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Hakon Leffler

Publications and source records attributed to Hakon Leffler.

At least 19 recordsLinked to original sources
PubMed2006

Enhancement of bound-state residual dipolar couplings: conformational analysis of lactose bound to Galectin-3.

Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free-state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin-3, and report enhanced RDCs that prove consistent with known bound-ligand geometries for this protein.

Alkylation↗
PubMed2006

Synthesis of a 3'-naphthamido-LacNAc fluorescein conjugate with high selectivity and affinity for galectin-3.

Described is the synthesis of a fluorescent LacNAc derivative appended with a 3'-deoxy-3'-naphthamido functionality, 2-(fluorescein-5/6-amido)ethyl 3-deoxy-3-(2-naphthamido)-beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside, which confers high affinity (Kd 170 nM) and selectivity for galectin-3 via a stacking interaction with Arg144. Its use as a selective and sensitive galectin-3 probe is demonstrated with fluorescence polarization measurements.

Disaccharides↗
PubMed2006

Synthesis of multivalent lactose derivatives by 1,3-dipolar cycloadditions: selective galectin-1 inhibition.

Acetylene derivatives of phenylalanine, phenethylamine and the multifunctional unnatural amino acids, phenyl-bis-alanine and phenyl-tris-alanine, were synthesized and functionalized with 2-azidoethyl beta-D-galactopyranosyl-(1-->4)-beta-D-glucopyranoside via regioselective copper(I)-mediated 1,3-dipolar cycloaddition to give a panel of mono-, di- and trivalent lactoside derivatives. Evaluation of the compounds as inhibitors against the tumour- and inflammation-related galectin-1, -3, -4N, -4C, -4, -7, -8N and -9N revealed a divalent compound with a Kd value as low as 3.2 microM for galectin-1, which corresponded to a relative potency of 30 per lactose unit as compared to the natural disaccharide ligand lactose. This divalent compound had at least one order of magnitude higher affinity for galectin-1 than for any of the other galectins investigated.

Acetylene↗
PubMed2005

Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells.

Galectins are implicated in a large variety of biological functions, many of which depend on their carbohydrate-binding ability. Fifteen members of the family have been identified in vertebrates based on binding to galactose (Gal) that is mediated by one or two, evolutionarily conserved, carbohydrate-recognition domains (CRDs). Variations in glycan structures expressed on glycoconjugates at the cell surface may, therefore, affect galectin binding and functions. To identify roles for different glycans in the binding of the three types of mammalian galectins to cells, we performed fluorescence cytometry at 4 degrees C with recombinant rat galectin-1, human galectin-3, and three forms of human galectin-8, to Chinese hamster ovary (CHO) cells and 12 different CHO glycosylation mutants. All galectin species bound to parent CHO cells and binding was inhibited >90% by 0.2 M lactose. Galectin-8 isoforms with either a long or a short inter-CRD linker bound similarly to CHO cells. However, a truncated form of galectin-8 containing only the N-terminal CRD bound only weakly to CHO cells and the C-terminal galectin-8 CRD exhibited extremely low binding. Binding of the galectins to the different CHO glycosylation mutants revealed that complex N-glycans are the major ligands for each galectin except the N-terminal CRD of galectins-8, and also identified some fine differences in glycan recognition. Interestingly, increased binding of galectin-1 at 4 degrees C correlated with increased propidium iodide (PI) uptake, whereas galectin-3 or -8 binding did not induce permeability to PI. The CHO glycosylation mutants with various repertoires of cell surface glycans are a useful tool for investigating galectin-cell interactions as they present complex and simple glycans in a natural mixture of multivalent protein and lipid glycoconjugates anchored in a cell membrane.

Animals↗
PubMed2005

3-(1,2,3-Triazol-1-yl)-1-thio-galactosides as small, efficient, and hydrolytically stable inhibitors of galectin-3.

Copper(I)-catalyzed addition of alkynes to methyl 3-azido-3-deoxy-1-thio-beta-D-galactopyranoside afforded stable and structurally simple 3-deoxy-3-(1H-1,2,3-triazol-1-yl)-1-thio-galactosides carrying a panel of substituents at the triazole C4 in high yields. The 3-(1H-[1,2,3]-triazol-1-yl)-1-thio-galactoside collection synthesized contained inhibitors of the tumor- and inflammation-related galectin-3 with Kd values as low as 107 microM, which is as potent as the natural disaccharide inhibitors lactose and N-acetyllactosamine.

Catalysis↗
PubMed2005

Synthesis of O-galactosyl aldoximes as potent LacNAc-mimetic galectin-3 inhibitors.

