Purification and characterization of structural and functional properties of two lectins from a marine sponge Spheciospongia vesparia.

The purification, structural and functional characterization of two different lectins (named Svl-1 and Svl-2) has been reported from the marine sponge Spheciospongia vesparia. Purification procedure includes ammonium sulfate precipitation, combined with chromatography including Octyl-Sepharose-(NH4)SO4 hydrophobic column and DEAE-Toyopearl anion-exchange column using a high performance liquid chromatography. The similarities in function, specificity for saccharides, molecular weight, amino acid content and the N-terminal sequence of two lectins suggest that these proteins are isolectins. Amino acid composition and fluorescence analyses reveal that they contain an intrachain disulfide bridge, which might contribute to their high thermal stability. Furthermore, the purified lectins exhibit antibacterial activity against the gram-negative bacteria Pseudomonas aeruginosa and E. coli, indicating that they may be involved in a recognition strategy and may play a role in the defense response function of the sponge. This is the first report on the isolation of lectins from the S. vesparia. The purified lectins represent a potential possible candidate for future application in the recognition or treatment of cancer cells.

Identification and localization of certain glycoconjugates during the development of thymus in the mice embryos Balb/C

Glycoconjugates molecules showed a programmed changes during the development. These components in the cell-surface and extracellular matrix plays important roles for different developmental processes. In this study lectin histochemistry technique was used for identification and localization of some glycoconjugates during the development of thymus. Balb/C mice embryos from days 10 to 15 and also day 18 fetuses iixed in formalin and provided 5m serial sections of these samples for histochemical study. Slices were incubated with three types of HRP-conjugated lectins [HRP] include: SBA specified for alpha, [beta-D-N-acetylgalactoseamin [alpha, beta-D-GalNAc], PNA specified for beta-D-Galactose -[l-3]-D-N-acetylgalactoseamin [beta-D-Gal-[l-3]-D-GalNAc], MPA specified for D-Galactose [D- Gal]. SBA lectin was presented with decreasing intensity in the Golgi zone [GZ] and cell surfaces of mesenchymal cells, epithelial cells and T-cells microenvironment in studying days. In first day [12 and 13] T-cells had high reaction with SBA in membranes and GZ. PNA lectin was revealed with several intensity in basal membranes and membranes of mesenchymal cells, epithelial and microenvironment of T-cells in studying days. MPA lectin weakly presented in the endodermal and mesenchymal cells and microenvironment of T-cells in all days. Results indicated that glycoconjugates molecules with terminal carbohydrate of GalNAc might have a role in the development of thymus gland and maturation of the T-cells. Glycoconjugates with terminal disaccharide of Gal/GalNAc probably have significant effect on the total developmental process of thymus gland. Further, Glycoconjugates with terminal carbohydrate of Gal probably had not a key role in the development of thymus gland and T-cells or with low quantity effect

Role of cell surface oligosaccharides of mouse mammary tumor cell lines in cancer metastasis.

Malignant transformation is associated with changes in the glycosylation of cell surface proteins and lipids. In tumor cells, alterations in cellular glycosylation may play a key role in their metastatic behaviour. In the present study, we have assessed the relationship between cell surface oligosaccharides and the metastasis ability of mouse mammary tumor cell lines 67NR and 4TO7. The cell surface oligosaccharides have been analyzed using specific binding assays with some plant lectins and the metastasis ability has been studied using transwell migration and invasion assays. In addition, we investigated the role of terminal sialic acids in the metastatic potential (cell adhesion on fibronectin, cell migration and invasion) in the 4TO7 cells on treatment with neuraminidase. The cell lines used in study have different metastasis abilities in vivo - the 67NR form primary tumors, but no tumor cells are detectable in any distant tissues, while cells of the 4TO7 line are able to spread to lung. In vitro metastasis experiments have revealed higher ability of adhesion, cell migration and invasion in the 4TO7 cells than the 67NR cells. Specific lectins binding assays show that the 4TO7 cells expressed more high-mannose type, multi-antennary complex-type N-glycans, beta-1,6-GlcNAc-branching, alpha-2,6-linked sialic acids, N-acetylgalactosamine and galactosyl(beta-1,3)-N-acetylgalactosamine. Removal of sialic acids on treatment with neuraminidase decreases adhesion, but increases the migration and has shown no significant change in the invasion ability of the 4TO7 cells. The study suggests that the sialic acids are not crucial for the cell migration and invasion in the 4TO7 cells. The findings provide the new insights in understanding the role of cell surface oligosaccharides in cancer metastasis.

