22 results on '"André, Sabine"'
Search Results
2. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner
- Author
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Berbís, Manuel Álvaro, André, Sabine, Cañada, F. Javier, Pipkorn, Rüdiger, Mayo, Kevin H., Kübler, Dieter, Gabius, Hans-Joachim, Jiménez-Barbero, Jesús, Berbís, Manuel Álvaro, André, Sabine, Cañada, F. Javier, Pipkorn, Rüdiger, Mayo, Kevin H., Kübler, Dieter, Gabius, Hans-Joachim, and Jiménez-Barbero, Jesús
- Abstract
Galectin-3 (Gal-3) is a multifunctional effector acting extracellularly after non-classical secretion, in the cytoplasm and the nucleus. Its modular display of a carbohydrate recognition domain (CRD) attached to a tail of collagen-like repeats (nine in the human protein) and an N-terminal peptide is unique in this lectin family and not yet fully explored, as is the physiological significance of serine and tyrosine phosphorylation. Using a series of nine synthetic (phospho)peptides and the 15N-labeled CRD of human Gal-3 as well as measuring chemical shift perturbations in mixtures, potential for peptide reactivity was revealed in Gal-3’s backface. Tyrosine phosphorylation reduced the affinity, while serine phosphorylation of the N-terminal peptide was essential. Controls with sequence scrambling underscored inherent specificity. These results detect capacity for distinct sites of intramolecular recognition in Gal-3, adjustable by phosphorylation, and thus prompt analysis using the full-length protein.
- Published
- 2014
3. Nitric oxide changes distinct aspects of the glycophenotype of human neuroblastoma NB69 cells
- Author
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Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, European Commission, University of Heidelberg, Wouwer, Marlies van de, André, Sabine, Gabius, Hans-Joachim, Villalobo, Antonio, Ministerio de Ciencia e Innovación (España), Comunidad de Madrid, European Commission, University of Heidelberg, Wouwer, Marlies van de, André, Sabine, Gabius, Hans-Joachim, and Villalobo, Antonio
- Abstract
It is an open question whether the presence of nitric oxide (NO) affects the cell glycophenotype. A panel of six plant lectins was used in this study to monitor distinct aspects of cell surface glycosylation under nitrosative stress. We determined that treating human neuroblastoma NB69 cells with the long-lived NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (DETA/NO) and monitoring the non-apoptotic adherent cell population significantly increases the presentation of N-glycans as detected by concanavalin A. Examining fine-structural features, bisected N-glycans and branch-end tailoring including ¿2,6-sialylation were found to be enhanced. Confocal fluorescence microscopy and cell permeabilization experiments pointed to a major effect of NO on the extent of cell surface N-glycan presentation. We also show that NO increases the level of protein O-GlcNAcylation, a multifunctional post-translational modification. Our results thus establish the first evidence for NO as modulator of distinct aspects of cell glycosylation.
- Published
- 2011
4. From lectin structure to functional glycomics: principles of the sugar code
- Author
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Gabius, Hans-Joachim, André, Sabine, Jiménez-Barbero, Jesús, Romero, Antonio, Solís, Dolores, Gabius, Hans-Joachim, André, Sabine, Jiménez-Barbero, Jesús, Romero, Antonio, and Solís, Dolores
- Abstract
Lectins are carbohydrate-binding proteins which lack enzymatic activity on their ligand and are distinct from antibodies and free mono- and oligosaccharide sensor/transport proteins. Emerging insights into the functional dimension of lectin binding to cellular glycans have strongly contributed to the shaping of the 'sugar code'. Fittingly, over a dozen folds and a broad spectrum of binding site architecture, ranging from shallow grooves to deep pockets, have developed sugar-binding capacity. A central question is how the exquisite target specificity of endogenous lectins for certain cellular glycans can be explained. In this regard, affinity regulation is first systematically dissected into six levels. Experimentally, the strategic combination of methods to monitor distinct aspects of the lectin glycan interplay offers a promising perspective to answer this question.
