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.
One galectin-3 function is to bind glycoproteins and cross-link them.
A glycoprotein engaged many more galectin-3 carbohydrate-binding sites than its number of relevant glycans.
The ligand induced binding of one galectin-3 to another galectin-3 to form oligomers in a previously unrecognized way.
This differs from previous models and provides a new framework to interpret biological effects of galectin-3.
Galectin-4 is a tandem-repeat galectin with two distinct carbohydrate recognition domains (CRD). Galectin-4 is expressed mainly in the alimentary tract and is proposed to function as a lipid raft and ...adherens junction stabilizer by its glycan cross-linking capacity. Galectin-4 plays divergent roles in cancer and inflammatory conditions, either promoting or inhibiting each disease progression, depending on the specific pathological condition. The study of galectin-4's ligand-binding profile may help decipher its roles under specific conditions. Here we present the X-ray structures of human galectin-4 N-terminal CRD (galectin-4N) bound to different saccharide ligands. Galectin-4's overall fold and its core interactions to lactose are similar to other galectin CRDs. Galectin-4N recognises the sulfate cap of 3'-sulfated glycans by a weak interaction through Arg45 and two water-mediated hydrogen bonds via Trp84 and Asn49. When galectin-4N interacts with the H-antigen mimic, 2'-fucosyllactose, an interaction is formed between the ring oxygen of fucose and Arg45. The extended binding site of galectin-4N may not be well suited to the A/B-antigen determinants, α-GalNAc/α-Gal, specifically due to clashes with residue Phe47. Overall, galectin-4N favours sulfated glycans whilst galectin-4C prefers blood group determinants. However, the two CRDs of galectin-4 can, to a less extent, recognise each other's ligands.
Galectins are among organisms' most abundantly expressed lectins (carbohydrate-binding proteins) that specifically bind β-galactosides. They act not only outside the cell, where they bind to ...extracellular matrix glycans, but also inside the cell, where they have a significant impact on signaling pathways. Galectin-8 is a galectin family protein encoded by the LGALS8 gene. Its role is evident in both T- and B-cell immunity and in the innate immune response, where it acts directly on dendritic cells and induces some pro-inflammatory cytokines. Galectin-8 also plays an important role in the defense against bacterial and viral infections. It is known to promote antibacterial autophagy by recognizing and binding glycans present on the vacuolar membrane, thus acting as a danger receptor. The most important role of galectin-8 is the regulation of cancer growth, metastasis, tumor progression, and tumor cell survival. Importantly, the expression of galectins is typically higher in tumor tissues than in noncancerous tissues. In this review article, we focus on galectin-8 and its function in immune response, microbial infections, and cancer. Given all of these functions of galectin-8, we emphasize the importance of developing new and selective galectin-8 inhibitors and report the current status of their development.
Some complex plant-derived polysaccharides, such as modified citrus pectins and galactomannans, have been shown to have promising anti-inflammatory and anti-cancer effects. Most reports propose or ...claim that these effects are due to interaction of the polysaccharides with galectins because the polysaccharides contain galactose-containing side chains that might bind this class of lectin. However, their direct binding to and/or inhibition of the evolutionarily conserved galactoside-binding site of galectins has not been demonstrated. Using a well established fluorescence anisotropy assay, we tested the direct interaction of several such polysaccharides with physiological concentrations of a panel of galectins. The bioactive pectic samples tested were very poor inhibitors of the canonical galactoside-binding site for the tested galectins, with IC50 values >10 mg/ml for a few or in most cases no inhibitory activity at all. The galactomannan Davanat® was more active, albeit not a strong inhibitor (IC50 values ranging from 3 to 20 mg/ml depending on the galectin). Pure synthetic oligosaccharide fragments found in the side chains and backbone of pectins and galactomannans were additionally tested. The most commonly found galactan configuration in pectins had no inhibition of the galectins tested. Galactosylated tri- and pentamannosides, representing the structure of Davanat®, had an inhibitory effect of galectins comparable with that of free galactose. Further evaluation using cell-based assays, indirectly linked to galectin-3 inhibition, showed no inhibition of galectin-3 by the polysaccharides. These data suggest that the physiological effects of these plant polysaccharides are not due to inhibition of the canonical galectin carbohydrate-binding site.
