purification of GNA starting from dry bulbs. Using a combination of ion exchange chromatography and affinity chromatography on mannose-Sepharose, a highly pure preparation of GNA can be obtained.
Asunto(s)
Galanthus/metabolismo , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Plantas/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Manosa/química , Raíces de Plantas/metabolismo , Sefarosa/químicaRESUMEN
Pisum sativum lectin (Psl) being a high-value protein has marked its application in the biomedical and therapeutic field. Aqueous two phase extraction (ATPE) was implemented as a selective partitioning technique for the partial purification of Psl from its seeds. PEG/citrate based biodegradable aqueous two phase system (ATPS) was screened and the factors such as the type and concentration of citrate salts, molar mass and concentration of polyethylene glycol (PEG), tie line length (TLL) and additive (NaCl) concentration, pH, crude load and volume ratio were studied for the selective partition of Psl. The Psl was successfully extracted to the top phase in the ATPS formed with 18% PEG 6000/16% sodium citrate at 41.01% TLL, 2% NaCl and pH of 7.5. A volume ratio of 0.76 and a crude load of 20% showed maximum activity yield of 122.12% with the purification factor of 16.26. The subunits of Psl namely α and ß were identified with a molecular weight of 6 and 18â¯kDa respectively during the purity analysis using SDS PAGE and HPLC.
Asunto(s)
Extracción Líquido-Líquido/métodos , Lectinas de Unión a Manosa/aislamiento & purificación , Pisum sativum/química , Lectinas de Plantas/aislamiento & purificación , Semillas/química , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Lectinas de Unión a Manosa/análisis , Lectinas de Unión a Manosa/química , Lectinas de Plantas/análisis , Lectinas de Plantas/química , Polietilenglicoles/química , Citrato de Sodio/químicaAsunto(s)
Dioclea/química , Lectinas de Unión a Manosa/química , Lectinas de Plantas/química , Semillas/química , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cristalización , Glioma , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/farmacología , Simulación del Acoplamiento Molecular , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/farmacología , Unión Proteica , Conformación Proteica , RatasRESUMEN
A lectin with insecticidal property against the stored product pest, Callosobruchus maculatus was successfully isolated from the seeds of Canavalia virosa using standard affinity chromatography. The isolated molecule typically behaved like a lectin in its characteristics. It agglutinated indicator red blood cells (RBC) in its native as well as enzyme treated conditions. The enzyme treated RBC types exhibited a very high hemagglutination (HA) titre values and this property of isolated molecule behaved like arcelin, the lectin-like molecules reported from several species of Phaseolus. As a characteristic feature of a lectin, the isolated molecule effectively inhibited the agglutination of indicator RBC types with simple and complex carbohydrates including glycoproteins. This nature of the isolated molecule also relate with characteristic feature of arcelin isoforms in inhibiting HA activity with complex glycoproteins as reported in many studies. Most interestingly, the present study disclosed trehalose as a potent inhibitor of C. virosa lectin. Therefore, feeding insect pests on the lectin like arcelin could serve as antibiosis factor/anti-insect activity. The molecular characteristics of this isolated molecule and its mass studies too revealed its homology with arcelin, arcelin-1, 2 and 6 isoforms of P. vulgaris and lectin from Canavalia cathartica, C. lineata and C. brasiliensis.