- Published
- 2011
5. Diffusion nuclear magnetic resonance spectroscopy detects substoichiometric concentrations of small molecules in protein samples
- Author
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Ribeiro, João P., Palczewska, Małgorzata, André, Sabine, Cañada, F. Javier, Gabius, Hans-Joachim, Jiménez-Barbero, Jesús, Mellström, Britt, Naranjo, José R., Scheffers, Dirk-Jan, Groves, Patrick, Ribeiro, João P., Palczewska, Małgorzata, André, Sabine, Cañada, F. Javier, Gabius, Hans-Joachim, Jiménez-Barbero, Jesús, Mellström, Britt, Naranjo, José R., Scheffers, Dirk-Jan, and Groves, Patrick
- Abstract
Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 microM imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 microM) by 1D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1D (1)H NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers.
- Published
- 2010
6. Identification of peptide ligands for malignancy- and growth-regulating galectins using random phage-display and designed combinatorial peptide libraries
- Author
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Dep Farmaceutische wetenschappen, Afd Chemical Biology and Drug Discovery, André, Sabine, Arnusch, Christopher J., Kuwabara, Ichiro, Russwurm, Roland, Kaltner, Herbert, Gabius, Hans-Joachim, Pieters, Roland J., Dep Farmaceutische wetenschappen, Afd Chemical Biology and Drug Discovery, André, Sabine, Arnusch, Christopher J., Kuwabara, Ichiro, Russwurm, Roland, Kaltner, Herbert, Gabius, Hans-Joachim, and Pieters, Roland J.
- Published
- 2005
7. Interference of the galactose-dependent binding of lectins by novel pentapeptide ligands
- Author
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Dep Farmaceutische wetenschappen, Afd Chemical Biology and Drug Discovery, Arnusch, Christopher J., André, Sabine, Valentini, Paola, Lensch, Martin, Russwurm, Roland, Siebert, Hans-Christian, Fischer, Marcel J. E., Gabius, Hans-Joachim, Pieters, Roland J., Dep Farmaceutische wetenschappen, Afd Chemical Biology and Drug Discovery, Arnusch, Christopher J., André, Sabine, Valentini, Paola, Lensch, Martin, Russwurm, Roland, Siebert, Hans-Christian, Fischer, Marcel J. E., Gabius, Hans-Joachim, and Pieters, Roland J.
- Published
- 2004
8. Combining glycocluster synthesis with protein engineering: an approach to probe into the significance of linker length in a tandem-repeat-type lectin (galectin-4).
- Author
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André S, Wang GN, Gabius HJ, and Murphy PV
- Subjects
- Amino Acid Sequence, Chemistry Techniques, Synthetic, Collagen chemistry, Glycoproteins chemistry, Humans, Models, Molecular, Molecular Sequence Data, Protein Conformation, Galectin 4 chemistry, Glycoproteins chemical synthesis, Glycoproteins genetics, Protein Engineering, Repetitive Sequences, Amino Acid
- Abstract
Complementarity in lectin-glycan interactions in situ is assumed to involve spatial features in both the lectin and the glycan, giving a functional meaning to structural aspects of the lectin beyond its carbohydrate-binding site. In combining protein engineering with glycocluster synthesis, it is shown that the natural linker length of a tandem-repeat-type human lectin (galectin-4) determines binding properties in two binding assays (using surface-presented glycoprotein and cell surface assays). The types of glycocluster tested included bivalent lactosides based on tertiary amides of terephthalic, isophthalic, 2,6-naphthalic and oxalic acids as well as bivalent H(type 2) trisaccharides grafted on secondary/tertiary terephthalamides and two triazole-linker-containing cores. The presented data reveal a marked change in susceptibility to the test compounds when turning the tandem-repeat-type to a proto-type-like display. The testing of glycoclusters is suggested as a general strategy to help to delineate the significance of distinct structural features of lectins beyond their contact sites to the glycan., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
