The mannose-binding protein from Agaricus bisporus inhibits the growth of MDA-MB-231 spheroids.

A mannose-binding protein from the mushroom Agaricus bisporus (Abmb) inhibits the growth of MDA-MB-231 cells, which is of an aggressive breast cancer subtype. This ability was observed in a monolayer cell (2D) culture setup, which often is unable to capture changes in cell morphology, polarity and division. That shortcoming may overestimate Abmb potency for its development as a pharmaceutical agent and its use in a therapy. Hence, Abmb's inhibition to the cell growth was performed in the 3D cell (spheroid) culture, which is more representative to the situation in vivo. The result showed that, although the presence of Abmb at ~14.7 µM already disrupted the MDA-MB-231 cell morphology in the 2D culture, its presence at ~16.5 µM only ceased the growth of the MDA-MB-231 spheroid. Further, Abmb is unique because structurally it belongs to the R-type lectin (RTL) family; most of mannose-binding protein is of the C-type lectin (CTL). As the natural ligand of Abmb is unknown thus the mechanism of action is unclear, Abmb effect on the cancer cells was assessed via observation of the altered expression of genes involved in the Wnt/ß-catenin signalling, which is one of the canonical pathways in the proliferation of cancer cells. The results suggested that Abmb did not alter the pathway upon exerting its anti-proliferative activity to the MDA-MB-231 cells.

Mannan-Binding Lectin via Interaction With Cell Surface Calreticulin Promotes Senescence of Activated Hepatic Stellate Cells to Limit Liver Fibrosis Progression.

BACKGROUND & AIMS: Liver fibrosis represents a hallmark of most chronic liver diseases (CLD) triggered by recurrent liver injury and subsequent myofibroblast transdifferentiations of resident hepatic stellate cells (HSCs). Mannan-binding lectin (MBL) is potentially involved in hepatic fibrosis in CLD through unclear mechanisms. Therefore, we investigated the crosstalk between MBL and HSCs, and the consequent effects on fibrosis progression. METHODS: Samples from patients with liver cirrhosis were collected. MBL deficiency (MBL-/-) and wild-type (WT) C57BL/6J mice were used to construct a CCl4-induced liver fibrosis model. Administration of MBL-expressing, liver-specific, adeno-associated virus was performed to restore hepatic MBL expression in MBL-/- mice. The human HSC line LX-2 was used for in vitro experiments. RESULTS: MBL levels in patients with liver cirrhosis were correlated with disease severity. In the CCl4-induced liver fibrosis model, MBL-/- mice showed severer liver fibrosis accompanied by reduced senescent activated HSCs in liver tissue compared with WT mice, which could be inhibited by administering MBL-expressing, liver-specific, adeno-associated virus. Moreover, depleting senescent cells with senolytic treatment could abrogate these differences owing to MBL absence. Furthermore, MBL could interact directly with calreticulin associated with low-density lipoprotein receptor-related protein 1 on the cell surface of HSCs, which further promotes senescence in HSCs by up-regulating the mammalian target of rapamycin/p53/p21 signaling pathway. CONCLUSIONS: MBL as a newfound senescence-promoting modulator and its crosstalk with HSCs in the liver microenvironment is essential for the control of hepatic fibrosis progression, suggesting its potential therapeutic use in treating CLD associated with liver fibrosis.

The synergistic proapoptotic effect of PARP-1 and HDAC inhibition in cutaneous T-cell lymphoma is mediated via Blimp-1.