Asunto(s)
Canavalia/química , Escarabajos , Maltosa/metabolismo , Lectinas de Unión a Manosa/análisis , Lectinas de Unión a Manosa/aislamiento & purificación , Trehalosa/metabolismo , Adsorción , Secuencia de Aminoácidos , Animales , Bioensayo , Ácido Edético/química , Hemaglutinación/efectos de los fármacos , Concentración de Iones de Hidrógeno , Insecticidas/análisis , Insecticidas/aislamiento & purificación , Insecticidas/metabolismo , Insecticidas/farmacología , Lectinas de Unión a Manosa/metabolismo , Lectinas de Unión a Manosa/farmacología , Estabilidad Proteica , Conejos , Semillas/química , Especificidad por Sustrato , TemperaturaRESUMEN
CONTEXT: Antibacterial resistance has dramatically increased and resulted in serious health problems worldwide. One appealing strategy to overcome this resistance problem is the use of combinations of antibacterial compounds to increase their potency. OBJECTIVE: The objective of this study is to determine the synergistic effects of artocarpin for ampicillin, norfloxacin, and tetracycline against methicillin-resistant Staphylococcus aureus (MRSA) as well as the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli. MATERIALS AND METHODS: A broth microdilution method (1.95-250 µg/mL) was used to determine the minimum inhibitory concentration (MIC) of artocarpin and the antibiotics. Any synergistic effects were evaluated at their own MIC using the checkerboard method and a time-kill assay at 37 °C for 24 h. RESULTS AND DISCUSSION: Artocarpin showed antibacterial activity against MRSA and E. coli with an MIC value of 62.5 µg/mL, and against P. aeruginosa with an MIC value of 250 µg/mL. The interaction of artocarpin with all tested antibiotics produced synergistic effects against MRSA with a fractional inhibitory concentration index (FICI) of 0.15-0.37. In addition, a combination of artocarpin and norfloxacin showed a synergistic effect against E. coli with an FICI value of 0.37, while the combinations of artocarpin and tetracycline as well as artocarpin and norfloxacin exhibited synergy interactions against P. aeruginosa with FICI values of 0.24 and 0.37, respectively. Time-kill assays indicated that artocarpin enhanced the antimicrobial activities of tetracycline, ampicillin, and norfloxacin against MRSA as well as Gram-negative bacteria.
Asunto(s)
Antibacterianos/administración & dosificación , Escherichia coli/efectos de los fármacos , Lectinas de Unión a Manosa/administración & dosificación , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Lectinas de Plantas/administración & dosificación , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/aislamiento & purificación , Artocarpus , Combinación de Medicamentos , Sinergismo Farmacológico , Escherichia coli/fisiología , Humanos , Lectinas de Unión a Manosa/aislamiento & purificación , Meticilina/administración & dosificación , Staphylococcus aureus Resistente a Meticilina/fisiología , Pruebas de Sensibilidad Microbiana/métodos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/aislamiento & purificación , Lectinas de Plantas/aislamiento & purificación , Pseudomonas aeruginosa/fisiologíaRESUMEN
This study aimed to purify and characterize a novel mannose-binding lectin from the seeds of Centrolobium microchaete. Centrolobium microchaete lectin (CML) was purified by affinity chromatography in mannose-Sepharose-4B column. CML agglutinated rabbit erythrocytes and was inhibited by D-mannose, α-methyl-D-mannoside, D-glucose, N-Acetyl-D-glucosamine and sucrose. The lectin was stable at pH 7.0 and 8.0 and temperatures up to 60°C. The monomeric form of CML showed approximately 28kDa, and its native form is probably a homodimer, as determined by gel filtration chromatography. The primary structure of CML was determined by tandem mass spectrometry that showed CML as a protein with two distinct forms (isolectins CML-1 and CML-2) with 246 and 247 residues, respectively. CML-2 possesses one residue of Asn more than CML-1 in C-terminal. The primary structure of CML agrees with the molecular weights found by electrospray ionization mass spectrometry: 27,224 and 27,338Da for CML-1 and CML-2, respectively. CML is a metal-dependent glycoprotein. Moreover, the glycan composition of CML and its structure were predicted.
Asunto(s)
Fabaceae/química , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/aislamiento & purificación , Semillas/química , Secuencia de Aminoácidos , Carbohidratos/química , Hemaglutinación , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Alineación de Secuencia , TemperaturaRESUMEN
Lectin sensitivity of the recent pandemic influenza A virus (H1N1-2009) was screened for 12 lectins with various carbohydrate specificity by a neutral red dye uptake assay with MDCK cells. Among them, a high mannose (HM)-binding anti-HIV lectin, ESA-2 from the red alga Eucheuma serra, showed the highest inhibition against infection with an EC50 of 12.4 nM. Moreover, ESA-2 exhibited a wide range of antiviral spectrum against various influenza strains with EC50s of pico molar to low nanomolar levels. Besides ESA-2, HM-binding plant lectin ConA, fucose-binding lectins such as fungal AOL from Aspergillus oryzae and AAL from Aleuria aurantia were active against H1N1-2009, but the potency of inhibition was of less magnitude compared with ESA-2. Direct interaction between ESA-2 and a viral envelope glycoprotein, hemagglutinin (HA), was demonstrated by ELISA assay. This interaction was effectively suppressed by glycoproteins bearing HM-glycans, indicating that ESA-2 binds to the HA of influenza virus through HM-glycans. Upon treatment with ESA-2, no viral antigens were detected in the host cells, indicating that ESA-2 inhibited the initial steps of virus entry into the cells. ESA-2 would thus be useful as a novel microbicide to prevent penetration of viruses such as HIV and influenza viruses to the host cells.
