Lectins from the Red Marine Algal Species Bryothamnion seaforthii and Bryothamnion triquetrum as Tools to Differentiate Human Colon Carcinoma Cells

The carbohydrate-binding activity of the algal lectins from the closely related red marine algal species Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL) was used to differentiate human colon carcinoma cell variants with respect to their cell membrane glyco-receptors. These lectins interacted with the cells tested in a dose-dependent manner. Moreover, the fluorescence spectra of both lectins clearly differentiated the cells used as shown by FACS profiles. Furthermore, as observed by confocal microscopy, BTL and BSL bound to cell surface glycoproteins underwent intense internalization, which makes them possible tools in targeting strategies

Morphology, ultrastructure and immunocytochemistry of Hypnea cervicornis and Hypnea musciformis-(Hypneaceae, Rhodophyta) from the coastal waters of Ceará, Brazil

AbstractBased on their morphological and physiological features, red algae comprise a complex and variable group of multiple genera, including Hypnea. In particular, the genus Hypnea J.V. Lamouroux (Cystocloniaceae, Rhodophyta) consists of approximately 54 species, including Hypnea cervicornis and H. musciformis. Lectins were described for both species; however, the localization of these proteins is still unclear. Therefore, this work aimed to characterize the morphology and ultrastructure of Hypnea cervicornis and H. musciformis, as well as localize their lectins at the subcellular level. Samples were collected at Praia do Pacheco (Fortaleza-CE) and processed for light, scanning and transmission electron microscopy, in addition to immunocytochemistry. The studied species presented cortical cell layers, subcortical cells and medullary cells. Based on ultrastructural analysis, these species presented vacuolated cortical cells, with a dense cytoplasm containing chloroplasts. The cell wall consisted of concentric microfibrils embedded in an amorphous matrix. Immunochemistry analysis showed the expression of lectins in the cytoplasm and cell walls. While the structure of the studied algae was similar to the description of other species of the genera under different conditions, this is the first record of algae lectin localization

Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

<p>Abstract</p> <p>Background</p> <p>An interaction between lectins from marine algae and PLA₂from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from <it>Bryothamnion triquetrum</it>, on the pharmacological and biological activities of a PLA₂isolated from rattlesnake venom (<it>Crotalus durissus cascavella</it>), to better understand the enzymatic and pharmacological mechanisms of the PLA₂and its complex.</p> <p>Results</p> <p>This PLA₂consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA₂s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity.</p> <p>The PLA₂and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA₂activity, 23% higher than that of PLA₂alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, <it>Clavibacter michiganensis michiganensis </it>(Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, <it>Xanthomonas axonopodis </it>pv <it>passiflorae </it>(Xap). PLA₂decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA₂-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm.</p> <p>PLA₂significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA₂. In addition, PLA₂exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound.</p> <p>Conclusion</p> <p>The unexpected results observed for the PLA₂-BTL-2 complex strongly suggest that the pharmacological activity of this PLA₂is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules.</p

Insights into the structural basis of the pH-dependent dimer-tetramer equilibrium through crystallographic analysis of recombinant Diocleinae lectins

International audienceThe structural ground underlying the pH-dependency of the dimer-tetramer transition of Diocleinae lectins was investigated by equilibrium sedimentation and X-ray crystal structure determination of wild-type and site-directed mutants of recombinant lectins. Synthetic genes coding for the full-length α-chains of the seed lectins of Dioclea guianensis (r-αDguia) and Dioclea grandiflora (r-αDGL) were designed and expressed in E. coli. This pioneer approach, which will be described in detail, yielded recombinant lectins displaying carbohydrate-binding activity, dimer-tetramer equilibria, and crystal structures indistinguisable from their natural homologues. Conversion of the pH-stable tetrameric r-αDGL into a structure exhibiting pH-dependent dimer-tetramer transition was accomplished through mutations that abolished the interdimeric interactions at the central cavity of the tetrameric lectins. Both, the central and the peripheral interacting regions bear structural information for formation of the canonical legume lectin tetramer. We hypothesize that the strength of the ionic contacts at these sites may be modulated by the pH, leading to dissociation of those lectin structures that are not locked into a pH-stable tetramer through interdimeric contacs networking the central cavity loops

The potent anti-cancer activity of Dioclea lasiocarpa lectin

The lectin DLasiL was isolated from seeds of the Dioclea lasiocarpa collected from the northeast coast of Brazil and characterized for the first time by mass spectrometry, DNA sequencing, inductively coupled plasma-mass spectrometry, electron paramagnetic resonance, and fluorescence spectroscopy. The structure of DLasiL lectin obtained by homology modelling suggested strong conservation of the dinuclear Ca/Mn and sugar-binding sites, and dependence of the solvent accessibility of tryptophan-88 on the oligomerisation state of the protein. DLasiL showed highly potent (low nanomolar) antiproliferative activity against several human carcinoma cell lines including A2780 (ovarian), A549 (lung), MCF-7 (breast) and PC3 (prostate), and was as, or more, potent than the lectins ConBr (Canavalia brasiliensis), ConM (Canavalia maritima) and DSclerL (Dioclea sclerocarpa) against A2780 and PC3 cells. Interestingly, DLasiL lectin caused a G2/M arrest in A2780 cells after 24 h exposure, activating caspase 9 and delaying the on-set of apoptosis. Confocal microscopy showed that fluorescently-labelled DLasiL localized around the nuclei of A2780 cells at lectin doses of 0.5–2 × IC50 and gave rise to enlarged nuclei and spreading of the cells at high doses. These data reveal the interesting antiproliferative activity of DLasiL lectin, and suggest that further investigations to explore the potential of DLasiL as a new anticancer agent are warranted

Crystallization and preliminary X-ray diffraction analysis of HML, a lectin from the red marine alga Hypnea musciformis

The crystallization and preliminary X-ray diffraction analysis of a red marine alga lectin isolated from H. musciformis is reported

Hemagglutinating/Hemolytic activities in extracts of marine invertebrates from the Brazilian coast and isolation of two lectins from the marine sponge Cliona varians and the sea cucumber Holothuria grisea

Twenty species of marine invertebrates from the Brazilian coast were screened for hemagglutinating/hemolytic activity. In at least twelve tested species, hemagglutinating activity was different for different blood types, suggesting the presence of lectins. Extracts from four species showed hemolytic activity. Two new lectins were purified from the marine sponge Cliona varians (CvL-2) and sea cucumber Holothuria grisea (HGL). CvL-2 was able to agglutinate rabbit erythrocytes and was inhibited by galactosides. The hemagglutinating activity was optimal in pH neutral and temperatures below 70 °C. CvL-2 is a trimeric protein with subunits of 175 kDa. On the other hand, HGL showed both hemagglutinating and hemolytic activity in human and rabbit erythrocytes, but hemolysis could be inhibited by osmotic protection, and agglutination was inhibited by mucin. HGL was stable in pH values ranging from 4 to 10 and temperatures up to 90 °C. In electrophoresis and gel filtration, HGL was a monomeric protein with 15 kDa. CvL-2 and HGL showed different levels of toxicity to Artemia naplii. CvL-2 showed LC50 of 850.1 μg/mL, whereas HGL showed LC50 of 9.5 µg/mL