Multifunctional Cell Regulation Activities of the Mussel Lectin SeviL: Induction of Macrophage Polarization toward the M1 Functional Phenotype.

SeviL, a galactoside-binding lectin previously isolated from the mussel Mytilisepta virgata, was demonstrated to trigger apoptosis in HeLa ovarian cancer cells. Here, we show that this lectin can promote the polarization of macrophage cell lines toward an M1 functional phenotype at low concentrations. The administration of SeviL to monocyte and basophil cell lines reduced their growth in a dose-dependent manner. However, low lectin concentrations induced proliferation in the RAW264.7 macrophage cell line, which was supported by the significant up-regulation of TOM22, a component of the mitochondrial outer membrane. Furthermore, the morphology of lectin-treated macrophage cells markedly changed, shifting from a spherical to an elongated shape. The ability of SeviL to induce the polarization of RAW264.7 cells to M1 macrophages at low concentrations is supported by the secretion of proinflammatory cytokines and chemokines, as well as by the enhancement in the expression of IL-6- and TNF-α-encoding mRNAs, both of which encode inflammatory molecular markers. Moreover, we also observed a number of accessory molecular alterations, such as the activation of MAP kinases and the JAK/STAT pathway and the phosphorylation of platelet-derived growth factor receptor-α, which altogether support the functional reprogramming of RAW264.7 following SeviL treatment. These results indicate that this mussel ß-trefoil lectin has a concentration-dependent multifunctional role in regulating cell proliferation, phenotype, and death in macrophages, suggesting its possible involvement in regulating hemocyte activity in vivo.

Carbohydrate-Binding Properties and Antimicrobial and Anticancer Potential of a New Lectin from the Phloem Sap of Cucurbita pepo.

A Cucurbita phloem exudate lectin (CPL) from summer squash (Cucurbita pepo) fruits was isolated and its sugar-binding properties and biological activities were studied. The lectin was purified by affinity chromatography and the hemagglutination assay method was used to determine its pH, heat stability, metal-dependency and sugar specificity. Antimicrobial and anticancer activities were also studied by disc diffusion assays and in vivo and in vitro methods. The molecular weight of CPL was 30 ± 1 KDa and it was stable at different pH (5.0 to 9.0) and temperatures (30 to 60 °C). CPL recovered its hemagglutination activity in the presence of Ca2+. 4-nitrophenyl-α-D-glucopyranoside, lactose, rhamnose and N-acetyl-D-glucosamine strongly inhibited the activity. With an LC50 value of 265 µg/mL, CPL was moderately toxic and exhibited bacteriostatic, bactericidal and antibiofilm activities against different pathogenic bacteria. It also exhibited marked antifungal activity against Aspergillus niger and agglutinated A. flavus spores. In vivo antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice was observed when CPL exerted 36.44% and 66.66% growth inhibition at doses of 3.0 mg/kg/day and 6.0 mg/kg/day, respectively. A 12-day treatment by CPL could reverse their RBC and WBC counts as well as restore the hemoglobin percentage to normal levels. The MTT assay of CPL performed against human breast (MCF-7) and lung (A-549) cancer cell lines showed 29.53% and 18.30% of inhibitory activity at concentrations of 128 and 256 µg/mL, respectively.

Flavonoids as potential KRAS inhibitors: DFT, molecular docking, molecular dynamics simulation and ADMET analyses.

KRAS mutations linked with cancer. Flavonoids were docked against KRAS G12C and G12D receptors. Abyssinone III, alpha naphthoflavone, beta naphthoflavone, abyssinone I, abyssinone II and beta naphthoflavone, genistin, daidzin showed good docking scores against KRAS G12C and G12D receptors, respectively. The MD simulation data revealed that Rg, RMSD, RMSF, and SASA values were within acceptable limits. Alpha and beta naphthoflavone showed good binding energies with KRAS G12C and G12D receptors. DFT and MEP analysis highlighted the nucleophilic and electrophilic zones of best-docked flavonoids. A novel avenue for the control of KRAS G12C and G12D mutations is made possible by flavonoids.