The therapy of advanced mycosis fungoides (MF) presents a therapeutic challenge, and the search for new therapeutic targets is ongoing. Poly(ADP-ribose) polymerase 1 was shown to be upregulated in patients with advanced MF and could be druggable by a new class of chemotherapeutic agents, PARP-1 inhibitors, which are already in clinical trials for other malignancies; however, the role of PARP-1 inhibitors in MF has never been established. We examined the efficacy of talazoparib in the murine model of cutaneous T-cell lymphoma. The cytotoxic effect of talazoparib on Moloney MuLV-induced T-cell lymphoma (MBL2) cells was a result of G2/M cell cycle arrest via the upregulation of p53. The in vivo experiments confirmed the clinical impact of talazoparib on MF tumors. When talazoparib was combined with the histone deacetylase (HDAC) inhibitor, romidepsin, the cytotoxic effect was synergized via downregulation of the DNA-repair genes Fanconianemia complementation group A (FANCA), Fanconi anemia complementation group D2 (FANCD2), and DNA topoisomerase II binding protein 1(TOPBP1)and stimulation of apoptosis via Blimp-1 (PRDM1)/Bax axis. Romidepsin increased the expression of IRF8 and Bcl-6, leading to upregulation of Blimp1and Bax; whereas talazoparib upregulated Blimp-1 and Bax via upregulation of interferon regulatory factor 4 (IRF4), leading to cleavage of caspases 6 and 7. Thus, a combination of talazoparib with romidepsin demonstrated the synergistic antilymphoma effect and warranted further investigation in a clinical trial.

Antimicrobial activity of mannose binding lectin in grass carp (Ctenopharyngodon idella) in vivo and in vitro.

Mannose-binding lectin (MBL) is a crucial pattern recognition receptor in the host innate immune system. Previously, we reported the biological function of Ctenopharyngodon idella MBL (CiMBL) in initiating the lectin pathway of the complement system. In the present study, we further explored its biological function including the agglutinating ability, binding capacity and protective role in vitro and in vivo. After Aeromonas hydrophila infection, western blot analysis revealed that the CiMBL were fluctuated and expressed in the serum and major immune-related tissues. The result of quantitative PCR (qPCR) showed that the recombinant CiMBL (rCiMBL) significantly inhibited the mRNA expression of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in liver, spleen and hepatic cells. Due to rCiMBL bound to d-mannose, d-galactose, d-glucose, N-acetyl-d-glucosamine (GlcNAc), lipopolysaccharide (LPS), peptidoglycan (PGN) and Agar in the presence of Ca2+, herein gram-positive (Staphylococcus aureus and Micrococcus luteus) and gram-negative (A. hydrophila and Vibrio anguillarum) bacteria were agglutinated by rCiMBL in a Ca2+-dependent manner. More importantly, rCiMBL enhanced the survival rate of grass carp following bacterial infection. Overall, the results provide an evidence that CiMBL can protect grass carp against A. hydrophila infection in aquaculture.

Agaricus bisporus mannose binding protein is not an agglutinating protein.

Agaricus bisporus mannose binding protein (Abmb) demonstrates permeability to epithelial monolayer barrier of the intestine, resistance to gastrointestinal tract conditions and to proteolysis therefore it holds potential as a drug carrier for oral route administration. Abmb also display antiproliferative activity to breast cancer cells and stimulation of immune system thus could potentially be also developed for therapeutic purpose. It is not immunogenic or toxic thereby safe for use. In this paper we further provide evidence that Abmb also lacks of agglutinating activity despite sharing high structural homology to lectins. Abmb is thereby the only mannose specific binding protein that is not member of lectin family. This evidence provides further support on the use of Abmb as pharmaceutical or medicinal agent. Its molecular globularity that may contribute to its lack of agglutination capacity was also evaluated.

Mannan-binding lectin promotes keratinocyte to produce CXCL1 and enhances neutrophil infiltration at the early stages of psoriasis.

Psoriasis is a chronic, relapsing inflammatory skin disorder. Numerous experimental evidence and therapeutic evidence have shown that the innate immune response is critical for the pathogenesis and development of psoriasis. Mannan-binding lectin (MBL), a prototypic pattern recognition molecule of the innate immune system, plays an essential role in the host defense against certain infections and also appears to be a major regulator of inflammation. In this study, we investigated the function of MBL on the course of experimental murine imiquimod (IMQ)-induced psoriasis. Our data showed that MBL-deficient (MBL-/- ) mice exhibited attenuated skin damage characterized by greatly decreased erythema compared with wild-type control mice during the early stages of IMQ-induced psoriasis-like skin inflammation. The reduced skin inflammation in MBL-/- mice was associated with the decreased infiltration of neutrophils. Furthermore, we have determined that MBL deficiency limited the chemokine CXCL1 production from skin keratinocytes upon IMQ stimulation, which might be responsible for the impaired skin recruitment of neutrophils. Additionally, we have provided the data that MBL protein promotes the IMQ-induced expression of CXCL1 and activation of MAPK/NF-κB signalling pathway in human keratinocyte HaCaT cells in vitro. In summary, our study revealed an unexpected role of MBL on keratinocyte function in skin, thus offering a new insight into the pathogenic mechanisms of psoriasis.

