Effects of Urtica dioica agglutinin on glycotargeting of the vasculature: an in ovo study on chicken embryo.

The angiogenesis process is a pivotal cellular process involved in both developmental and pathological circumstances. In this study we investigated effect of Urtica dioica agglutinin (UDA), as an unusual phyto-lectin from the chitin-binding protein family, on the angiogenesis of chicken embryos. The UDA was extracted from plant rhizomes and purified by affinity chromatography column. The activity of this lectin was assayed by hemagglutination test on the human RBCs. Anti-angiogenic effect of UDA on the extra-embryonic layer of the chick egg was studied in the different concentrations. Our results showed that the minimum concentration of UDA for agglutination were 48.00 and 15.00 µg mL-1 in macro- and microscopic studies, respectively. Because the number and length of the vessels were dramatically decreased at 100 µg kg-1 of UDA, the lectin had an inhibitory effect on angiogenesis of the embryonic vasculature of the chick. We concluded that UDA might target the vascularization events through binding to GlcNAc-conjugates. More investigations are needed to clarify the angiogenesis-related therapeutic roles of this interesting biomolecule.

Cuantificación de la actividad específica de lectina en teosinte Zea diploperennis / Quantification of specific lectin activity in teosinte Zea diploperennis

Objetivo: Evaluar la actividad específica de lectina en teosinte Zea diploperennis sano e infectado con Ustilago maydis.Materiales y métodos: Plántulas de Zea diploperennis de 6 días de crecimiento fueron inoculadas con Ustilago maydis. Se evaluó la concentración de proteínas totales y la actividad hemaglutinante de extractos crudos de teosinte sano e infectado en placas de microtitulación con eritrocitos tipo O al 3% siguiendo la técnica de diluciones dobles seriadas durante 6 días. Resultados: La concentración de proteínas totales se incrementa en coleoptilo sano durante cada día de su crecimiento. No así en teosinte infectado donde la curva presenta una tendencia a la baja desde el momento de la inoculación. La actividad específica de lectina disminuye en ambos casos desde el primer día de cuantificación. Conclusiones: La evidente reducción en la actividad de lectina en teosinte infectado en comparación con teosinte sano podría explicar la susceptibilidad de este teosinte a dicho fitopatógeno. Si bien, la participación de las lectinas de teosinte y maíz en el mecanismo de defensa a Ustilago maydis todavía no ha sido esclarecida, los resultados obtenidos contribuyen a la comprensión del efecto que puede tener la concentración de lectina y proteína sobre la resistencia en teosinte.(AU)

Objective: To evaluate the specific activity of lectin in healthy and infected teosinte Zea diploperennis with Ustilago maydis. Materials and methods: Zea diploperennis seedlings of 6 days of growth were inoculated with Ustilago maydis. Total protein concentration and hemagglutinating activity of crude extracts of healthy and infected teosinte were evaluated in microtiter plates with 3% type O red cells following the technique of serial double dilutions for 6 days. Results: The concentration of total proteins increases in healthy coleoptile during each day of its growth. Not so in infected teosinte where the curve shows a downward trend from the moment of inoculation. The specific lectin activity decreases in both cases from the first day of quantification. Conclusions: The evident reduction in lectin activity in infected teosinte compared to healthy teosinte could explain the susceptibility of this teosinte to said phytopathogen. Although the participation of the teosinte and corn lectins in the defense mechanism against Ustilago maydis has not yet been clarified, the results obtained contribute to the understanding of the effect that the concentration of lectin and protein can have on resistance in teosinte.(AU)

A highly stable, non-digestible lectin from Pomacea diffusa unveils clade-related protection systems in apple snail eggs.