In the present study, we computationally established the role of flavonoids as KRAS G12C and G12D inhibitors.Initially we selected 93 flavonoids and docked against 8AFB (KRAS G12C) and 7RT1 (KRAS G12D) using Sotorasib and MRTX 1133 as standards.A 100 ns MD simulation revealed that the radius of gyration, RMSD, RMSF, and SASA values were within acceptable limits and that there were a greater number of donors and acceptors for hydrogen bonds.In addition to the KRAS G12C 8AFB receptor, the maximum binding energy was shown by alpha Naphthoflavone (−26.471 kJ/mol), and for the KRAS G12D 7RT1 receptor, the maximum binding energy was shown by beta Naphthoflavone (−15.433 kJ/mol).FMO and MEP analysis data highlighted the best-docked flavonoids' potential areas for nucleophilic and electrophilic attacks.ADMET properties have been calculated and provide safe use and low toxicity for both aquatic and non-aquatic species.

In Silico Pharmacokinetics, Molecular Docking and Molecular Dynamics Simulation Studies of Nucleoside Analogs for Drug Discovery- A Mini Review.

Nucleoside analogs have been widely used as antiviral, antitumor, and antiparasitic agents due to their ability to inhibit nucleic acid synthesis. Adenosine, cytidine, guanosine, thymidine and uridine analogs such as didanosine, vidarabine, remdesivir, gemcitabine, lamivudine, acyclovir, abacavir, zidovusine, stavudine, and idoxuridine showed remarkable anticancer and antiviral activities. In our previously published articles, our main intention was to develop newer generation nucleoside analogs with acylation-induced modification of the hydroxyl group and showcase their biological potencies. In the process of developing nucleoside analogs, in silico studies play an important role and provide a scientific background for biological data. Molecular interactions between drugs and receptors followed by assessment of their stability in physiological environments, help to optimize the drug development process and minimize the burden of unwanted synthesis. Computational approaches, such as DFT, FMO, MEP, ADMET prediction, PASS prediction, POM analysis, molecular docking, and molecular dynamics simulation, are the most popular tools to culminate all preclinical study data and deliver a molecule with maximum bioactivity and minimum toxicity. Although clinical drug trials are crucial for providing dosage recommendations, they can only indirectly provide mechanistic information through researchers for pathological, physiological, and pharmacological determinants. As a result, in silico approaches are increasingly used in drug discovery and development to provide mechanistic information of clinical value. This article portrays the current status of these methods and highlights some remarkable contributions to the development of nucleoside analogs with optimized bioactivity.

Macromolecules: Synthesis, antimicrobial, POM analysis and computational approaches of some glucoside derivatives bearing acyl moieties.

Macromolecules i.e., carbohydrate derivatives are crucial to biochemical and medical research. Herein, we designed and synthesized eight methyl α-D-glucopyranoside (MGP) derivatives (2-8) in good yields following the regioselective direct acylation method. The structural configurations of the synthesized MGP derivatives were analyzed and verified using multiple physicochemical and spectroscopic techniques. Antimicrobial experiments revealed that almost all derivatives demonstrated noticeable antifungal and antibacterial efficacy. The synthesized derivatives showed minimum inhibitory concentration (MIC) values ranging from 0.75 µg/mL to 1.50 µg/mL and minimum bactericidal concentrations (MBCs) ranging from 8.00 µg/mL to 16.00 µg/mL. Compound 6 inhibited Ehrlich ascites carcinoma (EAC) cell proliferation by 10.36% with an IC50 of 2602.23 µg/mL in the MTT colorimetric assay. The obtained results were further rationalized by docking analysis of the synthesized derivatives against 4URO and 4XE3 receptors to explore the binding affinities and nonbonding interactions of MGP derivatives with target proteins. Compound 6 demonstrated the potential to bind with the target with the highest binding energy. In a stimulating environment, a molecular dynamics study showed that MGP derivatives have a stable conformation and binding pattern. The MGP derivatives were examined using POM (Petra/Osiris/Molinspiration) bioinformatics, and as a result, these derivatives showed good toxicity, bioavailability, and pharmacokinetics. Various antifungal/antiviral pharmacophore (Oδ-, O'δ-) sites were identified by using POM investigations, and compound 6 was further tested against other pathogenic fungi and viruses, such as Micron and Delta mutants of SARS-CoV-2.

