Mannose-Binding Lectin Possesses Agglutination Activity and Promotes Opsonophagocytosis of Macrophages with Calreticulin Interaction in an Early Vertebrate.

The innate immune system is an ancient defense system in the process of biological evolution, which can quickly and efficiently resist pathogen infection. In mammals, mannose-binding lectin (MBL) is a key molecule in the innate immune and plays an essential role in the first line of host defense against pathogenic bacteria. However, the evolutionary origins and ancient roles of immune defense of MBL and its mechanism in clearance of microbial pathogens are still unclear, especially in early vertebrates. In this study, Oreochromis niloticus MBL (OnMBL) was successfully isolated and purified from the serum of Nile tilapia (O. niloticus). The OnMBL was able to bind and agglutinate with two important pathogens of tilapia, Streptococcus agalactiae and Aeromonas hydrophila Interestingly, the OnMBL was able to significantly inhibit the proliferation of pathogenic bacteria and reduce the inflammatory response. Upon bacterial challenge, the downregulation of OnMBL expression by RNA interference could lead to rapid proliferation of the pathogenic bacteria, ultimately resulting in tilapia death. However, the phenotype was rescued by reinjection of the OnMBL, which restored the healthy status of the knockdown tilapia. Moreover, a mechanistic analysis revealed that the OnMBL could clear pathogenic bacteria by collaborating with cell-surface calreticulin to facilitate phagocytosis in a complement activation-independent manner. To our knowledge, these results provide the first evidence on the antibacterial response mechanism of MBL performing evolutionary conserved function to promote opsonophagocytosis of macrophages in early vertebrates and reveals new insights into the understanding of the evolutionary origins and ancient roles basis of the C-type lectins in the innate immune defense.

Purification, Quantification, and Functional Analysis of Collectins.

Native and recombinant collectins are purified by using mannan-agarose and an anti-collectin antibody column. The use of sandwich enzyme-linked immunosorbent assay (ELISA) with two antibodies against human mannan-binding lectin (MBL) enables elucidation of the collectin concentration in the blood, serum, and plasma. The collectin sugar specificity is demonstrated by determining the concentration of saccharide required to inhibit sugar binding by 50% in a saccharide-binding assay. Biological analyses including the complement-dependent hemolysis test and several other methods are used to evaluate collectin.

Induction of Recombinant Lectin Expression by an Artificially Constructed Tandem Repeat Structure: A Case Study Using Bryopsis plumosa Mannose-Binding Lectin.

Lectin is an important protein in medical and pharmacological applications. Impurities in lectin derived from natural sources and the generation of inactive proteins by recombinant technology are major obstacles for the use of lectins. Expressing recombinant lectin with a tandem repeat structure can potentially overcome these problems, but few studies have systematically examined this possibility. This was investigated in the present study using three distinct forms of recombinant mannose-binding lectin from Bryopsis plumosa (BPL2)-i.e., the monomer (rD1BPL2), as well as the dimer (rD2BPL2), and tetramer (rD4BPL2) arranged as tandem repeats. The concentration of the inducer molecule isopropyl ß-D-1-thiogalactopyranoside and the induction time had no effect on the efficiency of the expression of each construct. Of the tested constructs, only rD4BPL2 showed hemagglutination activity towards horse erythrocytes; the activity of towards the former was 64 times higher than that of native BPL2. Recombinant and native BPL2 showed differences in carbohydrate specificity; the activity of rD4BPL2 was inhibited by the glycoprotein fetuin, whereas that of native BPL2 was also inhibited by d-mannose. Our results indicate that expression as tandem repeat sequences can increase the efficiency of lectin production on a large scale using a bacterial expression system.

Biochemical and structural characterization of a mannose binding jacalin-related lectin with two-sugar binding sites from pineapple (Ananas comosus) stem.