9. Phosphorylation of multifunctional galectins by protein kinases CK1, CK2, and PKA.
- Author
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Kübler D, Seidler J, André S, Kumar S, Schwartz-Albiez R, Lehmann WD, and Gabius HJ
- Subjects
- Amino Acid Sequence, Animals, Chickens, Chromatography, High Pressure Liquid, Galectins chemistry, Humans, Mice, Molecular Sequence Data, Phosphorylation, Rats, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Tandem Mass Spectrometry, Casein Kinase I metabolism, Casein Kinase II metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Galectins metabolism
- Abstract
Phosphorylation is known to have a strong impact on protein functions. We analyzed members of the lectin family of multifunctional galectins as targets of the protein kinases CK1, CK2, and PKA. Galectins are potent growth regulators able to bind both glycan and peptide motifs at intra- and extracellular sites. Performing in vitro kinase assays, galectin phosphorylation was detected by phosphoprotein staining and autoradiography. The insertion of phosphoryl groups varied to a large extent depending on the type of kinase applied and the respective galectin substrate. Sites of phosphorylation observed in the recombinant galectins were determined by a strategic combination of phosphopeptide enrichment and nano-ultra-performance liquid chromatography tandem mass spectrometry (nanoUPLC-MS/MS). By in silico modeling, phosphorylation sites were visualized three-dimensionally. Our results reveal galectin-type-specific Ser-/Thr-dependent phosphorylation beyond the known example of galectin-3. These data are the basis for functional studies and also illustrate the analytical sensitivity of the applied methods for further work on human lectins., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
10. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner.
- Author
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Berbís MÁ, André S, Cañada FJ, Pipkorn R, Ippel H, Mayo KH, Kübler D, Gabius HJ, and Jiménez-Barbero J
- Subjects
- Amino Acid Sequence, Carbohydrates chemistry, Galectin 3 chemistry, Galectin 3 genetics, Humans, Molecular Sequence Data, Peptides chemistry, Peptides genetics, Peptides metabolism, Phosphorylation, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Tyrosine chemistry, Tyrosine metabolism, Galectin 3 metabolism
- Abstract
Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with (15)N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
11. Nitric oxide changes distinct aspects of the glycophenotype of human neuroblastoma NB69 cells.
- Author
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Van de Wouwer M, André S, Gabius HJ, and Villalobo A
- Subjects
- Cell Line, Tumor drug effects, Glycosylation drug effects, Humans, Microscopy, Confocal, Neuroblastoma physiopathology, Plant Lectins chemistry, DEET pharmacology, Free Radical Scavengers pharmacology, Nitric Oxide pharmacology, Phenotype
- Abstract
It is an open question whether the presence of nitric oxide (NO) affects the cell glycophenotype. A panel of six plant lectins was used in this study to monitor distinct aspects of cell surface glycosylation under nitrosative stress. We determined that treating human neuroblastoma NB69 cells with the long-lived NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (DETA/NO) and monitoring the non-apoptotic adherent cell population significantly increases the presentation of N-glycans as detected by concanavalin A. Examining fine-structural features, bisected N-glycans and branch-end tailoring including α2,6-sialylation were found to be enhanced. Confocal fluorescence microscopy and cell permeabilization experiments pointed to a major effect of NO on the extent of cell surface N-glycan presentation. We also show that NO increases the level of protein O-GlcNAcylation, a multifunctional post-translational modification. Our results thus establish the first evidence for NO as modulator of distinct aspects of cell glycosylation., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
12. N-domain of human adhesion/growth-regulatory galectin-9: preference for distinct conformers and non-sialylated N-glycans and detection of ligand-induced structural changes in crystal and solution.
