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The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an H-antigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer.
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Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as ß-galactoside binding lectins. However, certain members of this family have shown selective affinity toward specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7, and -12) toward a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I versus type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity toward poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance experiments. Particularly, we identified oligosaccharide blood group A antigen tetraose 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited interleukin-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.
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The interactions of glycans with proteins modulate many events related to health and disease. In fact, the establishment of these recognition events and their biological consequences are intimately related to the three-dimensional structures of both partners, as well as to their dynamic features and their presentation on the corresponding cell compartments. NMR techniques are unique to disentangle these characteristics and, indeed, diverse NMR-based methodologies have been developed and applied to monitor the binding events of glycans with their associate receptors. This protocol outlines the procedures to acquire, process and analyze two of the most powerful NMR methodologies employed in the NMR-glycobiology field, 1H-Saturation transfer difference (STD) and 1H,15N-Heteronuclear single quantum coherence (HSQC) titration experiments, which complementarily offer information from the glycan and protein perspective, respectively. Indeed, when combined they offer a powerful toolkit for elucidating both the structural and dynamic aspects of molecular recognition processes. This comprehensive approach enhances our understanding of glycan-protein interactions and contributes to advancing research in the chemical glycobiology field.
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Polisacáridos , Polisacáridos/química , Polisacáridos/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Proteínas/metabolismoRESUMEN
Prototypic receptors for human influenza viruses are N-glycans carrying α2,6-linked sialosides. Due to immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that bind α2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) chains. Here, binding modes of such drifted hemagglutinin's (HAs) are examined by chemoenzymatic synthesis of N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans are employed in 2D STD-1H by 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computation and mutagenesis demonstrate that mutations distal to the receptor binding domain of recent HAs create an extended binding site that accommodates with the extended LacNAc chain. A fluorine containing sialoside is used as NMR probe to derive relative binding affinities and confirms the contribution of the extended LacNAc chain for binding.
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Subtipo H3N2 del Virus de la Influenza A , Gripe Humana , Humanos , Subtipo H3N2 del Virus de la Influenza A/genética , Polisacáridos/metabolismo , Monosacáridos/metabolismoRESUMEN
[This corrects the article DOI: 10.1021/acsomega.3c02921.].
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CD14 is an innate immune receptor that senses pathogen-associated molecular patterns, such as lipopolysaccharide, to activate the innate immune response. Although CD14 is known to be glycosylated, detailed understanding about the structural and functional significance of this modification is still missing. Herein, an NMR and MS-based study, assisted by MD simulations, has provided a 3D-structural model of glycosylated CD14. Our results reveal the existence of a key N-glycosylation site at Asn282 that exclusively contains unprocessed oligomannnose N-glycans that perfectly fit the concave cavity of the bent-solenoid shaped protein. This site is not accessible to glycosidases and is fundamental for protein folding and secretion. A second N-site at Asn151 displays mostly complex N-glycans, with the typical terminal epitopes of the host cell-line expression system (i.e. ßGal, α2,3 and α2,6 sialylated ßGal, here), but also particularities, such as the lack of core fucosylation. The glycan at this site points outside the protein surface, resulting in N-glycoforms fully exposed and available for interactions with lectins. In fact, NMR experiments show that galectin-4, proposed as a binder of CD14 on monocytes to induce their differentiation into macrophages-like cells, interacts in vitro with CD14 through the recognition of the terminal glycoepitopes on Asn151. This work provides key information about CD14 glycosylation, which helps to better understand its functional roles and significance. Although protein glycosylation is known to be dynamic and influenced by many factors, some of the features found herein (presence of unprocessed N-glycans and lack of core Fuc) are likely to be protein specific.
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Lectinas , Polisacáridos , Glicosilación , Polisacáridos/química , Lectinas/metabolismo , Línea Celular , Lipopolisacáridos/metabolismoRESUMEN
The urgency to find complementary therapies to current SARS-CoV-2 vaccines, whose effectiveness is preserved over time and not compromised by the emergence of new and emerging variants, has become a critical health challenge. We investigate the possibility of jamming the opening of the Receptor Binding Domain (RBD) of the spike protein of SARS-CoV-2 with small compounds. Through in silico screening, we identified two potential candidates that would lock the Receptor Binding Domain (RBD) in a closed configuration, preventing the virus from infecting the host cells. We show that two drugs already approved by the FDA, mithramycin and dihydroergotamine, can block infection using concentrations in the µM range in cell-based assays. Further STD-NMR experiments support dihydroergotamine's direct interaction with the spike protein. Overall, our results indicate that repurposing of these compounds might lead to potential clinical drug candidates for the treatment of SARS-CoV-2 infection.