A panel of anomeric oxime ether derivatives of beta-galactose were synthesized via the reaction of O-beta-D-galactopyranosylhydroxylamine with aldehydes. The oxime ethers were evaluated as inhibitors against galectin-3 in a competitive fluorescence polarization assay. The best inhibitor, [E]-O-(beta-D-galactopyranosyl)-indole-3-carbaldoxime (E-52), had a Kd value of 180 microM, which is 24 times better than methyl beta-D-galactopyranoside (Kd=4400 microM) and in the same range as methyl lactoside (Kd=220 microM).

Drug Design↗
PubMed2005

Synthesis of a phenyl thio-beta-D-galactopyranoside library from 1,5-difluoro-2,4-dinitrobenzene: discovery of efficient and selective monosaccharide inhibitors of galectin-7.

The galectins are a family of [small beta]-galactoside-binding proteins that have been implicated in cancer and inflammation processes. Herein, we report the synthesis of a library of 28 compounds that was tested for binding to galectins-1, -3, -7, -8N and -9N. An aromatic nucleophilic substitution reaction between 1,5-difluoro-2,4-dinitrobenzene and a galacto thiol gave 5-fluoro-2,4-dinitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-galactopyranoside. This versatile intermediate was then modified in a two dimensional manner: either by further substitution of the second fluoride by amines or thiols, or by reduction of the nitro groups and acylation of the resulting amines, or both. Deacetylation then gave a library of aromatic beta-galactosides that showed variable inhibitory activity against the different galectins, as shown by screening with a fluorescence-polarisation assay. Particularly efficient inhibitors were found against galectin-7, while less impressive enhancements of inhibitor affinity over methyl beta-D-galactopyranoside were found for galectin-1, -3, -8N and -9N. The best inhibitors against galectin-7 showed significantly higher affinity (K(d) as low as 140 microM) than both beta-methyl galactoside (K(d) 4.8 mM) and the unsubstituted beta-phenyl thiogalactoside (non-inhibitory). The best inhibitors against galectin-7 were poor against the other galectins and thus have potential as structurally simple and selective tools for dissecting biological functions of galectin-7.

Dinitrofluorobenzene↗
PubMed2005

Fluorescent leukotriene B4: potential applications.

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.

Cell Membrane↗
PubMed2005

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.

The high-resolution X-ray crystal structures of the carbohydrate recognition domain of human galectin-3 were solved in complex with N-acetyllactosamine (LacNAc) and the high-affinity inhibitor, methyl 2-acetamido-2-deoxy-4-O-(3-deoxy-3-[4-methoxy-2,3,5,6-tetrafluorobenzamido]-beta-D-galactopyranose)-beta-D-glucopyranoside, to gain insight into the basis for the affinity-enhancing effect of the 4-methoxy-2,3,5,6-tetrafluorobenzamido moiety. The structures show that the side chain of Arg144 stacks against the aromatic moiety of the inhibitor, an interaction made possible by a reorientation of the side chain relative to that seen in the LacNAc complex. Based on these structures, synthesis of second generation LacNAc derivatives carrying aromatic amides at 3'-C, followed by screening with a novel fluorescence polarization assay, has led to the identification of inhibitors with further enhanced affinity for galectin-3 (K(d) > or = 320 nM). The thermodynamic parameters describing the binding of the galectin-3 C-terminal to selected inhibitors were determined by isothermal titration calorimetry and showed that the affinity enhancements were due to favorable enthalpic contributions. These enhancements could be rationalized by the combined effects of the inhibitor aromatic structure on a cation-Pi interaction and of direct interactions between the aromatic substituents and the protein. The results demonstrate that protein-ligand interactions can be significantly enhanced by the fine-tuning of arginine-arene interactions.

Amino Sugars↗
PubMed2004

Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions.

Galectins are a family of beta-galactose binding lectins associated with functions such as immunological and malignant events. To study the binding affinity of galectins for natural and artificial saccharides and glycoconjugates we have developed an assay using fluorescence polarization. A collection of fluorescein-conjugated saccharides was synthesized and used as probes with galectins-1 and -3 and the two carbohydrate recognition domains of galectin-4. Direct binding of a fixed probe amount with different amounts of each galectin defined specificity and selectivity and permitted selection of the optimal probe for inhibition studies. Then fixed amounts of galectin and selected probe were used to screen the inhibitory potency of a library of nonfluorescent compounds. As the assay is in solution and does not require separation of free and bound probe, it is simple and rapid and can easily be applied to different unlabeled galectins. As all interaction components are known, K(d) values for galectin-inhibitor interaction can be directly calculated without approximation other than the assumption of a simple one-site competition.

Animals↗
PubMed2004

Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe.

