F. Javier Cañada

Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase

Lactase phlorizin hydrolase (LPH; EC 3.2.1.62) is a membrane‐bound, family 1 β‐glycosidase found on the brush border of the mammalian small intestine. LPH, purified from sheep small intestine, was capable of hydrolysing a range of flavonol and isoflavone glycosides. The catalytic efficiency (k cat/K m) for the hydrolysis of quercetin‐4′‐glucoside, quercetin‐3‐glucoside, genistein‐7‐glucoside and daidzein‐7‐glucoside was 170, 137, 77 and 14 (mM−1 s−1) respectively. The majority of the activity occurred at the lactase and not phlorizin hydrolase site. The ability of LPH to deglycosylate dietary (iso)flavonoid glycosides suggests a possible role for this enzyme in the metabolism of these biologically active compounds.

15-Deoxy-Delta 12,14-prostaglandin J2 inhibition of NF-kappaB-DNA binding through covalent modification of the p50 subunit.

Cyclopentenone prostaglandins display anti-inflammatory activities and interfere with the signaling pathway that leads to activation of transcription factor NF-κB. Here we explore the possibility that the NF-κB subunit p50 may be a target for the cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2). This prostaglandin inhibited the DNA binding ability of recombinant p50 in a dose-dependent manner. The inhibition required the cyclopentenone moiety and could be prevented but not reverted by glutathione and dithiothreitol. Moreover, a p50 mutant with a C62S mutation was resistant to inhibition, indicating that the effect of 15d-PGJ2 was probably due to its interaction with cysteine 62 in p50. The covalent modification of p50 by 15d-PGJ2 was demonstrated by reverse-phase high pressure liquid chromatography and mass spectrometry analysis that showed an increase in retention time and in the molecular mass of 15d-PGJ2-treated p50, respectively. The interaction between p50 and 15d-PGJ2 was relevant in intact cells. 15d-PGJ2 effectively inhibited cytokine-elicited NF-κB activity in HeLa without reducing IκBα degradation or nuclear translocation of NF-κB subunits. 15d-PGJ2 reduced NF-κB DNA binding activity in isolated nuclear extracts, suggesting a direct effect on NF-κB proteins. Finally, treatment of HeLa with biotinylated-15d-PGJ2 resulted in the formation of a 15d-PGJ2-p50 adduct as demonstrated by neutravidin binding and immunoprecipitation. These results clearly show that p50 is a target for covalent modification by 15d-PGJ2 that results in inhibition of DNA binding.

Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-Δ12,14-prostaglandin J2

Cyclopentenone prostaglandins may interfere with cellular functions by multiple mechanisms. The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has been reported to inhibit the activity of the transcription factor AP-1 in several experimental settings. We have explored the possibility of a direct interaction of 15d-PGJ2 with AP-1 proteins. Here we show that 15d-PGJ2 covalently modifies c-Jun and directly inhibits the DNA binding activity of AP-1. The modification of c-Jun occurs both in vitro and in intact cells as detected by labeling with biotinylated 15d-PGJ2 and mass spectrometry analysis. Attachment of the cyclopentenone prostaglandin occurs at cysteine 269, which is located in the c-Jun DNA binding domain. In addition, 15d-PGJ2 can promote the oligomerization of a fraction of c-Jun through the formation of intermolecular disulfide bonds or 15d-PGJ2-bonded dimers. Our results identify a novel site of interaction of 15d-PGJ2 with the AP-1 activation pathway that may contribute to the complex effects of cyclopentenone prostaglandins on the cellular response to pro-inflammatory agents. They also show the first evidence for the induction of protein cross-linking by 15d-PGJ2.

Carbohydrate-protein interactions: a 3D view by NMR.

This review focuses on the application of NMR methods for understanding, at the molecular and atomic levels, the diverse mechanisms by which sugar molecules are recognised by the binding sites of lectins, antibodies and enzymes. Given the intrinsic chemical natures of sugars and their flexibility, it is well established that NMR parameters should be complemented by computational methods in attempts to unravel the structural and conformational features of the molecular recognition process unambiguously. We therefore aim here to describe new and significant advances in the knowledge of carbohydrate–protein interactions, obtained by employing state‐of‐the‐art NMR and molecular modelling. We have not attempted to prepare an exhaustive review but have tried to focus on describing the key aspects that should be considered when tackling a problem within this research topic.

Conformational Differences Between O- and C-Glycosides: Theα-O-Man-(1→1)-β-Gal/α-C-Man-(1→1)-β-Gal Case- A Decisive Demonstration of the Importance of theexo-Anomeric Effect on the Conformation of Glycosides

The conformational behavior of α-O-Man-(1→1)-β-Gal (1) and its C-analogue (2) has been studied using J/NOE NMR data, molecular mechanics, molecular dynamics, and ab initio calculations. The population distribution around the glycosidic linkages of 1 and 2 is rather different, especially for the α-Man linkage. A lower limit for the exo-anomeric effect in water has been experimentally determined.