Asunto(s)
Antivirales/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Lectinas de Unión a Manosa/farmacología , Rhodophyta/química , Animales , Antivirales/química , Antivirales/aislamiento & purificación , Perros , Ensayo de Inmunoadsorción Enzimática , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Gripe Humana/prevención & control , Gripe Humana/virología , Lectinas/química , Lectinas/aislamiento & purificación , Lectinas/farmacología , Células de Riñón Canino Madin Darby , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/aislamiento & purificación , Internalización del Virus/efectos de los fármacosRESUMEN
KEY MESSAGE: Trimeric Galanthus nivalis agglutinin-related lectin of Orchidaceae with two conformational forms was first studied in Dendrobium pendulum . It was highly expressed by stress factors. Using mannan-agarose column chromatography, a mannose-binding protein was purified from Dendrobium pendulum Roxb. pseudobulb. After heating in the presence of sodium dodecyl sulfate (SDS) with or without 2-mercaptoethanol, the protein showed one band with molecular mass of 14.0 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Without heating, three bands were found at positions of 14.0, 39.4, and 41.5 kDa, but a higher amount of 39.4 and 41.5 kDa protein bands were seen in the presence of 2-mercaptoethanol. Liquid chromatography-tandem mass spectrometry and database search indicated that the 14.0 kDa protein band contained three peptide fragments identical to parts of a lectin precursor from Dendrobiu m findleyanum Parish & Rchb.f. Native-PAGE and Ferguson plot showed that the purified protein had two native forms with molecular masses of 44.2 and 45.3 kDa, indicating three 14.0 kDa polypeptide subunits. The purified protein exhibited the agglutination activity with trypsinized chicken erythrocytes. It was then recognized as a Galanthus nivalis agglutinin-related lectin and named D. pendulum agglutinin (DPA). Using reverse transcription-polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of DPA precursor showed the highest homology (96.4%) with a lectin precursor of D. findleyanum and contained three mannose-binding sites. Greater amounts of DPA were found when the pseudobulbs were treated with stress factors including ultraviolet light, abscisic acid, hydrogen peroxide, and acetylene gas.
Asunto(s)
Dendrobium/química , Lectinas/aislamiento & purificación , Lectinas de Unión a Manosa/química , Lectinas de Plantas/química , Multimerización de Proteína , Estrés Fisiológico , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Calor , Lectinas/química , Lectinas/metabolismo , Lectina de Unión a Manosa/metabolismo , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/metabolismo , Mercaptoetanol/farmacología , Datos de Secuencia Molecular , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/metabolismo , Multimerización de Proteína/efectos de los fármacos , Estándares de Referencia , Homología de Secuencia de Aminoácido , Estrés Fisiológico/efectos de los fármacosRESUMEN
Various dermatological disorders and microbial skin infection can cause hyperpigmentation. Therefore, screenings for whitening and antimicrobial agents from Thai medicinal plants have been of research interest. Seventy-seven ethanol plant extracts were investigated for antityrosinase activity, eleven samples showed the tyrosinase inhibition more than 50 % were further preliminary screening for antimicrobial activity by agar disc diffusion and broth micro-dilution methods. Artocarpus integer (Thunb.) Merr. (Moraceae) root extract, which showed the potential of tyrosinase inhibition with 90.57 ± 2.93 % and antimicrobial activity against Staphylococcus aureus, S. epidermidis, Propionibacterium acnes and Trichophyton mentagophytes with inhibition zone as 9.10 ± 0.00, 10.67 ± 0.09, 15.25 ± 0.05 and 6.60 ± 0.17 mm, respectively was selected for phytochemical investigation. Three pure compounds were isolated as artocarpin, cudraflavone C and artocarpanone. And artocarpanone exhibited anti-tyrosinase effect; artocarpin and cudraflavone C also showed the potential of antibacterial activity against S. aureus, S. epidermidis and P. acnes with MIC at 2, 4 and 2 µg/ml, respectively and MBC at 32 µg/ml for these bacteria. So, these pure compounds are interesting for further study in order to provide possibilities of new whitening and antibacterial development. This will be the first report of phytochemical investigation of A. integer root.