Synthesis, Antimicrobial, Anticancer, PASS, Molecular Docking, Molecular Dynamic Simulations & Pharmacokinetic Predictions of Some Methyl ß-D-Galactopyranoside Analogs.

A series of methyl ß-D-galactopyranoside (MGP, 1) analogs were selectively acylated with cinnamoyl chloride in anhydrous N,N-dimethylformamide/triethylamine to yield 6-O-substitution products, which was subsequently converted into 2,3,4-tri-O-acyl analogs with different acyl halides. Analysis of the physicochemical, elemental, and spectroscopic data of these analogs revealed their chemical structures. In vitro antimicrobial testing against five bacteria and two fungi and the prediction of activity spectra for substances (PASS) showed promising antifungal functionality comparing to their antibacterial activities. Minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) tests were conducted for four compounds (4, 5, 6, and 9) based on their activity. MTT assay showed low antiproliferative activity of compound 9 against Ehrlich's ascites carcinoma (EAC) cells with an IC50 value of 2961.06 µg/mL. Density functional theory (DFT) was used to calculate the thermodynamic and physicochemical properties whereas molecular docking identified potential inhibitors of the SARS-CoV-2 main protease (6Y84). A 150-ns molecular dynamics simulation study revealed the stable conformation and binding patterns in a stimulating environment. In-silico ADMET study suggested all the designed molecules to be non-carcinogenic, with low aquatic and non-aquatic toxicity. In summary, all these antimicrobial, anticancer and in silico studies revealed that newly synthesized MGP analogs possess promising antiviral activity, to serve as a therapeutic target for COVID-19.

Antiproliferative and Antimicrobial Potentials of a Lectin from Aplysia kurodai (Sea Hare) Eggs.

In recent years, there has been considerable interest in lectins from marine invertebrates. In this study, the biological activities of a lectin protein isolated from the eggs of Sea hare (Aplysia kurodai) were evaluated. The 40 kDa Aplysia kurodai egg lectin (or AKL-40) binds to D-galacturonic acid and D-galactose sugars similar to previously purified isotypes with various molecular weights (32/30 and 16 kDa). The N-terminal sequence of AKL-40 was similar to other sea hare egg lectins. The lectin was shown to be moderately toxic to brine shrimp nauplii, with an LC50 value of 63.63 µg/mL. It agglutinated Ehrlich ascites carcinoma cells and reduced their growth, up to 58.3% in vivo when injected into Swiss albino mice at a rate of 2 mg/kg/day. The morphology of these cells apparently changed due to AKL-40, while the expression of apoptosis-related genes (p53, Bax, and Bcl-XL) suggested a possible apoptotic pathway of cell death. AKL-40 also inhibited the growth of human erythroleukemia cells, probably via activating the MAPK/ERK pathway, but did not affect human B-lymphoma cells (Raji) or rat basophilic leukemia cells (RBL-1). In vitro, lectin suppressed the growth of Ehrlich ascites carcinoma and U937 cells by 37.9% and 31.8%, respectively. Along with strong antifungal activity against Talaromyces verruculosus, AKL showed antibacterial activity against Staphylococcus aureus, Shigella sonnei, and Bacillus cereus whereas the growth of Escherichia coli was not affected by the lectin. This study explores the antiproliferative and antimicrobial potentials of AKL as well as its involvement in embryo defense of sea hare.

In silico DFT study, molecular docking, and ADMET predictions of cytidine analogs with antimicrobial and anticancer properties.

Nucleoside analogs contribute in pharmaceutical and clinical fields as medicinal agents and approved drugs. This work focused to investigate the antimicrobial, anticancer activities, and structure-activity relationship (SAR) of cytidine and its analogs with computational studies. Microdilution was used to determine the antimicrobial activity, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the modified analogs against human and phytopathogenic strains. Compounds (7), (10), and (14) were the most potent against Escherichia coli and Salmonella abony strains with MIC and MBC values from 0.316 ± 0.02 to 2.50 ± 0.03 and 0.625 ± 0.04 to 5.01 ± 0.06 mg/ml, respectively. The highest inhibitory activity was observed against gram-positive bacteria. Numerous analogs (10), (13), (14), and (15) exhibited good activity against the tested fungi Aspergillus niger and Aspergillus flavus. Anticancer activity of the cytidine analogs was examined through MTT colorimetric assay against Ehrlich's ascites carcinoma (EAC) tumor cells whereas compound 6 showed the maximum antiproliferative activity with an IC50 value of 1168.97 µg/ml. To rationalize this observation, their quantum mechanical and molecular docking studies have been performed against urate oxidase of A. flavus 1R51 to investigate the binding mode, binding affinity, and non-bonding interactions. It was observed that most of the analogs exhibited better binding properties than the parent drug. In silico ADMET prediction was attained to evaluate the drug-likeness properties that revealed the improved pharmacokinetic profile with lower acute oral toxicity of cytidine analogs. Based on the in vitro and in silico analysis, this exploration can be useful to develop promising cytidine-based antimicrobial drug(s). SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s40203-021-00102-0.