Orf239342 from the mushroom Agaricus bisporus is a mannose binding protein.

A recently discovered lectin-like protein from mushroom tyrosinase designated as orf239342 inhibits proliferation of the MCF-7 breast cancer cells. This characteristic is likely derived from its ability to recognize sugar entity on the cell surface. Thereby, the binding specificity of orf239342 to sugars was studied. Orf239342 was found to bind specifically to mannose upon analysis with the surface plasmon resonance technique. Finally, our in vitro study showed that mannose impeded orf239342 ability to inhibit proliferation of the MCF-7 breast cancer cells, providing further evidence for the mannose binding onto the protein. Our finding is a breakthrough to characterise orf239342 i.e. to define its functioning in the mushroom, association to the tyrosinase, or even possible application in breast cancer therapy. In addition, the finding allows the more appropriate designation of the protein as Agaricus bisporus mannose binding-protein (AbMb).

High mannose N-glycan binding lectin from Remusatia vivipara (RVL) limits cell growth, motility and invasiveness of human breast cancer cells.

Breast cancer known for its high metastatic potential is responsible for large mortality rate amongst women; hence it is imperative to search for effective anti-metastatic molecules despite anticancer drugs. The current study describes the potential of Remusatia vivipara lectin (RVL), inducing apoptosis in breast cancer cells there by limiting motility and invasiveness. RVL binds to the cell surface glycans of MDA-MB-468 and MCF-7 cells, exhibiting strong glycan mediated cytotoxic effect, but show marginal effect on non-tumorigenic MCF-10A cells. RVL elicits increased cellular stress, apoptotic vacuoles and nuclear disintegration in both MDA-MB-468 and MCF-7 cells accompanied by depletion of G0/G1, S and G2/M phases. Lectin interaction induced production of reactive oxygen species through altering mitochondrial membrane potential progressing to apoptosis. Further, RVL strongly elicited reproductive cell death in MDA-MB-468 cells and showed strong inhibitory effect on neovascularization demonstrated in chorioallantoic membrane assay. Treatment of MDA-MB-468 cells with RVL, suppress the motility and invasive property as shown by scratch wound heal and Boyden chamber transwell assays respectively. These results provide an insight into significance of interaction of RVL with specific cell surface high mannose N-glycans resulting in curtailing the metastatic ability of cancer cells.

Purification, characterization and biological significance of mannose binding lectin from Dioscorea bulbifera bulbils.

Dioscorea bulbifera or air potato has been used as a folk remedy to treat cancer. A mannose binding lectin from bulbils of D. bulbifera was purified in a single step by affinity chromatography on mucin coupled Sepharose 4B column, determined by its fine sugar specificity by glycan array analysis and studied for its clinical potential in cancer and HIV research. SDS-PAGE showed that lectin is a monomer of Mr 24kDa. DBL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, fetuin, asialofetuin and transferrin but not by any monosaccharides. Glycan array analysis of DBL revealed its affinity toward high mannose N-linked glycans with enhanced affinity for terminal mannose including N-linked glycans of HIV envelope glycoprotein gp120 and has strong anti-reverse transcriptase activity. DBL showed strong binding to non-metastatic human colon epithelial cancer HT 29, metastatic SW 620 and hepatocellular HepG2 cell lines. DBL showed dose and time dependent growth inhibitory effects on all the three cell lines HT 29, SW 620 and HepG2 with IC50 of 110µg, 9.8µg, 40µg respectively at 72h. Inhibitory effect of DBL was effectively blocked in presence of competing glycans like mucin. DBL has promising clinical potential both in cancer and HIV research.

Tri- and tetravalent mannoclusters cross-link and aggregate BC2L-A lectin from Burkholderia cenocepacia.