Galectin-3 is a carbohydrate binding protein which has important roles in cancer and immunity. Potent galectin-3 inhibitors have been synthesized, for experimental purposes and potential clinical ...use. As galectin-3 is implicated in both intra- and extracellular activities, permeability of galectin-3 inhibitors is an important parameter determining biological effects. We compared the cellular uptake of galectin-3 inhibitors and their potency in the intracellular or extracellular space. The inhibitors differed in their polar surface area (PSA), but had similar affinities for galectin-3. Using a well-established permeability assay, we confirmed that the uptake was significantly higher for the inhibitor with the lowest PSA, as expected. To analyze intracellular activity of the inhibitors, we developed a novel assay based on galectin-3 accumulation around damaged intracellular vesicles. The results show striking differences between the inhibitors intracellular potency, correlating with their PSAs. To test extracellular activity of the inhibitors, we analyzed their potency to block binding of galectin-3 to cell surfaces. All inhibitors were equally able to block galectin-3 binding to cells and this was proportional to their affinity for galectin-3. These inhibitors may serve as useful tools in exploring biological roles of galectin-3 and may further our understanding of intracellular versus extracellular roles of 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 beta-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)-beta and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-beta 1 induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of beta-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin -3, TD139, blocked TGF-beta-induced beta-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.
Corneal neovascularization and scarring commonly lead to significant vision loss. This study was designed to determine whether a small-molecule inhibitor of galectin-3 can inhibit both corneal ...angiogenesis and fibrosis in experimental mouse models.
Animal models of silver nitrate cautery and alkaline burn were used to induce mouse corneal angiogenesis and fibrosis, respectively. Corneas were treated with the galectin-3 inhibitor, 33DFTG, or vehicle alone and were processed for whole-mount immunofluorescence staining and Western blot analysis to quantify the density of blood vessels and markers of fibrosis. In addition, human umbilical vein endothelial cells (HUVECs) and primary human corneal fibroblasts were used to analyze the role of galectin-3 in the process of angiogenesis and fibrosis in vitro.
Robust angiogenesis was observed in silver nitrate-cauterized corneas on day 5 post injury, and markedly increased corneal opacification was demonstrated in alkaline burn-injured corneas on days 7 and 14 post injury. Treatment with the inhibitor substantially reduced corneal angiogenesis and opacification with a concomitant decrease in α-smooth muscle actin (α-SMA) expression and distribution. In vitro studies revealed that 33DFTG inhibited VEGF-A-induced HUVEC migration and sprouting without cytotoxic effects. The addition of exogenous galectin-3 to corneal fibroblasts in culture induced the expression of fibrosis-related proteins, including α-SMA and connective tissue growth factor.
Our data provide proof of concept that targeting galectin-3 by the novel, small-molecule inhibitor, 33DFTG, ameliorates pathological corneal angiogenesis as well as fibrosis. These findings suggest a potential new therapeutic strategy for treating ocular disorders related to pathological angiogenesis and fibrosis.