A mannose binding jacalin-related lectin from Ananas comosus stem (AcmJRL) was purified and biochemically characterized. This lectin is homogeneous according to native, SDS-PAGE and N-terminal sequencing and the theoretical molecular mass was confirmed by ESI-Q-TOF-MS. AcmJRL was found homodimeric in solution by size-exclusion chromatography. Rat erythrocytes are agglutinated by AcmJRL while no agglutination activity is detected against rabbit and sheep erythrocytes. Hemagglutination activity was found more strongly inhibited by mannooligomannosides than by D-mannose. The carbohydrate-binding specificity of AcmJRL was determined in some detail by isothermal titration calorimetry. All sugars tested were found to bind with low affinity to AcmJRL, with Ka values in the mM range. In agreement with hemagglutination assays, the affinity increased from D-mannose to di-, tri- and penta-mannooligosaccharides. Moreover, the X-ray crystal structure of AcmJRL was obtained in an apo form as well as in complex with D-mannose and methyl-α-D-mannopyranoside, revealing two carbohydrate-binding sites per monomer similar to the banana lectin BanLec. The absence of a wall separating the two binding sites, the conformation of ß7ß8 loop and the hemagglutinating activity are reminiscent of the BanLec His84Thr mutant, which presents a strong anti-HIV activity in absence of mitogenic activity.

Isolation of a methylated mannose-binding protein from terrestrial worm Enchytraeus japonensis.

To elucidate a biological role of the methylated mannose residues found in N-glycans of terrestrial worm Enchytraeus japonensis, we first synthesized 3-O-methyl mannose- and 4-O-methyl mannose-derivatives and immobilized them to Sepharose 4B beads in order to isolate the sugar-binding protein. When whole protein extracts from the worms was applied to a series of the columns immobilized with the modified and unmodified mannose-derivatives, respectively, a protein with a molecular weight of 25,000 was isolated by 4-O-methyl mannose-immobilized column chromatography, and termed as a methylated mannose-binding protein (mMBP). mMBP bound weakly to a mannose-immobilized column and moderately to a 3-O-methyl mannose-immobilized column. The N-terminal amino acid sequences of mMBP and its endoprotease-digested peptides were determined. Using the degenerate first primers synthesized based on the primary sequence, a genomic DNA fragment was isolated. Then, the second primers were synthesized based on the genomic DNA fragment, and with use of them two cDNA fragments were obtained by the 3'- and 5'-RACE methods. Finally, the third primers were synthesized based on the sequences of the two cDNA fragments and one genomic DNA fragment, and with use of them a full-length cDNA of mMBP was isolated and shown to comprise a putative 633 bp open reading frame encoding 210 amino acid residues. BLAST analysis revealed that mMBP has identities by 26 ~ 55% to several proteins including the regeneration-upregulated protein 3 from the same species. Whether mMBP is involved in the regeneration of the worm is under investigation.

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.

Expression and characterization of recombinant chicken mannose binding lectin.

Mannose binding lectin (MBL) is a serum collagenous C-type lectin that plays an important role in the innate immune protection against pathogens. Previously, human and mouse studies have demonstrated that MBL binds a broad range of pathogens that results in their neutralization through agglutination, enhanced phagocytosis, and/or complement activation via the lectin pathway. The role of MBL in chicken is not well understood although the MBL concentration in serum seems to correlate with protection against infections. To investigate the role of MBL in chicken further, recombinant chicken MBL (RcMBL) was produced in HeLa R19 cells and purified using mannan affinity chromatography followed by gel filtration. RcMBL was shown to be structurally and functionally similar to native chicken MBL (NcMBL) isolated from serum. RcMBL is expressed as an oligomeric protein (mixture of trimers and oligomerized trimers) with a monomeric mass of 26kDa as determined by mass spectrometry, corresponding to the predicted mass. Glycan array analysis indicated that RcMBL bound most strongly to high-mannose glycans but also glycans with terminal fucose and GlcNac residues. The biological activity of RcMBL was demonstrated via its capacity to agglutinate Salmonella Typhimurium and to inhibit the hemagglutination activity of influenza A virus. The production of a structurally well-characterized and functionally active RcMBL will facilitate detailed studies into the protective role of MBL in innate defense against pathogens in chicken and other avian species.

