Disentangling Glycan-Protein Interactions: Nuclear Magnetic Resonance (NMR) to the Rescue.

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.

Antigenic determinants driving serogroup-specific antibody response to Neisseria meningitidis C, W, and Y capsular polysaccharides: Insights for rational vaccine design.

Meningococcal glycoconjugate vaccines sourced from capsular polysaccharides (CPSs) of pathogenic Neisseria meningitidis strains are well-established measures to prevent meningococcal disease. However, the exact structural factors responsible for antibody recognition are not known. CPSs of Neisseria meningitidis serogroups Y and W differ by a single stereochemical center, yet they evoke specific immune responses. Herein, we developed specific monoclonal antibodies (mAbs) targeting serogroups C, Y, and W and evaluated their ability to kill bacteria. We then used these mAbs to dissect structural elements responsible for carbohydrate-protein interactions. First, Men oligosaccharides were screened against the mAbs using ELISA to select putative lengths representing the minimal antigenic determinant. Next, molecular interaction features between the mAbs and serogroup-specific sugar fragments were elucidated using STD-NMR. Moreover, X-ray diffraction data with the anti-MenW CPS mAb enabled the elucidation of the sugar-antibody binding mode. Our findings revealed common traits in the epitopes of all three sialylated serogroups. The minimal binding epitopes typically comprise five to six repeating units. Moreover, the O-acetylation of the neuraminic acid moieties was fundamental for mAb binding. These insights hold promise for the rational design of optimized meningococcal oligosaccharides, opening new avenues for novel production methods, including chemical or enzymatic approaches.

Solid-Phase-Supported Chemoenzymatic Synthesis and Analysis of Chondroitin Sulfate Proteoglycan Glycopeptides.

Proteoglycans (PGs), consisting of glycosaminoglycans (GAGs) linked with the core protein through a tetrasaccharide linkage region, play roles in many important biological events. The chemical synthesis of PG glycopeptides is extremely challenging. In this work, the enzymes required for synthesis of chondroitin sulfate (CS) PG (CSPG) have been expressed and the suitable sequence of enzymatic reactions has been established. To expedite CSPG synthesis, the peptide acceptor was immobilized on solid phase and the glycan units were directly installed enzymatically onto the peptide. Subsequent enzymatic chain elongation and sulfation led to the successful synthesis of CSPG glycopeptides. The CS dodecasaccharide glycopeptide was the longest homogeneous CS glycopeptide synthesized to date. The enzymatic synthesis was much more efficient than the chemical synthesis of the corresponding CS glycopeptides, which could reduce the total number of synthetic steps by 80%. The structures of the CS glycopeptides were confirmed by mass spectrometry analysis and NMR studies. In addition, the interactions between the CS glycopeptides and cathepsin G were studied. The sulfation of glycan chain was found to be important for binding with cathepsin G. This efficient chemoenzymatic strategy opens new avenues to investigate the structures and functions of PGs.

A Multidisciplinary Structural Approach to the Identification of the Haemophilus influenzae Type b Capsular Polysaccharide Protective Epitope.

Glycoconjugate vaccines so far licensed are generally composed of a native or size-reduced capsular polysaccharide conjugated to carrier proteins. Detailed information on the structural requirements necessary for CPS recognition is becoming the key to accelerating the development of next-generation improved glycoconjugate vaccines. Structural glycobiology studies using oligosaccharides (OS) complexed with functional monoclonal antibodies represent a powerful tool for gaining information on CPS immunological determinants at the atomic level. Herein, the minimal structural epitope of Haemophilus influenzae type b (Hib) CPS recognized by a functional human monoclonal antibody (hmAb) is reported. Short and well-defined Hib oligosaccharides originating from the depolymerization of the native CPS have been used to elucidate saccharide-mAb interactions by using a multidisciplinary approach combining surface plasmon resonance (SPR), saturation transfer difference-nanomagnetic resonance (STD-NMR), and X-ray crystallography. Our study demonstrates that the minimal structural epitope of Hib is comprised within two repeating units (RUs) where ribose and ribitol are directly engaged in the hmAb interaction, and the binding pocket fully accommodates two RUs without any additional involvement of a third one. Understanding saccharide antigen structural characteristics can provide the basis for the design of innovative glycoconjugate vaccines based on alternative technologies, such as synthetic or enzymatic approaches.

