Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
J Biol Chem
2010 Nov 05;28545:35079-91. doi: 10.1074/jbc.M109.098160.
Show Gene links
Show Anatomy links
Mutational tuning of galectin-3 specificity and biological function.
Salomonsson E
,
Carlsson MC
,
Osla V
,
Hendus-Altenburger R
,
Kahl-Knutson B
,
Oberg CT
,
Sundin A
,
Nilsson R
,
Nordberg-Karlsson E
,
Nilsson UJ
,
Karlsson A
,
Rini JM
,
Leffler H
.
???displayArticle.abstract???
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.
FIGURE 1. Galectin-3 CRD in complex with saccharide ligands. a, the galectin-CRD is shown as a solvent-accessible surface with bound LacNAc as a stick model based on the x-ray crystal structure (PDB code 1KJL) (38). The positions of sites A-D are indicated above the binding groove and the loosely defined site E to the right. The conserved six amino acids making up site C are shown in white (with black text for Trp-181), and the surrounding mutated residues (Arg-144, Ala-146, Lys-176, Asn-180, Gly-182, Arg-186) are in black with white text; Asp-148 is labeled for reference but was not mutated. The Gal residue of LacNAc is shown in black with position 3 labeled, and the GlcNAc is shown in white with position 1 labeled. b and c, shown is a close-up of the core carbohydrate recognition site (C-D) with bound LacNAc (PDB code 1KJL) (b) or a model of Galβ1–3GlcNAc (c). The white numbers indicate the positions where interactions with the conserved binding site are made and where substitutions would sterically hinder binding. Other labeling is as for panel a. Note the difference in orientation of the reducing hydroxyl in b–c (labeled 1), which would direct structures linked here in a different way. The images were made by PyMOL (DeLano Scientific LLC, Palo Alto, CA).
FIGURE 2. Glycan array analysis of wild type galectin-3 and mutants. Each diagram shows binding (as thin bars) of fluorescein-tagged galectin (wild type or mutant, final concentration 200 μg/ml) to each of the 179 potential galectin binding glycans, expressed as RFU ×10−4 on y axis. The binding data are shown twice, with the glycans grouped by the disaccharide that is likely to be in site C-D of the galectin (panel a) or the monosaccharide that is likely to be in site B, i.e. that is linked to the 3-position of the galactose in site C (panel b). In both panels the glycans are ordered by their intensity of binding to wild type galectin-3 (from left to right). The features by which glycans are grouped are shown under the x axis using the symbols recommended by the Consortium for Functional Glycomics. For site C-D they are, from left to right, Galβ1–4GlcNAc, Galβ1–3GlcNAc, Galβ1–4Glc, Galβ1–3GalNAc, and other. For site B they are sulfate-3, NeuAcα2–3, GalNAcα1–3, Galα1–3, GlcNAcβ1–3, and nothing (that is the Gal in site C is terminal). The bottom right diagram of panel a shows the very low background binding of wild type galectin-3 and all of the mutants to the remaining 227 glycans on the array, which are not expected to bind galectins either because they do not contain β-galactose or all of the β-galactoside-containing disaccharides that they do contain are blocked by substitutions sterically preventing binding in site C and D (2) as explained in Fig. 1, b and c. The R186I mutant gave higher background (five peaks >1000 RFU, maximum 2674) than the others (four peaks >500 RFU, maximum 810) but not high enough to confound interpretation. The numerical values of binding, glycan structures and original glycan numbering are given in supplemental Tables S2, a and b.
FIGURE 3. Fluorescence anisotropy analysis of wild type galectin-3 and mutants. Each panel shows a bar diagram of the relative affinity ((Ka mutant)/(Ka galectin-3 wt) = (Kd galectin-3 wt/Kd mutant) of 1 mutant for 15 different fluorescein-labeled saccharide probes (1–15). Average values of relative affinities are based on analysis using a fluorescence anisotropy assay at chilled conditions (∼ 4 °C), n ≥ 4, as described in more detail in supplemental Fig. S2 and Table S3. The saccharide probes are shown in a table below the figure with the disaccharide most likely to bind in site C-D shown in bold.
FIGURE 4. Modeling of interactions in site B. The monosaccharide attached to position 3 of Gal in site C and selected amino acid side chains are shown as stick models, with some positions and functional groups labeled and hydrogen bonds denoted by dotted lines. A transparent surface of the galectin-3 CRD is shown as background. a, shown is one possible position of NeuAc in NeuAca2–3Galβ1–4Glc based on the complex of NeuAcα2–3Lac with the N-CRD of galectin-8 (19); an alternative similar position was found in an x-ray crystal structure of a complex of NeuAcα2–3Lac with a fungal galectin (40), although the structure of site B is different in this case. b, shown is the position of GlcNAcβ in lacto-N-neotetraose (Galβ1–4GlcNAcβ1–3Galβ1–4Glc) and the amino acid side chains from the x-ray crystal structure of a galectin-3 CRD complex with lacto-N-neotetraose.5 c, shown is the position of GalNAcα in a blood group A tetrasaccharide (GalNAcα1–3(Fucα1–2)Galβ1–4Glc) based on a complex with a fungal galectin (39). The models were generated by MacroModel (9.7 edition), and the images were generated by PyMOL.