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The dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is an innate immune C-type lectin receptor that recognizes carbohydrate-based pathogen associated with molecular patterns of various bacteria, fungi, viruses and protozoa. Although a range of highly mannosylated glycoproteins have been shown to induce signaling via DC-SIGN, precise structure of the recognized oligosaccharide epitope is still unclear. Using the array of oligosaccharides related to selected fragments of main fungal antigenic polysaccharides we revealed a highly specific pentamannoside ligand of DC-SIGN, consisting of α-(1 â 2)-linked mannose chains with one inner α-(1 â 3)-linked unit. This structural motif is present in Candida albicans cell wall mannan and corresponds to its antigenic factors 4 and 13b. This epitope is not ubiquitous in other yeast species and may account for the species-specific nature of fungal recognition via DC-SIGN. The discovered highly specific oligosaccharide ligands of DC-SIGN are tractable tools for interdisciplinary investigations of mechanisms of fungal innate immunity and anti-Candida defense. Ligand- and receptor-based NMR data demonstrated the pentasaccharide-to-DC-SIGN interaction in solution and enabled the deciphering of the interaction topology.
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Lectin-glycan interactions are at the heart of a multitude of biological events. Glycans are usually presented in a multivalent manner on the cell surface as part of the so-called glycocalyx, where they interact with other entities. This multivalent presentation allows us to overcome the typical low affinities found for individual glycan-lectin interactions. Indeed, the presentation of glycans may drastically impact their binding by lectins, highly affecting the corresponding binding affinity and even selectivity. In this context, we herein present the study of the interaction of a variety of homo- and heteromultivalent lactose-functionalized glycomacromolecules and their lipid conjugates with two human galectins. We have employed as ligands the glycomacromolecules, as well as liposomes decorated with those structures, to evaluate their interactions in a cell-mimicking environment. Key details of the interaction have been unravelled by NMR experiments, both from the ligand and receptor perspectives, complemented by cryo-electron microscopy methods and molecular dynamics simulations.
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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.
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Carbohidratos , Oligosacáridos , Carbohidratos/química , Oligosacáridos/química , Oligosacáridos/metabolismo , Polisacáridos/química , Espectroscopía de Resonancia Magnética , LectinasRESUMEN
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 170â 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.
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Biomimética , Disacáridos , Polisacáridos/química , Carbohidratos/química , GlicómicaRESUMEN
Fluorine (19 F) incorporation into glycan-binding proteins (lectins) has been achieved and exploited to monitor the binding to carbohydrate ligands by nuclear magnetic resonance (NMR) spectroscopy. Galectins are a family of lectins that bind carbohydrates, generally with weak affinities, through a combination of intermolecular interactions including a key CH-π stacking involving a conserved tryptophan residue. Herein, Galectin-3 (Gal3) and Galectin-8 (Gal8) with one and two carbohydrate recognition domains (CRDs), respectively, were selected. Gal3 contains one Trp, whereas Gal8 contains three, one at each binding site and a third one not involved in sugar binding; these were substituted by the corresponding F-Trp analogues. The presence of fluorine did not significantly modify the affinity for glycan binding, which was in slow exchange on the 19 F NMR chemical-shift timescale, even for weak ligands, and allowed binding events taking place at two different binding sites within the same lectin to be individualized.
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Flúor , Galectinas , Galectinas/metabolismo , Carbohidratos , Polisacáridos/química , Sitios de Unión , Espectroscopía de Resonancia Magnética , Galectina 3/metabolismoRESUMEN
The molecular recognition features of LSECtin toward asymmetric N-glycans have been scrutinized by NMR and compared to those occurring in glycan microarrays. A pair of positional glycan isomers (LDN3 and LDN6), a nonelongated GlcNAc4Man3 N-glycan (G0), and the minimum binding epitope (the GlcNAcß1-2Man disaccharide) have been used to shed light on the preferred binding modes under both experimental conditions. Strikingly, both asymmetric LDN3 and LDN6 N-glycans are recognized by LSECtin with similar affinities in solution, in sharp contrast to the results obtained when those glycans are presented on microarrays, where only LDN6 was efficiently recognized by the lectin. Thus, different results can be obtained using different experimental approaches, pointing out the tremendous difficulty of translating in vitro results to the in vivo environment.
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The Cost Action "Innovation with glycans: new frontiers from synthesis to new biological targets" (INNOGLY) hosted the Workshop "Neuroglycoproteins in health and disease", in Alicante, Spain, on March 2022. This event brought together an european group of scientists that presented novel insights into changes in glycosylation in diseases of the central nervous system and cancer, as well as new techniques to study protein glycosylation. Herein we provide the abstracts of all the presentations.