Polymeric nanoparticles is an established and efficient means to achieve controlled release of drugs. Incorporation of minocycline, an antibiotic with anti-inflammatory and neuroprotective ...properties, into biodegradable nanoparticles may therefore provide an efficient means to combat foreign body reactions to implanted electrodes in the brain. However, minocycline is commonly associated with poor encapsulation efficiencies and/or fast release rates due to its high solubility in water. Moreover, minocycline is unstable under conditions of low and high pH, heat and exposure to light, which exacerbate the challenges of encapsulation. In this work drug loaded PLGA nanoparticles were prepared by a modified emulsification-solvent-diffusion technique and characterized for size, drug encapsulation and in vitro drug release. A novel hydrophobic ion pair complex of minocycline, Ca(2+) ions and the anionic surfactant AOT was developed to protect minocycline from degradation and prolong its release. The optimized formulation resulted in particle sizes around 220 nm with an entrapment efficiency of 43% and showed drug release over 30 days in artificial cerebrospinal fluid. The present results constitute a substantial increase in release time compared to what has hitherto been achieved for minocycline and indicate that such particles might provide useful for sustained drug delivery in the CNS.
Molecular recognition
is fundamental to biological signaling. A
central question is how individual interactions between molecular
moieties affect the thermodynamics of ligand binding to proteins and
...how these effects might propagate beyond the immediate neighborhood
of the binding site. Here, we investigate this question by introducing
minor changes in ligand structure and characterizing the effects of
these on ligand affinity to the carbohydrate recognition domain of
galectin-3, using a combination of isothermal titration calorimetry,
X-ray crystallography, NMR relaxation, and computational approaches
including molecular dynamics (MD) simulations and grid inhomogeneous
solvation theory (GIST). We studied a congeneric series of ligands
with a fluorophenyl-triazole moiety, where the fluorine substituent
varies between the
ortho
,
meta
,
and
para
positions (denoted O, M, and P). The M and
P ligands have similar affinities, whereas the O ligand has 3-fold
lower affinity, reflecting differences in binding enthalpy and entropy.
The results reveal surprising differences in conformational and solvation
entropy among the three complexes. NMR backbone order parameters show
that the O-bound protein has reduced conformational entropy compared
to the M and P complexes. By contrast, the bound ligand is more flexible
in the O complex, as determined by
19
F NMR relaxation,
ensemble-refined X-ray diffraction data, and MD simulations. Furthermore,
GIST calculations indicate that the O
-
bound complex
has less unfavorable solvation entropy compared to the other two complexes.
Thus, the results indicate compensatory effects from ligand conformational
entropy and water entropy, on the one hand, and protein conformational
entropy, on the other hand. Taken together, these different contributions
amount to entropy–entropy compensation among the system components
involved in ligand binding to a target protein.
Pseudomonas aeruginosa provokes a painful, sight-threatening corneal infection. It progresses rapidly and is difficult to treat. In this study, using a mouse model of P. aeruginosa keratitis, we ...demonstrate the importance of a carbohydrate-binding protein, galectin-8 (Gal-8), in regulation of the innate immune response. First, using two distinct strains of P. aeruginosa, we established that Gal-8-/- mice are resistant to P. aeruginosa keratitis. In contrast, mice deficient in Gal-1, Gal-3, and Gal-9 were fully susceptible. Second, the addition of exogenous rGal-8 to LPS (TLR4 ligand)-stimulated bone marrow-derived macrophages suppressed 1) the activation of the NF-κB pathway, and 2) formation of the MD-2/TLR4 complex. Additionally, the expression level of reactive oxygen species was substantially higher in infected Gal-8-/- bone marrow neutrophils (BMNs) compared with the Gal-8+/+ BMNs, and the P. aeruginosa killing capacity of Gal-8-/- BMNs was considerably higher compared with that of Gal-8+/+ BMNs. In the bacterial killing assays, the addition of exogenous rGal-8 almost completely rescued the phenotype of Gal-8-/- BMNs. Finally, we demonstrate that a subconjunctival injection of a Gal-8 inhibitor markedly reduces the severity of infection in the mouse model of P. aeruginosa keratitis. These data lead us to conclude that Gal-8 downmodulates the innate immune response by suppressing activation of the TLR4 pathway and clearance of P. aeruginosa by neutrophils. These findings have broad implications for developing novel therapeutic strategies for treatment of conditions resulting from the hyperactive immune response both in ocular as well as nonocular tissues.
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