- Author
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Solís D, Maté MJ, Lohr M, Ribeiro JP, López-Merino L, André S, Buzamet E, Cañada FJ, Kaltner H, Lensch M, Ruiz FM, Haroske G, Wollina U, Kloor M, Kopitz J, Sáiz JL, Menéndez M, Jiménez-Barbero J, Romero A, and Gabius HJ
- Subjects
- Animals, CHO Cells, Cell Adhesion, Cell Growth Processes, Cricetinae, Cricetulus, Crystallization, Crystallography, X-Ray, Galectins chemistry, Glycosylation, Humans, Lactose chemistry, Ligands, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Protein Stability, Galectins metabolism, Lactose metabolism, Protein Conformation
- Abstract
Human tandem-repeat-type galectin-9 is a potent adhesion/growth-regulatory effector via lectin capacity of its N- and C-terminal domains. This bioactivity prompted further crystallographic study of the N-domain, combined with analysis in solution. Binding of lactose markedly increased the N-domain's resistance to thermal denaturation. Crystallography revealed its intimate contact profile, besides detecting an extension of the beta-sandwich fold by an antiparallel beta-strand F0 aligned to the C-terminal F1 strand. Ligand accommodation in its low-energy conformation leads to a movement of Arg87's side chain. As consequence, the ligand's glucose moiety and Arg87 become hydrogen bonded. The resulting predictions for spatial parameters in solution were verified by determining (a) the pattern of magnetization transfer from the protein to protons of lactose and Forssman disaccharide by NMR spectroscopy and (b) the ellipticity changes at wavelengths characteristic for Trp/Tyr residues in near-UV CD spectroscopy. Whereas solid-phase assays confirmed a previously noted tendency for homo- and heterotypic aggregation, gel filtration and ultracentrifugation disclosed monomeric status in solution, in line with crystallographic data. Using cell mutants with defects in glycosylation, this lectin domain was shown to preferentially bind N-glycans without alpha2,3-sialylation. Since proximal promoter sequences were delineated to diverge markedly among galectin genes and resulting differences in expression profiles were exemplarily documented immunohistochemically, the intrafamily diversification appears to have assigned this protein to a characteristic expression and activity profile among galectins. Our data thus take the crystallographic information to the level of the lectin in solution and in tissues by a strategic combination of spectroscopic and cell/histochemical assays., (Copyright 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
13. WITHDRAWN: Corrigendum to "A lectin from the Chinese bird-hunting spider binds sialic acids"
- Author
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Siebert HC, Lu SY, Wechselberger R, Born K, Eckert T, Liang S, der Lieth CW, Jiménez-Barbero J, Schauer R, G Vliegenthart JF, Lütteke T, André S, Kaltner H, Gabius HJ, and Kožár T
- Abstract
The Publisher regrets that this article is an accidental duplication of an article that has already been published, doi: 10.1016/j.carres.2010.01.003. The duplicate article has therefore been withdrawn., (Copyright © 2009. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
14. Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages.
- Author
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Barrès C, Blanc L, Bette-Bobillo P, André S, Mamoun R, Gabius HJ, and Vidal M
- Subjects
- Animals, Antigens, Surface metabolism, Biological Transport physiology, Lysosomal-Associated Membrane Protein 2 metabolism, Lysosomal-Associated Membrane Protein 2 physiology, Protein Binding physiology, Protein Transport physiology, Rats, Rats, Sprague-Dawley, Secretory Pathway physiology, Transport Vesicles metabolism, Exosomes metabolism, Galectins metabolism, Galectins physiology, Macrophages metabolism, Reticulocytes metabolism
- Abstract
Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.
- Published
- 2010
- Full Text
- View/download PDF
15. Diffusion nuclear magnetic resonance spectroscopy detects substoichiometric concentrations of small molecules in protein samples.
- Author
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Ribeiro JP, Palczewska M, André S, Cañada FJ, Gabius HJ, Jiménez-Barbero J, Mellström B, Naranjo JR, Scheffers DJ, and Groves P
- Subjects
- Animals, Chickens, Computer Simulation, Diffusion, Galectin 1 analysis, Humans, Reference Standards, Imidazoles analysis, Magnetic Resonance Spectroscopy methods, Proteins analysis
- Abstract
Small molecules are difficult to detect in protein solutions, whether they originate from elution during affinity chromatography (e.g., imidazole, lactose), buffer exchange (Tris), stabilizers (e.g., beta-mercaptoethanol, glycerol), or excess labeling reagents (fluorescent reagents). Mass spectrometry and high-pressure liquid chromatography (HPLC) often require substantial efforts in optimization and sample manipulation to provide sufficient sensitivity and reliability for their detection. One-dimensional (1D) (1)H nuclear magnetic resonance (NMR) could, in principle, detect residual amounts of small molecules in protein solutions down to equimolecular concentrations with the protein. However, at lower concentrations, the NMR signals of the contaminants can be hidden in the background spectrum of the protein. As an alternative, the 1D diffusion difference protocol used here is feasible. It even improves the detection level, picking up NMR signals from small-molecule contaminants at lower concentrations than the protein itself. We successfully observed 30 microM imidazole in the presence of four different proteins (1-1.5 mg/ml, 6-66 kDa, 25-250 microM) by 1D diffusion-ordered spectroscopy (DOSY) difference and 1-h total acquisition time. Of note, imidazole was not detected in the corresponding 1D (1)H NMR spectra. This protocol can be adapted to different sample preparation procedures and NMR acquisition methods with minimal manipulation in either deuterated or nondeuterated buffers.