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Neoplasias , Polisacáridos , Glicosilación , Humanos , Polisacáridos/metabolismoRESUMEN
Two years after its emergence, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several vaccines. The extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin converting enzyme 2 (ACE2) through its receptor binding domain (RBD), is the major target of neutralizing antibodies. Like to many other viral fusion proteins, the SARS-CoV-2 spike protein utilizes a glycan shield to thwart the host immune response. To grasp the influence of chemical signatures on carbohydrate mobility and reconcile the cryo-EM density of specific glycans we combined our cryo-EM map of the S ectodomain to 4.1 Å resolution, reconstructed from a limited number of particles, and all-atom molecular dynamics simulations. Chemical modifications modeled on representative glycans (defucosylation, sialylation and addition of terminal LacNAc units) show no significant influence on either protein shielding or glycan flexibility. By estimating at selected sites the local correlation between the full density map and atomic model-based maps derived from molecular dynamics simulations, we provide insight into the geometries of the α-Man-(1â3)-[α-Man-(1â6)-]-ß-Man-(1â4)-ß-GlcNAc(1â4)-ß-GlcNAc core common to all N-glycosylation sites.
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Galectins are a family of glycan binding proteins that stand out for the wide range of biological phenomena in which they are involved. Most galectin functions are associated with their glycan binding capacities, which are generally well characterized at the oligosaccharide level, but not at the glycoprotein or glycolipid level. Glycolipids form the part of cell membranes where they can act as galectin cellular receptors. In this scenario, glycan presentation as well as the membrane chemical and structural features are expected to have a strong impact in these molecular association processes. Herein, liposomes were used as membrane mimicking scaffolds for the presentation of glycosphingolipids (GSLs) and to investigate their interaction with Galectin-3 and the N-domain of Galectin-8 (Gal8N). The binding towards GM3 and GM1 and their non-silaylated GSLs was compared to the binding to the free glycans, devoid of lipid. The analysis was carried out using a combination of NMR methods, membrane perturbation studies, and molecular modeling. Our results showed a different tendency of the two galectins in their binding capacities towards the glycans, depending on whether they were free oligosaccharides or as part of GSL inserted into a lipid bilayer, highlighting the significance of GSL glycan presentation on membranes in lectin binding.
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The interaction of the SARS CoV2 spike glycoprotein with two sialic acid-containing trisaccharides (α2,3 and α2,6 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 α2,3 and α2,6 sialyl N-acetyllactosamine ligands has been achieved by synthesizing uniformly 13 C-labelled trisaccharides at the sialic acid and galactose moieties. STD-1 H,13 C-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 α2,3-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.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Sitios de Unión , Galactosa , Humanos , Ácido N-Acetilneuramínico/química , SARS-CoV-2 , Ácidos Siálicos/química , Glicoproteína de la Espiga del Coronavirus/química , TrisacáridosRESUMEN
Acyl group migration is a fundamental phenomenon in carbohydrate chemistry, recently shown to take place also between two non-adjacent hydroxyl groups, across the glycosidic bond, in a ß-(1â4)-linked mannan trisaccharide model compound. With the central mannoside unit containing acetyl groups at the O2 and O3 positions, the O2-acetyl was in the earlier study shown to migrate to O6 of the reducing end. Potential implications of the general acyl migration process on cell signaling events and plant growth in nature are intriguing open questions. In the present work, migration kinetics in this original trisaccharide model system were studied in more detail together with potential interactions of the model compound and the migration products with DC-SIGN lectin. Furthermore, we demonstrate here for the first time that similar migration may also take place in native polysaccharides, here represented by galactoglucomannan from Norway spruce.
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Glicósidos/química , Mananos/química , Oligosacáridos/química , Conformación de Carbohidratos , CinéticaRESUMEN
The interaction of multi-LacNAc (Galß1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.
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Proteínas Sanguíneas/química , Galectina 1/química , Galectinas/química , Metacrilatos/química , Polímeros/química , Acrilamidas/química , Acrilamidas/farmacología , Sitios de Unión/efectos de los fármacos , Proteínas Sanguíneas/genética , Carbohidratos/química , Microscopía por Crioelectrón , Galectina 1/genética , Galectinas/genética , Humanos , Ligandos , Metacrilatos/farmacología , Polímeros/farmacología , Unión Proteica/efectos de los fármacosRESUMEN
The importance of multivalency for N-glycan-protein interactions has primarily been studied by attachment of minimal epitopes to artificial multivalent scaffold and not in the context of multi-antennary glycans. N-glycans can be modified by bisecting GlcNAc, core xylosides and fucosides, and extended N-acetyl lactosamine moieties. The impact of such modifications on glycan recognition are also not well understood. We describe here a chemoenzymatic methodology that can provide N-glycans expressed by the parasitic worm S. mansoni having unique epitopes at each antenna and containing core xyloside. NMR, computational and electron microscopy were employed to investigate recognition of the glycans by the human lectin DC-SIGN. It revealed that core xyloside does not influence terminal epitope recognition. The multi-antennary glycans bound with higher affinity to DC-SIGN compared to mono-valent counterparts, which was attributed to proximity-induced effective concentration. The multi-antennary glycans cross-linked DC-SIGN into a dense network, which likely is relevant for antigen uptake and intracellular routing.