José L. de Paz

Langerin-Heparin Interaction: Two Binding Sites for Small and Large Ligands as revealed by a combination of NMR Spectroscopy and Cross-Linking Mapping Experiments

Langerin is a C-type lectin present on Langerhans cells that mediates capture of pathogens in a carbohydrate-dependent manner, leading to subsequent internalization and elimination in the cellular organelles called Birbeck granules. This mechanism mediated by langerin was shown to constitute a natural barrier for HIV-1 particle transmission. Besides interacting specifically with high mannose and fucosylated neutral carbohydrate structures, langerin has the ability to bind sulfated carbohydrate ligands as 6-sulfated galactosides in the Ca(2+)-dependent binding site. Very recently langerin was demonstrated to interact with sulfated glycosaminoglycans (GAGs), in a Ca(2+)-independent way, resulting in the proposal of a new binding site for GAGs. On the basis of those results, we have conducted a structural study of the interactions of small heparin (HEP)-like oligosaccharides with langerin in solution. Heparin bead cross-linking experiments, an approach specifically designed to identify HEP/heparan sulfate binding sites in proteins were first carried out and experimentally validated the previously proposed model for the interaction of langerin extracellular domain with 6 kDa HEP. High-resolution NMR studies of a set of eight synthetic HEP-like trisaccharides harboring different sulfation patterns demonstrated that all of them bound to langerin in a Ca(2+)-dependent way. The binding epitopes were determined by saturation transfer difference NMR and the bound conformations by transferred NOESY experiments. These experimental data were combined with docking and molecular dynamics and resulted in the proposal of a binding mode characterized by the coordination of calcium by the two equatorial hydroxyl groups, OH3 and OH4, at the non-reducing end. The binding also includes the carboxylate group at the adjacent iduronate residue. This epitope is shared by all eight ligands, explaining the absence of any impact on binding from differences in their substitution patterns. Finally, in contrast to the small trisaccharides, we demonstrated that a longer HEP-like hexasaccharide, bearing an additional O-sulfate group at the non-reducing end, which precludes binding to the Ca(2+) site, interacts with langerin in the previously identified Ca(2+)-independent binding site.

Recent Advances and Future Challenges in Glycan Microarray Technology

Glycan microarrays, carrying hundreds of different sugars on chip surfaces, have become a standard tool for the study of interactions of biomolecules with carbohydrates. The chip-based format offers important advantages, including the ability to screen in parallel several thousand binding events on a single slide, the minimal amount of sample required for one experiment, and the multivalent display of sugars on the chip that mimics the presentation of carbohydrates in nature. This chapter presents recent advances and future challenges in glycan microarray technology. We describe different immobilization and detection methods as well as applications in glycomics, drug discovery, and biomedicine.

Synthesis of chondroitin/dermatan sulfate-like oligosaccharides and evaluation of their protein affinity by fluorescence polarization.

Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligosaccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good yields (74-91%) with our protecting group distribution. Using this approach, we have synthesized, for the first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids, D-glucuronic and L-iduronic acid. Moreover, we have employed a fluorescence polarization competition assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast growth factor). Our results show that this method, using standard instrumentation and minimal sample consumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution.

Insights into the Glycosaminoglycan-Mediated Cytotoxic Mechanism of Eosinophil Cationic Protein Revealed by NMR

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECPs toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-D-GlcNS6S-α(1-4)-L-IdoA2S-α(1-4)-D-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The (2)S(0) conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34-Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparins inhibition of ECPs ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECPs toxicity implicated in eosinophil pathologies.

Importance of the polarity of the glycosaminoglycan chain on the interaction with FGF-1.

Heparin-like saccharides play an essential role in binding to the fibroblast growth factor (FGF)-1 and to their membrane receptors fibroblast growth factor receptor forming a ternary complex that is responsible of the internalization of the signal, via the dimerization of the intracellular regions of the receptor. In this study, we report the binding affinities between five synthetic hexasaccharides with human FGF-1 obtained by surface plasmon resonance experiments, and compare with the induced mitogenic activity previously obtained. These five oligosaccharides differ in sulfation pattern and in sequence. We have previously demonstrated that all the five hexasaccharides have similar 3D structure of the backbone. Consequently, the differences in binding affinity should have their origin in the substitution pattern. Subsequently, the different capacity for induction of mitogenic activity can be, at least partially, explained from these binding affinities. Interestingly, one of the oligosaccharides lacking axially symmetry ( 3: ) was biologically inactive, whereas the other ( 2: ) was the most active. The difference between both compounds is the order of the FGF-binding motifs along the chain relative to the carbohydrate polarity. We can conclude that the directionality of the GAG chain is essential for the binding and subsequent activation. The relative biological activity of the compounds with regular substitution pattern can be inferred from their values of IC50. Remarkably, the sulfate in position 6 of d-glucosamine was essential for the mitogenic activity but not for the interaction with FGF-1.