Human neutrophils are activated by the beta-galactoside-binding lectin galectin-3, provided that the cells are primed by in vivo extravasation or by in vitro preactivation with, for example, LPS. Removal of terminal sialic acid can change neutrophil functionality and responsiveness due to exposure of underlying glycoconjugate receptors or change in surface charge. Here, we investigated whether such alteration of the cell surface carbohydrate composition can alter the responsiveness of the cells to galectin-3. Neutrophils were treated with neuraminidases (NA) of different origins: Clostridium perfringens (CP), Salmonella typhimurium, Vibrio cholerae, and Newcastle disease virus (NDV). In the presence of NDV-NA, but no other NA, the otherwise non-responding neutrophils responded readily to galectin-3 by activation of the NADPH-oxidase. The galectin-3 priming effect was inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid. Earlier studies have shown that priming of the neutrophil response to galectin-3 with, for example, LPS is paralleled by degranulation of intracellular vesicles and granules and upregulation of potential galectin-3 receptors. Also, NDV-NA (but not CP-NA) treatment induced degranulation, shown as an upregulation of complement receptor 3. Since not only the galectin response but also the response to the chemoattractant fMLF was primed, NDV-NA appears to induce a general priming phenomenon, possibly due to receptor upregulation by degranulation.

Cell Degranulation↗
PubMed2004

Phylogenetic analysis of the vertebrate galectin family.

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.

Animals↗
PubMed2004

Galectin-4 in normal tissues and cancer.

Galectin-4 belongs to a subfamily of galectins composed of two carbohydrate recognition domains within the same peptide chain. The two domains have all the conserved galectin signature amino acids, but their overall sequences are only approximately 40% identical. Both domains bind lactose with a similar affinity as other galectins, but their respective preferences for other disaccharides, and larger saccharides, are distinctly different. Thus galectin-4 has a property of a natural cross-linker, but in a modified sense since each domain prefers a different subset of ligands. Similarly to other galectins, galectin-4 is synthesized as a cytosolic protein, but can be externalized. During development and in adult normal tissues, galectin-4 is expressed only in the alimentary tract, from the tongue to the large intestine. It is often found in relatively insoluble complexes, as a component of either adherens junctions or lipid rafts in the microvillus membrane, and it has been proposed to stabilize these structures. Strong expression of galectin-4 can be induced, however, in cancers from other tissues including breast and liver. Within a collection of human epithelial cancer cell lines, galectin-4 is overexpressed and soluble in those forming highly differentiated polarized monolayers, but absent in less differentiated ones. In cultured cells, intracellular galectin-4 may promote resistance to nutrient starvation, whereas--as an extracellular protein--it can mediate cell adhesion. Because of its distinct induction in breast and other cancers, it may be a valuable diagnostic marker and target for the development of inhibitory carbohydrate-based drugs.

Biological Transport, Active↗
PubMed2003

Conformational differences in liganded and unliganded states of Galectin-3.

The conformation of the carbohydrate recognition domain of Galectin-3, a lectin known to bind galactose containing oligosaccharides in mammalian systems, has been investigated in the absence of ligand and in the presence of N-acetylactosamine. A new methodology based on the measurement of residual dipolar couplings from NMR spectra has been used to characterize differences in protein structure along the backbone in the presence and absence of ligand, as well as the binding geometry of the ligand itself. The data on the ligand are consistent with the ligand binding geometry found in a crystal structure of the complexed state. However, a significant rearrangement of backbone loops near the binding site appears to occur in the absence of ligand. The implications for ligand specificity and protein functionality are discussed.

Amino Sugars↗
PubMed2003

Truncated galectin-3 inhibits tumor growth and metastasis in orthotopic nude mouse model of human breast cancer.

PURPOSE: The goal of this research was to evaluate a potential therapeutic agent for breast cancer based on galectin-3 that has been implicated in tumorigenicity and metastasis of breast cancer. The hypothesis was that therapy with NH(2)-terminally truncated form of galectin-3 (galectin-3C) will be efficacious for reduction in tumor growth and for inhibition of metastases. EXPERIMENTAL DESIGN: Recombinant human galectin-3 was produced in Escherichia coli from which galectin-3C was derived by collagenase enzyme digestion. Toxicity, pharmacokinetic, and organ biodistribution studies were performed in nude mice. For efficacy studies, nude mice bearing orthotopically implanted tumors derived from breast cancer cell line MDA-MB-435 were treated with galectin-3C or a vehicle control i.m. twice daily for 90 days. RESULTS: The maximum tolerated dose of galectin-3C in nude mice was determined to be >125 mg/kg without overt adverse effects. The elimination half-life when administered i.m. was found to be 3.0 h in the serum and 4.3 h in the cellular fraction of the blood. Organ biodistribution studies revealed that galectin-3C localized in the liver, kidneys, and spleen but not in the heart or lungs. We found that the mean tumor volumes and weights were statistically significantly less in mice treated with galectin-3C compared with control mice, and that fewer numbers of mice exhibited lymph node metastases in the treated group compared with the control group. CONCLUSIONS: Galectin-3C is not overtly toxic, and is efficacious in reducing metastases and tumor volumes and weights in primary tumors in an orthotopic nude mouse model of human breast cancer.

Animals↗