A novel peptide derived from the mannose binding lectin inhibits LPS-activated TLR4/NF-κB signaling and suppresses ocular inflammation.

Uveitis is a major cause of vision impairment worldwide. Current treatments have limited effectiveness but severe complications. Mannose binding lectin (MBL) is an important protein of the innate immune system that binds to TLR4 and suppresses LPS-induced inflammatory cytokine secretion. MBL-mediated inhibition of inflammation via the TLR4 pathway and MBL-derived peptides might be a potential therapeutics. In this study, we designed a novel MBL-derived peptide, WP-17, targeting TLR4. Bioinformatics analysis was conducted for the sequence, structure and biological properties of WP-17. The binding of WP-17 to THP-1 cells was analyzed using flow cytometry. Signaling molecules were analyzed by western blotting, and activation of NF-κB was measured by immunofluorescence-histochemical analysis. Effects of WP-17 were studied in vitro using LPS-stimulated THP-1 cells and in vivo in endotoxin-induced uveitis (EIU). Our results showed that WP-17 could bind to TLR4 expressed on macrophages, thus downregulating the expression levels of MyD88, IRAK-4, and TRAF-6, and inhibiting the downstream NF-kB signaling pathway and LPS-induced expression of TNF-α and IL-6 in THP-1 cells. Moreover, in EIU rats, intravitreal pretreatment with WP-17 demonstrated significant inhibitory effects on ocular inflammation, attenuating the clinical and histopathological manifestations of uveitis, reducing protein leakage and cell infiltration into the aqueous humor, and suppressing TNF-α and IL-6 production in ocular tissues. In summary, our study provides the first evidence of a novel MBL-derived peptide that suppressed activation of the NF-кB pathway by targeting TLR4. The peptide effectively inhibited rat uveitis and may be a promising candidate for the management of ocular inflammatory diseases.

Capture and neutralization of SARS-CoV-2 and influenza virus by algae-derived lectins with high-mannose and core fucose specificities.

We first investigated the interactions between several algae-derived lectins and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). We created lectin columns using high-mannose (HM)-type glycan-specific lectins OAA and KAA-1 or core fucose-specific lectin hypninA-2 and conducted binding experiments with SARS-CoV-2. The results showed that these lectins were capable of binding to the virus. Furthermore, when examining the neutralization ability of nine different lectins, it was found that KAA-1, ESA-2, and hypninA-2 were effective in neutralizing SARS-CoV-2. In competitive inhibition experiments with glycoproteins, neutralization was confirmed to occur through HM-type or core fucose-type glycans. However, neutralization was not observed with other lectins, such as OAA. This trend of KAA-1 and ESA-2 having the neutralizing ability and OAA not having it was also similar to influenza viruses. Electron microscopy observations revealed that KAA-1 and hypninA-2 strongly aggregated SARS-CoV-2 particles, while OAA showed a low degree of aggregation. It is believed that the neutralization of SARS-CoV-2 involves multiple factors, such as glycan attachment sites on the S protein, the size of lectins, and their propensity to aggregate, which cause inhibition of receptor binding or aggregation of virus particles. This study demonstrated that several algae-derived lectins could neutralize SARS-CoV-2 and that lectin columns can effectively recover and concentrate the virus.

Anticancer Activity of Mannose-Specific Lectin, BPL2, from Marine Green Alga Bryopsis plumosa.

Lectin is a carbohydrate-binding protein that recognizes specific cells by binding to cell-surface polysaccharides. Tumor cells generally show various glycosylation patterns, making them distinguishable from non-cancerous cells. Consequently, lectin has been suggested as a good anticancer agent. Herein, the anticancer activity of Bryopsis plumosa lectins (BPL1, BPL2, and BPL3) was screened and tested against lung cancer cell lines (A549, H460, and H1299). BPL2 showed high anticancer activity compared to BPL1 and BPL3. Cell viability was dependent on BPL2 concentration and incubation time. The IC50 value for lung cancer cells was 50 µg/mL after 24 h of incubation in BPL2 containing medium; however, BPL2 (50 µg/mL) showed weak toxicity in non-cancerous cells (MRC5). BPL2 affected cancer cell growth while non-cancerous cells were less affected. Further, BPL2 (20 µg/mL) inhibited cancer cell invasion and migration (rates were ˂20%). BPL2 induced the downregulation of epithelial-to-mesenchymal transition-related genes (Zeb1, vimentin, and Twist). Co-treatment with BPL2 and gefitinib (10 µg/mL and 10 µM, respectively) showed a synergistic effect compared with monotherapy. BPL2 or gefitinib monotherapy resulted in approximately 90% and 70% cell viability, respectively, with concomitant treatment showing 40% cell viability. Overall, BPL2 can be considered a good candidate for development into an anticancer agent.