3D structure of a heparin mimetic analogue of a FGF-1 activator. A NMR and molecular modelling study

The motional behaviour of heparin oligosaccharides in solution is best described as a top rotor having two perpendicular rotation axes. This prevents an accurate extraction of interprotonic distances by NOESY/ROESY based methods. In this paper, we describe the solution structure of the hexasaccharide 1 calculated from high exactitude distance data obtained from off-resonance ROESY combined with a long MD simulation of 500 ns. In previous studies, we have found that two synthetic hexasaccharides having the sulphate groups directed towards one side of its central plane have an opposite biological activity, while 1 is unable to activate the FGF-1 signalling pathway, the other (2) is even more active than the regular region derived hexasaccharide (3) that mimics the natural active compound, heparin. From the structural analysis it was concluded that 1 has similar three-dimensional characteristics to 2 or 3 and therefore the differences in the activity should be due to the arrangement of the sulphate groups within the hexasaccharidic sequence.

Effect of the Substituents of the Neighboring Ring in the Conformational Equilibrium of Iduronate in Heparin‐like Trisaccharides

Based on the structure of the regular heparin, we have prepared a smart library of heparin-like trisaccharides by incorporating some sulfate groups in the sequence α-D-GlcNS- (1-4)-α-L-Ido2S-(1-4)-α-D-GlcN. According to the 3D structure of heparin, which features one helix turn every four residues, this fragment corresponds to the minimum binding motif. We have performed a complete NMR study and found that the trisaccharides have a similar 3D structure to regular heparin itself, but their spectral properties are such that allow to extract very detailed information about distances and coupling constants as they are isotropic molecules. The characteristic conformational equilibrium of the central iduronate ring has been analyzed combining NMR and molecular dynamics and the populations of the conformers of the central iduronate ring have been calculated. We have found that in those compounds lacking the sulfate group at position 6 of the reducing end glucosamine, the population of (2)S(0) of the central iduronate residue is sensitive to the temperature decreasing to 19% at 278 K. On the contrary, the trisaccharides with 6-O-sulfate in the reducing end glucosamine keep the level of population constant with temperature circa 40% of (2)S(0) similar to that observed at room temperature. Another structural feature that has been revealed through this analysis is the larger flexibility of the L-IdoAS- D-GlcN glycosidic linkage, compared with the D-GlcNS-L-IdoA. We propose that this is the point where the heparin chain is bended to form structures far from the regular helix known as kink that have been proposed to play an important role in the specificity of the heparin-protein interaction.

Regio- and stereoselective synthesis of 3- and 5-(C-glycosyl)-4-nitroisoxazolidines by nitrone–nitroalkene [3+2] cycloaddition reactions

Abstract Cycloaddition reactions of nitrones, including sugar nitrones, with nitroalkenes, including sugar nitroolefins, led with complete regioselectivity and stereospecificity to 4,5- trans -4-nitroisoxazolidines in 51–78% global yields. The endo/exo stereoselectivity depends on the type of sugar derivative used. As expected, the best π-diastereofacial selectivity was observed when both partners were sugar derivatives. Isomerisation of the first formed diastereomers by the action of silica gel was observed in some cases. Absolute configurations for two crystalline products were assigned by X-ray diffraction methods.

Conformations of the iduronate ring in short heparin fragments described by time-averaged distance restrained molecular dynamics

The polyconformational behavior of L-iduronic acid (L-IdoA2S) in heparin derivatives has been previously analyzed in terms of intra-ring proton-proton vicinal coupling constants ((3)JHH) through mathematical fit of experimental and theoretical values (Ferro DR, Provasoli A, Ragazzi M, Casu B, Torri G, Bossennec V, Perly B, Sinay P, Petitou M, Choay J. 1990. Conformer Populations of L-Iduronic Acid Residues in Glycosaminoglycan Sequences. Carbohydr Res. 195:157-167; Muñoz-García JC, López-Prados J, Angulo J, Díaz-Contreras I, Reichardt N, de Paz JL, Martín-Lomas M, Nieto PM. 2012. Effect of the substituents of the neighboring ring in the conformational equilibrium of iduronate in heparin-like trisaccharides. Chemistry. 18:16319-16331.). However, this methodology is subjected to the experimental uncertainties of the J-coupling measurements, the force field deviations and the goodness of the least-squares fit. In the present work, we have used time-averaged distance restrained molecular dynamics (tar-MD) to largely reduce these errors, which enables accurate quantification of the population of conformers, or puckers, of the L-IdoA2S residue, in a set of eight heparin-like trisaccharides following the general sequence d-glucosamine (GlcN)-IdoA-GlcN, directly from the time evolution of the puckering coordinates θ and . Thus, by carrying out tar-MD simulations in explicit water, with the exclusive nuclear overhauser enhancement (NOE)-derived distance H2-H5 of the L-IdoA2S (2)SO conformer as the unique imposed constraint, we have been able to accurately and easily determine the different extents to which the iduronate ring populates the polar ((1)C4 chair) and equatorial ((2)SO skew-boat) areas of the puckering sphere depending on the sulfation pattern of the flanking GlcN residues, under low temperature conditions (278 K). The results indicate that 6-O-sulfation at the reducing-end GlcN residue facilitates the (1)C4 to (2)SO transitions of the iduronate ring by augmenting the flexibility of the C2-C3 torsion, driving the conformational equilibrium toward a majority of equatorial conformers.

Exploration of the use of an acylsulfonamide safety-catch linker for the polymer-supported synthesis of hyaluronic acid oligosaccharides.

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected D-glucosamine (GlcN) and D-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.