PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-12 (12)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Regulation of Transforming Growth Factor-β1–driven Lung Fibrosis by Galectin-3 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
doi:10.1164/rccm.201106-0965OC
PMCID: PMC3410728  PMID: 22095546
fibrosis; epithelial cells; fibroblasts
2.  Ligand Induced Galectin-3 Protein Self-association* 
The Journal of Biological Chemistry  2012;287(26):21751-21756.
Background: One galectin-3 function is to bind glycoproteins and cross-link them.
Results: A glycoprotein engaged many more galectin-3 carbohydrate-binding sites than its number of relevant glycans.
Conclusion: The ligand induced binding of one galectin-3 to another galectin-3 to form oligomers in a previously unrecognized way.
Significance: This differs from previous models and provides a new framework to interpret biological effects of galectin-3.
Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in cross-linking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here we studied interaction of galectin-3 with the model glycoprotein asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concentration of free galectin carbohydrate recognition sites in solution. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concentrations, showing that many more galectin-3 molecules were engaged than expected due to the about nine known glycan-based binding sites per ASF molecule. This suggests that after ASF-induced nucleation, galectin-3 associates with itself by the carbohydrate recognition site binding to another galectin-3 molecule, possibly forming oligomers. We named this type-C self-association to distinguish it from the previously proposed models (type-N) where galectin-3 molecules bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-association can result in precipitates, as measured here by turbidimetry and dynamic light scattering. Type-C self-association and precipitation occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required much higher concentration (∼50 μm) as compared with wild type (∼1 μm). ASF also induced weaker type-C self-association of galectin-3 lacking its N-terminal domains, but as expected, no precipitation. Neither a monovalent nor a divalent N-acetyl-d-lactosamine-containing glycan induced type-C self-association, even if the latter gave precipitates with high concentrations of galectin-3 (>∼50 μm) in agreement with published results and perhaps due to type-N self-association.
doi:10.1074/jbc.C112.358002
PMCID: PMC3381137  PMID: 22549776
Carbohydrate-binding Protein; Endocytosis; Galectin; Glycoprotein; Protein Cross-linking; Fluorescence Polarization; Oligomerization
3.  The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics 
Biochemistry  2011;51(1):296-306.
The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate–protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultra-high-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 Å at 100 K, 1.25 Å at 298 K) and in complex with lactose (0.86 Å) or glycerol (0.9 Å). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of β-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design.
doi:10.1021/bi201459p
PMCID: PMC3255464  PMID: 22111949
4.  Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins 
Journal of Clinical Immunology  2011;32(2):246-255.
Background
Immunoglobulin A nephritis (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2–3-sialylated galactosides (NeuAcα2-3Gal).
Purpose
The purpose of this study was to analyze whether altered glycosylation of IgA would lead to an altered binding to galectin-8, an endogenous lectin with strong affinity for 2–3-sialylated galactosides. Galectins are a family of β-galactoside-binding proteins; by binding various glycoproteins, they play important roles in the regulation of cellular functions in inflammation and immunity. Hence, an altered binding of IgA to galectin-8 could lead to pathologic immune functions, such as glomerulonephritis.
Methods
Affinity chromatography of serum glycoproteins on the human sialogalactoside-binding lectin galectin-8N permitted quantitation of bound and unbound fractions, including IgA.
Results
Analysis of ∼100 IgA nephritis sera showed that the galectin-8N unbound fraction of IgA increased compared to ∼100 controls, consistent with the known loss of galactosylation. A subgroup of ∼15% of the IgAN patients had a ratio of galectin-8 bound/unbound IgA <0.09, not found for any of the controls. Unexpectedly, the galectin-8N-binding fraction of serum glycoproteins other than IgA increased in the sera of IgAN patients but not in controls, suggesting a previously unrecognized change in this disease.
Conclusion
This is the first study that relates a galectin, an endogenous lectin family, to IgA nephritis and thus should stimulate new avenues of research into the pathophysiology of the disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s10875-011-9618-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s10875-011-9618-3
PMCID: PMC3305883  PMID: 22173878
IgA nephropathy; galectin-8; affinity chromatography; NeuAcα2-3Gal
5.  Galectin-1-Binding Glycoforms of Haptoglobin with Altered Intracellular Trafficking, and Increase in Metastatic Breast Cancer Patients 
PLoS ONE  2011;6(10):e26560.
Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7–2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8–3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20–80) in cancer sera and about 30% (range 25–50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells.
doi:10.1371/journal.pone.0026560
PMCID: PMC3196588  PMID: 22028908
6.  The Anti-angiogenic Peptide Anginex Greatly Enhances Galectin-1 Binding Affinity for Glycoproteins* 
The Journal of Biological Chemistry  2011;286(16):13801-13804.
