Purification, characterization of an entomopathogenic fungal lectin from Purpureocillium lilacinum and its involvement in pathogenesis leading to mycotic keratitis.

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.

Cephalosporium curvulum lectin causes mycotic keratitis by initiating infection through MyD88 dependent cellular proliferation and apoptosis in human corneal epithelial cells.

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.

Rhizoctonia bataticola lectin induces apoptosis and inhibits metastasis in ovarian cancer cells by interacting with CA 125 antigen differentially expressed on ovarian cells.

A N-glycan specific lectin from Rhizoctonia bataticola [RBL] was shown to induce growth inhibitory and apoptotic effect in human ovarian, colon and leukemic cells but mitogenic effect on normal PBMCs as reported earlier, revealing its clinical potential. RBL has unique specificity for high mannose tri and tetra antennary N-glycans, expressed in ovarian cancer and also recognizes glycans which are part of CA 125 antigen, a well known ovarian cancer marker. Hence, in the present study diagnostic and therapeutic potential of RBL was investigated using human ovarian epithelial cancer SKOV3 and OVCAR3 cells known for differentially expressing CA 125. RBL binds differentially to human ovarian normal, cyst and cancer tissues. Flow cytometry, western blot analysis of membrane proteins showed the competitive binding of RBL and CA 125 antibody for the same binding sites on SKOV3 and OVCAR3 cells. RBL has strong binding to both SKOV3 and OVCAR3 cells with MFI of 173 and 155 respectively. RBL shows dose and time dependent growth inhibitory effect with IC50 of 2.5 and 8 µg/mL respectively for SKOV3 and OVCAR3 cells. RBL induces reproductive cell death, morphological changes, nuclear degradation and increased release of ROS in SKOV3 and OVCAR3 cells leading to cell death. This is also supported by increase in hypodiploid population, altered MMP leading to apoptosis possibly involving intrinsic pathway. Adhesion, wound healing, invasion and migration assays demonstrated anti-metastasis effect of RBL apart from its growth inhibitory effect. These results show the promising potential of RBL both as a diagnostic and therapeutic agent.

The fucose-specific lectin ANL from Aspergillus niger possesses anti-cancer activity by inducing the intrinsic apoptosis pathway in hepatocellular and colon cancer cells.

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.

A core fucose specific lectin from Cephalosporium curvulum induces cellular apoptosis in hepatocellular and pancreatic cancer cells and effective in detecting AFP.

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.

Sclerotium rolfsii lectin induces opposite effects on normal PBMCs and leukemic Molt-4 cells by recognising TF antigen and its variants as receptors.

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10 µg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25 µg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen-Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.

A lectin with anti-microbial and anti proliferative activities from Lantana camara, a medicinal plant.

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 14.49 kDa and 17.4 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 HT29 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 HT29 cells with IC50 of 3.75  µg/ml at 48 h. LCL has potent antibacterial and anti fungal activity. CONCLUSION: LCL can be explored for its clinical potential.

Investigation of TF-binding lectins from dietary sources and SRL on proliferation and cell cycle progression in human colon HT29 and SW620 cells.

TF antigen binding lectins from dietary sources PNA, ACA, ABL, JAC, and SRL from Sclerotium rolfsii have been reported to induce diverse effects on cancer cell proliferation by different mechanisms. This study aimed to compare effects of these lectins on growth and cell cycle progression in colon cancer HT29 and SW620 cells. As reported SRL, ABL, and JAC inhibited while PNA and ACA increased cell proliferation. ABL and JAC treated HT29 cells showed increased cell population in G0/G1 phase. PNA, ACA, ABL, and JAC increased SW620 cell population in S and decreased in G2/M phase. In contrast, SRL and JAC increased hypodiploid population in both the cells. PNA and ACA reduced whereas SRL and ABL diminished cell cyclin D1 expression. SRL, PNA, and ACA also reduced cellular cyclin D3 level while SRL, ABL, and JAC reduced cyclin E levels. ABL decreased CDK5 levels while SRL and ACA completely abolished CDK5 expression. All the lectins completely abolished cyclin D2 expression. These results not only confirms growth regulatory effects of TF-binding lectins but also indicates different effects of these lectins on cell growth is associated with regulation on expression of cell cycle associated proteins in G1-S phase and on cell cycle progression.