Glycosylation reduces the glycan-independent immunomodulatory effect of recombinant Orysata lectin in Drosophila S2 cells.

Several plant lectins, or carbohydrate-binding proteins, interact with glycan moieties on the surface of immune cells, thereby influencing the immune response of these cells. Orysata, a mannose-binding lectin from rice, has been reported to exert immunomodulatory activities on insect cells. While the natural lectin is non-glycosylated, recombinant Orysata produced in the yeast Pichia pastoris (YOry) is modified with a hyper-mannosylated N-glycan. Since it is unclear whether this glycosylation can affect the YOry activity, non-glycosylated rOrysata was produced in Escherichia coli (BOry). In a comparative analysis, both recombinant Orysata proteins were tested for their carbohydrate specificity on a glycan array, followed by the investigation of the carbohydrate-dependent agglutination of red blood cells (RBCs) and the carbohydrate-independent immune responses in Drosophila melanogaster S2 cells. Although YOry and BOry showed a similar carbohydrate-binding profiles, lower concentration of BOry were sufficient for the agglutination of RBCs and BOry induced stronger immune responses in S2 cells. The data are discussed in relation to different hypotheses explaining the weaker responses of glycosylated YOry. In conclusion, these observations contribute to the understanding how post-translational modification can affect protein function, and provide guidance in the selection of the proper expression system for the recombinant production of lectins.

Prevention by the Natural Artocarpin of Morphological and Biochemical Alterations on UVB-Induced HaCaT Cells.

Natural substances have gained considerable attention for skin protection against UV light reactions. Artocarpus altilis plant's heartwood extract is comprised of artocarpin as a major substance, already known for its interesting biological attributes as an antimicrobial, an anti-inflammatory, an antioxidant, and a melanogenesis inhibitor. The present work clarified the mechanism of natural artocarpin (NAR) with a purity of approximately 99% against the effects of UVB-induced HaCaT keratinocyte apoptosis. The indicated results showed that NAR suppresses free radical production (ROS and nitrite) and apoptosis-related molecule activation (caspase-3, p-p53, p-p38, and NF-κB p65) and secretion (TNF-α). Additionally, NAR prevented structural damages (nuclei condensation and fragmentation, apoptotic body formation, impaired cell adherence and round cell shape, disruption of F-actin filament, and clustering of cell death receptor CD95/Fas) and biophysical changes (plasma membrane rigidification). Thus, NAR acts directly from scavenging free radicals generated by UV and indirectly by suppressing morphological and biochemical UV-induced cell damages. Its biological effects are mainly attributed to antioxidant and antiapoptotic properties. Taken together, NAR could be considered as an effective natural product for photoprotective formulations.

The lectin Orysata induces phosphatase-mediated and carbohydrate-independent aggregation of insect cells.

Lectins, or carbohydrate-binding proteins, can cause agglutination of particular cells. This process is mediated by the interaction of the carbohydrate-binding domain with sugar structures on the cell surface, and this binding can be inhibited by pre-incubation of the lectin with its specific sugars. However, when incubated with insect cells, Orysata, a mannose-binding lectin from rice, caused aggregation of the cells, independent from carbohydrate binding activity. This phenomenon was observed for multiple insect cell lines, confirming the robustness of this phenotype. While the carbohydrate-dependent agglutination of red blood cells happens within minutes, the carbohydrate-independent aggregation of insect cells requires longer incubation times. Further analysis with the galactose-binding lectins SSA and Jacalin, validated the robustness of this lectin-induced, carbohydrate-independent aggregation in different insect cell lines. Since proteomic analysis revealed no changes in the proteome after treatment with the lectins, this cell aggregation is likely caused by the (in) activation or re-organization of the existing surface proteins. The use of inhibitors of phosphorylation and dephosphorylation, staurosporine (STS) and a phosphatase inhibitor (PPI) cocktail, pointed to dephosphorylation as a key mechanism in the lectin-induced, carbohydrate-independent aggregation of insect cells. Similar to contact inhibition, cell proliferation in cell aggregates was decreased. Analysis of the marker for cell proliferation, cyclin E, confirmed that aggregated cells enter a quiescent state. The current data offer a new perspective on the mechanism by which lectins execute their activities, specifically through lectin-induced phosphatase-mediated cell aggregation and proliferation inhibition, independent from their carbohydrate-binding activity.

