Spatial Structure of Lectin from the Mussel Mytilus trossulus: In-Sights from Molecular Modelling and Practical Proof.

Most proteins have the ability to self-associate into homooligomeric protein complexes, which consist of two or more identical subunits. Today, modern methods of molecular modeling are an integral part of the study of many biologically active molecules. In silico methods are widely used in structure establishing and function and activity prediction of lectins - carbohydrate-binding proteins. Here, we described by computer simulation the spatial organization of lectin isolated from the mantle of the mussel Mytilus trossulus (MTL). It was shown that the dimerization of MTL gives a total of six ligand binding sites that may be important for the manifestation its biological properties. The ability of MTL to form a dimeric and oligomeric structure was confirmed by dynamic light scattering and SDS-PAGE methods.

Activity Dependence of a Novel Lectin Family on Structure and Carbohydrate-Binding Properties.

A GalNAc/Gal-specific lectins named CGL and MTL were isolated and characterized from the edible mussels Crenomytilus grayanus and Mytilus trossulus. Amino acid sequence analysis of these lectins showed that they, together with another lectin MytiLec-1, formed a novel lectin family, adopting ß-trefoil fold. In this mini review we discuss the structure, oligomerization, and carbohydrate-binding properties of a novel lectin family. We describe also the antibacterial, antifungal, and antiproliferative activities of these lectins and report about dependence of activities on molecular properties. Summarizing, CGL, MTL, and MytiLec-1 could be involved in the immunity in mollusks and may become a basis for the elaboration of new diagnostic tools or treatments for a variety of cancers.

A Multivalent Marine Lectin from Crenomytilus grayanus Possesses Anti-cancer Activity through Recognizing Globotriose Gb3.

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a ß-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.

Quantitative proteomic analysis of the effects of a GalNAc/Man-specific lectin CSL on yeast cells by label-free LC-MS.

A Ca(2+)-dependent GalNAc/Man-specific lectin (CSL) from Cyclina sinensis was isolated, and its stimulatory action was characterized in yeast. CSL showed a potent effect on the production of ethanol by Saccharomyces cerevisiae. In this work, the changes in the protein expression profiles of S. cerevisiae after 24h of incubation with CSL were analyzed using label-free quantitative proteomics. A total of 1410 proteins were identified, but only 117 proteins showed significant differences in normalized volume (p<0.05). Among the latter proteins, 24 proteins were up-regulated, and 93 were down-regulated. Analysis of the proteome revealed that CSL triggered changes in the concentrations of some enzymes, such as increased expression of hexokinase, glyceraldehyde 3-phosphate dehydrogenase and enolase and decreased expression of dihydrolipoamide dehydrogenase and aldehyde dehydrogenase. These results indicate that CSL can cause some changes in the metabolic pathway involved in ethanol synthesis in S. cerevisiae. These data may help us understand the stimulatory mechanism of lectin in the fermentation process.

Purification of a secreted lectin from Andrias davidianus skin and its antibacterial activity.

A lectin secreted from Andrias davidianus skin (ADL) was purified by affinity chromatography on porcine stomach mucin (type III) (PSM)-crosslinked albumin, followed by gel filtration on Sephadex G-100 and HPLC on TSK gel G3000PWXL. The purified lectin was found to be a dimeric protein, as revealed by SDS-PAGE and MALDI-TOF analysis. SDS-PAGE showed that the ADL protein had a molecular mass of 17 kDa. ADL produced an 8.5 kDa band when examined using SDS-PAGE under reducing conditions. ADL agglutinated native and trypsinized human B erythrocytes. The hemagglutination activity was inhibited by glycoproteins, such as PSM and asialo-PSM, but not by any of the monosaccharides tested. The activity was stable between 4 °C and 50 °C. Significant ADL activity was observed between pH 4­5. The lectin reaction did not depend on the presence of the divalent cation Ca2+ or Mg2+. The N-terminal ADL sequence was determined to be VGYTVGATPM. The lectin exhibited antibacterial activity, involving growth and respiration inhibition in Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus, Bacillus subtilis and Shewanella sp. Furthermore, ADL showed inhibition activity against the yeast Saccharomyces cerevisiae. These findings suggest that ADL plays an important role in the innate immunity of A. davidianus on the body surface.

A lectin with antifungal activity from the mussel Crenomytilus grayanus.

Lectins (carbohydrate-binding proteins) are well known to actively participate in the defense functions of vertebrates and invertebrates where they play an important role in the recognition of foreign particles. In this study, we investigated of in vitro antifungal activity of lectin from the mussel Crenomytilus grayanus (CGL). Enzyme-linked immunosorbent assay indicated that CGL was predominantly detectable in tissues of mantle and to a lesser degree in the tissues of muscle, hepatopancreas, gill and hemocytes. After challenged by Pichia pastoris the level of CGL was upregulated and reached the maximum level at 12 h post challenge and recovered to the original level at 24 h. The lectin was capable of inhibiting the germination of spores and hyphal growth in the fungi. All these results indicated that CGL is involved in the innate immune response in mollusc animals.

cDNA cloning and structural characterization of a lectin from the mussel Crenomytilus grayanus with a unique amino acid sequence and antibacterial activity.

