Azucares y Proteínas: el papel de la dinámica en las interacciones moleculares

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Farmacia, leída el 18/10/2016Glycans are among the most varied and complex molecules in biological systems. The different branches of the tree of life could be differentiated on the basis of the glycan composition of the own glycoconjugate molecules. However, how much we already know about glycans and glycoconjugate function and distribution is still an open question. Not so many years ago our knowledge about protein N-glycosylation was considerably scarce. In fact, while protein N-glycosylation was once believed to be limited to eukaryotes, it is now firmly established that this complex modification also occurs in bacteria and archaea. Consequently, in the past 10 years, the field of protein glycosylation has witnessed enormous strides in the discovery of new and unusual carbohydrates, in the elucidation of the enzymes involved in glycan assembly and processing, and in the understanding the biological impact that these glycan modifications have on the structure and function of target protein. The reason for this “late” discovery probably lies in the intrinsic structural complexity, heterogeneity and flexibility of glycans. As counterweight, numerous and exhaustive works in glycomics have demonstrated that it is due to their structural complexity, heterogeneity and flexibility why glycans have been selected as key intermediates for cell proliferation, differentiation, adhesion, infection, communication, etc. With this thesis we have tried to look inside into glycan structure, with the aim to reconcile their structural features at the atomic level with the reasons of their molecular flexibility at a more complex scale. When glycans are recognized by their receptors, their intrinsic flexibility and the plasticity of the whole system has enormous effects in the molecular recognition phenomenon. In fact, both partners involved in the intermolecular interaction could adapt their contact surface in a way that enhances enthalpy-based favourable intermolecular interactions. Alternatively or simultaneously, the glycan and the receptor could strategically keep internal molecular motions, even in the bound state, in a way that minimizes the entropy penalty to the binding event. As consequence, the role of enthalpic/entropic compensation is not easy to predict and even, to assess. Along this thesis we have explored these features, focusing our attention on sugar protein interactions, starting from the sugar flexibility at the monosaccharide level, passing then to the study of disaccharides, and later investigating the complex motions within a sugar receptor. Finally, CH/ intermolecular interactions, which essentially contribute to the stability of sugar-protein complexes, have also been discussed and a new strategy for their direct detection has been proposed...Los glicanos constituyen unos de los tipos de moléculas más variadas y complejas entre los sistemas biológicos. Las diferentes ramas del árbol de la vida se pueden distinguir en base a la composición de los glicanos de los propios glicoconjugados. Sin embargo, cuánto conocemos acerca de la función y distribución de los glicanos y glicoconjugados es una cuestión todavía abierta. Hace no demasiados años, nuestro conocimiento acerca de las glicoproteinas era considerablemente escaso. De hecho, durante mucho tiempo se creyó que la N-glicosilación de proteínas se daba sólo en los organismos eucariotas. Sin embargo, hoy está firmemente establecido que esta compleja modificación ocurre también en bacterias y arqueas. Por lo tanto, en los últimos 10 años, el campo de las glicoproteínas ha sido testigo de enormes avances en el descubrimiento de nuevos e inusuales carbohidratos, así como en la elucidación de las enzimas responsables de la construcción y procesamiento de glicanos y en la comprensión del impacto biológico que estas modificaciones tienen sobre la estructura y función de la proteína diana. La razón de este “retraso”, probablemente radica en la intrínseca complejidad estructural, heterogeneidad y flexibilidad de los glicanos. Como contrapeso, numerosos y exhaustivos trabajos en el campo de la glicómica han demostrado que es exactamente gracias a su complejidad estructural, heterogeneidad y flexibilidad el porqué los glicanos han sido seleccionados, entre otras biomoléculas, como intermedios claves para los procesos celulares de proliferación, diferenciación, adhesión, infección, comunicación, etc. En esta tesis, hemos intentado mirar profundamente en el interior de la estructura de los glicanos, con el objetivo de conciliar sus propiedades estructurales intrínsecas, a nivel atómico, con los motivos de su flexibilidad molecular. Cuando los glicanos interaccionan con sus receptores, esta flexibilidad se une a la plasticidad del sistema global para dar al lugar al proceso de reconocimiento molecular. De hecho, las dos partes que participan en la interacción intermolecular pueden adaptar su superficie de contacto de manera que se maximice la entalpia de unión. De manera alternativa o complementaria, el glicano y el receptor podrían mantener movimientos moleculares internos, incluso en el estado unido, de tal manera que se minimizase la penalización entrópica del proceso de unión. Como consecuencia de ello, el papel de la compensación entálpico/entrópica no es fácil de evaluar ni de medir...Fac. de FarmaciaTRUEunpu