Catalytic and lectin domains in neuraminidase A from Streptococcus pneumoniae are capable of an intermolecular assembly: Implications for biofilm formation.

Neuraminidase A from Streptococcus pneumoniae (NanA) is a cell wall-bound modular enzyme containing one lectin and one catalytic domain. Unlike homologous NanB and NanC expressed by the same bacterium, the two domains within one NanA molecule do not form a stable interaction and are spatially separated by a 16-amino acid-long flexible linker. In this work, the ability of NanA to form intermolecular assemblies was characterized using the methods of molecular modeling and bioinformatic analysis based on crystallographic data and by bringing together previously published experimental data. It was concluded that two catalytic domains, as well as one catalytic and one lectin domain, originating from two cell wall-bound NanA molecules, can interact through a previously uncharacterized interdomain interface to form complexes stabilized by a network of intermolecular hydrogen bonds and salt bridges. Supercomputer modeling strongly indicated that artocarpin, an earlier experimentally discovered inhibitor of the pneumococcal biofilm formation, is able to bind to a site located in the catalytic domain of one NanA entity and prevent its interaction with the lectin or catalytic domain of another NanA entity, thus directly precluding the generation of intermolecular assemblies. The revealed structural adaptation is discussed as one plausible mechanism of noncatalytic participation of this potentially key pathogenicity enzyme in pneumococcal biofilm formation.

Photoprotective Potential of the Natural Artocarpin against In Vitro UVB-Induced Apoptosis.

Apoptosis, a well-known pattern of programmed cell death, occurs in multicellular organisms not only for controlling tissue homeostasis but also for getting rid of severely damaged cells in order to protect the redundant growth of abnormal cells undergoing cancerous cells. The epidermis of the human skin, composed largely of keratinocytes (KCs), is renewed continuously. Therefore, KCs apoptosis plays a critical role in the maintenance of epidermis structure and function. However, regulated cell death can be disturbed by environmental factors especially ultraviolet radiation (UV) B, leading to the formation of sunburn cells (KCs undergoing UVB-induced apoptosis) and impairing the skin integrity. In the present study, we firstly reported the potential of the natural artocarpin (NAR) to regulate UVB-induced human KCs apoptosis. The NAR showed antilipid peroxidation with an IC50 value of 18.2 ± 1.6 µg/mL, according to TBARS assay while the IC50 value of trolox, a well-known antioxidant, was 7.3 ± 0.8 µg/mL. For cell-based studies, KCs were pretreated with 3.1 µg/mL of the NAR for 24 hr and then exposed to UVB at 55 mJ/cm2. Our data indicated that the NAR pretreatment reduces UVB-induced oxidative stress by scavenging free radicals and nitric oxide and therefore prevents reactive oxygen species (ROS) and reactive nitrogen species- (RNS-) mediated apoptosis. The NAR pretreatment has been shown also to reduce the UVB-induced cyclobutane pyrimidine dimer (CPD) lesions by absorbing UVB radiation and regulating the cell cycle phase. Additionally, the NAR pretreatment was found to modulate the expression of cleaved caspases-3 and 8 that trigger different signalling cascades leading to apoptosis. Thus, these results provide a basis for the investigation of the photoprotective effect of the NAR isolated from A. altilis heartwood and suggest that it can be potentially used as an agent against UVB-induced skin damages.

A novel mannose-binding lectin from Liparis nervosa with anti-fungal and anti-tumor activities.

