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1.
Cell Rep ; 10(9): 1626-1638, 2015 Mar 10.
Article in English | MEDLINE | ID: mdl-25753426

ABSTRACT

Inflammatory response induced by microglia plays a critical role in the demise of neuronal populations in neuroinflammatory diseases. Although the role of toll-like receptor 4 (TLR4) in microglia's inflammatory response is fully acknowledged, little is known about endogenous ligands that trigger TLR4 activation. Here, we report that galectin-3 (Gal3) released by microglia acts as an endogenous paracrine TLR4 ligand. Gal3-TLR4 interaction was further confirmed in a murine neuroinflammatory model (intranigral lipopolysaccharide [LPS] injection) and in human stroke subjects. Depletion of Gal3 exerted neuroprotective and anti-inflammatory effects following global brain ischemia and in the neuroinflammatory LPS model. These results suggest that Gal3-dependent-TLR4 activation could contribute to sustained microglia activation, prolonging the inflammatory response in the brain.

2.
J Lab Autom ; 19(2): 208-14, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24051516

ABSTRACT

Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated high-fidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type-dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.


Subject(s)
Automation, Laboratory/methods , Culture Techniques/methods , High-Throughput Screening Assays/methods , Optical Imaging/methods , Spheroids, Cellular
3.
J Biol Chem ; 287(26): 21751-6, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22549776

ABSTRACT

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.


Subject(s)
Galectin 3/chemistry , Anisotropy , Binding Sites , Cell Membrane/metabolism , Cross-Linking Reagents/chemistry , Endocytosis , Fluorescence Polarization , Galectin 3/metabolism , Galectins/chemistry , Glycoproteins/chemistry , Humans , Kinetics , Ligands , Protein Binding , Protein Structure, Tertiary
4.
PLoS One ; 6(10): e26560, 2011.
Article in English | MEDLINE | ID: mdl-22028908

ABSTRACT

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.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Galectin 1/metabolism , Haptoglobins/metabolism , Intracellular Space/metabolism , Aged , Binding Sites , Case-Control Studies , Cell Line, Tumor , Endocytosis , Female , Galectin 1/chemistry , Galectin 1/immunology , Haptoglobins/chemistry , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Immunoglobulin M/immunology , Macrophages/cytology , Macrophages/metabolism , Middle Aged , Models, Molecular , N-Acetylneuraminic Acid , Neoplasm Metastasis , Polysaccharides/metabolism , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Transport , Substrate Specificity
5.
J Biol Chem ; 286(16): 13801-4, 2011 Apr 22.
Article in English | MEDLINE | ID: mdl-21372130

ABSTRACT

Angiogenesis is a key event in cancer progression and therefore a promising target in cancer treatment. Galectin-1, a ß-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.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Galectin 1/biosynthesis , Glycoproteins/chemistry , Peptides/pharmacology , Angiogenesis Inhibitors/chemistry , Disaccharides/chemistry , Fluorescence Polarization/methods , Galectins/chemistry , Glycoconjugates/chemistry , Haptoglobins/chemistry , Humans , Kinetics , Lectins/chemistry , Microscopy, Fluorescence/methods , Neoplasms/metabolism , Peptides/chemistry , Protein Binding , alpha-Fetoproteins/chemistry
6.
J Biol Chem ; 285(45): 35079-91, 2010 Nov 05.
Article in English | MEDLINE | ID: mdl-20807768

ABSTRACT

Galectins are defined by a conserved ß-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 ß-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 ß-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 ß-galactosides substituted with GlcNAcß1-3, 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.