- Published
- 2010
- Full Text
- View/download PDF
16. A lectin from the Chinese bird-hunting spider binds sialic acids.
- Author
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Siebert HC, Lu SY, Wechselberger R, Born K, Eckert T, Liang S, von der Lieth CW, Jiménez-Barbero J, Schauer R, Vliegenthart JF, Lütteke T, André S, Kaltner H, Gabius HJ, and Kozár T
- Subjects
- Amino Acid Sequence, Animals, Magnetic Resonance Spectroscopy, Mass Spectrometry, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Structure, Secondary, Spider Venoms metabolism, Lectins chemistry, Lectins metabolism, Sialic Acids chemistry, Sialic Acids metabolism, Spiders metabolism
- Abstract
The affinity to sialic acid-containing oligosaccharides of the small-animal lectin SHL-I isolated from the venom of the Chinese bird-hunting spider Selenocosmia huwena is here described for the first time. By a strategic combination of NMR techniques, molecular modeling, and data mining tools it was possible to identify the crucial amino acid residues that are responsible for SHL-I's ability to bind sialic acid residues in a specific way. Furthermore, we are able to discuss the role of the functional groups of sialic acid when bound to SHL-I. Also the impact of Pro31 in its cis- or trans-form on SHL-I's ligand affinity is of special interest, since it answers the question if Trp32 is a crucial amino acid for stabilizing complexes between SHL-I and sialic acid. SHL-I can be considered as a proper model system that provides further insights into the binding mechanisms of small-animal lectins to sialic acid on a sub-molecular level.
- Published
- 2009
- Full Text
- View/download PDF
17. Homodimeric chicken galectin CG-1B (C-14): Crystal structure and detection of unique redox-dependent shape changes involving inter- and intrasubunit disulfide bridges by gel filtration, ultracentrifugation, site-directed mutagenesis, and peptide mass fingerprinting.
- Author
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López-Lucendo MF, Solís D, Sáiz JL, Kaltner H, Russwurm R, André S, Gabius HJ, and Romero A
- Subjects
- Amino Acid Sequence, Animals, Chromatography, Gel, Crystallography, X-Ray, Dimerization, Disulfides, Galectins genetics, Mass Spectrometry, Molecular Sequence Data, Mutagenesis, Site-Directed, Oxidation-Reduction, Peptide Mapping, Protein Conformation, Protein Structure, Quaternary, Sequence Alignment, Ultracentrifugation, Chickens, Galectins chemistry
- Abstract
Intrafamily gene diversification has led to three prototype galectins in chicken [i.e., chicken galectin (CG)-1A, CG-1B, and CG-2] that show distinct expression profiles and developmental regulation. In order to pinpoint structural disparities among them, we determined the crystal structure of CG-1B. Alteration of the position of the Trp ring in the lectin site and the presence of only two ordered water molecules therein, as well as changes in the interface region between the two subunits, set the structure of CG-1B clearly apart from that of CG-1A. Intriguingly, the unique presence of two Cys residues at positions 2 and 7 in the N-terminal region translated into formation of an intersubunit disulfide bridge between the Cys7 residues of the homodimer in the crystal. In solution, oxidation is associated with significant shape changes in the dimeric protein and the additional occurrence of a compacted form with an intrasubunit disulfide bridge between Cys2 and Cys7. The single-site mutant C7S/C7V was not subjected to such changes, supporting the crucial role of Cys7 in redox-dependent shape changes. These results point to the functional significance of the distinctive presence of the two Cys residues in the N-terminal region of CG-1B.