Plant lectins are carbohydrate-binding proteins with nonimmune origin, which can reversibly bind with carbohydrates, agglutinate cells, and precipitate polysaccharides and glycoconjugates. Plant lectins have attracted much attention for their anti-virus, anti-proliferation, and pro-apoptosis properties. Thus the exploration of new lectins has received special attention. Here we purified a mannose-binding lectin from the rhizomes of Liparis nervosa by ion exchange chromatography on DEAE-Sepharose, affinity chromatography on Mannose-Sepharose 4B, and gel filtration chromatography on Sephacryl S-100. The purified L. nervosa lectin (LNL) was identified to be a monomeric protein with a molecular mass of 13 kDa. LNL exhibited hemagglutinating activity towards rabbit erythrocytes, and its activity could be strongly inhibited by D-mannose, N-acetyl glucosamine and thyroglobulin. In vitro experiments showed that LNL exhibited a comparable anti-fungal activity against Piricularia oryzae (Cavara), Bipolaris maydis, Fusarium graminearum, and Sclerotium rolfsii, and anti-proliferation activity against tumor cells by inducing apoptosis. The full-length cDNA sequence of LNL is 715 bp in length and contains a 525 bp open reading frame (ORF) encoding a 110-residue mature protein. It was predicted to have three mannose-binding conserved motifs 'QXDXNXVXY'. The binding pattern of LNL was further revealed by homology modeling and molecular docking. We demonstrated that LNL is not only a potential therapeutic candidate against tumor but also a new anti-fungal agent.

Exploring the carbohydrate-binding ability of Canavalia bonariensis lectin in inflammation models.

Lectins are a group of proteins of non-immune origin recognized for their ability to bind reversibly to carbohydrates. Researchers have been intrigued by oligosaccharides and glycoconjugates for their involvement as mediators of complex cellular events and then many biotechnological applications of lectins are based on glycocode decoding and their activities. Here, we report a structural and biological study of a ConA-like mannose/glucose-specific lectin from Canavalia bonariensis seeds, CaBo. More specifically, we evaluate the binding of CaBo with α-methyl-D-mannoside (MMA) and mannose-1,3-α-D-mannose (M13) and the resultant in vivo effects on a rat model of acute inflammation. A virtual screening was also carried out to cover a larger number of possible bindings of CaBo. In silico analysis demonstrated the stability of CaBo interaction with mannose-type ligands, and the lectin was able to induce acute inflammation in rats with the participation of the carbohydrate recognition domain (CRD) and histamine release. These results confirm the ability of CaBo to interact with hybrid and high-mannose N-glycans, supporting the hypothesis that CaBo's biological activity occurs primarily through its interaction with cell surface glycosylated receptors.

Discovery of antitumor lectins from rainforest tree root transcriptomes.

Glycans are multi-branched sugars that are displayed from lipids and proteins. Through their diverse polysaccharide structures they can potentiate a myriad of cellular signaling pathways involved in development, growth, immuno-communication and survival. Not surprisingly, disruption of glycan synthesis is fundamental to various human diseases; including cancer, where aberrant glycosylation drives malignancy. Here, we report the discovery of a novel mannose-binding lectin, ML6, which selectively recognizes and binds to these irregular tumor-specific glycans to elicit potent and rapid cancer cell death. This lectin was engineered from gene models identified in a tropical rainforest tree root transcriptome and is unusual in its six canonical mannose binding domains (QxDxNxVxY), each with a unique amino acid sequence. Remarkably, ML6 displays antitumor activity that is >105 times more potent than standard chemotherapeutics, while being almost completely inactive towards non-transformed, healthy cells. This activity, in combination with results from glycan binding studies, suggests ML6 differentiates healthy and malignant cells by exploiting divergent glycosylation pathways that yield naïve and incomplete cell surface glycans in tumors. Thus, ML6 and other high-valence lectins may serve as novel biochemical tools to elucidate the glycomic signature of different human tumors and aid in the rational design of carbohydrate-directed therapies. Further, understanding how nature evolves proteins, like ML6, to combat the changing defenses of competing microorganisms may allow for fundamental advances in the way we approach combinatorial therapies to fight therapeutic resistance in cancer.

Regulation of neutrophil function by selective targeting of glycan epitopes expressed on the integrin CD11b/CD18.

Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases.

Proliferative Effect of Tilapia Fish (Oreochromis niloticus) Lectin on BALB/c Mice Splenocytes.

BACKGROUND: Lectins have been studied in recent years due to their immunomodulatory activities. OBJECTIVE: We purified a lectin named OniL from tilapia fish (Oreochromis niloticus) and here we analyzed the cell proliferation and cytokine production in Balb/c mice splenocytes. METHODS: Cells were stimulated in vitro in 24, 48, 72 hours and 6 days with different concentrations of OniL and Con A. Evaluation of cell proliferation was performed through [3H]-thymidine incorporation, cytokines were investigated using ELISA assay and cell viability assay was performed by investigation of damage through signals of apoptosis and necrosis. RESULTS: OniL did not promote significant cell death, induced high mitogenic activity in relation to control and Con A and stimulated the cells to release high IL-2 and IL-6 cytokines. CONCLUSION: These findings suggest that, like Con A, OniL lectin can be used as a mitogenic agent in immunostimulatory assays.