Subject(s)
Galactosides/metabolism , Galectin 3/metabolism , Mutation, Missense , Neutrophil Activation/physiology , Neutrophils/metabolism , Amino Acid Substitution , Animals , Galactosides/genetics , Galectin 3/genetics , Galectin 3/pharmacology , Humans , Mice , Neutrophil Activation/drug effects , Substrate Specificity , Xenopus laevis
7.
Biochemistry ; 49(44): 9518-32, 2010 Nov 09.
Article in English | MEDLINE | ID: mdl-20873803

ABSTRACT

Galectin-1, a ß-galactoside binding lectin involved in immunoregulation and cancer, binds natural and many synthetic multivalent glycoconjugates with an apparent glycoside cluster effect, that is, affinity above and beyond what would be expected from the concentration of the determinant sugar. Here we have analyzed the mechanism of such cluster effects in solution at physiological concentration using a fluorescence anisotropy assay with a novel fluorescent high-affinity galectin-1 binding probe. The interaction of native dimeric and monomeric mutants of rat and human galectin-1 with mono- and divalent small molecules, fetuin, asialofetuin, and human serum glycoproteins was analyzed. Surprisingly, high-affinity binding did not depend much on the dimeric state of galectin-1 and thus is due mainly to monomeric interactions of a single carbohydrate recognition domain. The mechanism for this is unknown, but one possibility includes additional interactions that high-affinity ligands make with an extended binding site on the carbohydrate recognition domain. It follows that such weak additional interactions must be important for the biological function of galectin-1 and also for the design of galectin-1 inhibitors.


Subject(s)
Galectin 1/metabolism , Glycoproteins/metabolism , Animals , Asialoglycoproteins/metabolism , Binding Sites , Blood Proteins/metabolism , Crystallography, X-Ray , Fetuins , Fluorescence Polarization , Galectin 1/chemistry , Humans , Models, Molecular , Protein Binding , Protein Multimerization , Rats , alpha-Fetoproteins/metabolism
8.
Chembiochem ; 11(10): 1430-42, 2010 Jul 05.
Article in English | MEDLINE | ID: mdl-20549756

ABSTRACT

Multimeric lactosides based on carbohydrate scaffolds with valencies ranging from 1 to 4 and different linker lengths were synthesized by a copper-catalyzed azide-alkyne cycloaddition (CuAAC). The binding affinities and crosslinking abilities of the new "click clusters" toward biologically relevant galectins (gal-1, gal-3) and peanut lectin were evaluated by fluorescent polarization assay (FPA) and enzyme-linked lectin assay (ELLA), respectively. FPA indicated that the binding affinities of the synthetic multilactosides towards the galectins increased proportionally with their lactosyl content, without significant differences due to the spacer length. ELLA evidenced a modest cluster effect for the multivalent conjugates, with a relative potency per lactoside ranging from 2.1 to 3.2. Nearly identical binding affinities were recorded for derivatives differing in the length of the linkers, in agreement with the FPA data. These results demonstrate that this parameter does not significantly influence the recognition process when interactions occur at a single lectin site. Molecular dynamics revealed that glycoconjugates adopt a pseudoglobular structure with a random localization of the lactoside residues. These spatial distributions were observed irrespective of the linker length; this explains the virtually equal affinities recorded by ELLA. In contrast, two-site "sandwich" ELLA clearly revealed that multivalent derivatives bearing the longest spacers were more efficient for crosslinking lectins. Intrinsic affinities, devoid of aggregation effects, and crosslinking capabilities are, therefore, not directly related phenomena that must be taking into consideration in neoglycoconjugate design for specific applications.


Subject(s)
Galectin 1/chemistry , Galectin 3/chemistry , Glycosides/chemistry , Peanut Agglutinin/chemistry , Alkynes/chemistry , Arachis/metabolism , Azides/chemistry , Catalysis , Copper/chemistry , Enzyme Assays , Fluorescence Polarization Immunoassay , Molecular Dynamics Simulation
9.
Glycobiology ; 19(1): 16-20, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18849325