FIGURE 5. Fluorescence anisotropy analysis of the affinity of Xenopus (left) and mouse (right) galectin-3 relative to human galectin-3. Analysis was done, and results are presented as for Fig. 3, showing bar diagrams of the relative affinity of Xenopus and mouse galectin-3 ((Ka Xenopus or mouse galectin-3)/(Ka human galectin-3 wt)) for 15 different fluorescein labeled saccharide probes (1–15). Below each diagram is a model of human galectin-3 made as in Fig. 1a but colored black for amino acids that differ in the respective animal galectin and white for those that are identical. The residues in site B that differ are labeled.
FIGURE 6. Dose response of activation of TNFα-primed neutrophils by wild type galectin-3 and mutants. The dose (x axis) response (y axis) of wild type and mutants are shown in gray and black, respectively, in all panels. Extra and intracellular release of reactive oxygen species was measured using a luminescence assay. Each data point is the peak (reactive oxygen species) response from a time curve (as given in supplemental Fig. S3), normalized by defining 100% as the peak response to the highest concentration (3.5 μm) of wild type galectin-3 run in parallel. n = 3.
FIGURE 7. Binding of wild type galectin-3 and mutants to unprimed and TNFα-primed neutrophils. FITC-labeled galectin was incubated with freshly prepared neutrophils that had been pretreated by incubation for 30 min at 37 °C without (white bars) or with TNFα (gray bars) and at 4 °C without TNFα (black bars) as a control to prevent granule mobilization. The amount of bound galectin was measured by flow cytometry and is given as the mean and S.E. of the geometric mean of fluorescence intensity (y axis) from three experiments on different batches of neutrophils. The p values were calculated from paired one-tailed t tests; paired, to discount the variability between the neutrophils, and one-tailed, to specifically test the hypothesis of an increased binding after incubation with TNFα.
FIGURE 8. Expression and localization of GFP-galectin-3 wt and two mutants in SKBR3 breast carcinoma cells. The human breast cancer cell line SKBR3 was stably transfected with the pEGFP-C1 vector carrying the gene for EGFP-galectin-3 wild type (panel a), EGFP-R186S (panel b), and EGFP-G182A (panel c), respectively. Cells were cultured on coverslips, fixed in 4% paraformaldehyde, mounted with Prolong Antifade Reagent with DAPI, and analyzed by fluorescence microscopy. The number of galectin containing vesicles (seen as fluorescent dots) per cell was counted in 300 cells for each case and are represented as bar diagrams under each sample microscope panel.
Agrwal,
Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains.
1993, Pubmed
Agrwal,
Carbohydrate-binding protein 35. I. Properties of the recombinant polypeptide and the individuality of the domains.
1993,
Pubmed
Akahani,
Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family.
1997,
Pubmed
Almkvist,
Lipopolysaccharide-induced gelatinase granule mobilization primes neutrophils for activation by galectin-3 and formylmethionyl-Leu-Phe.
2001,
Pubmed
Amano,
The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering, phosphatase modulation, and T cell death.
2003,
Pubmed
Asada,
Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18.
1991,
Pubmed
Ban,
Structural basis of a fungal galectin from Agrocybe cylindracea for recognizing sialoconjugate.
2005,
Pubmed
Barondes,
Galectins. Structure and function of a large family of animal lectins.
1994,
Pubmed
Böyum,
Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g.
1968,
Pubmed
Cabrera,
Haploinsufficiency of C2GnT-I glycosyltransferase renders T lymphoma cells resistant to cell death.
2006,
Pubmed
Carlsson,
Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface.
2007,
Pubmed
Carlsson,
Intracellular sorting of galectin-8 based on carbohydrate fine specificity.
2007,
Pubmed
Cederfur,
Different affinity of galectins for human serum glycoproteins: galectin-3 binds many protease inhibitors and acute phase proteins.
2008,
Pubmed
Cumpstey,
Double affinity amplification of galectin-ligand interactions through arginine-arene interactions: synthetic, thermodynamic, and computational studies with aromatic diamido thiodigalactosides.
2008,
Pubmed
Dahlgren,
Respiratory burst in human neutrophils.
1999,
Pubmed
Delacour,
Apical sorting by galectin-3-dependent glycoprotein clustering.
2007,
Pubmed
Delacour,
Loss of galectin-3 impairs membrane polarisation of mouse enterocytes in vivo.
2008,
Pubmed
Delacour,
The role of galectins in protein trafficking.
2009,
Pubmed
Diehl,
Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3.
2009,
Pubmed
Dumic,
Galectin-3: an open-ended story.
2006,
Pubmed
Feuk-Lagerstedt,
Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils.
1999,
Pubmed
Finne,
Novel polyfucosylated N-linked glycopeptides with blood group A, H, X, and Y determinants from human small intestinal epithelial cells.
1989,
Pubmed
Forsman,
The beta-galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPH-oxidase and inactivation of the agonist.
2008,
Pubmed
Fukuda,
Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells.