Insights into anti-pathogenic activities of mannose lectins.

Carbohydrate-binding proteins, also known as lectins, are valuable tools for biotechnology, including pharmacological uses. Mannose lectins obtained from plant and animal sources are applied to protection and characterization of autoimmune diseases as well as defense proteins against pathogens. The presence of mannose-binding lectins in plants that also recognize glucose could be entitled Man/Glc lectins; such specificity has allowed employing these vegetal lectins for several applications. Animal mannose-binding lectins are synthesized in the liver and secreted into the blood stream where both concentration and activity are greatly affected due to gene polymorphisms; these serum proteins play important roles in the immune system by recognizing mannose-like carbohydrate ligands found exclusively on pathogenic microorganisms. Mannose lectins already showed strong binding to relevant bacteria, viruses, protozoa and helminth species, initiating potent host defense mechanisms by inducing growth inhibition or death of such organisms; the ability to prevent the formation or destruction of microbial biofilms has also been reported. Mannose-binding lectins have attracted considerable attention against carcinogenesis and atherogenesis. The aim of this review article is to approach biotechnology characteristics of these lectins from different sources and microorganism/cell surface interactions with mannose; in addition, aspects of mechanisms associated to lectin antipathogenic activities are described.

Mannose-Specific Lectins from Marine Algae: Diverse Structural Scaffolds Associated to Common Virucidal and Anti-Cancer Properties.

To date, a number of mannose-specific lectins have been isolated and characterized from seaweeds, especially from red algae. In fact, man-specific seaweed lectins consist of different structural scaffolds harboring a single or a few carbohydrate-binding sites which specifically recognize mannose-containing glycans. Depending on the structural scaffold, man-specific seaweed lectins belong to five distinct structurally-related lectin families, namely (1) the griffithsin lectin family (ß-prism I scaffold); (2) the Oscillatoria agardhii agglutinin homolog (OAAH) lectin family (ß-barrel scaffold); (3) the legume lectin-like lectin family (ß-sandwich scaffold); (4) the Galanthus nivalis agglutinin (GNA)-like lectin family (ß-prism II scaffold); and, (5) the MFP2-like lectin family (MFP2-like scaffold). Another algal lectin from Ulva pertusa, has been inferred to the methanol dehydrogenase related lectin family, because it displays a rather different GlcNAc-specificity. In spite of these structural discrepancies, all members from the five lectin families share a common ability to specifically recognize man-containing glycans and, especially, high-mannose type glycans. Because of their mannose-binding specificity, these lectins have been used as valuable tools for deciphering and characterizing the complex mannose-containing glycans from the glycocalyx covering both normal and transformed cells, and as diagnostic tools and therapeutic drugs that specifically recognize the altered high-mannose N-glycans occurring at the surface of various cancer cells. In addition to these anti-cancer properties, man-specific seaweed lectins have been widely used as potent human immunodeficiency virus (HIV-1)-inactivating proteins, due to their capacity to specifically interact with the envelope glycoprotein gp120 and prevent the virion infectivity of HIV-1 towards the host CD4+ T-lymphocyte cells in vitro.

Artocarpin induces cell apoptosis in human osteosarcoma cells through endoplasmic reticulum stress and reactive oxygen species.

Osteosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. However, because of side effects and drug resistance in chemotherapy and the insufficiency of an effective adjuvant therapy for osteosarcoma, it is necessary to research novel treatments. This study was the first to investigate the anticancer effects of the flavonoid derivative artocarpin in osteosarcoma. Artocarpin induced cell apoptosis in three human osteosarcoma cell lines-U2OS, MG63, and HOS. Artocarpin was also associated with increased intracellular reactive oxygen species (ROS). Mitochondrial dysfunction was followed by the release of cytochrome c from mitochondria and accompanied by decreased antiapoptotic Bcl-2 and Bcl-xL and increased proapoptotic protein Bak and Bax. Artocarpin triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels and increased glucose-regulated protein 78 and 94 expressions, and also increased calpains expression and activity. Animal studies revealed a dramatic 40% reduction in tumor volume after 18 days of treatment. This study demonstrated a novel anticancer activity of artocarpin against human osteosarcoma cells and in murine tumor models. In summary, artocarpin significantly induced cell apoptosis through ROS, ER stress, mitochondria, and the caspase pathway, and may thus be a novel anticancer treatment for osteosarcoma.