Asunto(s)
Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Monofenol Monooxigenasa/antagonistas & inhibidores , Extractos Vegetales/farmacología , Plantas Medicinales/química , Flavonas/aislamiento & purificación , Flavonas/farmacología , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/farmacología , Pruebas de Sensibilidad Microbiana , Lectinas de Plantas/aislamiento & purificación , Lectinas de Plantas/farmacología , TailandiaRESUMEN
Lectin from the seeds of Dioclea lasiophylla (DlyL) was purified in a single step by affinity chromatography on a Sephadex® G-50 column. DlyL strongly agglutinated rabbit erythrocytes and was inhibited by monosaccharides (D-mannose and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Similar to other Diocleinae lectins, DlyL has three chains, α, ß and γ, with mass of 25,569 ± 2, 12,998 ± 1 and 12,588 ± 1 Da, respectively, and has no disulfide bonds. The hemagglutinating activity of DlyL was optimal in pH 8.0, stable at a temperature of 70 °C and decreased in EDTA solution, indicating that lectin activity is dependent on divalent metals. DlyL exhibited low toxicity on Artemia sp. nauplii, but this effect was dependent on the concentration of lectin in solution. DlyL immobilized on cyanogen bromide-activated Sepharose® 4B bound 0.917 mg of ovalbumin per cycle, showing the ability to become a tool for glycoproteomics studies.
Asunto(s)
Dioclea/química , Hemaglutininas/farmacología , Lectinas de Unión a Manosa/farmacología , Extractos Vegetales/farmacología , Semillas/química , Animales , Artemia , Quelantes/química , Cromatografía de Afinidad , Ácido Edético/química , Eritrocitos/efectos de los fármacos , Hemaglutinación , Hemaglutininas/química , Hemaglutininas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Dosificación Letal Mediana , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/aislamiento & purificación , Ovalbúmina/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Unión Proteica , Conejos , Sefarosa/químicaRESUMEN
A new mannose/glucose-specific lectin, named DigL, was purified from seeds of Dialium guineense by a single step using a Sepharose 4b-Mannose affinity chromatography column. DigL strongly agglutinated rabbit erythrocytes and was inhibited by d-mannose, d-glucose, and derived sugars, especially α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine. DigL has been shown to be a stable protein, maintaining its hemagglutinating activity after incubation at a wide range of temperature and pH values and after incubation with EDTA. DigL is a glycoprotein composite by approximately 2.9% of carbohydrates by weight. By sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, the purified DigL exhibited an electrophoretic profile consisting of a broad band of 28-30 kDa. Analysis using electrospray ionization mass spectrometry indicated that purified DigL possesses a molecular average mass of 28 452 ± 2 Da and shows the presence of possible glycoforms. In addition, DigL exhibited an intermediary toxic effect on Artemia sp. nauplii, and this effect was both dependent on native structure and mediated by a carbohydrate-binding site.
Asunto(s)
Fabaceae/química , Glucosa/metabolismo , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/toxicidad , Semillas/química , Animales , Artemia/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Hemaglutinación/efectos de los fármacos , Concentración de Iones de Hidrógeno , Lectinas de Unión a Manosa/química , Espectrometría de Masas , Peso Molecular , Oligosacáridos/farmacología , Conejos , Temperatura , Pruebas de ToxicidadRESUMEN
Saccharomyces cerevisiae flocculation is governed by FLO genes, encoding Flo proteins (flocculins). Flo proteins are cell wall proteins consisting of three domains, sticking out of the cell wall and interacting with other yeast cells using their N-terminal mannose-binding domain. Until recently, flocculation research was focused on the genetic and cellular level. To extend the knowledge about flocculation to the protein level, we isolated the N-terminal domain of the Flo1p (N-Flo1p) that contains the mannose-binding domain, which is responsible for the strong interaction (flocculation) of S. cerevisiae cells. To obtain a high production yield and a more uniform and lower glycosylation of N-Flo1p, it was cloned in Pichia pastoris. The expression and the purification of N-Flo1p were optimised towards a one-step purification protocol. The activity of the protein, i.e. the binding of the purified protein to mannose using fluorescence spectroscopy, was demonstrated.
