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Giovanni Bottiroli - One of the best experts on this subject based on the ideXlab platform.
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Lectin-Binding Sites involved in Paramecium primaurelia mating pair formation
Journal of Eukaryotic Microbiology, 2007Co-Authors: Maria Umberta Delmonte Corrado, Donata Locatelli, Laura Paleari, Giovanni BottiroliAbstract:Mating pair formation in Paramecium primaurelia was shown to be inhibited by incubating mating-competent mating type (mt) I and mt II cells with Limulus polyphemus agglutinin (LPA) or wheat germ agglutinin (WGA). Preincubation of LPA and WGA with their specific Binding-monosaccharides, N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc), respectively, prevented the Lectin effect on pair formation. Addition either of NeuAc or GlcNAc resulted in a reversal of cell pairing inhibition by LPA or WGA, respectively. Both NeuAc and GlcNAc monosaccharides inhibited pair formation when their concentration exceeded a threshold value. The pattern of the relative distribution of NeuAe and GlcNAc molecules on the cell surface was analyzed using fluorescence resonance energy transfer techniques combined with imaging systems. Mt II cells showed a high Lectin-Binding Site density localized just on the surface region engaged in conjugation. The pattern of mt I cells, consisting of a quite homogeneous Lectin-Binding Site density spread on the cell surface, was also common to nonmating-competent cells and to immature cells. These findings suggest that in P. primaurelia pair formation involves both NeuAc and GlcNAc residues and that the development of mating-competence is related to a modification in NeuAc and GlcNAc relative distribution on the cell surface of mt II cells.
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Application of fluorescence resonance energy transfer techniques to the study of Lectin-Binding Site distribution on Paramecium primaurelia (Protista, Ciliophora) cell surface.
European journal of histochemistry : EJH, 1998Co-Authors: Donata Locatelli, Maria Umberta Delmonte Corrado, Huguette Politi, Giovanni BottiroliAbstract:Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon occurring between the molecules of two fluorochromes with suitable spectral characteristics (donor-acceptor dye pair), and consisting in an excitation energy migration through a non-radiative process. Since the efficiency of the process is strictly dependent on the distance and reciprocal orientation of the donor and acceptor molecules, FRET-based techniques can be successfully applied to the study of biomolecules and cell component organisation and distribution. These techniques have been employed in studying Paramecium primaurelia surface membrane for the reciprocal distribution of N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc) glycosidic residues, which were found to be involved in mating cell pairing. NeuAc and GlcNAc were detected by their specific Binding Lectins, Limulus polyphemus agglutinin (LPA) and wheat germ agglutinin (WGA), respectively. Microspectrofluorometric analysis afforded the choice of fluorescein isothiocyanate and Texas red conjugated with LPA and WGA, respectively, as a suitable donor-acceptor couple efficiently activating FRET processes. Studies performed both in solution and in cells allowed to define the experimental conditions favourable for a FRET analysis. The comparative study carried out both on the conjugating-region and the non conjugating region of the surface membrane, indicates that FRET distribution appears quite homogeneous in mating-competent mating type (mt) I, whereas, in mating-competent mt II cells, FRET distribution seems to be preferentially localised on the conjugating-region functionally involved in mating cell pairing. This difference in the distribution of Lectin-Binding Sites is suggested to be related to mating-competence acquisition.
Ana Maria Sampaio Assreuy - One of the best experts on this subject based on the ideXlab platform.
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Native crystal structure of a nitric oxide-releasing Lectin from the seeds of Canavalia maritima.
Journal of Structural Biology, 2005Co-Authors: Carlos Alberto De Almeida Gadelha, Frederico Bruno Mendes Batista Moreno, Nilson Vieira Pinto, Ana Maria Sampaio Assreuy, Tatiane Santi-gadelha, João Batista Cajazeiras, Bruno A.m. Rocha, Mário Rogério Lima Mota, A. V. P. Meireles, Júlio C. BorgesAbstract:Here, we report the crystallographic study of a Lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal Binding Sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described Lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific Lectin-Binding Site on the endothelium, resulting in a release of nitric oxide.
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Red marine alga Bryothamnion triquetrum Lectin induces endothelium-dependent relaxation of the rat aorta via release of nitric oxide.