Internalization of a sunflower mannose-binding lectin into phytopathogenic fungal cells induces cytotoxicity.

Lectins are carbohydrate-affinity proteins with the ability to recognize and reversibly bind specific glycoconjugates. We have previously isolated a bioactive sunflower mannose-binding lectin belonging to the jacalin-related family called Helja. Despite of the significant number of plant lectins described in the literature, only a small group exhibits antifungal activity and the mechanism by which they kill fungi is still not understood. The aim of this work was to explore Helja activity on plant pathogenic fungi, and provide insights into its mechanism of action. Through cellular and biochemical experimental approaches, here we show that Helja exerts an antifungal effect on Sclerotinia sclerotiorum, a sunflower pathogen. The lectin interacts with the fungal spore surface, permeabilizes its plasma membrane, can be internalized into the cell and induces oxidative stress, finally leading to the cell death. On the other hand, Helja is inactive towards Fusarium solani, a non-pathogen of sunflower, showing the selective action of the lectin. The mechanistic basis for the antifungal activity of an extracellular jacalin lectin is presented, suggesting its initial interaction with fungal cell wall carbohydrates and further internalization. The implication of our findings for plant defense is discussed.

A Novel High-Mannose Specific Lectin from the Green Alga Halimeda renschii Exhibits a Potent Anti-Influenza Virus Activity through High-Affinity Binding to the Viral Hemagglutinin.

We have isolated a novel lectin, named HRL40 from the green alga Halimeda renschii. In hemagglutination-inhibition test and oligosaccharide-binding experiment with 29 pyridylaminated oligosaccharides, HRL40 exhibited a strict binding specificity for high-mannose N-glycans having an exposed (α1-3) mannose residue in the D2 arm of branched mannosides, and did not have an affinity for monosaccharides and other oligosaccharides examined, including complex N-glycans, an N-glycan core pentasaccharide, and oligosaccharides from glycolipids. The carbohydrate binding profile of HRL40 resembled those of Type I high-mannose specific antiviral algal lectins, or the Oscillatoria agardhii agglutinin (OAA) family, which were previously isolated from red algae and a blue-green alga (cyanobacterium). HRL40 potently inhibited the infection of influenza virus (A/H3N2/Udorn/72) into NCI-H292 cells with half-maximal effective dose (ED50) of 2.45 nM through high-affinity binding to a viral envelope hemagglutinin (KD, 3.69 × 10-11 M). HRL40 consisted of two isolectins (HRL40-1 and HRL40-2), which could be separated by reverse-phase HPLC. Both isolectins had the same molecular weight of 46,564 Da and were a disulfide -linked tetrameric protein of a 11,641 Da polypeptide containing at least 13 half-cystines. Thus, HRL40, which is the first Type I high-mannose specific antiviral lectin from the green alga, had the same carbohydrate binding specificity as the OAA family, but a molecular structure distinct from the family.

Lectin from Canavalia villosa seeds: A glucose/mannose-specific protein and a new tool for inflammation studies.

With important carbohydrate binding properties, lectins are proteins able to decipher the glycocode, and as such, they can be used in bioassays involving cell-cell communication, protein targeting, inflammation, and hypernociception, among others. In this study, a new glucose/mannose-specific lectin from Canavalia villosa seeds (Cvill) was isolated by a single affinity chromatography step in a Sephadex® G-50 column, with a purification yield of 19.35mg of lectin per gram of powdered seed. Analysis of intact protein by mass spectrometry showed the lectin is composed of three polypeptide chains, including a 25.6kDa α chain, 12.9KDa ß, and 12.6 KDa γ fragments, similar to the profile of ConA-like glucose/mannose-specific lectins. Partial sequence of the protein was obtained by MS-MALDI TOF/TOF covering 41.7% of its primary structure. Cvill presented sugar specificity to d-glucose, α-methyl-d-mannoside, d-mannose, and glycoproteins fetuin and ovoalbumin. The lectin characterization showed that Cvill presents high stability within a broad range of pH and temperature, also showing average toxicity against Artemia nauplii. The proinflammatory effect of Cvill was observed by induction of paw edema and hypernociception in mice, with the participation of the carbohydrate binding site, showing its potential to be used as tool in inflammation studies.