Exploring multivalent carbohydrate–protein interactions by NMR

Nuclear Magnetic Resonance (NMR) has been widely employed to assess diverse features of glycan–protein molecular recognition events. Different types of qualitative and quantitative information at different degrees of resolution and complexity can be extracted from the proper application of the available NMR-techniques. In fact, affinity, structural, kinetic, conformational, and dynamic characteristics of the binding process are available. Nevertheless, except in particular cases, the affinity of lectin-sugar interactions is weak, mostly at the low mM range. This feature is overcome in biological processes by using multivalency, thus augmenting the strength of the binding. However, the application of NMR methods to monitor multivalent lectin–glycan interactions is intrinsically challenging. It is well known that when large macromolecular complexes are formed, the NMR signals disappear from the NMR spectrum, due to the existence of fast transverse relaxation, related to the large size and exchange features. Indeed, at the heart of the molecular recognition event, the associated free-bound chemical exchange process for both partners takes place in a particular timescale. Thus, these factors have to be considered and overcome. In this review article, we have distinguished, in a subjective manner, the existence of multivalent presentations in the glycan or in the lectin. From the glycan perspective, we have also considered whether multiple epitopes of a given ligand are presented in the same linear chain of a saccharide (i.e., poly-LacNAc oligosaccharides) or decorating different arms of a multiantennae scaffold, either natural (as in multiantennae N-glycans) or synthetic (of dendrimer or polymer nature). From the lectin perspective, the presence of an individual binding site at every monomer of a multimeric lectin may also have key consequences for the binding event at different levels of complexity.We thank generous funding by the European Research Council (RECGLYCANMR, Advanced Grant No. 788143), the Agencia Estatal de Investigación (Spain) for grant PDI2021-1237810B-C21, and CIBERES, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. We also thank Marie-Skłodowska-Curie actions (TN BactiVax, under grant agreement No. 860325)

Biomimetic Tweezers for N-Glycans: Selective Recognition of the Core GlcNAc(2) Disaccharide of the Sialylglycopeptide SGP

In recent years, glycomics have shown how pervasive the role of carbohydrates in biological systems is and how chemical tools are essential to investigate glycan function and modulate carbohydrate-mediated processes. Biomimetic receptors for carbohydrates can carry out this task but, although significant affinities and selectivities toward simple saccharides have been achieved, targeting complex glycoconjugates remains a goal yet unattained. In this work we report the unprecedented recognition of a complex biantennary sialylglycopeptide (SGP) by a tweezers-shaped biomimetic receptor, which selectively binds to the core GlcNAc2 disaccharide of the N-glycan with an affinity of 170 μM. Because of the simple structure and the remarkable binding ability, this biomimetic receptor can represent a versatile tool for glycoscience, opening the way to useful applications.We thank MIUR-Italy “Progetto Dipartimenti di Eccellenza 2018–2022” allocated to Department of Chemistry Ugo Schiff, COST Action (CA18132), MIUR-Italy PRIN2017 (2017XZ2ZBK) for granting a fellowship to F.M. and Ente Cassa di Risparmio di Firenze (Italy) is acknowledged for granting an ITC nanocalorimeter and a high-field NMR spectrometer. Open Access funding provided by Università degli Studi di Firenze within the CRUI-CARE Agreement

Mono and Di-Fucosylated Glycans of the Parasitic Worm S. Mansoniare Recognized Differently by the Innate Immune Receptor DC-SIGN

The parasitic worm,Schistosoma mansoni, expresses unusual fucosylated glycans in a stage-dependent manner that can be recognized by the human innate immune receptor DC-SIGN, thereby shaping host immune responses. We have developed a synthetic approach for mono- and bis-fucosylated LacdiNAc (LDN-F and LDN-DF, respectively), which are epitopes expressed on glycolipids and glycoproteins ofS. mansoni. It is based on the use of monosaccharide building blocks having carefully selected amino-protecting groups, facilitating high yielding and stereoselective glycosylations. The molecular interaction between the synthetic glycans and DC-SIGN was studied by NMR and molecular modeling, which demonstrated that the alpha 1,3-fucoside of LDN-F can coordinate with the Ca2+-ion of the canonical binding site of DC-SIGN allowing for additional interactions with the underlying LDN backbone. The 1,2-fucoside of LDN-DF can be complexed in a similar manner, however, in this binding mode GlcNAc and GalNAc of the LDN backbone are placed away from the protein surface resulting in a substantially lower binding affinity. Glycan microarray binding studies showed that the avidity and selectivity of binding is greatly enhanced when the glycans are presented multivalently, and in this format Le(x)and LDN-F gave strong responsiveness, whereas no binding was detected for LDN-DF. The data indicates thatS. mansonihas developed a strategy to avoid detection by DC-SIGN in a stage-dependent manner by the addition of a fucoside to a number of its ligands.This research was supported by the Netherlands Organization for Scientific Research (NWO; TOP-PUNT grant 718.015.003 to G.-J.B.), the Human Frontier Science Program Organization (HFSP; grant LT000747/2018-C to L.U.), the European Research Council (ERC-2017-AdG, project number 788143-RECGLYC-ANMR to J.J.-B.), the Agencia Estatal Investigacion of Spain (AEI; grant RTI2018-094751-B-C21 to J.J.-B.) and the Severo Ochoa Excellence Accreditation (SEV-2016-0644 to J.J.-B.)