The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata.

SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac[Formula: see text](2-3)Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc) and its precursor, asialo-GM1 (Gal[Formula: see text](1-3)GalNAc[Formula: see text](1-4)Gal[Formula: see text](1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the [Formula: see text]-trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

Catfish egg lectin affects influx and efflux rates of sunitinib in human cervical carcinoma HeLa cells.

New treatment protocols are aiming to reduce the dose of the multitargeted tyrosine kinase inhibitor sunitinib, as sunitinib elicits many adverse effects depending on its dosage. Silurus asotus egg lectin (SAL) has been reported to enhance the incorporation of propidium iodide as well as doxorubicin into Burkitt's lymphoma Raji cells through binding to globotriaosylceramide (Gb3) on the cell surface. The objective of this study was to examine whether SAL enhances the cytotoxic effect of sunitinib in Gb3-expressing HeLa cells. Although the treatment with SAL delayed the cell growth and enhanced the propidium iodide uptake, cell death accompanied by membrane collapse was not observed. The viability of sunitinib-treated HeLa cells was significantly reduced when the treatment occurred in combination with SAL compared to their separate usage. Sunitinib uptake significantly increased for 30 min in SAL-treated cells, and this increment was almost completely abolished by the addition of L-rhamnose, a hapten sugar of SAL, but not by D-glucose. After removal of SU from the medium, the intracellular sunitinib level in SAL-treated cells was higher than in untreated cells for 24 h, which was not observed in Gb3-deficient HeLa cells. Furthermore, we observed that SAL promoted the formation of lysosome-like structures, which are LAMP1 positive but not acidic in HeLa cells, which can trap sunitinib. Interestingly, SAL-induced vacuolation in HeLa cells was not observed in another Gb3 positive Raji cells. Our findings suggest that SAL/Gb3 interaction promoted sunitinib uptake and suppressed sunitinib excretion and that sunitinib efficiently exerted cytotoxicity against HeLa cells.

Purification and Functional Characterization of the Effects on Cell Signaling of Mytilectin: A Novel ß-Trefoil Lectin from Marine Mussels.

In the 2010s, a novel lectin family with ß-trefoil folding has been identified in marine mussels from the family Mytilidae (phylum Mollusca). "MytiLec-1," the lectin described in this chapter, was the first member of this family to be isolated and characterized from the Mediterranean mussel Mytilus galloprovincialis, a commercially and ecologically important species, spread in marine coastal areas worldwide. MytiLec-1 bound to the sugar moiety of globotriose (Gb3: Galα1-4Galß1-4Glc), an α-galactoside, leading to apoptosis of Gb3-expressing Burkitt's lymphoma cells. Although the primary structure of MytiLec-1 was quite unusual, its three-dimensional structure was arranged as a ß-trefoil fold, which is the typical architecture of "Ricin B chain (or R)-type" lectins, which are found in a broad range of organisms. To date, MytiLec-1-like lectins have been exclusively found in a few species of the mollusk family Mytilidae (M. galloprovincialis, M. trossulus, M. californianus, and Crenomytilus grayanus) and in the phylum Brachiopoda. Transcriptome data revealed the presence of different structural forms of mytilectin in mussels, which included prototype and chimera-type proteins. The primary sequence of these lectins did not match any previously described known protein family, leading to their assignment to the new "mytilectin family." We here report the method of purification of this lectin and describe its use in cell biology.

