RESUMEN
A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non-reducing conditions. PCL is a blood group non-specific lectin and has highest affinity towards chitin, mucin, asialomucin, fetuin with a MIC of 0.15 µg/mL and also recognizes L-fucose, galactose, lactose, N-acetyl galactosamine, hyaluronic acid. PCL is stable up to 60 °C and within the pH range 4-8. To understand its role in pathogenesis, effect of PCL was evaluated on human corneal epithelial cells (HCECs). PCL showed strong glycan mediated binding to HCECs and PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.
Asunto(s)
Antígenos de Grupos Sanguíneos , Queratitis , Humanos , Lectinas , Fucosa , Galactosa , Lactosa , Sulfato de Amonio/metabolismo , Sefarosa , Ácido Hialurónico , Interleucina-6 , Queratitis/microbiología , Quitina/metabolismo , Fetuínas , Mucinas/metabolismo , Mezclas Complejas , GalactosaminaRESUMEN
BACKGROUND: Altered expression of N-glycans such as polylactosamine is observed in colon cancer. AHL, a polylactosamine specific lectin from Adenia hondala from a medicinal plant from the Passifloraceae family has been reported earlier. OBJECTIVE: The aim of the present study is to study the interaction of AHL with human colon cancer epithelial HT-29 cells and colon cancer tissues. METHODS: Cell viability was determined by MTT [3-[4, 5- dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide] assay, while cell surface binding, apoptosis by Annexin-V-PI assay and ROS production using DCFDA [2',7' - dichlorofluorescindiacetate] kit method were analysed by flowcytometry, immunohistochemistry was performed using biotinylated AHL, protein purification by affinity chromatography using asialofetuin-coupled Sepharose -4B column. RESULTS: AHL strongly binds to HT-29 cells with a Mean Fluorescence Intensity of 12.4, which could be blocked by competing glycoprotein asialofetuin. AHL inhibits HT-29 cell growth in a dose and time-dependent manner with IC50 of 2.5 µg/mL and differentially binds to human normal and cancerous tissues. AHL induces apoptosis and slight necrosis in HT-29 cells with an increase in the early apoptotic population of 25.1 and 36% for 24 h and 48 h respectively and necrotic population of 1.5 and 4.6% at 24 h and 48 h respectively as revealed by Annexin-V-PI assay. AHL induces the release of Reactive Oxygen Species in HT-29 cells in a dose-dependent manner. CONCLUSION: To the best of knowledge, this is the first report on lectin from Adenia hondala which is not a RIP with apoptotic and necrotic effects. These findings support the promising potential of AHL in cancer research.
Asunto(s)
Amino Azúcares/química , Neoplasias del Colon/tratamiento farmacológico , Lectinas/química , Necrosis/tratamiento farmacológico , Passifloraceae/química , Extractos Vegetales/química , Polisacáridos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HT29 , Humanos , Lectinas/farmacología , Extractos Vegetales/farmacología , Especies Reactivas de OxígenoRESUMEN
Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3 µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5 µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.
Asunto(s)
Acremonium/metabolismo , Células Epiteliales/efectos de los fármacos , Epitelio Corneal/citología , Queratitis/microbiología , Lectinas/toxicidad , Apoptosis , Línea Celular , Proliferación Celular , Humanos , Queratitis/patología , Lectinas/inmunología , Factor 88 de Diferenciación MieloideRESUMEN
The L-fucose-specific lectin from Aspergillus niger (ANL), isolated from the corneal smears of a keratitis patient was reported earlier. Here, we examined the interaction of ANL with human hepatocellular and colon cancer cells, evaluated its anti-cancer activity and diagnostic potential to detect aberrantly glycosylated tumour-associated serum glycoproteins such as alpha-fetoprotein (AFP). We observed that ANL strongly bound to both HepG2 and HT-29 cell-lines and this interaction was effectively blocked with L-fucose and mucin in a dose and time-dependent manner with an IC50 of 1.25 and 5 µg/mL for HepG2 and HT-29 cells respectively at 48 hours. ANL treatment increased hypodiploidy and decreased the number of HepG2 cell in G0 -G1 phase at both 24 and 48 hours. Furthermore, ANL increased the level of apoptosis in both HepG2 and HT-29 cells in a time-dependent manner via enhanced production of reactive oxygen species and altered mitochondrial membrane potential, indicative of intrinsic apoptotis pathway activation. Immunoblot analysis confirmed the time-dependent elevation of levels of cytochrome c, initiator caspase-9 and activation of caspase-3. ANL immunohistochemistry on colon cancer tissue and quantification of AFP in HCC patient serum samples by developing an ANL-anti-AFP antibody sandwich enzyme-linked immunosorbent assay confirmed the diagnostic potential of ANL. Here, interaction of ANL with AFP could be effectively blocked in the presence of competing fucose-bearing glycans. We found ANL to be more sensitive than Lens culinaris lectin, a well-known fucose-specific lectin and currently used diagnostic agent. ANL can be further explored as a diagnostic and anti-cancer agent.