The opportunistic Gram-negative bacterium Burkholderia cenocepacia causes lethal infections in cystic fibrosis patients. Multivalent mannoside derivatives were prepared as potential inhibitors of lectin BC2L-A, one of the virulence factors deployed by B. cenocepacia in the infection process. An (α1→2)-thio-linked mannobioside mimic bearing an azide functionalized aglycon was conjugated to different multivalent scaffolds such as propargylated calix[4]arenes, methyl gallate and pentaerythritol by azide-alkyne 1,3-dipolar cycloaddition. The interaction between the glycoclusters and the mannose binding BC2L-A lectin from B. cenocepacia was examined by isothermal microcalorimetry, surface plasmon resonance, inhibition of yeast agglutination and analytical ultracentrifugation.

High mannose-specific lectin Msl mediates key interactions of the vaginal Lactobacillus plantarum isolate CMPG5300.

To characterize the interaction potential of the human vaginal isolate Lactobacillus plantarum CMPG5300, its genome was mined for genes encoding lectin-like proteins. cmpg5300.05_29 was identified as the gene encoding a putative mannose-binding lectin. Phenotypic analysis of a gene knock-out mutant of cmpg5300.05_29 showed that expression of this gene is important for auto-aggregation, adhesion to the vaginal epithelial cells, biofilm formation and binding to mannosylated glycans. Purification of the predicted lectin domain of Cmpg5300.05_29 and characterization of its sugar binding capacity confirmed the specificity of the lectin for high- mannose glycans. Therefore, we renamed Cmpg5300.05_29 as a mannose-specific lectin (Msl). The purified lectin domain of Msl could efficiently bind to HIV-1 glycoprotein gp120 and Candida albicans, and showed an inhibitory activity against biofilm formation of uropathogenic Escherichia coli, Staphylococcus aureus and Salmonella Typhimurium. Thus, using a combination of molecular lectin characterization and functional assays, we could show that lectin-sugar interactions play a key role in host and pathogen interactions of a prototype isolate of the vaginal Lactobacillus microbiota.

Inhibition of Hepatocarcinogenesis by ArtinM via Anti-proliferative and Pro-apoptotic Mechanisms.

ArtinM is a d-mannose-binding lectin found in the seeds of Artocarpus heterophyllus (jackfruit) that interacts with N-glycans, that is associated with receptors on the surface of phagocytic cells and induces the production of inflammatory mediators. Some of them are especially important because they may be required for antitumor immune response. This study aimed to evaluate the effect of ArtinM on hepatocellular preneoplastic foci. Wistar rats received 50 mg/kg of diethyl-nitrosamine (DEN) intraperitoneal weekly for 12 weeks. From the 14th week, the treated animals received 50 µg/kg of ArtinM subcutaneous every 2 weeks until the 18th week, whereas control animals were injected with vehicle alone. Preneoplastic-related factors were estimated using histological, western blotting and RT-PCR analysis. In comparison to the groups exposed to DEN, the ArtinM-treated rats showed diminution of preneoplastic foci, decreased expression of proliferating cell nuclear antigen (PCNA), increased number of nuclear p21 and p27 stained cells, augmented number of apoptotic cells, increased expression of p53, p42/44 MAPK and p21 proteins, reduced cyclin D1 (CCND1) protein levels and increased expression of TNFα and IFNγ genes. No difference was observed in interleukin 12 (IL12) protein levels. These findings indicate that ArtinM may provide protection against hepatocarcinogenesis as a result of the induction of cell-cycle blockage and pro-apoptotic mechanisms.

[Structure and Function of a Novel Class of High Mannose-binding Proteins with Anti-viral or Anti-tumor Activity].