A GM1b/asialo-GM1 oligosaccharide-binding R-type lectin from purplish bifurcate mussels Mytilisepta virgata and its effect on MAP kinases.

A 15-kDa lectin, termed SeviL, was isolated from Mytilisepta virgata (purplish bifurcate mussel). SeviL forms a noncovalent dimer that binds strongly to ganglio-series GM1b oligosaccharide (Neu5Acɑ2-3Galß1-3GalNAcß1-4Galß1-4Glc) and its precursor, asialo-GM1 (Galß1-3GalNAcß1-4Galß1-4Glc). SeviL also interacts weakly with the glycan moiety of SSEA-4 hexaose (Neu5Acα2-3Galß1-3GalNAcß1-3Galα1-4Galß1-4Glc). A partial protein sequence of the lectin was determined by mass spectrometry, and the complete sequence was identified from transcriptomic analysis. SeviL, consisting of 129 amino acids, was classified as an R(icin B)-type lectin, based on the presence of the QxW motif characteristic of this fold. SeviL mRNA is highly expressed in gills and, in particular, mantle rim tissues. Orthologue sequences were identified in other species of the family Mytilidae, including Mytilus galloprovincialis, from which lectin MytiLec-1 was isolated and characterized in our previous studies. Thus, mytilid species contain lectins belonging to at least two distinct families (R-type lectins and mytilectins) that have a common ß-trefoil fold structure but differing glycan-binding specificities. SeviL displayed notable cytotoxic (apoptotic) effects against various cultured cell lines (human breast, ovarian, and colonic cancer; dog kidney) that possess asialo-GM1 oligosaccharide at the cell surface. This cytotoxic effect was inhibited by the presence of anti-asialo-GM1 oligosaccharide antibodies. With HeLa ovarian cancer cells, SeviL showed dose- and time-dependent activation of kinase MKK3/6, p38 MAPK, and caspase-3/9. The transduction pathways activated by SeviL via the glycosphingolipid oligosaccharide were triggered apoptosis. DATABASE: Nucleotide sequence data have been deposited in the GenBank database under accession numbers MK434191, MK434192, MK434193, MK434194, MK434195, MK434196, MK434197, MK434198, MK434199, MK434200, and MK434201.

MytiLec-1 Shows Glycan-Dependent Toxicity against Brine Shrimp Artemia and Induces Apoptotic Death of Ehrlich Ascites Carcinoma Cells In Vivo.

MytiLec-1, a 17 kDa lectin with ß-trefoil folding that was isolated from the Mediterranean mussel (Mytilus galloprovincialis) bound to the disaccharide melibiose, Galα(1,6) Glc, and the trisaccharide globotriose, Galα(1,4) Galß(1,4) Glc. Toxicity of the lectin was found to be low with an LC50 value of 384.53 µg/mL, determined using the Artemia nauplii lethality assay. A fluorescence assay was carried out to evaluate the glycan-dependent binding of MytiLec-1 to Artemia nauplii. The lectin strongly agglutinated Ehrlich ascites carcinoma (EAC) cells cultured in vivo in Swiss albino mice. When injected intraperitoneally to the mice at doses of 1.0 mg/kg/day and 2.0 mg/kg/day for five consecutive days, MytiLec-1 inhibited 27.62% and 48.57% of cancer cell growth, respectively. Antiproliferative activity of the lectin against U937 and HeLa cells was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro in RPMI-1640 medium. MytiLec-1 internalized into U937 cells and 50 µg/mL of the lectin inhibited their growth of to 62.70% whereas 53.59% cell growth inhibition was observed against EAC cells when incubated for 24 h. Cell morphological study and expression of apoptosis-related genes (p53, Bax, Bcl-X, and NF-κB) showed that the lectin possibly triggered apoptosis in these cells.

Functional Characterization of OXYL, A SghC1qDC LacNAc-specific Lectin from The Crinoid Feather Star Anneissia Japonica.