Recent Developments in Synthetic Carbohydrate-Based Diagnostics, Vaccines, and Therapeutics

Glycans are everywhere in biological systems, being involved in many cellular events with important implications for medical purposes. Building upon a detailed understanding of the functional roles of carbohydrates in molecular recognition processes and disease states, glycans are increasingly being considered as key players in pharmacological research. On the basis of the important progress recently made in glycochemistry, glycobiology, and glycomedicine, we provide a complete overview of successful applications and future perspectives of carbohydrates in the biopharmaceutical and medical fields. This review highlights the development of carbohydrate-based diagnostics, exemplified by glycan imaging techniques and microarray platforms, synthetic oligosaccharide vaccines against infectious diseases (e.g., HIV) and cancer, and finally carbohydrate-derived therapeutics, including glycomimetic drugs and glycoproteins.

Symmetric dithiodigalactoside: strategic combination of binding studies and detection of selectivity between a plant toxin and human lectins

Thioglycosides offer the advantage over O-glycosides to be resistant to hydrolysis. Based on initial evidence of this recognition ability for glycosyldisulfides by screening dynamic combinatorial libraries, we have now systematically studied dithiodigalactoside on a plant toxin (Viscum album agglutinin) and five human lectins (adhesion/growth-regulatory galectins with medical relevance e.g. in tumor progression and spread). Inhibition assays with surface-presented neoglycoprotein and in solution monitored by saturation transfer difference NMR spectroscopy, flanked by epitope mapping, as well as isothermal titration calorimetry revealed binding properties to VAA (K(a): 1560 ± 20 M(-1)). They were reflected by the structural model and the affinity on the level of toxin-exposed cells. In comparison, galectins were considerably less reactive, with intrafamily grading down to very minor reactivity for tandem-repeat-type galectins, as quantitated by radioassays for both domains of galectin-4. Model building indicated contact formation to be restricted to only one galactose moiety, in contrast to thiodigalactoside. The tested glycosyldisulfide exhibits selectivity between the plant toxin and the tested human lectins, and also between these proteins. Therefore, glycosyldisulfides have potential as chemical platform for inhibitor design.

The Conformational Behaviour of Non-Hydrolizable Lactose Analogues: The Thioglycoside, Carbaglycoside, and Carba-Iminoglycoside Cases

-glycosidic linkages.In fact, the C2S bond length (1.78 A˚ ) and C2S2C bondangle (99°) strongly differ from the values for C2O (1.41A˚ ) and C2O2C (116°). On the other hand, the C2N dis-tance and C2N2C bond angle values are 1.47 A˚ and be-tween 116.52119.6°, according to MM3* calculations. Onthis basis, we report here the conformational study of thethioglycoside- (

Heparin modulates the mitogenic activity of fibroblast growth factor by inducing dimerization of its receptor. a 3D view by using NMR.

In vitro mitogenesis assays have shown that sulfated glycosaminoglycans (GAGs; heparin and heparan sulfate) cause an enhancement of the mitogenic activity of fibroblast growth factors (FGFs). Herein, we report that the simultaneous presence of FGF and the GAG is not an essential requisite for this event to take place. Indeed, preincubation with heparin (just before FGF addition) of cells lacking heparan sulfate produced an enhancing effect equivalent to that observed when the GAG and the protein are simultaneously added. A first structural characterization of this effect by analytical ultracentrifugation of a soluble preparation of the heparin‐binding domain of fibroblast growth factor receptor 2 (FGFR2) and a low molecular weight (3 kDa) heparin showed that the GAG induces dimerization of FGFR2. To derive a high resolution structural picture of this molecular recognition process, the interactions of a soluble heparin‐binding domain of FGFR2 with two different homogeneous, synthetic, and mitogenically active sulfated GAGs were analyzed by NMR spectroscopy. These studies, assisted by docking protocols and molecular dynamics simulations, have demonstrated that the interactions of these GAGs with the soluble heparin‐binding domain of FGFR induces formation of an FGFR dimer; its architecture is equivalent to that in one of the two distinct crystallographic structures of FGFR in complex with both heparin and FGF1. This preformation of the FGFR dimer (with similar topology to that of the signaling complex) should favor incorporation of the FGF component to form the final assemblage of the signaling complex, without major entropy penalty. This cascade of events is probably at the heart of the observed activating effect of heparin in FGF‐driven mitogenesis.

Structural aspects of binding of α-linked digalactosides to human galectin-1

By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH-π stacking. These issues are resolved by a combination of (15)N-(1)H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH-π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH-π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.