Asunto(s)
Antineoplásicos , Aspergillus niger/química , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Proteínas Fúngicas , Lectinas , Neoplasias Hepáticas/tratamiento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacología , Células HT29 , Células Hep G2 , Humanos , Lectinas/química , Lectinas/farmacología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologíaRESUMEN
An L-fucose lectin, ANL from the corneal smears of a mycotic keratitis patient was reported earlier. Interaction of ANL with immortalized Human Corneal Epithelial Cells (HCECs) was studied in order to assign the role of ANL in pathogenesis. ANL showed strong binding to HCECs which could be blocked by L-fucose and mucin. At concentrations below 0.6 µg/mL ANL showed proliferative effect and highest at 0.07 µg/mL leading to expression of proinflammatory cytokines IL-6 and IL-8. ANL induced proinflammatory response is mediated by TLR-2,-4, MyD88, NFkB and C-Jun dependent signaling. In contrast, ANL at concentrations above 0.6 µg/mL showed growth inhibitory effect at 48 h with an IC50 of 2.75 µg/mL. Western blot analysis revealed that HCECs treated with ANL at lower concentration induced the expression of proinflammatory signaling proteins TLR-2, 4, MyD88, NFkB and C-Jun which maintain high cell proliferating state. At higher concentration ANL induced apoptotic effect in HCECs with an increase in early apoptotic population as demonstrated by Annexin V-PI assay. ANL induced the expression of apoptotic proteins FADD, Caspase 8 and -3 mediated by MyD88. These findings demonstrate implication of ANL in pathogenesis and the findings are of clinical significance in developing strategy for controlling the infection leading to mycotic keratitis.
Asunto(s)
Apoptosis/efectos de los fármacos , Aspergillus niger/química , Células Epiteliales/patología , Epitelio Corneal/patología , Lectinas/toxicidad , Factor 88 de Diferenciación Mieloide/metabolismo , Línea Celular Transformada , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Fucosa/metabolismo , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Interleucinas/metabolismo , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Cephalosporium curvulum lectin (CSL), a lectin from pathogenic fungus has exquisite specificity towards α1-6 linkage of core fucosylated glycans, expressed in hepatocellular and pancreatic cancer. Interaction and effect of CSL and other fucose specific lectins LCA and AOL on HepG2 and PANC-1 cells was investigated. CSL, LCA and AOL exhibited strong binding to PANC-1 cells which could be effectively blocked by competing glycoprotein mucin. Effect of CSL, LCA and AOL on PANC-1 and HepG2 cells was determined by MTT assay and all the three lectins inhibited the cell growth which could be blocked by mucin, cell cycle analysis revealed that CSL increased hypodiploid HepG2 cell population indicating cellular apoptosis. CSL induced apoptosis in HepG2 cells was confirmed by Annexin V/PI assay. CSL induced increase in early apoptotic HepG2 cell population, a time dependent increase in the expression of caspases-3, 9 and cytochrome-c was observed by western blotting suggesting the possible involvement of intrinsic caspase dependent apoptosis. Increase in ROS and decrease in MMP demonstrated involvement of intrinsic caspase dependent apoptosis. Quantification of AFP in HCC patients using CSL lectin-antibody sandwich ELISA, supports diagnostic potential of CSL.