We identified a lectin (carbohydrate-binding protein) belonging to the complement 1q(C1q) family in the feather star Anneissia japonica (a crinoid pertaining to the phylum Echinodermata). The combination of Edman degradation and bioinformatics sequence analysis characterized the primary structure of this novel lectin, named OXYL, as a secreted 158 amino acid-long globular head (sgh)C1q domain containing (C1qDC) protein. Comparative genomics analyses revealed that OXYL pertains to a family of intronless genes found with several paralogous copies in different crinoid species. Immunohistochemistry assays identified the tissues surrounding coelomic cavities and the arms as the main sites of production of OXYL. Glycan array confirmed that this lectin could quantitatively bind to type-2 N-acetyllactosamine (LacNAc: Galß1-4GlcNAc), but not to type-1 LacNAc (Galß1-3GlcNAc). Although OXYL displayed agglutinating activity towards Pseudomonas aeruginosa, it had no effect on bacterial growth. On the other hand, it showed a significant anti-biofilm activity. We provide evidence that OXYL can adhere to the surface of human cancer cell lines BT-474, MCF-7, and T47D, with no cytotoxic effect. In BT-474 cells, OXYL led to a moderate activation of the p38 kinase in the MAPK signaling pathway, without affecting the activity of caspase-3. Bacterial agglutination, anti-biofilm activity, cell adhesion, and p38 activation were all suppressed by co-presence of LacNAc. This is the first report on a type-2 LacNAc-specific lectin characterized by a C1q structural fold.

Histochemical localization of N-acetylhexosamine-binding lectin HOL-18 in Halichondria okadai (Japanese black sponge), and its antimicrobial and cytotoxic anticancer effects.

We studied localization and physiological activities of a lectin showing specific binding to N-acetylhexosamines, termed HOL-18, purified from Japanese black sponge (Halichondria okadai). Antiserum against the lectin was generated in rabbit and applied for immunohistochemical analyses. HOL-18 was expressed specifically around water pores and on spicules of sponge tissues. It showed strong binding to a variety of N-acetylhexosamines: N-acetyl D-glucosamine, N-acetyl D-galactosamine, N-acetyl mannosamine, N-acetyl muramic acid, and N-acetyl neuraminic acid. Hemagglutination induced by the lectin was inhibited by lipopolysaccharides and a peptidoglycan. HOL-18 inhibited growth of a gram-positive bacterium (Listeria monocytogenes), gram-negative bacteria (Escherichia coli, Shigella boydii, Pseudomonas aeruginosa), and a fungus (Aspergillus niger). It displayed anti-biofilm activity against P. aeruginosa. HOL-18 was internalized into conidiophores of A. niger, and displayed notable antifungal activity. Fluorescence microscopy revealed binding and incorporation of the lectin into human cancer cell lines HeLa, MCF-7, and T47D, but not Caco-2. HOL-18 displayed dose-dependent cytotoxic effects against HeLa, MCF-7, and T47D, with respective IC50 values 40, 52, and 63 µg/mL. In HeLa cells, it activated phosphorylation of MAPK pathway molecule (ERK1/2) and activated caspase-3 to trigger apoptosis. HOL-18 thus has the potential to upregulate metabolic pathways in higher animal cells through binding to N-acetylhexosamines.

Computational design of a symmetrical ß-trefoil lectin with cancer cell binding activity.

Computational protein design has advanced very rapidly over the last decade, but there remain few examples of artificial proteins with direct medical applications. This study describes a new artificial ß-trefoil lectin that recognises Burkitt's lymphoma cells, and which was designed with the intention of finding a basis for novel cancer treatments or diagnostics. The new protein, called "Mitsuba", is based on the structure of the natural shellfish lectin MytiLec-1, a member of a small lectin family that uses unique sequence motifs to bind α-D-galactose. The three subdomains of MytiLec-1 each carry one galactose binding site, and the 149-residue protein forms a tight dimer in solution. Mitsuba (meaning "three-leaf" in Japanese) was created by symmetry constraining the structure of a MytiLec-1 subunit, resulting in a 150-residue sequence that contains three identical tandem repeats. Mitsuba-1 was expressed and crystallised to confirm the X-ray structure matches the predicted model. Mitsuba-1 recognises cancer cells that express globotriose (Galα(1,4)Galß(1,4)Glc) on the surface, but the cytotoxicity is abolished.

Catfish rhamnose-binding lectin induces G0/1 cell cycle arrest in Burkitt's lymphoma cells via membrane surface Gb3.