Asunto(s)
Lectinas de Unión a Manosa/genética , Lectinas de Unión a Manosa/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Saccharomyces cerevisiae/genética , Clonación Molecular , Floculación , Glicosilación , Manosa/metabolismo , Lectinas de Unión a Manosa/biosíntesis , Pichia , Unión Proteica , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/biosíntesis , Espectrometría de FluorescenciaRESUMEN
The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.
Asunto(s)
Antineoplásicos/farmacología , Benzoatos/farmacología , Diseño de Fármacos , Lectinas de Unión a Manosa/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Simulación de Dinámica Molecular , Piperidinas/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzoatos/síntesis química , Benzoatos/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/metabolismo , Estructura Molecular , Peso Molecular , Piperidinas/síntesis química , Piperidinas/química , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Superficie Celular/metabolismo , Relación Estructura-ActividadRESUMEN
Monocot mannose-binding lectins (MMBLs) or agglutinins are an extended superfamily of structurally and evolutionarily related proteins. They play important roles in plant defenses. Here we describe the synthesis of full-length cDNA of monocot mannose-binding insecticidal agglutinin isolated from Allium sativum, a traditional herb known to be of great applications in Africa, using reverse transcription polymerase chain reaction (RT-PCR) with specific primers designed based on the insecticidal sequence (NCBI primary accession no. AY866499.1). Sequence analysis revealed a 327bp open reading frame (ORF) encoding a putative 108-aa agglutinin precursor with a C-terminal domain. Multiple alignments of BLEC1 amino acids with those of eight other MMBLs revealed three highly conserved domains among them, indicating BLEC1 belongs to a member of the MMBL superfamily. Tertiary structure analysis showed that BLEC1 had three potential equal mannose-binding sites. Phylogenetic analysis indicated that 20 MMBLs including BLEC1 belonged to an extended superfamily. Gene ontology analyses indicate one biological process with GO ID: 0006952 representing defense response, with two secondary IDs GO: 0002217 GO: 0042829. The child terms has both negative and positive regulation some of which include GO: 0002242 defense response to parasitic plant and GO: 0002213 defense response to insect. The cloning and characterization of BLEC1 will enable us to study its potential use in plant genetic engineering in the development of insect resistance plant.
Asunto(s)
Resistencia a la Enfermedad/genética , Ajo/genética , Lectinas de Unión a Manosa/genética , Lectinas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Biología Computacional , Ajo/inmunología , Lectinas de Unión a Manosa/química , Lectinas de Unión a Manosa/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Enfermedades de las Plantas/inmunología , Lectinas de Plantas/química , Lectinas de Plantas/aislamiento & purificación , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Mannose-specific lectin Concanavalin A (Con A) was purified from Canavalia ensiformis seeds. For this purpose, mannose attached poly(hydroxyethyl methacrylate) (PHEMA) cryogel was prepared by cryopolymerization. Mannose was used as the affinity ligand and was covalently attached onto the PHEMA cryogel via carbodiimide activation. The PHEMA cryogel containing 23.3 mmol mannose/g polymer were used in the binding studies. Con A binding with the mannose attached PHEMA cryogel from Con A aqueous solution was 5.2 mg/g at pH 7. Maximum binding capacity for Con A from C. ensiformis seed extract was 39 mg/g. Con A was eluted with 0.3 M galactose, and the purity of Con A was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was observed that the mannose attached PHEMA cryogel can be used without significant decrease in Con A binding capacity after six binding-elution cycles.
Asunto(s)
Canavalia/embriología , Criogeles , Lectinas de Unión a Manosa/aislamiento & purificación , Polihidroxietil Metacrilato/química , Semillas/química , Electroforesis en Gel de Poliacrilamida , Microscopía Electrónica de Rastreo , Concentración Osmolar , TemperaturaRESUMEN
A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5 kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528 bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manß1-4GlcNAcß1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.