The acquisition of egg protection is vital for species survival. Poisonous eggs from Pomacea apple snails have defensive macromolecules for protection. Here we isolated and characterized a novel lectin called PdPV1 that is massively accumulated in the eggs of Pomacea diffusa and seems part of its protective cocktail. The native protein, an oligomer of ca 256 kDa, has high structural stability, withstanding 15 min boiling and denaturing by SDS. It resists in vitro proteinase digestion and displays structural stability between pH 2.0 and pH 12.0, and up to 85°C. These properties, as well as its subunit sequences, glycosylation pattern, presence of carotenoids, size and global shape resemble those of its orthologs from other Pomacea. Furthermore, like members of the canaliculata clade, PdPV1 is recovered unchanged in feces of mice ingesting it, supporting an anti-nutritive defensive function. PdPV1 also displays a strong hemagglutinating activity, specifically recognizing selected ganglioside motifs with high affinity. This activity is only shared with PsSC, a perivitelline from the same clade (bridgesii clade). As a whole, these results indicate that species in the genus Pomacea have diversified their egg defenses: those from the bridgesii clade are protected mostly by non-digestible lectins that lower the nutritional value of eggs, in contrast with protection by neurotoxins of other Pomacea clades, indicating that apple snail egg defensive strategies are clade specific. The harsh gastrointestinal environment of predators would have favored their appearance, extending by convergent evolution the presence of plant-like highly stable lectins, a strategy not reported in other animals.

Functional Analysis of Botulinum Hemagglutinin (HA).

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum, is the most potent toxin and produced as a complex with non-toxic components. Food-borne botulism is caused by the ingestion of these BoNT complexes. Hemagglutinin (HA), one of the non-toxic components, is known to have lectin (carbohydrate binding) activity and E-cadherin-binding activity. These activities promote the intestinal absorption of BoNT. To elucidate the mechanism of the onset of food-borne botulism, we focused on the role of HA in the intestinal absorption of BoNT. We describe the functional analysis methods for HA, including the expression of recombinant proteins, binding to glycoproteins and epithelial cells, and localization in mouse intestinal tissue.

MIC4 from Toxoplasma gondii: A Lectin Acting as a Toll-Like Receptor Agonist.

Tachyzoites, which are infective forms of Toxoplasma gondii, use their actinomyosin system to move over surfaces and invade host cells. Central to this process is the regulated release of micronemes organelles contents. The microneme protein 4 (MIC4) has the property to recognize galactosides residues linked to glycoproteins on the host cell surface. This property allows that MIC4 binds to TLR2- and TLR4 N-linked glycans and promote the activation of cell innate immune cells and secretion of inflammatory cytokines, acting on resistance against the parasite. Obtention of MIC4 from T. gondii requires several purification steps, is time-consuming and provides low yield. Therefore, this section details the protocol for prokaryotic expression, production, and purification of recombinant MIC4 (rMIC4) and for experimental assays to confirm its biological activity.

Exploring lectin-like activity of the S-layer protein of Lactobacillus acidophilus ATCC 4356.

The surface layer (S-layer) protein of Lactobacillus acidophilus is a crystalline array of self-assembling, proteinaceous subunits non-covalently bound to the outmost bacterial cell wall envelope and is involved in the adherence of bacteria to host cells. We have previously described that the S-layer protein of L. acidophilus possesses anti-viral and anti-bacterial properties. In this work, we extracted and purified S-layer proteins from L. acidophilus ATCC 4356 cells to study their interaction with cell wall components from prokaryotic (i.e., peptidoglycan and lipoteichoic acids) and eukaryotic origin (i.e., mucin and chitin), as well as with viruses, bacteria, yeast, and blood cells. Using chimeric S-layer fused to green fluorescent protein (GFP) from different parts of the protein, we analyzed their binding capacity. Our results show that the C-terminal part of the S-layer protein presents lectin-like activity, interacting with different glycoepitopes. We further demonstrate that lipoteichoic acid (LTA) serves as an anchor for the S-layer protein. Finally, a structure for the C-terminal part of S-layer and possible binding sites were predicted by a homology-based model.