An amino acid sequence of GalNAc/Gal-specific lectin from the mussel Crenomytilus grayanus (CGL) was determined by cDNA sequencing. CGL consists of 150 amino acid residues, contains three tandem repeats with high sequence similarities to each other (up to 73%) and does not belong to any known lectins family. According to circular dichroism results CGL is a ß/α-protein with the predominance of ß-structure. CGL was predicted to adopt a ß-trefoil fold. The lectin exhibits antibacterial activity and might be involved in the recognition and clearance of bacterial pathogens in the shellfish.

A new lectin from the sea worm Serpula vermicularis: isolation, characterization and anti-HIV activity.

A GlcNAc-specific lectin was isolated from the sea worm Serpula vermicularis (SVL) (Annelida) and purified by ion-exchange, affinity and gel permeation chromatography. SVL was a homotetrameric protein with native molecular mass of about 50 kDa, and consisted of identical subunits of 12.7 kDa. The carbohydrate content of 1.9% suggested that the lectin was a glycoprotein, and mainly composed by aspartic and glutamic acids, glycine, valine and serine; with relatively lower content of basic amino acids and cysteine. The first 15 residues of the N-terminal region were determined as ADTPCQMLGSRYGWR. It was stable at pH 6-9 and at temperatures up to 40 degrees C. SVL was Ca(2+)-independent lectin that agglutinated native and trypsinized human erythrocytes. Hapten inhibition studies indicated that SVL showed binding specificity only for N-acetyl-d-glucosamine and its derivatives among the monosaccharides tested and required the presence of hydroxyl group at the C-3 of GlcNAc. The presence of hydrophobic p-nitrophenyl aglycone improved inhibitory potency of N-acetyl-d-glucosamine. Ovomucoid and ovalbumin were found to be inhibitors among the glycoproteins used for inhibition assay. The anti-HIV-1 (human immunodeficiency virus) activity of SVL in vitro was determined: SVL inhibited the production of viral p24 antigen and cytopathic effect induced by HIV-1. The EC(50) values were 0.23 and 0.15 microg x mL(-1) respectively.

Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer.

The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we determined that galectin-3, a beta-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we determined by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concentrated and form multimers or interact covalently with other proteins.

Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A.

Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media. We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion. Interestingly, the cells can also divide and attain confluency under these conditions. Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca2+ ions. Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes. Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion. Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against beta1 integrins substantially reduced the adhesion. Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.

Members of the cystatin superfamily interact with MMP-9 and protect it from autolytic degradation without affecting its gelatinolytic activities.

Matrix metalloproteinases (MMPs), like other proteinases, can undergo autolytic degradation once activated in vivo. Whereas the activities of these enzymes are tightly regulated by tissue inhibitors of matrix metalloproteinases (TIMPs), it is not clear mechanistically how these enzymes are protected from autolysis in their active state. We previously reported that MMPs particularly MMP-9 and MMP-2 interact with the serum glycoprotein fetuin-A [Arch. Biochem. Biophys. (1995) 322, 250], a member of the cystatin superfamily. In the present analyses, we demonstrate that this interaction protects MMP-9 from autolytic degradation without interfering with its enzymatic activity, allowing it to efficiently digest gelatin. Our data demonstrate that MMP-9 binds to members of the cystatin family with K(diss) ranging from 25 to 58 nM for fetuin-A and 1.5-1.9 microM for cystatin C. The ability of fetuin-A to protect MMP-9 from autolysis requires a molar ratio of at least 8:1 (fetuin-A/MMP-9). More interestingly, our data show that the other members of the cystatin also have the ability to protect MMP-9 from autolysis, provided they are in molar excess relative to MMP-9. Taken together, our data suggest that cystatins, particularly fetuin-A, in any cellular compartment including the circulatory system, efficiently protect MMP-9 and possibly other MMPs from autolysis. This mechanism ensures the digestion of the preferred substrate for MMP-9 without sacrificing the enzyme in the process.

Extracellular functions of galectin-3.

Galectin-3 has been suspected of modulating cell to extracellular matrix interactions in a novel fashion ever since it was first described. However, the rapid accumulation of research data in just the last 8 years alone has completely changed our perspective of this multifunctional protein. Its chimeric nature (consists of carbohydrate recognition and collagen like domains) somehow makes it suited to interact with a plethora of interesting extracellular matrix proteins some of which might enable it to cross the plasma membrane despite its lack of appropriate signal peptides. It is now becoming established as a mediator of signal transduction events on the cell surface as well as a mediator of a variety of extra-cellular processes such as kidney development, angiogenesis, neuronal functions, tumor metastasis, autoimmune disorders, endocytosis and possibly exocytosis. Nevertheless, it still retains its unique position as a mediator/modulator of cell to extracellular matrix adhesive interactions. Cells, particularly epithelial cells which lack galectin-3 expression, interact poorly with their extracellular matrices. In some of these processes, it functions as a matricellular protein, displaying both pro- and anti-adhesive properties.