The recently discovered high mannose (HM)-binding lectin family in lower organisms such as bacteria, cyanobacteria, and marine algae represents a novel class of anti-viral or anti-tumor compounds. This lectin family shows unique carbohydrate binding properties with exclusive high specificity for HM glycans with core trisaccharide comprising Manα(1-3)Manα(1-6)Man at the D2 arm. At low nanomolar levels, these lectins exhibit potent antiviral activity against HIV and influenza viruses through the recognition of HM glycans on virus spike glycoproteins. In addition, some of these lectins, such as bacterial PFL, show cytotoxicity for various cancer cells at low micromolar levels. Cell surface molecules to which PFL bound were identified as integrin alpha 2 and epidermal growth factor receptor (EGFR) by peptide mass finger printing with MALDI-TOF MS. Upon PFL binding, these molecules were rapidly internalized to cytoplasm. EGFR was time dependently degraded in the presence of PFL, and this process was largely responsible for autophagy. Furthermore, PFL sensitizes cancer cells to the EGFR kinase inhibitor, gefitinib. In vivo experiments showed that intratumoral injection of PFL significantly inhibited the growth of tumors in nude mice. PFL-mediated down regulation of integrin/EGFR ultimately contributed to the inhibition of tumor growth both in vitro and in vivo. Thus, the novel anti-cancer mechanism of PFL suggests that this lectin is potentially useful as an anti-cancer drug or as an adjuvant for other drugs. This class of proteins will likely have beneficial impact as a tool for biochemical and biomedical research because of its unique carbohydrate specificity and various biological activities.

Mannan-binding lectin at supraphysiological concentrations inhibits differentiation of dendritic cells from human CD14+ monocytes.

Mannan-binding lectin (MBL), a circulating C-type lectin, is an important member of the defense collagen family. It exhibits a high potential for recognizing broad categories of pathogen-associated molecular patterns and initiating complement cascade responses. DCs are well-known specialist antigen-presenting cells that significantly trigger specific T cell-mediated immune responses. In our previous study, it was observed that high concentrations of MBL significantly attenuate LPS-induced maturation of monocyte-derived DCs (MoDCs). In the current study, it was postulated that MBL at similar supraphysiological concentrations would affect early differentiation of MoDCs in some way. CD14(+) monocytes from human peripheral blood mononuclear cells were cultured with granulocyte-macrophage colony-stimulating factor and IL-4 in the presence or absence of physiological (1 µg/mL) and supraphysiological concentrations (20 µg/mL) of MBL protein, respectively. Phenotypic analysis indicated that the differentiated DCs incubated with high concentrations of MBL expressed MHC class II and costimulatory molecules (e.g., CD80 and CD40) more weakly than did control groups. The secretion of IL-10 and IL-6 increased markedly, whereas their mixed lymphocyte reaction-stimulating capacity decreased. Members of the signal transducer and activator of transcription family were also found to be differentially regulated. Thus, beyond the role of MBL as an opsonin, our data reveal a possible inhibitory effect of MBL at high concentrations in monocyte-DC transition, which probably provides one way of regulating adaptive immune responses by strict regulation of DCs, making MBL a better prospect for controlling relevant pathological events such as autoimmune diseases.

Recombinant human mannose-binding lectin dampens human alveolar macrophage inflammatory responses to influenza A virus in vitro.

IAV pneumonia remains a serious global health problem, and preventative and therapeutic strategies remain limited. AM are critical effector cells in the control of influenza, impairing IAV replication, promoting IAV clearance, and promoting efferocytosis and resolution of lung inflammation. MBL, an innate immune pattern recognition molecule, present in the lungs, binds IAV, and plasma MBL deficiency is associated with increased susceptibility to IAV, although the mechanism remains incompletely understood, and the influence of MBL on the IAV-AM interaction has not been established. In the current study, focusing on human macrophages (U937 cell line and clinically relevant human AM), data demonstrated that unopsonized IAV is readily internalized, induced release of TNF and ROS, and promoted macrophage apoptosis. In contrast, IAV, opsonized with rhMBL, reduced IAV uptake and macrophage apoptosis and dramatically reduced TNF release and ROS. Macrophage host-defense responses were reduced further in the presence of MASPs. Taken together, these data support the concept that rhMBL may serve a protective innate host response and a critical biological response modifier function by limiting AM inflammation, oxidative injury, and AM apoptosis, which may allow effective IAV clearance while limiting collateral damage to vital organs, such as the lungs.

Efficacy of recombinant chimeric lectins, consisting of mannose binding lectin and L-ficolin, against influenza A viral infection in mouse model study.