Challenges and opportunities in fractionation of recombinant human mannan-binding lectin.

Mannan-binding lectin is an oligomeric protein of the innate immune system. Both natural MBL as well as recombinant protein is a heterogeneous composition of oligomers containing from two up to at least eight structural units. Recombinant synthesis in mammalian cell lines often leads to a composition containing small oligomers not abundant in MBL from natural sources such as human plasma. Here, two procedures for fractionation of MBL oligomers are described. In one case, affinity chromatography is employed to enrich the abundance of large MBL oligomers to produce a composition of oligomers resembling natural MBL. In a second case, large-volume ion exchange chromatography is employed to separate these oligomers into fractions with an enhanced size homogeneity. Finally, a well-established procedure for estimating the MBL protein concentration is detailed.

Neutrophil-infiltrated paw edema induced by mannose-binding Dioclea violacea lectin.

BACKGROUND: The potential edematogenic effect and the pharmacological characterization of a glucose-mannose-binding lectin from Dioclea violacea (DvL) were investigated. METHODS: Paw edema was induced with DvL in control animals, and in animals pretreated with glucocorticoid or with blockers of histamine, nitric oxide synthase, cyclooxygenase, platelet activating factor (PAF), bradykinin and lipoxygenase. RESULTS: DvL-induced paw edema paralleled with an increase in vascular permeability and myeloperoxidase (MPO) activity. DvL-induced edema could be prevented by pre-treatment with the lectin-binding sugar α-D-methyl mannoside. Dexamethasone, meclizine and Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) inhibited this effect. CONCLUSIONS: DvL induces edema, increase in vascular permeability and neutrophil infiltration. The edematogenic activity involves the lectin mannose-binding sites and is associated with histamine, cytokines and nitric oxide, since it could be treated with meclizine, dexamethasone and L-NAME.

Recombinant expression of the autocatalytic complement protease MASP-1 is crucially dependent on co-expression with its inhibitor, C1 inhibitor.

MASP-1 is a protease of the lectin pathway of complement. It is homologous with MASP-2, previously thought both necessary and sufficient for lectin pathway activation. Recently MASP-1 has taken centre stage with the observation that it is crucial to the activation of MASP-2 and thus central to complement activation. Numerous additional functions have been suggested for MASP-1 and its importance is obvious. Yet, thorough analyses of proteolytic activities and physiological roles in the human scenario have been hampered by difficulties in purifying or producing full-length human MASP-1. We present the successful expression of full-length recombinant human MASP-1 entirely in the zymogen form in a mammalian expression system. We found that the catalytic activity of MASP-1 suppresses its expression through rapid auto-activation and auto-degradation. This auto-degradation was not inhibited by the addition of inhibitors to the culture medium, and it was subsequently found to occur intracellularly. Numerous mutations aimed at attenuating auto-activation or preventing auto-degradation failed to rescue expression, as did also attempts at stabilizing the protease by co-expression with MBL or ficolins or expression in hepatocyte cell lines, representing the natural site of synthesis. The active protease was finally produced through co-expression with the serine protease inhibitor C1 inhibitor. We demonstrate that the expressed protease is capable of binding MBL and auto-activating, and is catalytically active. We have generalized the concept to the expression also of MASP-2 entirely in its zymogen form and with improved yields. We suggest a general advantage of expressing aggressive, autocatalytic proteases with their cognate inhibitors.

Mannan-binding lectin of the sea urchin Strongylocentrotus nudus.

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/ß-protein with eight ß-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.

Overproduction of recombinant human mannose-binding lectin (MBL) in Chinese hamster ovary cells.