A mitogenic lectin from Rhizoctonia bataticola arrests growth, inhibits metastasis, and induces apoptosis in human colon epithelial cancer cells.

The correlation between colorectal cancer (CRC) progression and altered expression of N-glycans can be considered in search for new biomarkers and anticancer agents to control CRC. Earlier N-glycan specific mitogenic lectin from Rhizoctonia bataticola (RBL) has been reported which has growth inhibitory and apoptotic effect on human ovarian and leukemic cells, but mitogenic effect on normal PBMCs revealing its clinical potential. Here, we report the effect of RBL on human colon cancer HT 29, SW480, and SW620 cell growth and its differential binding to human normal colon and cancer tissues. RBL has strong binding to both primary and metastatic colon cancer cells with MFI of 403, 404, and 192, respectively for HT 29, SW480, and SW620 cells. RBL shows dose and time dependent growth inhibitory effect with IC50 of 5, 6.4, and 6.8 µg/mL, respectively for HT 29, SW480, and SW620 cells. RBL inhibited the clonogenicity of colon carcinoma cells. RBL arrests metastatic SW620 cell growth at S phase, increased hypodiploid population by 6.1%, 14.3%, and 23.2%, respectively at 12, 24, and 36 h. Further, RBL induces SW620 cell apoptosis in time dependent manner, showed increased release of ROS and nuclear degradation compared to lectin untreated control. Adhesion, wound healing, invasion, and migration assays demonstrated anti-metastasis effect of RBL in SW620 cells apart from its growth inhibitory effect. Anti angiogenic effect of RBL was demonstrated by CAM assay. All these results show the promising potential of RBL both as diagnostic and therapeutic agent.

Purification, characterization and fine sugar specificity of a N-Acetylgalactosamine specific lectin from Adenia hondala.

Plant lectins are gaining interest because of their interesting biological properties. Several Adenia species, that are being used in traditional medicine to treat many health ailments have shown presence of lectins or carbohydrate binding proteins. Here, we report the purification, characterization and biological significance of N-Acetyl galactosamine specific lectin from Adenia hondala (AHL) from Passifloraceae family. AHL was purified in a single step by affinity chromatography on asialofetuin Sepharose 4B column, characterized and its fine sugar specificity determined by glycan array analysis. AHL is human blood group non specific and also agglutinates rabbit erythrocytes. AHL is a glycoprotein with 12.5% of the carbohydrate, SDS-PAGE, MALDI-TOF-MS and ESI-MS analysis showed that AHL is a monomer of 31.6 kDa. AHL is devoid of DNase activity unlike other Ribosome inactivating proteins (RIPs). Glycan array analysis of AHL revealed its highest affinity for terminal lactosamine or polylactosamine of N- glycans, known to be over expressed in hepatocellular carcinoma and colon cancer. AHL showed strong binding to human hepatocellular carcinoma HepG2 cells with MFI of 59.1 expressing these glycans which was effectively blocked by 93.1% by asialofetuin. AHL showed dose and time dependent growth inhibitory effects on HepG2 cells with IC50 of 4.8 µg/ml. AHL can be explored for its clinical potential.

An overview of lectin-glycan interactions: a key event in initiating fungal infection and pathogenesis.