Illuminating a Solvent-Dependent Hierarchy for Aromatic CH/π Complexes with Dynamic Covalent Glyco-Balances.

CH/π interactions are prevalent among aromatic complexes and represent invaluable tools for stabilizing well-defined molecular architectures. Their energy contributions are exceptionally sensitive to various structural and environmental factors, resulting in a context-dependent nature that has led to conflicting findings in the scientific literature. Consequently, a universally accepted hierarchy for aromatic CH/π interactions has remained elusive. Herein, we present a comprehensive experimental investigation of aromatic CH/π complexes, employing a novel approach that involves isotopically labeled glyco-balances generated in situ. This innovative strategy not only allows us to uncover thermodynamic insights but also delves into the often less-accessible domain of kinetic information. Our analyses have yielded more than 180 new free energy values while considering key factors such as solvent properties, the interaction geometry, and the presence and nature of accompanying counterions. Remarkably, the obtained results challenge conventional wisdom regarding the stability order of common aromatic complexes. While it was believed that cationic CH/π interactions held the highest strength, followed by polarized CH/π, nonpolarized CH/π, and finally anionic CH/π interactions, our study reveals that this hierarchy can be subverted depending on the environment. Indeed, the performance of polarized CH/π interactions can match or even outcompete that of cationic CH/π interactions making them a more reliable stabilization strategy across the entire spectrum of solvent polarity. Overall, our results provide valuable guidelines for the selection of optimal interacting partners in every chemical environment, allowing the design of tailored aromatic complexes with applications in supramolecular chemistry, organocatalysis, and/or material sciences.

Unraveling Molecular Recognition of Glycan Ligands by Siglec-9 via NMR Spectroscopy and Molecular Dynamics Modeling.

Human sialic-acid-binding immunoglobulin-like lectin-9 (Siglec-9) is a glycoimmune checkpoint receptor expressed on several immune cells. Binding of Siglec-9 to sialic acid containing glycans (sialoglycans) is well documented to modulate its functions as an inhibitory receptor. Here, we first assigned the amino acid backbone of the Siglec-9 V-set domain (Siglec-9d1), using well-established triple resonance three-dimensional nuclear magnetic resonance (NMR) methods. Then, we combined solution NMR and molecular dynamic simulation methods to decipher the molecular details of the interaction of Siglec-9 with the natural ligands α2,3 and α2,6 sialyl lactosamines (SLN), sialyl Lewis X (sLeX), and 6-O sulfated sLeX and with two synthetically modified sialoglycans that bind with high affinity. As expected, Neu5Ac is accommodated between the F and G ß-strands at the canonical sialic acid binding site. Addition of a heteroaromatic scaffold 9N-5-(2-methylthiazol-4-yl)thiophene sulfonamide (MTTS) at the C9 position of Neu5Ac generates new interactions with the hydrophobic residues located at the G-G' loop and the N-terminal region of Siglec-9. Similarly, the addition of the aromatic substituent (5-N-(1-benzhydryl-1H-1,2,3-triazol-4-yl)methyl (BTC)) at the C5 position of Neu5Ac stabilizes the conformation of the long and flexible B'-C loop present in Siglec-9. These results expose the underlying mechanism responsible for the enhanced affinity and specificity for Siglec-9 for these two modified sialoglycans and sheds light on the rational design of the next generation of modified sialoglycans targeting Siglec-9.

Correction to "Targeting the Spike: Repurposing Mithramycin and Dihydroergotamine to Block SARS-CoV-2 Infection".

[This corrects the article DOI: 10.1021/acsomega.3c02921.].

Evolution of human H3N2 influenza virus receptor specificity has substantially expanded the receptor-binding domain site.