Galectinas: estructura, especificidad glicídica y ligandos específicos / Galectins: structure, sugar specificity and specific ligands

Las galectinas se definen por dos propiedades: secuencias de aminoácidos características compartidas y afinidad por azúcares ß-galactosídicos. Numerosas galactinas de mamíferos fueron secuenciadas y bien caracterizadas en diferentes especies, siendo clasificadas como galectina-1 a galectina-10, según sus homologías de secuencia. La identidad entre dominios que ligan carbohidratos de distintas galectinas de una especie de mamífero oscila entre 20-40 por ciento, mientras que la identidad de galectina-1, por ejemplo, entre distintas especies es de 80-90 por ciento. En la presente revisión, se describen las principales propiedades distintivas de las galectinas de mamífero en cuanto a estructura proteica, estructura cristalina, especificidad glicídica y ligandos específicos

Glutaraldehyde cross-linking of lectins to marker enzymes: protection of binding site by specific sugars.

The role of bound specific sugars in protecting the sugar binding activity of several galactose binding proteins during their covalent conjugation to horse radish peroxidase by glutaraldehyde-mediated cross-linking was examined by: a) affinity matrix binding of the conjugate, b) enzyme linked lectin assay and c) hemagglutination assay. During conjugation using 1% glutaraldehyde, protection of jack fruit (Artocarpus integrifolia) lectin (jacalin) activity depended on concentration of specific sugar present during conjugation; optimum protection was offered by 50 mM galactose. This indicated the presence of one or more primary groups at the binding site of jacalin, which is (are) essential for sugar binding. On the other hand, such essential amino group(s) was not indicated at the sugar binding site of the peanut lectin, bovine heart galectin or of the human serum anti alpha-galactoside antibody, since exclusion of sugar during their conjugation to HRP did not diminish sugar binding activity. The differential behavior is discussed in the light of reported differences in sugar specificities. Results indicated that sugar mediated blocking of active site may be used in characterization of the latter in lectins.

Identification of amino groups in the carbohydrate binding activity of winged bean acidic agglutinin.

Chemical modification studies reveal that the modification of amino groups in WBA II leads to a complete loss in the hemagglutinating and saccharide binding activities. Since WBA II is a dimeric molecule and contains two binding sites, one amino group in each of the binding sites is inferred to be essential for its activity. The presence of amino group which has a potential to form hydrogen bonded interactions with the ligand, substantiates our observation regarding the forces involved in WBA II-receptor and WBA II-simple sugar interactions.

Characterization of a lectin from goat peripheral blood lymphocytes.

A D-glucose specific lectin was isolated from goat peripheral blood lymphocytes by affinity chromatography on N-acetyl D-glucosamine agarose gel. The fluorescence intensity of 4 methyl umbelliferyl D-glucose was quenched to about 62% on addition of the lectin. This lectin gave a single band corresponding to 112 kDa in SDS-PAGE irrespective of treatment with 2-mercaptoethanol. The molecular weight and the Stoke's radius of the lectin in the native conditions were found to be 114 kDa and 4.54 nm, respectively, as determined by gel filtration on Sephacryl S 500 column. The lectin was found to be a glycoprotein with 5.6% of neutral hexose content and 5.5% of sialic acid. The lectin agglutinated trypsinized rabbit erythrocytes and human type A erythrocytes. The hemagglutinating activity was dependent on the presence of divalent cations like Mn2+ and Ca2+. Optimum pH, ionic strength and temperature for rebinding of lectin to acid treated Sephadex G200 were found to be 7.5, 0.16 and 30-37 degrees C, respectively.

Purification, physicochemical characterization and biological properties of a lectin from Erythrina velutina forma aurantiaca seeds

A lectin was purified from seeds of Erythrina velutina forma aurantiaca by affinity chromatography on cross-linked guargum. The lectin is a potent agglutinin for human (minimal concentration of protein able to cause visible agglutination of a 2 per cent erythrocyte suspension varying from 1 to 4 mug/ml), rabbit(4 mug/ml) and chicken erythrocytes (8 mug/ml) but presented low activity against cow (250 mug/ml) or sheep (333 mug/ml) blood cells. Hemagglutination of human O+ erythrocytes was inhibited by D-lactose (0.2 mM) > D-galactose(0.8 mM) > D-raffinose (2.1 mM). At pH 7.5, chromatography on a Superose 12 HR 10/30 column showed that the lectin was primarily a dimer (56.0 kDa) composed of two identical subunits (31.6 kDa each). A small amount of a tetrameric form was also apparently present. The lectin is a glycoprotein (7.3 per cent carbohydrate), has a pI of 4.5, contains high levels of acidic (Asp and Glu, 64.2 and 51.6 residues/mol, respectively) and hydroxy amino acids (Ser and Thr, 42.9 and 38.5 residues/mol, respectively) but relatively low amounts of sulfur amino acids (Cys and Met, 1.0 and 5.0 residues/mol, respectively) and has an N-terminal sequence of Val-Glu-Thr-Ile/Leu-Pro-Phe-Ser. Its hemagglutinating activity was abolished by heating at 70 degrees Celsius for 10 min. The activation energy (delta G') required for denaturation measured by loss of hemagglutination activity was 24.87 kcal/mol. In rats, the purified lectin (100 mug) induced neutrophil migration into the peritoneal cavity (3.7 ñ 0.6 x 10(6) neutrophils/ml) or into the air pouch (2.75 ñ 0.25 x 10(6) neutrophils/ml), 8 and 10 times greater than the negative control, respectively.