- Published
- 2009
- Full Text
- View/download PDF
18. Detection and chromatographic removal of lipopolysaccharide in preparations of multifunctional galectins.
- Author
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Sarter K, André S, Kaltner H, Lensch M, Schulze C, Urbonaviciute V, Schett G, Herrmann M, and Gabius HJ
- Subjects
- Animals, Cell Line, Chromatography, Dendritic Cells drug effects, Dendritic Cells metabolism, Galectin 3 biosynthesis, Galectin 3 pharmacology, Galectins biosynthesis, Galectins pharmacology, Humans, Lipopolysaccharides isolation & purification, Mice, Polymyxin B pharmacology, Protein Binding, Reagent Kits, Diagnostic, Recombinant Proteins pharmacology, Tumor Necrosis Factor-alpha metabolism, Galectins chemistry, Lipopolysaccharides analysis, Recombinant Proteins chemistry
- Abstract
The functional spectrum of human galectins is currently explored, with a wide range of activities being described. The role of galectin-3 as adhesin for bacteria is based on its strong binding to lipopolysaccharides (LPSs), which brings the possibility of such a contamination in galectin preparations to awareness. This assumption was verified in three independent functional assay systems using polymyxin B as inhibitor of LPS-dependent effects. Moreover, a commercial LPS quantification kit also revealed LPS in galectin preparations. Chromatography was effective in removing LPS, suggesting that such a technique needs to be applied to prevent assigning cellular responses to galectins rather than LPS.
- Published
- 2009
- Full Text
- View/download PDF
19. Assessing the inhibitory potency of galectin ligands identified from combinatorial (glyco)peptide libraries using surface plasmon resonance spectroscopy.
- Author
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Maljaars CE, André S, Halkes KM, Gabius HJ, and Kamerling JP
- Subjects
- Amino Acid Sequence, Animals, Asialoglycoproteins chemistry, Asialoglycoproteins metabolism, Cattle, Fetuins, Galectin 1 chemistry, Galectin 3 chemistry, Glycopeptides chemistry, Humans, Ligands, Molecular Sequence Data, Protein Binding, alpha-Fetoproteins chemistry, alpha-Fetoproteins metabolism, Galectin 1 metabolism, Galectin 3 metabolism, Glycopeptides metabolism, Peptide Library, Surface Plasmon Resonance methods
- Abstract
Combinatorial (glyco)peptide libraries offer the possibility to define effective inhibitors of protein (lectin)-glycan interactions. If a (glyco)peptide surpasses the inhibitory potency of the free sugar, then the new peptide-lectin contacts underlying the affinity enhancement may guide further rational drug design. Focusing on the adhesion/growth regulatory human galectins 1 and 3, a screening of three combinatorial solid-phase (glyco)peptide libraries, containing Gal(beta1-O)Thr, Gal(beta1-S)Cys/Gal(beta1-N)Asn, and Lac(beta1-O)Thr, with the fluorescently labeled lectins had led to a series of lead compounds. To define the inhibitory potency of a selection of resynthesized (glyco)peptides systematically, a surface plasmon resonance-based inhibition assay with immobilized asialofetuin was set up. (Glyco)Peptides with up to 66-fold potency relative to free lactose as inhibitor were characterized. The presence of lactose in the most effective glycopeptides indicated the presence of affinity-enhancing peptide-lectin contacts. In addition to drug design, they may be helpful for fine-structural analysis of the binding sites.
- Published
- 2008
- Full Text
- View/download PDF
20. The conformation of the C-glycosyl analogue of N-acetyl-lactosamine in the free state and bound to a toxic plant agglutinin and human adhesion/growth-regulatory galectin-1.
- Author
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García-Aparicio V, Sollogoub M, Blériot Y, Colliou V, André S, Asensio JL, Cañada FJ, Gabius HJ, Sinaÿ P, and Jiménez-Barbero J
- Subjects
- Acetylglucosamine chemistry, Carbohydrate Conformation, Cell Adhesion, Cell Division, Disaccharides chemistry, Humans, Magnetic Resonance Spectroscopy, Models, Molecular, Plant Lectins chemistry, Amino Sugars chemistry, Galectin 1 chemistry, Glycosides chemistry, Plant Lectins toxicity
- Abstract
The conformational behavior of the C-glycoside analogue of N-acetyl-lactosamine, beta-C-Gal-(1-->4)-beta-GlcNAc-OMe, 1, has been studied using a combination of molecular mechanics calculations and NMR spectroscopy (J and NOE data). It is shown that the C-disaccharide populates three distinctive conformational families in solution, the major one being the anti-psi conformation. Of note, this conformation is only marginally populated for the O-disaccharide. Due to its conspicuous role in the regulation of adhesion, growth and tissue invasion of tumors and its avid binding to N-acetyl-lactosamine human, galectin-1 was tested as a receptor. This endogenous lectin recognizes a local minimum of 1, the syn-PhiPsi conformer, and thus a conformational selection process is correlated with the molecular recognition event.