Hemagglutinins (HAs) from human influenza viruses descend from avian progenitors that bind α2-3-linked sialosides and must adapt to glycans with α2-6-linked sialic acids on human airway cells to transmit within the human population. Since their introduction during the 1968 pandemic, H3N2 viruses have evolved over the past five decades to preferentially recognize human α2-6-sialoside receptors that are elongated through addition of poly-LacNAc. We show that more recent H3N2 viruses now make increasingly complex interactions with elongated receptors while continuously selecting for strains maintaining this phenotype. This change in receptor engagement is accompanied by an extension of the traditional receptor-binding site to include residues in key antigenic sites on the surface of HA trimers. These results help explain the propensity for selection of antigenic variants, leading to vaccine mismatching, when H3N2 viruses are propagated in chicken eggs or cells that do not contain such receptors.

Strategy for the Enzymatic Acylation of the Apple Flavonoid Phloretin Based on Prior α-Glucosylation.

The acylation of flavonoids serves as a means to alter their physicochemical properties, enhance their stability, and improve their bioactivity. Compared with natural flavonoid glycosides, the acylation of nonglycosylated flavonoids presents greater challenges since they contain fewer reactive sites. In this work, we propose an efficient strategy to solve this problem based on a first α-glucosylation step catalyzed by a sucrose phosphorylase, followed by acylation using a lipase. The method was applied to phloretin, a bioactive dihydrochalcone mainly present in apples. Phloretin underwent initial glucosylation at the 4'-OH position, followed by subsequent (and quantitative) acylation with C8, C12, and C16 acyl chains employing an immobilized lipase from Thermomyces lanuginosus. Electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) confirmed that the acylation took place at 6-OH of glucose. The water solubility of C8 acyl glucoside closely resembled that of aglycone, but for C12 and C16 derivatives, it was approximately 3 times lower. Compared with phloretin, the radical scavenging capacity of the new derivatives slightly decreased with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and was similar to 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+). Interestingly, C12 acyl-α-glucoside displayed an enhanced (3-fold) transdermal absorption (using pig skin biopsies) compared to phloretin and its α-glucoside.

Dissecting the Conformational Stability of a Glycan Hairpin.

Systematic structural studies of model oligopeptides revealed important aspects of protein folding and offered design principles to access non-natural materials. In the same way, the rules that regulate glycan folding could be established by studying synthetic oligosaccharide models. However, their analysis is often limited due to the synthetic and analytical complexity. By utilizing a glycan capable of spontaneously folding into a hairpin conformation as a model system, we investigated the factors that contribute to its conformational stability in aqueous solution. The modular design of the hairpin model featured a trisaccharide turn unit and two ß-1,4-oligoglucoside stacking strands that allowed for systematic chemical modifications of the glycan sequence, including the introduction of NMR labels and staples. Nuclear magnetic resonance assisted by molecular dynamics simulations revealed that stereoelectronic effects and multiple glycan-glycan interactions are the major determinants of folding stabilization. Chemical modifications in the glycan primary sequence (e.g., strand elongation) can be employed to fine-tune the rigidity of structural motifs distant from the modification sites. These results could inspire the design of other glycan architectures, with implications in glycobiology and material sciences.

The impact of glycosylation on the structure, function, and interactions of CD14.

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.

Targeting the Spike: Repurposing Mithramycin and Dihydroergotamine to Block SARS-CoV-2 Infection.

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.

Synthesis of a glycan hairpin.

The primary sequence of a biopolymer encodes the essential information for folding, permitting to carry out sophisticated functions. Inspired by natural biopolymers, peptide and nucleic acid sequences have been designed to adopt particular three-dimensional (3D) shapes and programmed to exert specific functions. In contrast, synthetic glycans capable of autonomously folding into defined 3D conformations have so far not been explored owing to their structural complexity and lack of design rules. Here we generate a glycan that adopts a stable secondary structure not present in nature, a glycan hairpin, by combining natural glycan motifs, stabilized by a non-conventional hydrogen bond and hydrophobic interactions. Automated glycan assembly enabled rapid access to synthetic analogues, including site-specific 13C-labelled ones, for nuclear magnetic resonance conformational analysis. Long-range inter-residue nuclear Overhauser effects unequivocally confirmed the folded conformation of the synthetic glycan hairpin. The capacity to control the 3D shape across the pool of available monosaccharides has the potential to afford more foldamer scaffolds with programmable properties and functions.