The Journal of pharmacy and pharmacology, 2004Co-Authors: Ricardo F. Lima, Alexandre Holanda Sampaio, David N. Criddle, Emmanuel P. Souza, Kyria S. Nascimento, Benildo Sousa Cavada, Ana Maria Sampaio AssreuyAbstract:We have investigated the vascular relaxant effects of the Lectin from a red marine alga Bryothamnion triquetrum (BTL), in particular, the endothelial-dependency and the participation of a specific glycoprotein-Binding Site. BTL (1-100 microg mL(-1)) was applied to rat isolated aortic rings, with or without endothelium, tonically precontracted with phenylephrine (0.1 microM). Endothelium-dependent relaxation was assessed in the presence of indometacin (10 microM), L-nitro arginine methyl ester (L-NAME, 100 microM) and tetraethylammonium (TEA, 500 microM). For the involvement of the glycoprotein-Binding Site, BTL was assayed in presence of mucin (300 microg mL(-1)) or N-acetyl D-glucosamine (GlcNAc; 300 microg mL(-1)), a specific and non-specific Lectin-Binding sugar, respectively. BTL fully and concentration dependently relaxed preparations that possessed an intact endothelium (IC50 (concn producing 50% contraction) = 12.1 +/- 1.6 microg mL(-1)), whereas no significant relaxation was observed in endothelial-denuded tissue. L-NAME, but not indometacin or TEA, completely inhibited the Lectin relaxation, suggesting the involvement of nitric oxide (NO). The Lectin in association with mucin, but not with GlcNAc, inhibited BTL-induced relaxation, implicating the involvement of the Lectin Binding Site. Our data suggest that the relaxant effect of the red marine alga Bryothamnion triquetrumLectin on isolated aorta occurs via interaction with a specific Lectin-Binding Site on the endothelium, resulting in a release of NO.
Anne Imberty - One of the best experts on this subject based on the ideXlab platform.
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role of water molecules in structure and energetics of pseudomonas aeruginosa Lectin i interacting with disaccharides
Journal of Biological Chemistry, 2010Co-Authors: Alessandra Nurisso, Bertrand Blanchard, Aymeric Audfray, Lina Rydner, Annabelle Varrot, Stefan Oscarson, Anne ImbertyAbstract:Abstract Calcium-dependent Lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an α-galactose residue at their nonreducing end, such as the disaccharides αGal1–2βGalOMe, αGal1–3βGalOMe, and αGal1–4βGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL·αGal1–2βGalOMe complex, which was solved at 2.4 A resolution and reported herein. The role of a structural bridge water molecule in the Binding Site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the Lectin Binding Site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct Binding affinity trends with the 1–2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the Binding Site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.
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conformational analysis of blood group a trisaccharide in solution and in the Binding Site of dolichos biflorus Lectin using transient and transferred nuclear overhauser enhancement noe and rotating frame noe experiments
FEBS Journal, 1996Co-Authors: Florence Casset, Elena Korchagina, Thomas Peters, Marilynn Etzler, Nikolai Nifantev, Serge Perez, Anne ImbertyAbstract:The present study is concerned with the elucidation of the conformation of the blood group A trisaccharide (alpha-D-GalNAc(1-3)[alpha-L-Fuc(1-->2)] beta-D-Gal-O-R) in the combining Site of Dolichos biflorus seed Lectin by use of 400-MHz and 600-MHz NMR spectroscopy. D. biflorus Lectin displays a unique specificity for GalNAc residues. It occurs in solution as a tetrameric assembly having a molecular mass of 110 kDa, with two carbohydrate-Binding Sites per molecule. First, NOE build-up curves were obtained for the free blood group A trisaccharide from one-dimensional transient NOE experiments. Simulated NOE build-up curves were constructed from an ensemble of low-energy conformers derived from previous investigations. The comparison of theoretical and experimental data indicates that an equilibrium between two families of low-energy conformers most likely reflects the solution behavior of the trisaccharide in solution. Two-dimensional transferred NOE and rotating-frame enhancements (ROE) were subsequently measured for the trisaccharide complexed with the D. biflorus seed Lectin. In addition to the NOEs observed for the free trisaccharide, the transferred NOESY spectrum showed several new NOEs that were identified as spin diffusion using a rotating-frame NOESY (ROESY) experiment. Experimental interglycosidic transferred nuclear Overhauser effect (TRNOE) build-up curves were compared to theoretical curves calculated for both low-energy conformers located in the D. biflorus Lectin-Binding Site. Calculations of theoretical TRNOE were performed using a combination of the full relaxation matrix and the protein-ligand exchange matrix. Comparison between experimental and simulated TRNOE volumes leads to the conclusion that one conformation of blood group A trisaccharide is selected upon Binding by D. biflorus Lectin.