The SARS-CoV-2 Spike Glycoprotein Directly Binds Exogeneous Sialic Acids: A NMR View

[EN] The interaction of the SARS CoV2 spike glycoprotein with two sialic acid-containing trisaccharides (alpha 2,3 and alpha 2,6 sialyl N-acetyllactosamine) has been demonstrated by NMR. The NMR-based distinction between the signals of those sialic acids in the glycans covalently attached to the spike protein and those belonging to the exogenous alpha 2,3 and alpha 2,6 sialyl N-acetyllactosamine ligands has been achieved by synthesizing uniformly C-13-labelled trisaccharides at the sialic acid and galactose moieties. STD-H-1,C-13-HSQC NMR experiments elegantly demonstrate the direct interaction of the sialic acid residues of both trisaccharides with additional participation of the galactose moieties, especially for the alpha 2,3-linked analogue. Additional experiments with the spike protein in the presence of a specific antibody for the N-terminal domain and with the isolated receptor binding and N-terminal domains of the spike protein unambiguously show that the sialic acid binding site is located at the N-terminal domain.This research was funded by the European Research Council (ERC-2017-AdG, project number 788143-RECGLYCA NMR to J.J.B.) and Agencia Estatal de Investigacion (Spain), projects RTI2018-094751-B-C21 to J.J.B. & A.A. and PID2019-107770RA-I00 to J.E.O., and by the Human Frontier Science Program (HFSP; grant LT000747/2018-C to L.U.) and CIBER, an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spai

Synthetic O-Acetyl- N-glycolylneuraminic Acid Oligosaccharides Reveal Host-Associated Binding Patterns of Coronaviral Glycoproteins.

A panel of O-acetylated N-glycolylneuraminic acid oligosaccharides has been prepared by diversification of common synthetic precursors by regioselective de- O-acetylation by coronaviral hemagglutinin-esterase (HE) combined with C7-to-C9 acetyl ester migration. The resulting compound library was printed on streptavidin-coated glass slides to give a microarray to investigate receptor binding specificities of viral envelope glycoproteins, including spike proteins and HEs from animal and human coronaviruses. It was found that the binding patterns of the viral proteins for N-glycolylated sialosides differ considerable from those of the previously synthesized N-acetylated counterparts. Generally, the spike proteins tolerate N-glycolyl modification, but selectivities differ among viruses targeting different hosts. On the other hand, the lectin domain of the corresponding HEs showed a substantial decrease or loss of binding of N-glycolylated sialosides. MD simulations indicate that glycolyl recognition by HE is mediated by polar residues in a loop region (109-119) that interacts with the 5- N-glycolyl moiety. Collectively, the results indicate that coronaviruses have adjusted their receptor fine specificities to adapt to the sialoglycome of their host species

Synthetic Heparanase Inhibitors Can Prevent Herpes Simplex Viral Spread

Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1. A range of HS-oligosaccharides was prepared to examine the importance of chain length and 2-O-sulfation of iduronic moieties for Hpse inhibition. It was found that hexa- and octasaccharides potently inhibited the enzyme and that 2-O-sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure–activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV-1 with the hexa- and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties

The N-glycan structures of the antigenic variants of chlorovirus PBCV-1 major capsid protein help to identify the virus-encoded glycosyltransferases

The chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) is a large dsDNA virus that infects the microalga Chlorella variabilis NC64A. Unlike most other viruses, PBCV-1 encodes most, if not all, of the machinery required to glycosylate its major capsid protein (MCP). The structures of the four N-linked glycans from the PBCV-1 MCP consist of nonasaccharides, and similar glycans are not found elsewhere in the three domains of life. Here, we identified the roles of three virus-encoded glycosyltransferases (GTs) that have four distinct GT activities in glycan synthesis. Two of the three GTs were previously annotated as GTs but the third GT was identified in this study. We determined the GT functions by comparing the wild-type glycan structures from PBCV-1 with those from a set of PBCV-1 spontaneous GT genes mutants resulting in antigenic variants having truncated glycan structures. According to our working model, the virus gene a064r encodes a GT with three domains: domain 1 has a β-L-rhamnosyltransferase activity, domain 2 has an α -L-rhamnosyltransferase activity and domain 3 is a methyltransferase that decorates two positions in the terminal α -L-rhamnose (Rha) unit. The a075l gene encodes a β -xylosyltransferase that attaches the distal D-xylose (Xyl) unit to the L-fucose (Fuc) that is part of the conserved N-glycan core region. Lastly, gene a071r encodes a GT that is involved in the attachment of a semiconserved element, α-D-Rha, to the same L-Fuc in the core region. Our results uncover GT activities that assemble four of the nine residues of the PBCV-1 MCP N-glycans. Includes supplemental material