ABSTRACT

Galectin-3, a beta-galactoside binding, endogenous lectin, takes part in various inflammatory events and is produced in substantial amounts at inflammatory foci. We investigated whether extracellular galectin-3 could participate in the phagocytic clearance of apoptotic neutrophils by macrophages, a process of crucial importance for termination of acute inflammation. Using human leukocytes, we show that exogenously added galectin-3 increased the uptake of apoptotic neutrophils by monocyte-derived macrophages (MDM). Both the proportion of MDM that engulfed apoptotic prey and the number of apoptotic neutrophils that each MDM engulfed were enhanced in the presence of galectin-3. The effect was lactose-inhibitable and required galectin-3 affinity for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins, since a mutant lacking this activity was without effect. The enhanced uptake relied on the presence of galectin-3 during the cellular interaction and was paralleled by lectin binding to apoptotic cells as well as MDM in a lactose-dependent manner. These findings suggest that galectin-3 functions as a bridging molecule between phagocyte and apoptotic prey, acting as an opsonin. The process of clearance, whereby apoptotic neutrophils are removed by macrophages, is crucial for the resolution of acute inflammation and our data imply that the increased levels of galectin-3 often found at inflammatory sites could potently affect this process.


Subject(s)
Apoptosis/immunology , Galectin 3/metabolism , Macrophages/metabolism , Neutrophils/metabolism , Opsonin Proteins/metabolism , Binding Sites , Cells, Cultured , Humans , Macrophage Activation
10.
Glycobiology ; 18(11): 905-12, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18725453

ABSTRACT

Neutrophils interacting with a chemoattractant gradually become nonresponsive to further stimulation by the same agonist, a process known as desensitization. Receptor desensitization is a highly regulated process that involves different mechanisms depending on which receptor-ligand pair that is studied. Galectin-3, a member of a large family of beta-galactoside-binding lectins, has been suggested to be a regulator of the inflammatory process, augmenting or directly triggering the neutrophil functional repertoire. We show here that the desensitized state of neutrophils interacting with the chemotactic peptide fMLF is broken by galectin-3 and that this is achieved through an oxygen radical-mediated inactivation of the chemoattractant. The effect was inhibited by the competitor lactose and required the affinity of galectin-3 for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins. The latter was shown using a galectin-3 mutant that lacked N-acetyllactosamine binding activity, and this protein was not active. The mechanism behind the inactivation of the chemoattractant was found to depend on the ability of galectin-3 to induce a neutrophil generation/secretion of reactive oxygen species which in combined action with myeloperoxidase inactivated the peptides.


Subject(s)
Galectin 3/pharmacology , Immunologic Factors/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , NADPH Oxidases/metabolism , Neutrophils/enzymology , Reactive Oxygen Species/metabolism , Binding Sites , Enzyme Activation , Galactosides/metabolism , Galectin 3/metabolism , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Neutrophils/drug effects , Neutrophils/metabolism
11.
Chemistry ; 14(14): 4233-45, 2008.
Article in English | MEDLINE | ID: mdl-18366047

ABSTRACT

A series of aromatic mono- or diamido-thiodigalactoside derivatives were synthesized and studied as ligands for galectin-1, -3, -7, -8N terminal domain, and -9N terminal domain. The affinity determination in vitro with competitive fluorescence-polarization experiments and thermodynamic analysis by isothermal microcalorimetry provided a coherent picture of structural requirements for arginine-arene interactions in galectin-ligand binding. Computational studies were employed to explain binding preferences for the different galectins. Galectin-3 formed two almost ideal arene-arginine stacking interactions according to computer modeling and also had the highest affinity for the diamido-thiodigalactosides (K(d) below 50 nM). Site-directed mutagenesis of galectin-3 arginines involved in binding corroborated the importance of their interaction with the aromatic diamido-thiodigalactosides. Furthermore, the arginine mutants revealed distinct differences between free, flexible, and solvent-exposed arginine side chains and tightly ion-paired arginine side chains in interactions with aromatic systems.