Proteínas pulmonares asociadas a sustancias con actividad surfactante7 / Pulmonary proteins associated to substances with surfactant activity

La ausencia de surfactantes pulmonares trae como consecuencia el incremento de la tensión superficial a lo largo del epitelio alveolar, provocando un colapso alveolar y la lisis de las células epiteliales. Este proceso culmina con la aparición de un síndrome de insuficiencia respiratoria, que es la causa principal de morbimortalidad en niños prematuros. Recientemente, la aplicación de mezclas de agentes surfactantes con fines terapéuticos ha constituido un gran apoyo para la terapia respiratoria, ya que permite una evolución más rápida de los niños que padecen este síndrome. Por todo esto, resulta de gran importancia el conocimiento más detallado de la función, el metabolismo y la regulación de la expresión genética de las proteíinas surfactantes, para el diseño de nuevas y mejores estrategias terapéuticas para combatir este síndrome

Further characterization of glucose-specific peanut root lectin (PRA II).

Amino acid analysis of PRA II, a glucose-specific lectin isolated from 7 day-old peanut seedling roots shows that this lectin is rich in glycyl (103 per mole) and seryl residues (59 per mole), and poor in essential amino acids, the acidic amino acid content is higher than the basic amino acids and that its amino acid composition differs from its seed counterpart (PNA), although neither of the lectins contains cystein. PRA II has two carbohydrate binding sites per molecule as determined by equilibrium dialysis. Modifications of the specific amino acid residues of the lectin with group specific reagents indicate that hydroxyl group of tyrosine is involved in the binding of carbohydrate to PRA II.

A monomeric protein with hemagglutinating activity from seeds of Vigna mungo (Phaseolus mungo).

Black gram (Vigna mungo) seeds are shown to contain a lectin with certain unusual features. The lectin agglutinates only trypsinized red cells, and its sugar specificity is complex as none of the common sugars, oligosaccharides or complex polysaccharides exhibit any affinity for the lectin. The purified lectin has a molecular weight of 58 kDa and is a monomer. Unlike other plant lectins, antibodies to the P. mungo lectin do not exhibit any immunological cross reactivity. The clot forming ability of the lectin is unusual in that the clot once formed is rapidly disaggregated indicated that it induces, as yet undefined, certain membrane alterations.

Plant lectins, chemical and biological aspects

Lectins, carbohydrate-binding proteins of non-immune origin, that agglutinate cells or precipitate polysaccharides and glycoconjugates, are well distributed in nature, mainly in the Plant Kingdom. The great majority of the plante lectins are present in seed cotyledons where they are found in the cytoplasm or int he protein bodies, although they have also been found in roots, stems and leaves. Due to their peculiar properties, the lectins are used as a tool both for analytical and preparative purposes in biochemistry, cellular biology, immunology and related areas. In agriculture and medicine the use of lectins greatly improved in the last few years. The lextins, with few exceptions, are glycoproteins, need divalent cations to display full activity and are, in general, oligomers with variable molecular weight. Although the studies on lectins have completed a century, their role in nature is yet ynknown . Several hypotheses on their physiological functions have been suggested. Thus, lectins could play important roles in defense against pathogens, plant-microorganism symbiosis, cell organization, embryo morphogenesis, phagocytosis, cell wall elongation, pollen recognition and as reserve proteins. A brief review on the general properties and roles of the lectins is given.

Aspectos biológicos e moleculares das lectinas / Biological and molecular aspects of lectins

Alguns aspectos da estrutura e do modo de açäo das lectinas säo brevemente revisados. Especial ênfase é dada as funçöes biológicas dessas moléculas, bem como às suas aplicaçöes como ferramentas em diversas áreas