Showing papers by "Jesús Jiménez-Barbero published in 2017"

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences.

Abstract: The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- and/or C-terminal prior glycosylation (GalNAc-O-Ser/Thr) preferences modulated by the lectin domain. Here we report studies on GalNAc-T4 that reveal the origins of its unique N-terminal long-range glycopeptide specificity, which is the opposite of GalNAc-T2. The GalNAc-T4 structure bound to a monoglycopeptide shows that the GalNAc-binding site of its lectin domain is rotated relative to the homologous GalNAc-T2 structure, explaining their different long-range preferences. Kinetics and molecular dynamics simulations on several GalNAc-T2 flexible linker constructs show altered remote prior glycosylation preferences, confirming that the flexible linker dictates the rotation of the lectin domain, thus modulating the GalNAc-Ts' long-range preferences. This work for the first time provides the structural basis for the different remote prior glycosylation preferences of the GalNAc-Ts.

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch-Selective Interaction of a Tetra-Antennary N-Glycan with Lectins.

Abstract: The biological recognition of complex-type N-glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N-glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra-antennary N-glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N-acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N-glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N-acetyllactosamine-binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).

NMR and Molecular Recognition of N-Glycans: Remote Modifications of the Saccharide Chain Modulate Binding Features

Abstract: Glycans play a key role as recognition elements in the communication of cells and other organisms. Thus, the analysis of carbohydrate–protein interactions has gained significant importance. In particular, nuclear magnetic resonance (NMR) techniques are considered powerful tools to detect relevant features in the interaction between sugars and their natural receptors. Here, we present the results obtained in the study on the molecular recognition of different mannose-containing glycans by Pisum sativum agglutinin. NMR experiments supported by Corcema-ST analysis, isothermal titration calorimetry (ITC) experiments, and molecular dynamics (MD) protocols have been successfully applied to unmask important binding features and especially to determine how a remote branching substituent significantly alters the binding mode of the sugar entity. These results highlight the key influence of common structural modifications in natural glycans on molecular recognition processes and underscore their importance for the ...

The Use of Fluoroproline in MUC1 Antigen Enables Efficient Detection of Antibodies in Patients with Prostate Cancer.

Abstract: A structure-based design of a new generation of tumor-associated glycopeptides with improved affinity against two anti-MUC1 antibodies is described. These unique antigens feature a fluorinated proline residue, such as a (4S)-4-fluoro-l-proline or 4,4-difluoro-l-proline, at the most immunogenic domain. Binding assays using biolayer interferometry reveal 3-fold to 10-fold affinity improvement with respect to the natural (glyco)peptides. According to X-ray crystallography and MD simulations, the fluorinated residues stabilize the antigen-antibody complex by enhancing key CH/π interactions. Interestingly, a notable improvement in detection of cancer-associated anti-MUC1 antibodies from serum of patients with prostate cancer is achieved with the non-natural antigens, which proves that these derivatives can be considered better diagnostic tools than the natural antigen for prostate cancer.

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH-π Stacking Interactions by Different Fluorination Patterns.

Abstract: We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar-protein interactions by simple 1 H and/or 19 F NMR spectroscopy methods. In this process, we have chosen the binding of N,N'-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2 CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2 FCONH- group and the natural molecule (with the CH3 CONH- fragment). In contrast, the molecule with the CF3 CONH- isoster displayed the weakest intermolecular interaction (one order of magnitude weaker). Because sugar-aromatic CH-π interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.

Mechanistic Insight into the Binding of Multivalent Pyrrolidines to α-Mannosidases

Abstract: Novel pyrrolidine-based multivalent iminosugars, synthesized by a CuAAC approach, have shown remarkable multivalent effects towards jack bean α-mannosidase and a Golgi α-mannosidase from Drosophila melanogaster, as well as a good selectivity with respect to a lysosomal α-mannosidase, which is important for anticancer applications. STD NMR and molecular modeling studies supported a multivalent mechanism with specific interactions of the bioactive iminosugars with Jack bean α-mannosidase. TEM studies suggested a binding mode that involves the formation of aggregates, which result from the intermolecular cross-linked network of interactions between the multivalent inhibitors and two or more dimers of JBMan heterodimeric subunits.

Enzymatic Synthesis of a Novel Neuroprotective Hydroxytyrosyl Glycoside.

Abstract: The eco-friendly synthesis of non-natural glycosides from different phenolic antioxidants was carried out using a fungal β-xylosidase to evaluate changes in their bioactivities. Xylosides from hydroquinone and catechol were successfully formed, although the best results were obtained for hydroxytyrosol, the main antioxidant from olive oil. The formation of the new products was followed by thin-layer chromatography, liquid chromatography, and mass spectrometry. The hydroxytyrosyl xyloside was analyzed in more detail, to maximize its production and evaluate the effect of glycosylation on some hydroxytyrosol properties. The synthesis was optimized up to the highest production reported for a hydroxytyrosyl glycoside. The structure of this compound was solved by two-dimensional nuclear magnetic resonance and identified as 3,4-dihydroxyphenyl-ethyl-O-β-d-xylopyranoside. Evaluation of its biological effect showed an enhancement of both its neuroprotective capacity and its ability to ameliorate intracellular leve...