Identification of a new DC-SIGN binding pentamannoside epitope within the complex structure of Candida albicans mannan.

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.

Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b.

Sialic acid-binding Ig-like lectin 15 (Siglec-15) is an immune modulator and emerging cancer immunotherapy target. However, limited understanding of its structure and mechanism of action restrains the development of drug candidates that unleash its full therapeutic potential. In this study, we elucidate the crystal structure of Siglec-15 and its binding epitope via co-crystallization with an anti-Siglec-15 blocking antibody. Using saturation transfer-difference nuclear magnetic resonance (STD-NMR) spectroscopy and molecular dynamics simulations, we reveal Siglec-15 binding mode to α(2,3)- and α(2,6)-linked sialic acids and the cancer-associated sialyl-Tn (STn) glycoform. We demonstrate that binding of Siglec-15 to T cells, which lack STn expression, depends on the presence of α(2,3)- and α(2,6)-linked sialoglycans. Furthermore, we identify the leukocyte integrin CD11b as a Siglec-15 binding partner on human T cells. Collectively, our findings provide an integrated understanding of the structural features of Siglec-15 and emphasize glycosylation as a crucial factor in controlling T cell responses.

Highly Diastereoselective Multicomponent Synthesis of Spirocyclopropyl Oxindoles Enabled by Rare-Earth Metal Salts.

The synthesis of polysubstituted spirocyclopropyl oxindoles using a series of rare-earth metal (REM) salts is reported. REMs, in particular Sc(OTf)3, allowed access to the target compounds by a multicomponent reaction with high diastereoselectivity (≤94:6:0:0). Density functional theory calculations on the model reaction are consistent with the observed selectivity and revealed that the special coordinating capabilities and the oxophilicity of the metal are key factors in inducing the formation of one main diastereoisomer.

Blood pH Analysis in Combination with Molecular Medical Tools in Relation to COVID-19 Symptoms.

The global outbreak of SARS-CoV-2/COVID-19 provided the stage to accumulate an enormous biomedical data set and an opportunity as well as a challenge to test new concepts and strategies to combat the pandemic. New research and molecular medical protocols may be deployed in different scientific fields, e.g., glycobiology, nanopharmacology, or nanomedicine. We correlated clinical biomedical data derived from patients in intensive care units with structural biology and biophysical data from NMR and/or CAMM (computer-aided molecular modeling). Consequently, new diagnostic and therapeutic approaches against SARS-CoV-2 were evaluated. Specifically, we tested the suitability of incretin mimetics with one or two pH-sensitive amino acid residues as potential drugs to prevent or cure long-COVID symptoms. Blood pH values in correlation with temperature alterations in patient bodies were of clinical importance. The effects of biophysical parameters such as temperature and pH value variation in relation to physical-chemical membrane properties (e.g., glycosylation state, affinity of certain amino acid sequences to sialic acids as well as other carbohydrate residues and lipid structures) provided helpful hints in identifying a potential Achilles heel against long COVID. In silico CAMM methods and in vitro NMR experiments (including 31P NMR measurements) were applied to analyze the structural behavior of incretin mimetics and SARS-CoV fusion peptides interacting with dodecylphosphocholine (DPC) micelles. These supramolecular complexes were analyzed under physiological conditions by 1H and 31P NMR techniques. We were able to observe characteristic interaction states of incretin mimetics, SARS-CoV fusion peptides and DPC membranes. Novel interaction profiles (indicated, e.g., by 31P NMR signal splitting) were detected. Furthermore, we evaluated GM1 gangliosides and sialic acid-coated silica nanoparticles in complex with DPC micelles in order to create a simple virus host cell membrane model. This is a first step in exploring the structure-function relationship between the SARS-CoV-2 spike protein and incretin mimetics with conserved pH-sensitive histidine residues in their carbohydrate recognition domains as found in galectins. The applied methods were effective in identifying peptide sequences as well as certain carbohydrate moieties with the potential to protect the blood-brain barrier (BBB). These clinically relevant observations on low blood pH values in fatal COVID-19 cases open routes for new therapeutic approaches, especially against long-COVID symptoms.