Silurus asotus egg lectin (SAL), an α-galactoside-binding protein isolated from the eggs of catfish, is a member of the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1-4Galß1-4Glc). We have previously demonstrated that SAL reduces the proliferation of Gb3-expressing Burkitt's lymphoma Raji cells and confirm here that it does not reduce their viability, indicating that unlike other lectins, it is not cytotoxic. The aim of this study was to determine the signal transduction mechanism(s) underlying this novel SAL/Gb3 binding-mediated effect profile. SAL/Gb3 interaction arrested the cell cycle through increasing the G0/1 phase population of Raji cells. SAL suppressed the transcription of cell cycle-related factors such as c-MYC, cyclin D3, and cyclin-dependent protein kinase (CDK)-4. Conversely, the CDK inhibitors p21 and p27 were elevated by treatment with SAL. In particular, the production of p27 in response to SAL treatment increased steadily, whereas p21 production was maximal at 12 h and lower at 24 h. Activation of Ras-MEK-ERK pathway led to an increase in expression of p21. Notably, treatment of Raji cells with anti-Gb3 mAb alone did not produce the above effects. Taken together, our findings suggest that Gb3 on the Raji cell surface interacts with SAL to trigger sequential GDP-Ras phosphorylation, Ras-MEK-ERK pathway activation, p21 production, and cell cycle arrest at the G0/1 phase.

Crystal structure of MytiLec, a galactose-binding lectin from the mussel Mytilus galloprovincialis with cytotoxicity against certain cancer cell types.

MytiLec is a lectin, isolated from bivalves, with cytotoxic activity against cancer cell lines that express globotriaosyl ceramide, Galα(1,4)Galß(1,4)Glcα1-Cer, on the cell surface. Functional analysis shows that the protein binds to the disaccharide melibiose, Galα(1,6)Glc, and the trisaccharide globotriose, Galα(1,4)Galß(1,4)Glc. Recombinant MytiLec expressed in bacteria showed the same haemagglutinating and cytotoxic activity against Burkitt's lymphoma (Raji) cells as the native form. The crystal structure has been determined to atomic resolution, in the presence and absence of ligands, showing the protein to be a member of the ß-trefoil family, but with a mode of ligand binding unique to a small group of related trefoil lectins. Each of the three pseudo-equivalent binding sites within the monomer shows ligand binding, and the protein forms a tight dimer in solution. An engineered monomer mutant lost all cytotoxic activity against Raji cells, but retained some haemagglutination activity, showing that the quaternary structure of the protein is important for its cellular effects.

cDNA and Gene Structure of MytiLec-1, A Bacteriostatic R-Type Lectin from the Mediterranean Mussel (Mytilus galloprovincialis).

MytiLec is an α-d-galactose-binding lectin with a unique primary structure isolated from the Mediterranean mussel (Mytilus galloprovincialis). The lectin adopts a ß-trefoil fold that is also found in the B-sub-unit of ricin and other ricin-type (R-type) lectins. We are introducing MytiLec(-1) and its two variants (MytiLec-2 and -3), which both possess an additional pore-forming aerolysin-like domain, as members of a novel multi-genic "mytilectin family" in bivalve mollusks. Based on the full length mRNA sequence (911 bps), it was possible to elucidate the coding sequence of MytiLec-1, which displays an extended open reading frame (ORF) at the 5' end of the sequence, confirmed both at the mRNA and at the genomic DNA sequence level. While this extension could potentially produce a polypeptide significantly longer than previously reported, this has not been confirmed yet at the protein level. MytiLec-1 was revealed to be encoded by a gene consisting of two exons and a single intron. The first exon comprised the 5'UTR and the initial ATG codon and it was possible to detect a putative promoter region immediately ahead of the transcription start site in the MytiLec-1 genomic locus. The remaining part of the MytiLec-1 coding sequence (including the three sub-domains, the 3'UTR and the poly-A signal) was included in the second exon. The bacteriostatic activity of MytiLec-1 was determined by the agglutination of both Gram-positive and Gram-negative bacteria, which was reversed by the co-presence of α-galactoside. Altogether, these data support the classification of MytiLec-1 as a member of the novel mytilectin family and suggest that this lectin may play an important role as a pattern recognition receptor in the innate immunity of mussels.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galß1-4Glc). MytiLec revealed ß-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt's lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.