Subject(s)
Arginine/chemistry , Diamide/analogs & derivatives , Galectins/chemistry , Thioglycosides/chemistry , Amino Acid Sequence , Arginine/metabolism , Diamide/chemical synthesis , Diamide/chemistry , Galectins/genetics , Galectins/metabolism , Hydrocarbons, Aromatic/chemical synthesis , Hydrocarbons, Aromatic/chemistry , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Protein Isoforms , Protein Structure, Tertiary , Sequence Alignment , Structure-Activity Relationship , Thermodynamics , Thioglycosides/chemical synthesis
12.
Glycobiology ; 18(5): 384-94, 2008 May.
Article in English | MEDLINE | ID: mdl-18263896

ABSTRACT

Here we report the first survey of galectins binding to glycoproteins of human serum. Serum was subjected to affinity chromatography using immobilized galectins, and the bound glycoproteins were analyzed by electrophoresis, Western blotting, and mass spectrometry. Galectins-3, -8, and -9 bound a much broader range of ligands in serum than previously known, galectin-1 bound less, and galectins-2, -4, and -7 bound only traces or no serum ligands. Galectin-3 bound most major glycoproteins, including alpha-2-macroglobulin and acute phase proteins such as haptoglobin. It bound only a selected minor fraction of transferrin, and bound none or little of IgG. Galectins-8 and -9 bound a similar range of glycoproteins as galectin-3, but in lower amounts, and galectin-8 had a relative preference for IgA. Galectin-1 bound mainly a fraction of alpha-2-macroglobulin and only traces of other glycoproteins. The binding of galectin-3 to serum glycoproteins requires affinity for LacNAc, since a mutant (R186S), which has lost this affinity, did not bind any serum glycoproteins. The average affinity of galectin-3 for serum glycoproteins was estimated to correspond to K(d) approximately 1-5 muM by modeling of the affinity chromatography and a fluorescence anisotropy assay. Since galectins are expressed on endothelial cells and other cells exposed to serum components, this report gives new insight into function of galectins and the role of their different fine specificity giving differential binding to the serum glycoproteins.


Subject(s)
Acute-Phase Proteins/metabolism , Galectin 3/metabolism , Galectins/metabolism , Glycoproteins/blood , Glycoproteins/metabolism , Protease Inhibitors/metabolism , Binding Sites , Chromatography, Affinity , Fluorescence Polarization , Humans , Kinetics , Ligands
13.
Chembiochem ; 8(12): 1389-98, 2007 Aug 13.
Article in English | MEDLINE | ID: mdl-17631664

ABSTRACT

Aromatic lactose 2-O-esters were synthesized and used to probe arene-arginine interactions with the galectin family of proteins. They were found to be low microM inhibitors of galectin-1, -3, and -9N-terminal domain and moderate inhibitors of galectin-7, but not inhibitors of galectin-8N-terminal, which lacks an arginine residue close to the critical, esterified lactose 2-O-position. Molecular modeling of galectins in complex with aromatic lactose 2-O-esters, as well as binding studies with a galectin-3 R186S mutant, confirmed that the inhibitory efficiency of the lactose 2-O-esters was due to the formation of strong interactions between the aromatic ester moieties and the arginine guanidinium groups of galectin-1 and -3. An important common feature shared by galectin-1 and -3 was that the arginines formed in-plane ion pairs with two side-chain carboxylates, which resulted in extended planar pi-electron surfaces that did not require solvation by water; these surfaces were ideal for stacking with aromatic moieties of the ligands. The results provide a basis for the design of lectin inhibitors and drugs that exploit interactions with arginine side-chains via aromatic moieties, which are involved in intramolecular protein salt bridges.


Subject(s)
Esters , Lactose , Arginine/metabolism , Galectin 1 , Galectin 3 , Galectins/chemistry
14.
Traffic ; 8(4): 379-88, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17319896

ABSTRACT

Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid-raft-independent apical carrier vesicles harbour the beta-galactoside-binding lectin galectin-3, which interacts directly with apical cargo in a glycan-dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin-3-depleted cells, dedicating a central role to this lectin in raft-independent sorting as apical receptor. Here, we demonstrate that high-molecular-weight clusters are exclusively formed in the presence of galectin-3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation-deficient Madin-Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross-linking by galectin-3 is required for apical sorting of non-raft-associated cargo.


Subject(s)
Galectin 3/physiology , Glycoproteins/metabolism , Animals , Cell Line , Dogs , Glycosylation , Protein Transport , Receptor, Nerve Growth Factor/genetics , Receptor, Nerve Growth Factor/metabolism
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