Influenza A virus infection could result in fatal complications. Although immunization is the most effective prevention it is not effective to pandemic infection and is less effective or not approved for certain age groups. Some influenza virus strains have developed resistance to antiviral agents. Thus, new therapeutic agents are urgently needed. We focused on innate immune molecules, including mannose-binding lectin (MBL). In order to optimize its antiviral activities, we have previously generated three recombinant chimeric lectins (RCL), by introducing portions of L-ficolin, another innate immune lectin. Our in vitro characterizations previously selected RCL2 and RCL3 for further investigations against viruses, including influenza viruses. Here, we examined efficacy of these lectins against infection with PR8 (H1N1) influenza A virus using mouse model studies and a human tracheal epithelial cell system. Our results provide in vivo evidence that RCL3 is effective agent against influenza virus infection. The therapeutic mechanisms are in part by providing host protective responses mediated by cytokines. We conclude that RCL3 is a potential new innate immune anti-influenza virus therapeutic agent.

Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells.

It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response against bacterial lipopolysaccharide (LPS) stimulation. LPS-mediated pro-inflammatory cytokine productions on mBMMCs obtained from Toll-like receptor4 (TLR4)-deficient mice, TLR2-defficient mice, and their wildtype, were specifically attenuated by the addition of either mouse MBL-A or ficolin-A in a dose-dependent manner. However, the inhibitory effects by mouse MBL-A or ficolin-A were restored by the addition of mannose or N-acetylglucosamine, respectively. These results suggest that mouse MBL-A and ficolin-A bind to LPS via its carbohydrate-recognition domain and fibrinogen-like domain, respectively, whereby cytokine production by LPS-mediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells.

Dendritic cells previously exposed to mannan-binding lectin enhance cytokine production in allogeneic mononuclear cell cultures.

Mannan (or mannose)-binding lectin (MBL) can bind to monocytes and dendritic cells, but the significance of such interactions is unknown. We hypothesized that the presence of MBL might prevent the differentiation of monocytes into monocyte-derived dendritic cells or interfere with the development of dendritic cells in some way. We therefore investigated the influence of recombinant human MBL on surface antigen expression and on secretion of selected cytokines. By these means, no direct influence of rhMBL on dendritic cell differentiation or maturation was detected. However, mature dendritic cells prepared in the presence of rhMBL and subsequently co-cultured with allogeneic mononuclear cells, markedly promoted production of interleukin-1ß, interleukin-6, and tumor necrosis factor-α in vitro. In most dendritic cell-mononuclear cell combinations, IFN-γ production was also enhanced. This influence required the presence of rhMBL during dendritic cell maturation and was critically dependent on the presence of monocytes. This observation provides evidence that MBL can influence cellular immunity in addition to its established role as an opsonin.

Recombinant form of human wild type mannan-binding lectin (MBL/A) but not its structural variant (MBL/C) promotes phagocytosis of zymosan by activating complement.

Mannan-binding lectin (MBL) mediates innate immune responses, such as activation of the complement lectin pathway and phagocytosis, to help fight infections. In the present study, employing recombinant forms of human MBL (rMBL), the role of wild type MBL (rMBL/A) and its structural variant rMBL/C in mediating THP-1 phagocytosis of fluorescent-labeled zymosan was examined and compared to MBL purified from human plasma (pMBL/A). Flow cytometric analyses revealed that opsonization of zymosan with rMBL/A and pMBL/A (0.5-30microg/ml) resulted in a 1.9- and 2.7-fold enhancement in its uptake by THP-1 cells in the presence of serum that was depleted of both MBL and the classical pathway component, C1q (MBL/C1q Dpl serum). In contrast, no enhancement in phagocytosis was observed when zymosan was opsonized with rMBL/C. Addition of MBL monoclonal antibody, EDTA, or mannan to the opsonization reaction mixture inhibited THP-1 phagocytosis of pMBL/A opsonized zymosan. Heat inactivation of MBL/C1q Dpl serum abolished the 2-fold increase in phagocytosis and in the absence of serum the direct opsonic activity of MBL did not contribute significantly to the uptake of zymosan into THP-1 cells. Activation products of complement components C3 and C4 were deposited on zymosan opsonized with pMBL/A and rMBL/A but not rMBL/C indicating that MBL-mediated phagocytosis of zymosan requires activation of the complement lectin pathway. The findings imply that impaired MBL-mediated phagocytosis may put individuals homozygous for the mutant allele MBL/C but not wild type MBL/A at increased risk to infections such as yeast.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity.

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80 degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 microg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.