Asunto(s)
Acremonium/química , Ensayo de Inmunoadsorción Enzimática/métodos , Lectinas/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , alfa-Fetoproteínas/análisis , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fucosa/metabolismo , Células Hep G2 , Humanos , Lectinas/química , Lectinas/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Pancreáticas/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Lectins are known to possess interesting biological properties such as anti microbial, nematicidal, anti tumor and anti viral activities. Lantana camara from verbenaceae family is a medicinal plant known for possessing anti oxidant and anticancer activities. Since anticancer activity is reported in plant lectins, leaves of Lantana camara was used to check the presence of lectin. METHODS AND RESULTS: Here we report the purification, characterization and biological properties of a lectin from Lantana camara (LCL) leaves. LCL was purified by ion exchange chromatography on CM-cellulose column followed by affinity chromatography on mucin coupled Sepharose 4B column and gel filtration chromatography on Superdex G75 column. LCL is a glycoprotein with 10% of the carbohydrate and is blood group non specific. SDS-PAGE analysis of affinity purified LCL showed two proteins with apparent molecular weight of 14.49â¯kDa and 17.4â¯kDa which were subsequently separated by Gel filtration chromatography on Superdex G75 column. Hapten inhibition studies of LCL revealed its highest affinity for Chitin, Milibiose, α-D-Methyl galactopyranoside and glycoproteins like mucin, asialomucin. LCL showed strong binding to human colon adenocarcinoma HT29â¯cells with MFI of 242 which was effectively blocked by 68.1 and 62.5% by both mucin and milibiose. LCL showed dose and time dependent growth inhibitory effects on HT29â¯cells with IC50 of 3.75â¯â¯µg/ml at 48â¯h. LCL has potent antibacterial and anti fungal activity. CONCLUSION: LCL can be explored for its clinical potential.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Antineoplásicos/farmacología , Lantana/química , Lectinas de Plantas/farmacología , Antibacterianos/aislamiento & purificación , Antifúngicos/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Quitina/química , Quitina/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Hongos/efectos de los fármacos , Hongos/crecimiento & desarrollo , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/crecimiento & desarrollo , Células HT29 , Humanos , Melibiosa/química , Melibiosa/metabolismo , Metilgalactósidos/química , Metilgalactósidos/metabolismo , Pruebas de Sensibilidad Microbiana , Mucinas/química , Mucinas/metabolismo , Extractos Vegetales/química , Hojas de la Planta/química , Lectinas de Plantas/aislamiento & purificación , Plantas Medicinales , Unión ProteicaRESUMEN
An L-fucose specific lectin from pathogenic fungus Aspergillus niger isolated from the corneal smears of keratitis patient was purified in a single step using mucin coupled sepharose-4B column by 58-fold. The purified lectin, ANL has molecular mass of 30â¯kDa by SDS-PAGE and 31.6â¯kDa by ESI-MS. ANL is a glycoprotein with 2.59% carbohydrate. ANL is blood group nonspecific and also agglutinates rabbit erythrocytes. ANL is heat stable up to 50⯰C and over a pH range of 7-10. Hapten inhibition studies revealed that ANL is specific to L-fucose, galactose, lactose and glycoproteins, showing highest MIC of 3.125⯵g for L-fucose, mucin and fetuin. ANL has potent antibacterial activity against Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli and also it inhibits the biofilm formation by them. ANL showed strong binding to human pancreatic adenocarcinoma PANC-1 cells which was effectively blocked by L-fucose and mucin respectively by 76.2% and 84.2%. ANL showed dose and time dependent growth inhibitory effect on PANC-1 cells with IC50 of 1.25⯵g/ml at 48â¯h. Effect of ANL was compared with another fucose specific lectin AOL, from Aspergillus oryzae showing an IC50 of 1.85⯵g/ml at 48â¯h revealing promising clinical potential of ANL.
Asunto(s)
Aspergilosis/microbiología , Aspergillus niger/química , Fucosa/metabolismo , Queratitis/microbiología , Lectinas/aislamiento & purificación , Lectinas/metabolismo , Animales , Línea Celular Tumoral , Eritrocitos , Humanos , Concentración de Iones de Hidrógeno , Lectinas/química , Ratones , Peso Molecular , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TemperaturaRESUMEN
RATIONALE: The modes of cleavage of lanthionine/methyllanthionine bridges under electron transfer dissociation (ETD) were investigated using synthetic and natural lantipeptides. Knowledge of the mass spectrometric fragmentation of lanthionine/methyllanthionine bridges may assist in the development of analytical methods for the rapid discovery of new lantibiotics. The present study strengthens the advantage of ETD in the characterization of posttranslational modifications of peptides and proteins. METHODS: Synthetic and natural lantipeptides were obtained by desulfurization of peptide disulfides and cyanogen bromide digestion of the lantibiotic nisin, respectively. These peptides were subjected to electrospray ionization collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and ETD-MS/MS using an HCT ultra ETDII ion trap mass spectrometer. MS3 CID was performed on the desired product ions to prove cleavage of the lanthionine/methyllanthionine bridge during ETD-MS/MS. RESULTS: ETD has advantages over CID in the cleavage of the side chain of lanthionine/methyllanthionine bridges. The cleavage of the N-Cα backbone peptide bond followed by C-terminal side chain of the lanthionine bridge results in formation of câ¢+ and z+ ions. Cleavage at the preceding peptide bond to the C-terminal side chain of lanthionine/methyllanthionine bridges yields specific fragments with the cysteine/methylcysteine thiyl radical and dehydroalanine. CONCLUSIONS: ETD successfully cleaves the lanthionine/methyllanthionine bridges of synthetic and natural lantipeptides. Diagnostic fragment ions of ETD cleavage of lanthionine/methyllanthionine bridges are the N-terminal cysteine/methylcysteine thiyl radical and C-terminal dehydroalanine. Detection of the cysteine/methylcysteine thiyl radical and dehydroalanine in combined ETD-CID-MS may be used for the rapid identification of lantipeptide natural products.