KR-12 Derivatives Endow Nanocellulose with Antibacterial and Anti-Inflammatory Properties: Role of Conjugation Chemistry.

This work combines the wound-healing-related properties of the host defense peptide KR-12 with wood-derived cellulose nanofibrils (CNFs) to obtain bioactive materials, foreseen as a promising solution to treat chronic wounds. Amine coupling through carbodiimide chemistry, thiol-ene click chemistry, and Cu(I)-catalyzed azide-alkyne cycloaddition were investigated as methods to covalently immobilize KR-12 derivatives onto CNFs. The effects of different coupling chemistries on the bioactivity of the KR12-CNF conjugates were evaluated by assessing their antibacterial activities against Escherichia coli and Staphylococcus aureus. Potential cytotoxic effects and the capacity of the materials to modulate the inflammatory response of lipopolysaccharide (LPS)-stimulated RAW 245.6 macrophages were also investigated. The results show that KR-12 endowed CNFs with antibacterial activity against E. coli and exhibited anti-inflammatory properties and those conjugated by thiol-ene chemistry were the most bioactive. This finding is attributed to a favorable peptide conformation and accessibility (as shown by molecular dynamics simulations), driven by the selective chemistry and length of the linker in the conjugate. The results represent an advancement in the development of CNF-based materials for chronic wound care. This study provides new insights into the effect of the conjugation chemistry on the bioactivity of immobilized host defense peptides, which we believe to be of great value for the use of host defense peptides as therapeutic agents.

Molecular Recognition of Glycan-Bearing Glycomacromolecules Presented at Membrane Surfaces by Lectins: An NMR View.

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.

Development of synthetic, self-adjuvanting, and self-assembling anticancer vaccines based on a minimal saponin adjuvant and the tumor-associated MUC1 antigen.

The overexpression of aberrantly glycosylated tumor-associated mucin-1 (TA-MUC1) in human cancers makes it a major target for the development of anticancer vaccines derived from synthetic MUC1-(glyco)peptide antigens. However, glycopeptide-based subunit vaccines are weakly immunogenic, requiring adjuvants and/or additional immunopotentiating approaches to generate optimal immune responses. Among these strategies, unimolecular self-adjuvanting vaccine constructs that do not need coadministration of adjuvants or conjugation to carrier proteins emerge as a promising but still underexploited approach. Herein, we report the design, synthesis, immune-evaluation in mice, and NMR studies of new, self-adjuvanting and self-assembling vaccines based on our QS-21-derived minimal adjuvant platform covalently linked to TA-MUC1-(glyco)peptide antigens and a peptide helper T-cell epitope. We have developed a modular, chemoselective strategy that harnesses two distal attachment points on the saponin adjuvant to conjugate the respective components in unprotected form and high yields via orthogonal ligations. In mice, only tri-component candidates but not unconjugated or di-component combinations induced significant TA-MUC1-specific IgG antibodies able to recognize the TA-MUC1 on cancer cells. NMR studies revealed the formation of self-assembled aggregates, in which the more hydrophilic TA-MUC1 moiety gets exposed to the solvent, favoring B-cell recognition. While dilution of the di-component saponin-(Tn)MUC1 constructs resulted in partial aggregate disruption, this was not observed for the more stably-organized tri-component candidates. This higher structural stability in solution correlates with their increased immunogenicity and suggests a longer half-life of the construct in physiological media, which together with the enhanced antigen multivalent presentation enabled by the particulate self-assembly, points to this self-adjuvanting tri-component vaccine as a promising synthetic candidate for further development.