Insights on the Interaction between Transthyretin and Aβ in Solution. A Saturation Transfer Difference (STD) NMR Analysis of the Role of Iododiflunisal

Abstract: Several strategies against Alzheimer disease (AD) are directed to target Aβ-peptides. The ability of transthyretin (TTR) to bind Aβ-peptides and the positive effect exerted by some TTR stabilizers for modulating the TTR–Aβ interaction have been previously studied. Herein, key structural features of the interaction between TTR and the Aβ(12–28) peptide (3), the essential recognition element of Aβ, have been unravelled by STD-NMR spectroscopy methods in solution. Molecular aspects related to the role of the TTR stabilizer iododiflunisal (IDIF, 5) on the TTR–Aβ complex have been also examined. The NMR results, assisted by molecular modeling protocols, have provided a structural model for the TTR–Aβ interaction, as well as for the ternary complex formed in the presence of IDIF. This basic structural information could be relevant for providing light on the mechanisms involved in the ameliorating effects of AD symptoms observed in AD/TTR± animal models after IDIF treatment and eventually for designing new molec...

Protein–Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy

Abstract: Protein–glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1H15N-HSQC signals. The intensity of the Gal-3 1H15N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1H15N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein.

Contribution of Shape and Charge to the Inhibition of a Family GH99 endo-α-1,2-Mannanase

Abstract: Inhibitor design incorporating features of the reaction coordinate and transition-state structure has emerged as a powerful approach for the development of enzyme inhibitors. Such inhibitors find use as mechanistic probes, chemical biology tools, and therapeutics. Endo-α-1,2-mannosidases and endo-α-1,2-mannanases, members of glycoside hydrolase family 99 (GH99), are interesting targets for inhibitor development as they play key roles in N-glycan maturation and microbiotal yeast mannan degradation, respectively. These enzymes are proposed to act via a 1,2-anhydrosugar “epoxide” mechanism that proceeds through an unusual conformational itinerary. Here, we explore how shape and charge contribute to binding of diverse inhibitors of these enzymes. We report the synthesis of neutral dideoxy, glucal and cyclohexenyl disaccharide inhibitors, their binding to GH99 endo-α-1,2-mannanases, and their structural analysis by X-ray crystallography. Quantum mechanical calculations of the free energy landscapes reveal how ...

Development of a nucleotide exchange inhibitor that impairs Ras oncogenic signaling

Abstract: Despite more than three decades of intense effort, no anti-Ras therapies have reached clinical application. Contributing to this failure has been an underestimation of Ras complexity and a dearth of structural information. In this regard, recent studies have revealed the highly dynamic character of the Ras surface and the existence of transient pockets suitable for small-molecule binding, opening up new possibilities for the development of Ras modulators. Herein, a novel Ras inhibitor (compound 12) is described that selectively impairs mutated Ras activity in a reversible manner without significantly affecting wild-type Ras, reduces the Ras–guanosine triphosphate (GTP) levels, inhibits the activation of the mitogen-activated protein kinase (MAPK) pathway, and exhibits remarkable cytotoxic activity in Ras-driven cellular models. The use of molecular dynamics simulations and NMR spectroscopy experiments has enabled the molecular bases responsible for the interactions between compound 12 and Ras protein to be explored. The new Ras inhibitor binds partially to the GTP-binding region and extends into the adjacent hydrophobic pocket delimited by switch II. Hence, Ras inhibitor 12 could represent a new compound for the development of more efficacious drugs to target Ras-driven cancers; a currently unmet clinical need.

Direct Enzymatic Branch-End Extension of Glycocluster-Presented Glycans: An Effective Strategy for Programming Glycan Bioactivity

Abstract: The sequence of a glycan and its topology of presentation team up to determine the specificity and selectivity of recognition by saccharide receptors (lectins). Structure activity analysis would be furthered if the glycan part of a glycocluster could be efficiently elaborated in situ while keeping all other parameters constant. By using a bacterial alpha 2,6-sialyltransferase and a small library of bi- to tetravalent glycoclusters, we illustrate the complete conversion of scaffold-presented lactoside units into two different sialylated ligands based on N-acetyl/glycolyl-neuraminic acid incorporation. We assess the ensuing effect on their bioactivity for a plant toxin, and present an analysis of the noncovalent substrate binding contacts that the added sialic acid moiety makes to the lectin. Enzymatic diversification of a scaffold presented glycan can thus be brought to completion in situ, offering a versatile perspective for rational glycocluster engineering.