- Published
- 2007
- Full Text
- View/download PDF
21. Unique sequence and expression profiles of rat galectins-5 and -9 as a result of species-specific gene divergence.
- Author
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Lensch M, Lohr M, Russwurm R, Vidal M, Kaltner H, André S, and Gabius HJ
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chromosome Mapping, Galectins analysis, Gene Expression Profiling, Immunohistochemistry, Mice, Molecular Sequence Data, Promoter Regions, Genetic, RNA, Messenger analysis, RNA, Messenger metabolism, Rats, Tissue Distribution, Erythropoiesis genetics, Galectins genetics, Gene Expression Regulation, Genetic Variation
- Abstract
Presence of species-specific gene divergence in a protein family prompts to thoroughly study structural aspects and expression profiles of the products. We herein focus on two members of an adhesion/growth-regulatory group of endogenous lectins, i.e. galectins-5 and -9. After first ascertaining species specificity of occurrence of galectin-5, constituted by a short section of rat galectin-9's N-terminal part and its C-terminal carbohydrate recognition domain, by database mining, we next detected and defined sequence differences in the proximal promoter region between the two genes. The ensuing hypothesis for distinct expression profiles was tested first by RT-PCR and then by immunohistochemistry. For the latter purpose, we employed antibodies rigorously controlled for absence of cross-reactivity including assays with various other galectins and, if necessary, refined by chromatographic removal of bi- or oligospecific activities. Indeed, the galectins have non-identical expression profiles, qualitative differences, e.g. seen for galectin-5-positive bone marrow and erythrocytes or for hitherto unknown expression in cells of the theca folliculi and galectin-9-positive skin epidermis and esophageal epithelium. Lack of hepatocyte or renal cortex staining separates these two expression profiles in rat from localization of galectin-9 in mouse. Interspecies extrapolation in a case of a galectin involved in unique gene divergence may thus not be valid. The presented results on galectin-5 relative to galectin-9 intimate distinct functions especially in erythropoiesis and imply currently unknown mechanisms to compensate its absence from the galectin network in other mammals.
- Published
- 2006
- Full Text
- View/download PDF
22. Growth-regulatory human galectin-1: crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding.
- Author
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López-Lucendo MF, Solís D, André S, Hirabayashi J, Kasai K, Kaltner H, Gabius HJ, and Romero A
- Subjects
- Amino Acid Sequence, Carbohydrate Metabolism, Crystallography, X-Ray, Cysteine metabolism, Galectin 1 genetics, Galectin 1 metabolism, Humans, Ligands, Molecular Sequence Data, Mutation, Oxidation-Reduction, Protein Binding, Protein Structure, Tertiary, Static Electricity, Thermodynamics, Galectin 1 chemistry
- Abstract
Human galectin-1 is a potent multifunctional effector that participates in specific protein-carbohydrate and protein-protein (lipid) interactions. By determining its X-ray structure, we provide the basis to define the structure of its ligand-binding pocket and to perform rational drug design. We have also analysed whether single-site mutations introduced at some distance from the carbohydrate recognition domain can affect the lectin fold and influence sugar binding. Both the substitutions introduced in the C2S and R111H mutants altered the presentation of the loop, harbouring Asp123 in the common "jelly-roll" fold. The orientation of the side-chain was inverted 180 degrees and the positions of two key residues in the sugar-binding site of the R111H mutant were notably shifted, i.e. His52 and Trp68. Titration calorimetry was used to define the decrease in ligand affinity in both mutants and a significant increase in the entropic penalty was found to outweigh a slight enhancement of the enthalpic contribution. The position of the SH-groups in the galectin appeared to considerably restrict the potential to form intramolecular disulphide bridges and was assumed to be the reason for the unstable lectin activity in the absence of reducing agent. However, this offers no obvious explanation for the improved stability of the C2S mutant under oxidative conditions. The noted long-range effects in single-site mutants are relevant for the functional divergence of closely related galectins and in more general terms, the functionality definition of distinct amino acids.
- Published
- 2004
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