1985,
Pubmed
Fukuda,
Structure of sialylated fucosyl lactosaminoglycan isolated from human granulocytes.
1984,
Pubmed
Fukuda,
Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells.
1986,
Pubmed
Furukawa,
Structural study of the O-linked sugar chains of human leukocyte tyrosine phosphatase CD45.
1998,
Pubmed
Garner,
Galectin-glycan lattices regulate cell-surface glycoprotein organization and signalling.
2008,
Pubmed
Henderson,
The regulation of inflammation by galectin-3.
2009,
Pubmed
Henrick,
Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3.
1998,
Pubmed
Hirabayashi,
Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin.
1991,
Pubmed
Houzelstein,
Phylogenetic analysis of the vertebrate galectin family.
2004,
Pubmed
Hsu,
Galectin-3 regulates T-cell functions.
2009,
Pubmed
John,
Galectin-3 binds lactosaminylated lipooligosaccharides from Neisseria gonorrhoeae and is selectively expressed by mucosal epithelial cells that are infected.
2002,
Pubmed
Karlsson,
Galectin-3 activates the NADPH-oxidase in exudated but not peripheral blood neutrophils.
1998,
Pubmed
Karlsson,
Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils.
2009,
Pubmed
Karlsson,
Different glycosphingolipid composition in human neutrophil subcellular compartments.
2001,
Pubmed
Knibbs,
Carbohydrate-binding protein 35. II. Analysis of the interaction of the recombinant polypeptide with saccharides.
1993,
Pubmed
Koch,
Galectin-3, a novel centrosome-associated protein, required for epithelial morphogenesis.
2010,
Pubmed
Lau,
N-Glycans in cancer progression.
2008,
Pubmed
Leffler,
Specificity of binding of three soluble rat lung lectins to substituted and unsubstituted mammalian beta-galactosides.
1986,
Pubmed
Leffler,
Introduction to galectins.
2002,
Pubmed
Lin,
Galectin-3 targeted therapy with a small molecule inhibitor activates apoptosis and enhances both chemosensitivity and radiosensitivity in papillary thyroid cancer.
2009,
Pubmed
Liu,
Galectins as modulators of tumour progression.
2005,
Pubmed
Massa,
L-29, an endogenous lectin, binds to glycoconjugate ligands with positive cooperativity.
1993,
Pubmed
Nickel,
The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes.
2003,
Pubmed
Patnaik,
Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells.
2006,
Pubmed
Paz,
Galectin-3, a marker for vacuole lysis by invasive pathogens.
2010,
Pubmed
Rabinovich,
Turning 'sweet' on immunity: galectin-glycan interactions in immune tolerance and inflammation.
2009,
Pubmed
Rudd,
Glycosylation of natural human neutrophil gelatinase B and neutrophil gelatinase B-associated lipocalin.
1999,
Pubmed
Ruebel,
Effects of DNA methylation on galectin-3 expression in pituitary tumors.
2005,
Pubmed
Sato,
Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin.
1992,
Pubmed
Sato,
Structural study of the sugar chains of human leukocyte common antigen CD45.
1993,
Pubmed
Schneider,
Trafficking of galectin-3 through endosomal organelles of polarized and non-polarized cells.
2010,
Pubmed
Seelenmeyer,
Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1.
2005,
Pubmed
Seetharaman,
X-ray crystal structure of the human galectin-3 carbohydrate recognition domain at 2.1-A resolution.
1998,
Pubmed
Shalom-Feuerstein,
Galectin-3 regulates a molecular switch from N-Ras to K-Ras usage in human breast carcinoma cells.
2005,
Pubmed
Shoji,
Characterization of the Xenopus galectin family. Three structurally different types as in mammals and regulated expression during embryogenesis.
2003,
Pubmed
,
Xenbase
Sparrow,
Multiple soluble beta-galactoside-binding lectins from human lung.
1987,
Pubmed
Stowell,
Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens.
2008,
Pubmed
Stowell,
Innate immune lectins kill bacteria expressing blood group antigen.
2010,
Pubmed
Sörme,
Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions.
2004,
Pubmed
Sörme,
Structural and thermodynamic studies on cation-Pi interactions in lectin-ligand complexes: high-affinity galectin-3 inhibitors through fine-tuning of an arginine-arene interaction.
2005,
Pubmed
Theilgaard-Mönch,
Highly glycosylated alpha1-acid glycoprotein is synthesized in myelocytes, stored in secondary granules, and released by activated neutrophils.
2005,
Pubmed
Toscano,
Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death.
2007,
Pubmed
Vasta,
Roles of galectins in infection.
2009,
Pubmed
Walser,
Structure and functional analysis of the fungal galectin CGL2.
2004,
Pubmed
Yang,
Galectins: structure, function and therapeutic potential.
2008,
Pubmed
Yang,
Structural basis for the tumor cell apoptosis-inducing activity of an antitumor lectin from the edible mushroom Agrocybe aegerita.
2009,
Pubmed
Yu,
Galectin-3 interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes increased cancer cell endothelial adhesion.
2007,
Pubmed
van den Berg,
LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.
2004,
Pubmed