Angiogenesis is a key event in cancer progression and therefore a promising target in cancer treatment. Galectin-1, a β-galactoside binding lectin, is up-regulated in the endothelium of tumors of different origin and has been shown to be the target for anginex, a powerful anti-angiogenic peptide with anti-tumor activity. Here we show that when bound to anginex, galectin-1 binds various glycoproteins with hundred- to thousand-fold higher affinity. Anginex also interacts with galectin-2, -7, -8N, and -9N but not with galectin-3, -4, or -9C.
doi:10.1074/jbc.C111.229096
PMCID: PMC3077580  PMID: 21372130
Cancer Therapy; Carbohydrate; Fluorescence; Glycoprotein; Lectin; Peptide Interactions; Angiogenesis; Fluorescence Polarization; Galactose; Galectin
7.  Mutational Tuning of Galectin-3 Specificity and Biological Function* 
The Journal of Biological Chemistry  2010;285(45):35079-35091.
Galectins are defined by a conserved β-galactoside binding site that has been linked to many of their important functions in e.g. cell adhesion, signaling, and intracellular trafficking. Weak adjacent sites may enhance or decrease affinity for natural β-galactoside-containing glycoconjugates, but little is known about the biological role of this modulation of affinity (fine specificity). We have now produced 10 mutants of human galectin-3, with changes in these adjacent sites that have altered carbohydrate-binding fine specificity but that retain the basic β-galactoside binding activity as shown by glycan-array binding and a solution-based fluorescence anisotropy assay. Each mutant was also tested in two biological assays to provide a correlation between fine specificity and function. Galectin-3 R186S, which has selectively lost affinity for LacNAc, a disaccharide moiety commonly found on glycoprotein glycans, has lost the ability to activate neutrophil leukocytes and intracellular targeting into vesicles. K176L has increased affinity for β-galactosides substituted with GlcNAcβ1–3, as found in poly-N-acetyllactosaminoglycans, and increased potency to activate neutrophil leukocytes even though it has lost other aspects of galectin-3 fine specificity. G182A has altered carbohydrate-binding fine specificity and altered intracellular targeting into vesicles, a possible link to the intracellular galectin-3-mediated anti-apoptotic effect known to be lost by this mutant. Finally, the mutants have helped to define the differences in fine specificity shown by Xenopus, mouse, and human galectin-3 and, as such, the evidence for adaptive change during evolution.
doi:10.1074/jbc.M109.098160
PMCID: PMC2966122  PMID: 20807768
Carbohydrate Function; Galactose; Mutant; Neutrophil; Trafficking; Galectin; Specificity
8.  The role of integrin glycosylation in galectin-8-mediated trabecular meshwork cell adhesion and spreading 
Glycobiology  2008;19(1):29-37.
Primary open angle glaucoma (POAG) is a major blindness-causing disease, characterized by elevated intraocular pressure due to an insufficient outflow of aqueous humor. The trabecular meshwork (TM) lining the aqueous outflow pathway modulates the aqueous outflow facility. TM cell adhesion, cell–matrix interactions, and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. In a recent study, we demonstrated that galectin-8 (Gal8) modulates the adhesion and cytoskeletal arrangement of TM cells and that it does so through binding to β1 integrins and inducing Rho signaling. The current study is aimed at the characterization of the mechanism by which Gal8 mediates TM cell adhesion and spreading. We demonstrate here that TM cells adhere to and spread on Gal8-coated wells but not on galectin-1 (Gal1)- or galectin-3 (Gal3)-coated wells. The adhesion of TM cells to Gal8-coated wells was abolished by a competing sugar, β-lactose, but not by a noncompeting sugar, sucrose. Also, a trisaccharide, NeuAcα2-3Galβ1-4GlcNAc, which binds specifically to the N-CRD of Gal8, inhibited the spreading of TM cells to Gal8-coated wells. In contrast, NeuAcα2-6Galβ1-4GlcNAc which lacks affinity for Gal8 had no effect. Affinity chromatography of cell extracts on a Gal8-affinity column and binding experiments with plant lectins, Maakia Amurensis and Sambucus Nigra, revealed that α3β1, α5β1, and αvβ1 integrins are major counterreceptors of Gal8 in TM cells and that TM cell β1 integrins carry predominantly α2-3-sialylated glycans, which are high-affinity ligands for Gal8 but not for Gal1 or Gal3. These data lead us to propose that Gal8 modulates TM cell adhesion and spreading, at least in part, by interacting with α2-3-sialylated glycans on β1 integrins.
doi:10.1093/glycob/cwn100
PMCID: PMC2733777  PMID: 18849583
cell adhesion; galectin-8; glaucoma; integrins; trabecular meshwork
9.  Lipopolysaccharide-Induced Gelatinase Granule Mobilization Primes Neutrophils for Activation by Galectin-3 and Formylmethionyl-Leu-Phe 
Infection and Immunity  2001;69(2):832-837.
