Showing papers by "Hakon Leffler published in 2003"

Galectin-4 in normal tissues and cancer

Abstract: 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. Published in 2004.

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

Abstract: 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.

Design and Synthesis of Galectin Inhibitors

Abstract: Galectins, a lectin family, have shown binding affinities towards b-galactosides. Galectins have been proposed to be involved in a wide range of functions like for example, cell growth, adhesion, migration, chemo taxis and apoptosis. They have also been associated with various cancer types. However, the detailed functions of galectins are still very much unknown. High affinity inhibitors towards the galectins would thus be of value as research tools, as well as possible future pharmaceutical agents. Existing inhibitors have undesirable properties, for example high molecular weight and instability. This thesis concerns the synthesis of small high affinity galectin inhibitors. A previously published X-ray structure of galectin-3 together with LacNAc revealed an extended binding pocket close to 3´-C of the galactoside residue. Filling this pocket with additional chemical entities was hypothesized to allow for further interactions and hence creating higher affinity ligands as compared to the naturally occurring ligand. The hypothesis was probed by substituting the 3´-hydroxyl group on the galactose unit of LacNAc with an amine, which enables the introduction of functional groups under mild reaction conditions. We synthesised a collection of more than 60 LacNAc derivatives with various functional groups at 3´-C of the galactose unit. The measurements of inhibitor potencies towards galectins were made in a novel fluorescence polarisation (FP) assay, which is a solution phase method, as well as a general technique that do not need major re-optimisation to enable the study of other galectins. Hence, it enabled us to study the panel of synthetic inhibitors towards galectin-1, -3 and –4. Selective and high affinity inhibitors were discovered, which is of value as often more than one galectin is present in one and the same system. We found that aromatic amides in particular showed high affinity towards galectin-3. In addition, the X-ray structure of one of the best inhibitors (Kd 0.9 mM) revealed that Arg-144 on galectin-3 had moved 3.5 A to enable a face-to-face stacking interaction with a 4-methoxy-2,3,5,6-tetrafluorobenzamido substituent. The best inhibitor synthesised as of yet, carried a 2-naphthamido functionality at 3´of the galactose residue. This inhibitor had a Kd of 0.3 mM, which the strongest binding affinity achieved as compared to any monovalent inhibitor. It shows over 200 times higher affinity towards galectin-3 than the unfunctionalised LacNAc. (Less)

Fluorescence polarization to study galectin-ligand interactions.

Abstract: Publisher Summary Galectins are typically soluble cytosolic proteins that can be secreted from cells by nonclassic pathways to interact with external glycoconjugates and have a variety of activities both extra- and intracellularly, with important implicated roles in immunity, inflammation, and cancer. One obvious essential feature of galectins is their carbohydrate-binding specificity. Frontal affinity chromatography is presented as an elegant, highly efficient way to compare the binding of a large library of fluorescently tagged saccharides to immobilized galectins. One disadvantage of this technique is that each galectin needs to be immobilized, with possible confounding effects on interpreting binding data. A number of other techniques requiring immobilizing and/or labeling of either the galectin or ligand have been used by others, each with their advantages and disadvantages. Titration microcalorimetry has been applied to a few galectins and has given good information on solution-phase binding parameters but requires high galectin and saccharide amounts. This chapter describes fluorescence polarization (FP) as an alternative solution-phase binding assay for galectins. FP is an established technique and has been also used for studies of lectin–carbohydrate interactions. However, instruments permitting the application of this method to small volumes in microtiter plate format have become available only more recently. The method is gaining increased popularity for various types of screening in the pharmaceutical industry and clinical drug measurements.

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

Abstract: 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.

CHAPTER 15 – Carbohydrate Recognition and Signaling

Abstract: The interactions between lectins and carbohydrates are relatively weak in nature and, as such, carbohydrate-mediated interactions may play important roles where weak interactions are required—the leukocyte rolling phenomenon perhaps providing a good example. In many cases, however, type II lectins have employed multivalency as a means of conferring increased affinity and specificity on their binding interactions. Although similar in some ways, it is clear that proteincarbohydrate interactions differ from protein-protein interactions in ways that might confer on them unique signaling roles or properties. Because glycosylation is a posttranslation modification capable of modifying any molecule with the appropriate acceptor, the subsequent recognition of carbohydrate determinants differs fundamentally from that involving specific protein-protein interactions. In addition to the potential for triggering signalingevents, the formation of carbohydrate-mediated cross-linked arrays may also be important in receptor turnover, one way in which signaling events are modulated. Knowledge of lectin and glycoprotein structures shows that multivalent interactions are a recurring theme. Many lectins are oligomeric and/or membrane bound, and many glycoproteins (and certainly proteoglycans) possess multiple glycosylation sites. Their inherent ability to mediate cross-links make it certain that new examples of signaling roles will follow from the study of these complex and diverse molecules.