Júlio C. Borges - One of the best experts on this subject based on the ideXlab platform.
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Native crystal structure of a nitric oxide-releasing Lectin from the seeds of Canavalia maritima.
Journal of Structural Biology, 2005Co-Authors: Carlos Alberto De Almeida Gadelha, Frederico Bruno Mendes Batista Moreno, Nilson Vieira Pinto, Ana Maria Sampaio Assreuy, Tatiane Santi-gadelha, João Batista Cajazeiras, Bruno A.m. Rocha, Mário Rogério Lima Mota, A. V. P. Meireles, Júlio C. BorgesAbstract:Here, we report the crystallographic study of a Lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal Binding Sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described Lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific Lectin-Binding Site on the endothelium, resulting in a release of nitric oxide.
Maria Umberta Delmonte Corrado - One of the best experts on this subject based on the ideXlab platform.
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Lectin-Binding Sites involved in Paramecium primaurelia mating pair formation
Journal of Eukaryotic Microbiology, 2007Co-Authors: Maria Umberta Delmonte Corrado, Donata Locatelli, Laura Paleari, Giovanni BottiroliAbstract:Mating pair formation in Paramecium primaurelia was shown to be inhibited by incubating mating-competent mating type (mt) I and mt II cells with Limulus polyphemus agglutinin (LPA) or wheat germ agglutinin (WGA). Preincubation of LPA and WGA with their specific Binding-monosaccharides, N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc), respectively, prevented the Lectin effect on pair formation. Addition either of NeuAc or GlcNAc resulted in a reversal of cell pairing inhibition by LPA or WGA, respectively. Both NeuAc and GlcNAc monosaccharides inhibited pair formation when their concentration exceeded a threshold value. The pattern of the relative distribution of NeuAe and GlcNAc molecules on the cell surface was analyzed using fluorescence resonance energy transfer techniques combined with imaging systems. Mt II cells showed a high Lectin-Binding Site density localized just on the surface region engaged in conjugation. The pattern of mt I cells, consisting of a quite homogeneous Lectin-Binding Site density spread on the cell surface, was also common to nonmating-competent cells and to immature cells. These findings suggest that in P. primaurelia pair formation involves both NeuAc and GlcNAc residues and that the development of mating-competence is related to a modification in NeuAc and GlcNAc relative distribution on the cell surface of mt II cells.
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Application of fluorescence resonance energy transfer techniques to the study of Lectin-Binding Site distribution on Paramecium primaurelia (Protista, Ciliophora) cell surface.
European journal of histochemistry : EJH, 1998Co-Authors: Donata Locatelli, Maria Umberta Delmonte Corrado, Huguette Politi, Giovanni BottiroliAbstract:Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon occurring between the molecules of two fluorochromes with suitable spectral characteristics (donor-acceptor dye pair), and consisting in an excitation energy migration through a non-radiative process. Since the efficiency of the process is strictly dependent on the distance and reciprocal orientation of the donor and acceptor molecules, FRET-based techniques can be successfully applied to the study of biomolecules and cell component organisation and distribution. These techniques have been employed in studying Paramecium primaurelia surface membrane for the reciprocal distribution of N-acetylneuraminic acid (NeuAc) and N-acetylglucosamine (GlcNAc) glycosidic residues, which were found to be involved in mating cell pairing. NeuAc and GlcNAc were detected by their specific Binding Lectins, Limulus polyphemus agglutinin (LPA) and wheat germ agglutinin (WGA), respectively. Microspectrofluorometric analysis afforded the choice of fluorescein isothiocyanate and Texas red conjugated with LPA and WGA, respectively, as a suitable donor-acceptor couple efficiently activating FRET processes. Studies performed both in solution and in cells allowed to define the experimental conditions favourable for a FRET analysis. The comparative study carried out both on the conjugating-region and the non conjugating region of the surface membrane, indicates that FRET distribution appears quite homogeneous in mating-competent mating type (mt) I, whereas, in mating-competent mt II cells, FRET distribution seems to be preferentially localised on the conjugating-region functionally involved in mating cell pairing. This difference in the distribution of Lectin-Binding Sites is suggested to be related to mating-competence acquisition.