Mannose-binding lectin (MBL) is an important serum protein that functions in the innate immune system and has been considered to have therapeutic potential in MBL replacement therapies for patients with deficient or low levels of MBL. In this study, we established a Chinese hamster ovary (CHO) cell line that overexpresses the recombinant human MBL (rhMBL) protein. In an 11-day batch culture process using a 30-L bioreactor (20-L working volume) and serum-free medium, these cells could produce over 226 mg/L of rhMBL protein. The recombinant protein was then purified to homogeneity from the culture supernatant using a three-step chromatographic procedure that resulted in a recovery rate of approximately 55%. This purified rhMBL protein adopted oligomeric bouquet-like structures that were similar to those of native MBL present in human blood, and these oligomeric structures were reported to be critical in MBL functions. We further demonstrated in carbohydrate binding and complementation activation assays that this rhMBL protein was functionally active with very similar dissociation constants and half maximal effective concentrations to those of native MBL.

[Preparation of the trimeric subunits of recombinant human mannan-binding lectin and analysis of its bioactivity].

OBJECTIVE: To prepare the trimeric subunits of recombinant human mannan-binding lectin (MBL) with biological activities. METHODS: A prokaryotic expression vector containing human MBL N-terminal deletant (rhMBLδN) gene we previously constructed was transformed into E. coli for efficient expression of rhMBLδN fusion protein. Based on the principle that the collagen polypeptides tend to self-assembly into the tertiary structure of proteins by forming a triple helix due to the characteristic properties of the collagen proteins, rhMBLδN fusion protein was limitedly hydrolyzed with thrombin. The obtained rhMBLδN polypeptide was repeatedly dialyzed in 50 mmol/L PBS (pH7.2) and ddH(2)O, and the final product was analyzed for its bioactivities using a ligand-binding assay and a C4d deposition assay. RESULTS: rhMBLδN polypeptide with a relative molecular mass of about 20 000 was obtained by limited proteolysis of rhMBLδN fusion protein with thrombin. Repeated dialyses of rhMBLδN polypeptides in 50 mmol/L PBS and ddH(2)O resulted in the isolation of the trimeric subunit trhMBLδN (with a relative molecular mass of about 50 000), which contained a collagen-like helix. The trhMBLδN protein had a higher ligand-binding activity than rhMBLδN polypeptide, and acquired the activity to initiate the lectin pathway of complement activation, but the activities were lower than those of natural MBL. CONCLUSION: We have successfully obtained the bioactive trimeric subunit of rhMBL, trhMBLδN, and this structural subunit is also the functional subunit of the MBL molecule.

Purification and characterization of a mannose recognition lectin from Oreochromis niloticus (tilapia fish): cytokine production in mice splenocytes.

The aim of this work was to purify and partially characterize a mannose recognition lectin from Nile tilapia (Oreochromis niloticus) serum, named OniL. OniL was isolated through precipitation with ammonium sulfate and affinity chromatography (Concanavalin A-Sepharose 4B). In addition, we evaluated carbohydrate specificity, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profiles, and in vitro immunomodulatory activity on mice splenocyte experimental cultures through cytotoxic assays and cytokine production. The ammonium sulfate fraction F2 showed the highest specific hemagglutinating activity (331) and was applied to affinity matrix. Adsorbed proteins (OniL) were eluted with methyl-α-D: -mannopyranoside. OniL, a 17-kDa protein by SDS-PAGE constituted by subunits of 11 and 6.6 kDa, showed highest affinity for methyl-α-D: -mannopyranoside and D: -mannose. Immunological assays, in vitro, showed that OniL did not show cytotoxicity against splenocytes, induced higher IFN-γ production and lower IL-10 as well as nitrite release. In conclusion, OniL lectin was successfully purified and showed a preferential Th1 response in mice splenocytes.

Inhibition of hepatitis C virus 3a genotype entry through Glanthus Nivalis Agglutinin.

BACKGROUND: Hepatitis C Virus (HCV) has two envelop proteins E1 and E2 which is highly glycosylated and play an important role in cell entry. Inhibition of virus at entry step is an important target to find antiviral drugs against HCV. Glanthus Nivalis Agglutinin (GNA) is a mannose binding lectin which has tendency for specific recognition and reversible binding to the sugar moieties of a wide variety of glycoproteins of enveloped viruses. RESULTS: In the present study, HCV pseudoparticles (HCVpp) for genotype 3a were produced to investigate the ability of GNA to block the HCV entry. The results demonstrated that GNA inhibit the infectivity of HCVpp and HCV infected serum in a dose-dependent manner and resulted in 50% reduction of virus at 1 ± 2 µg concentration. Molecular docking of GNA and HCV glycoproteins (E1 and E2) showed that GNA inhibit HCV entry by binding N-linked glycans. CONCLUSION: These results demonstrated that targeting the HCV glycans is a new approach to develop antiviral drugs against HCV.

Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis.

The mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis has been cloned, expressed, purified and crystallized and the crystals have been characterized using X-ray diffraction. The Matthews coefficient suggests the possibility of two lectin domains in the triclinic cell. The amino-acid sequence of the domain indicates structural similarity to well characterized ß-prism II fold lectins.

Characterization of the complex between mannose-binding lectin trimer and mannose-binding lectin-associated serine proteases.

Mannose-binding lectin (MBL) is an oligomeric serum lectin involved in innate immunity. Human MBL is complexed with three types of serine proteases (MASP-1, MASP-2 and MASP-3) and two types of their truncated forms (sMAP and MAp44). When an MBL complex binds to carbohydrates of pathogens, the complement system is activated via the lectin pathway. Human MBL is a mixture of different sized oligomers that range mainly from trimers to hexamers. It has been suggested that different MBL oligomers may have distinct MASP compositions. In the present study, an MBL trimer (MBL-I) exclusive of other oligomers was isolated from human serum by chromatography. Immunoblot analysis of MBL-I revealed that it had been co-purified with MASP-1 and sMAP. This suggests that MASP-1 and sMAP are bound to each other in MBL-I. The MBL-I complex was found to activate C2, but to lack the ability to activate C4 due to the absence of MASP-2.

Mannan-binding lectin regulates dendritic cell maturation and cytokine production induced by lipopolysaccharide.

BACKGROUND: Mannan-binding lectin (MBL) is a pattern-recognition molecule present in serum, which is involved in the innate immune defense by activating complement and promoting opsonophagocytosis. Dendritic cells (DCs) are professional antigen presenting cells (APCs) that are crucial for the initiation of adaptive immunity. Lipopolysaccharide (LPS) has been shown to be a strong activator of the inflammatory response and immune regulation. We first examined whether MBL modulated LPS-induced cellular responses, then investigated possible mechanisms of its inhibitory effect. RESULTS: MBL at higher concentrations (10-20 µg/ml) significantly attenuated LPS-induced maturation of monocyte-derived DCs (MDCs) and production of proinflammatory cytokines (e.g., IL-12 and TNF-α), and inhibited their ability to activate allogeneic T lymphocytes. It bound to immature MDCs at physiological calcium concentrations, and was optimal at supraphysiological calcium concentrations. MBL also bound directly to immature MDCs and attenuated the binding of LPS to the cell surfaces, resulting in decreased LPS-induced nuclear factor-κB (NF-κB) activity in these cells. CONCLUSION: All these data suggest that MBL could affect the functions of DCs by modifying LPS-induced cellular responses. This study supports an important role for MBL in the regulation of adaptive immune responses and inflammatory responses.

Characterisation of a mannose-binding C-type lectin from Oxyuranus scutellatus snake venom.

C-type lectins are calcium-dependent sugar binding proteins and are distributed ubiquitously amongst vertebrate organisms. As part of a wider study on Australian snake venom components, we have identified and characterised a C-type lectin from the venom of Oxyuranus scutellatus (Australian coastal taipan) with mannose-binding activity. This protein exhibited a subunit molecular mass of 15 kDa and was found to bind mannose and also bind to and agglutinate erythrocytes in a Ca(2+)-dependent manner. cDNA transcripts coding for C-lectin proteins were cloned and sequenced from six Australian elapid snake species and an antibody generated against the O. scutellatus mannose-binding C-lectin identified C-lectin proteins in the venom of 13 Australian elapid snakes by immunoblotting. Experimental evidence and molecular modelling also suggest that this protein exhibits a unique dimeric structure. This is the first confirmed example of a snake venom C-lectin with mannose-binding activity.

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.