Infections due to microfungi are of serious concern in many parts of the world. Many species of microfungi are known to cause systemic infection in human beings. Pathogenic microorganisms employ various molecular strategies for colonizing a susceptible host. Recent studies have shown the importance of lectins from microfungi that enable the pathogen to interact with the host, resulting in host immune response. These fungal lectins or adhesins show specific affinities to the glycans present on the membrane proteins or lipids. Binding of the pathogen to the receptors, probably toll-like receptors or dectins, present on the host cell surface triggers/initiates a cascade of signalling pathways, leading to the activation of transcription factors such as NF-κB resulting in the release of proinflammatory cytokines which in turn recruit cells of the immune system to the site of microbial insult to combat the pathogen or resulting in pathogenesis. In this review, we will focus on the interaction between fungal lectins and the host glycans initiating pathogenesis and how the host immune system tries to suppress the pathogenesis.

Mitogenic lectins from Cephalosporium curvulum (CSL) and Aspergillus oryzae (AOL) mediate host-pathogen interactions leading to mycotic keratitis.

A core-fucose-specific lectin, CSL from Cephalosporium curvulum, has been reported earlier. Here we assign the role for CSL and another lectin AOL, from pathogenic fungus Aspergillus oryzae, in causing mycotic keratitis. CSL and AOL show strong binding to immortalized and primary human corneal epithelial cells (HCECs) which are inhibited by asialofetuin, confirming their glycan-mediated binding. CSL and AOL showed increase in viability at lower concentrations (0.07 µg/ml) whereas at higher concentrations (0.15 µg/ml and 0.30 µg/ml), have inhibitory effect on immortalized HCECs. Lectin-mediated effect was comparable with the effect induced by the Colony Forming Units (CFUs) of C. curvulum and A. oryzae. CFUs induced more than 1.5-fold increase in HCECs proliferation. Both lectins and fungal CFUs induce secretion of proinflammatory cytokines IL6 and IL8 implicated in ocular diseases. This was supported by upregulation of TLR2 and 4 by lectins as revealed by flow cytometry and RT-PCR. CSL and AOL mediate host-pathogen interactions leading to mycotic keratitis. The mechanism of pathogenesis is possibly initiated through surface binding of mycelia through the lectins to TLR2/4 followed by upregulation of proinflammatory cytokines IL6, IL8 and TLR2 and 4. Understanding the mechanism of pathogenesis is of clinical significance in designing and developing therapeutic strategy to control the infection.

Exquisite specificity of mitogenic lectin from Cephalosporium curvulum to core fucosylated N-glycans.

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.

Molecular mechanism of anticancer effect of Sclerotium rolfsii lectin in HT29 cells involves differential expression of genes associated with multiple signaling pathways: A microarray analysis.

Sclerotium rolfsii lectin (SRL) is a lectin isolated from fungus S. rolfsii and has high binding specificity toward the oncofetal Thomsen-Friedenreich carbohydrate antigen (Galß1-3GalNAc-α-O-Ser/Thr, T or TF), which is expressed in more than 90% of human cancers. Our previous studies have shown that binding of SRL to human colon, breast and ovarian cancer cells induces cell apoptosis in vitro and suppresses tumor growth in vivo. This study investigated the SRL-mediated cell signaling in human colon cancer HT29 cells by mRNA and miRNA microarrays. It was found that SRL treatment results in altered expression of several hundred molecules including mitogen-activated protein kinase (MAPK) and c-JUN-associated, apoptosis-associated and cell cycle and DNA replication-associated signaling molecules. Pathway analysis using GeneSpring 12.6.1 revealed that SRL treatment induces changes of MAPK and c-JUN-associated signaling pathways as early as 2 h while changes of cell cycle, DNA replication and apoptosis pathways were significantly affected only after 24 h. A significant change of cell miRNA expression was also observed after 12 h treatment of the cells with SRL. These changes were further validated by quantitative real time polymerase chain reaction and immunoblotting. This study thus suggests that the presence of SRL affects multiple signaling pathways in cancer cells with early effects on cell proliferation pathways associated with MAPK and c-JUN, followed by miRNA-associated cell activity and apoptosis. This provides insight information into the molecular mechanism of the anticancer activity of this fungal lectin.

Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants.

SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galß1→3GalNAc-α-Ser//Thr) expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr). The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis.

Crystal structure of a ß-prism II lectin from Remusatia vivipara.