Glycans in Infectious Diseases. A Molecular Recognition Perspective

Abstract: Background From the simplest bacteria to the highest complex mammals, including humans, every single cell is covered by a dense coat of glycans. Glycans are involved in almost every biological process that takes place in our body, playing a central role in the communication between cells and their environment. Glycans are also involved in infectious diseases, which arise from the specific interaction between glycans of the pathogen cell coat and specific receptors on the host cell or vice versa. Objective The understanding of the mechanisms governing these specific carbohydrateprotein interactions, at atomic and molecular levels, is crucial to develop new drugs able to block the infection and to avoid the disease. Methods Recent advances in biophysical techniques allow for a complete picture of the hostpathogen infection event, unveiling the key aspects of the molecular interaction and, thus, providing an opportunity to interfere with it. Conclusion In this general review, we discuss some recent contributions, providing a summary of what we consider the most innovative and inspiring research lines to the field.

Interactions between a Heparin Trisaccharide Library and FGF-1 Analyzed by NMR Methods.

Abstract: FGF-1 is a potent mitogen that, by interacting simultaneously with Heparan Sulfate Glycosaminoglycan HSGAG and the extracellular domains of its membrane receptor (FGFR), generates an intracellular signal that finally leads to cell division. The overall structure of the ternary complex Heparin:FGF-1:FGFR has been finally elucidated after some controversy and the interactions within the ternary complex have been deeply described. However, since the structure of the ternary complex was described, not much attention has been given to the molecular basis of the interaction between FGF-1 and the HSGAG. It is known that within the complex, the carbohydrate maintains the same helical structure of free heparin that leads to sulfate groups directed towards opposite directions along the molecular axis. The precise role of single individual interactions remains unclear, as sliding and/or rotating of the saccharide along the binding pocket are possibilities difficult to discard. The HSGAG binding pocket can be subdivided into two regions, the main one can accommodate a trisaccharide, while the other binds a disaccharide. We have studied and analyzed the interaction between FGF-1 and a library of trisaccharides by STD-NMR and selective longitudinal relaxation rates. The library of trisaccharides corresponds to the heparin backbone and it has been designed to interact with the main subsite of the protein.

Hidden α-helical propensity segments within disordered regions of the transcriptional activator CHOP.

Abstract: C/EBP-homologous protein (CHOP) is a key determinant of the apoptotic response to endoplasmic reticulum stress or DNA damage. As a member of the C/EBP family, CHOP contains a low complexity N-terminal region involved in transcriptional activation, followed by a bZIP that binds DNA after dimerization. However, in contrast to other C/EBPs, CHOP directs binding to non-canonical C/EBP sites due to unique substitutions in its DNA-binding domain. Herein, we show that the N-terminal region of CHOP is intrinsically unstructured but contains two segments presenting α-helical propensity. One of these segments is conserved in other C/EBPs and mediates essential roles of CHOP, including regulation through phosphorylation. The second segment is placed within a proteolytic-resistant portion of the protein and exhibits reduced flexibility. Moreover, the DNA-binding region of CHOP also contains a segment with α-helical character towards its most N-terminal part. Our results suggest that structure-prone segments scattered within disordered regions may be critical for macromolecular recognition during CHOP-mediated transcriptional activation.

The Folded Conformation of Perezone Revisited. Long Range nOe Interaction in Small Molecules: Interpretable Small Signals or Useless Large Artifacts?

Abstract: The nuclear Overhauser effect (nOe) is a consequence of the cross-relaxation and it involves the transfer of nuclear spin polarization from one population to another intra or inter-molecularly. It is generally accepted that a proton-proton distance between 4 and 5 A is the upper limit for the occurrence of measurable nOe´s in small molecules. However, we herein show how detectable nOe´s may, in fact, take place between alkyl groups of quinones with substitutions at 1-4 relative positions, showing distances longer than 6 A. Although the signals of interest are very small, of the order of 1% of a normal nOe, so a priori are considered artifacts originated by decoupling modulation, they maintain, properties that make them interesting and give them coherence as interpretable signals. If the signals of interest are not artifacts these observations represent an important breakthrough with impact on the standard protocols that are commonly used for determination of molecular structure and conformation.

Chapter 6:Computational Chemistry Tools in Glycobiology: Modelling of Carbohydrate–Protein Interactions

Abstract: Molecular modelling provides a major impact in the field of glycosciences, helping in the characterisation of the molecular basis of the recognition between lectins from pathogens and human glycoconjugates, and in the design of glycocompounds with anti-infectious properties. The conformational properties of oligosaccharides are complex, and therefore, the simulation of these properties is a challenging task. Indeed, the development of suitable force fields is required for the proper simulation of important problems in glycobiology, such as the interatomic interactions responsible for oligosaccharide and glycoprotein dynamics, including O-linkages in oligo- and polysaccharides, and N- and O-linkages in glycoproteins. The computational description of representative examples is discussed, herein, related to biologically active oligosaccharides and their interaction with lectins and other proteins, and the new routes open for the design of glycocompounds with promising biological activities.

CHAPTER 5:Lanthanide-Chelating Carbohydrate Conjugates to Detect Carbohydrate–Protein Interactions

Abstract: The synergy between paramagnetic and biomolecular NMR is currently setting the limits of carbohydrate NMR at levels difficult to imagine a few years ago Indeed, the use of properly designed lanthanide-binding-tags is currently allowing to deduce fine details of the conformational and recognition features of complex glycans that were rather challenging or impossible to uncover using conventional NMR techniques