We have earlier shown that galectin-3, a lactose-binding mammalian lectin that is secreted from activated macrophages, basophils, and mast cells, induces activation of the NADPH oxidase in exudated but not in peripheral blood neutrophils (A. Karlsson, P. Follin, H. Leffler, and C. Dahlgren, Blood 91:3430–3438, 1998). The alteration in responsiveness occurring during extravasation correlated with mobilization of the gelatinase and/or specific granules to the cell surface, indicating a role for mobilizable galectin-3 receptors. In this study we have investigated galectin-3-induced NADPH oxidase activation, measured as superoxide production, in lipopolysaccharide (LPS)-primed neutrophils. Upon galectin-3 challenge, the LPS-primed cells produced superoxide, both extracellularly and intracellularly. A primed extracellular response to formylmethionyl-Leu-Phe (fMLF) was also achieved. The exposure of complement receptors 1 and 3 as well as the formyl peptide receptor on the cell surface was markedly increased after LPS treatment, indicating that granule fusion with the plasma membrane had occurred. Further assessment of specific markers for neutrophil granules showed that the LPS treatment had mobilized the gelatinase granules but only a minor fraction of the specific granules. We thus suggest that the mechanism behind LPS priming lies at the level of granule (receptor) mobilization for galectin-3 as well as for fMLF.
doi:10.1128/IAI.69.2.832-837.2001
PMCID: PMC97959  PMID: 11159975
10.  Binding Specificity of Piliated Strains of Escherichia coli and Salmonella typhimurium to Epithelial Cells, Saccharomyces cerevisiae Cells, and Erythrocytes 
Infection and Immunity  1981;32(2):796-804.
The binding to mammalian cells of piliated enteric bacteria and the inhibition of the binding by antibodies to purified pili were studied. The target cells were epithelial cells from human bucca and human and rat urinary tracts, erythrocytes from various species, and Saccharomyces cerevisiae cells. The strains were selected to represent the two main agglutination patterns of enteric bacteria: mannose-resistant agglutination of human and other erythrocytes and mannose-sensitive agglutination of guinea pig and other erythrocytes. Escherichia coli 3669 caused only mannose-resistant agglutination, E. coli 6013 caused only mannose-sensitive agglutination, and E. coli 3048 caused both types of agglutination simultaneously. Salmonella typhimurium SH6749 exhibited only mannose-sensitive hemagglutination and was included to allow comparison of its pili with those of E. coli strains. The range of epithelial cells to which the bacteria adhered was related to their agglutination patterns. All four strains attached to human buccal cells. Only E. coli strains 3669 and 3048, which caused mannose-resistant agglutination, adhered to human urinary tract epithelial cells, and only those strains that caused mannose-sensitive agglutination adhered to rat urinary tract epithelial cells. The binding of S. typhimurium SH6749, but not of the other strains with mannose-sensitive agglutination, was significantly inhibited by d-mannose. Globotetraosylceramide, a glycolipid present in the human urinary tract epithelium, inhibited attachment to human uroepithelial cells of the two strains with mannose-resistant hemagglutination. As tested by the enzyme-linked immunosorbent assay, cross-reactions between type 1 pili of the E. coli strains were strong, but those between S. typhimurium and E. coli mannose-sensitive pili were weak. The two pili that induced mannose-resistant hemagglutination on E. coli did not cross-react. Significant inhibition of adhesion of all four strains was obtained with the homologous anti-pilus antiserum. The binding of bacteria to mammalian cells may thus be mediated by several types of bacterial pili reacting with different receptors on mammalian cells.
Images
PMCID: PMC351515  PMID: 6114036
11.  Protein Flexibility and Conformational Entropy in Ligand Design Targeting the Carbohydrate Recognition Domain of Galectin-3 
Journal of the American Chemical Society  2010;132(41):14577-14589.
Rational drug design is predicated on knowledge of the three-dimensional structure of the protein−ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. 15N and 2H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein−carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.
doi:10.1021/ja105852y
PMCID: PMC2954529  PMID: 20873837
12.  Inhibition mechanism of human galectin-7 by a novel galactose-benzylphosphate inhibitor 
The Febs Journal  2012;279(2):193-202.
Galectins are involved in many cellular processes due to their ability to bind carbohydrates. Understanding their functions has shown the necessity for potent and specific galectin inhibitors. Human galectin-7 (hGal-7), in particular, has been highlighted as an important marker in many types of cancer by either inhibiting or promoting tumour growth. Producing ligands able to selectively target hGal-7 will offer promising tools for deciphering cancer processes in which hGal-7 is involved as well as present potential solutions for future therapeutics. Here we report the high resolution crystal structure of hGal-7 in complex with a synthetic 2-O-benzylphosphate-galactoside inhibitor (which is > 60-fold more potent than its parent galactoside). The high resolution crystallographic analysis highlights the validity of using saccharide derivatives, conserving properties of the galactose binding, while enhanced affinity and specificity is provided by the added phosphate group. This structural information will allow the design of further inhibitors with improved potency and specificity.
Database
The atomic coordinates for the complex of human galectin-7 as well as for the free structure have been deposited with the Protein Data Bank (accession numbers 3ZXE and 3ZXF respectively)
Structured digital abstract
hGal-7 and hGal-7 bind by X-raycrystallography (Viewinteraction)
doi:10.1111/j.1742-4658.2011.08414.x
PMCID: PMC3328751  PMID: 22059385
drug design; galactoside inhibitor; galectin-7; lectin

Results 1-12 (12)