The crystal structure of a ß-prism II (BP2) fold lectin from Remusatia vivipara, a plant of traditional medicinal value, has been determined at a resolution of 2.4 Å. This lectin (RVL, Remusatia vivipara lectin) is a dimer with each protomer having two distinct BP2 domains without a linker between them. It belongs to the "monocot mannose-binding" lectin family, which consists of proteins of high sequence and structural similarity. Though the overall tertiary structure is similar to that of lectins from snowdrop bulbs and garlic, crucial differences in the mannose-binding regions and oligomerization were observed. Unlike most of the other structurally known proteins in this family, only one of the three carbohydrate recognition sites (CRSs) per BP2 domain is found to be conserved. RVL does not recognize simple mannose moieties. RVL binds to only N-linked complex glycans like those present on the gp120 envelope glycoprotein of HIV and mannosylated blood proteins like fetuin, but not to simple mannose moieties. The molecular basis for these features and their possible functional implications to understand the different levels of carbohydrate affinities in this structural family have been investigated through structure analysis, modeling and binding studies. Apart from being the first structure of a lectin to be reported from the Araceae/Arum family, this protein also displays a novel mode of oligomerization among BP2 lectins.

The TF-antigen binding lectin from Sclerotium rolfsii inhibits growth of human colon cancer cells by inducing apoptosis in vitro and suppresses tumor growth in vivo.

Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galß1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer.

CD45-mediated signaling pathway is involved in Rhizoctonia bataticola lectin (RBL)-induced proliferation and Th1/Th2 cytokine secretion in human PBMC.

We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.

Rhizoctonia bataticola lectin (RBL) induces mitogenesis and cytokine production in human PBMC via p38 MAPK and STAT-5 signaling pathways.

BACKGROUND: Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus Rhizoctonia bataticola is highly mitogenic towards human peripheral blood mononuclear cells (PBMC). The lectin has sugar specificity towards N-glycans and binds to glycoproteins containing complex N-glycans (Nagre et al., Glycoconj J. 2010). In this study, we investigated the role of Mitogen Activated Protein Kinase (MAPK) and Signal Transducers and Activators of Transcription (STAT)-5 signaling in RBL-induced proliferation and production of Th1/Th2 cytokines. METHODS: Human PBMC were stimulated with RBL and proliferation was determined by tritiated thymidine incorporation assay, cytokine profiles by ELISA and activation of MAPK and STAT-5 by western blotting. RBL binding was monitored by immunofluorescence staining. Expression of IL-2Rα (CD25) was measured by flow cytometry. RESULTS: The binding and mitogenic activities of RBL were inhibited by glycoproteins- mucin, asialofetuin and fetuin. RBL stimulated expression of IL-2Rα and production of Th1/Th2 cytokines- IL-2, IFN-γ, IL-4 and IL-10. RBL-induced phosphorylation of ERK1/2 and p38 MAPK was detected at 1h and 3h respectively. Significant phosphorylation of STAT-5 (tyr(694)) was observed at 12h. Pharmacological inhibitors of p38 MAPK (SB203580) and JAK/STAT (AG490) but not ERK (PD98059) abrogated proliferation. RBL-induced expression of IL-2Rα and secretion of cytokines were drastically inhibited by SB203580 and AG490. CONCLUSIONS: RBL-induced proliferation and production of Th1/Th2 cytokines are mediated via p38 MAPK and STAT-5 signaling. GENERAL SIGNIFICANCE: RBL, a lectin with complex sugar specificity, is strongly mitogenic to human PBMC and stimulates the production of Th1 and Th2 cytokines. The results identified the signaling mechanism underlying the immunostimulatory activity of RBL.

Exquisite binding specificity of Sclerotium rolfsii lectin toward TF-related O-linked mucin-type glycans.

Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galß1-3GalNAc-ser/thr, T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core structures. It binds with high specificity to "α-anomers" but not the "ß-anomers" of the TF structure. The axial C4-OH group of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures. Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of the cellular glycans in cancer studies.