Exploring galectin interactions with human milk oligosaccharides and blood group antigens identifies BGA6 as a functional galectin-4 ligand.

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.

Proceedings of workshop: "Neuroglycoproteins in health and disease", INNOGLY cost action.

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.

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.

Targeting Galectins With Glycomimetics.

Among glycan-binding proteins, galectins, ß-galactoside-binding lectins, exhibit relevant biological roles and are implicated in many diseases, such as cancer and inflammation. Their involvement in crucial pathologies makes them interesting targets for drug discovery. In this review, we gather the last approaches toward the specific design of glycomimetics as potential drugs against galectins. Different approaches, either using specific glycomimetic molecules decorated with key functional groups or employing multivalent presentations of lactose and N-acetyl lactosamine analogs, have provided promising results for binding and modulating different galectins. The review highlights the results obtained with these approximations, from the employment of S-glycosyl compounds to peptidomimetics and multivalent glycopolymers, mostly employed to recognize and/or detect hGal-1 and hGal-3.

Fluorinated carbohydrates as chemical probes for molecular recognition studies. Current status and perspectives.

This review provides an extensive summary of the effects of carbohydrate fluorination with regard to changes in physical, chemical and biological properties with respect to regular saccharides. The specific structural, conformational, stability, reactivity and interaction features of fluorinated sugars are described, as well as their applications as probes and in chemical biology.

Molecular Recognition in C-Type Lectins: The Cases of DC-SIGN, Langerin, MGL, and L-Sectin.

Carbohydrates play a pivotal role in intercellular communication processes. In particular, glycan antigens are key for sustaining homeostasis, helping leukocytes to distinguish damaged tissues and invading pathogens from healthy tissues. From a structural perspective, this cross-talk is fairly complex, and multiple membrane proteins guide these recognition processes, including lectins and Toll-like receptors. Since the beginning of this century, lectins have become potential targets for therapeutics for controlling and/or avoiding the progression of pathologies derived from an incorrect immune outcome, including infectious processes, cancer, or autoimmune diseases. Therefore, a detailed knowledge of these receptors is mandatory for the development of specific treatments. In this review, we summarize the current knowledge about four key C-type lectins whose importance has been steadily growing in recent years, focusing in particular on how glycan recognition takes place at the molecular level, but also looking at recent progresses in the quest for therapeutics.

Glycans in drug discovery.

Glycans are key players in many biological processes. They are essential for protein folding and stability and act as recognition elements in cell-cell and cell-matrix interactions. Thus, being at the heart of medically relevant biological processes, glycans have come onto the scene and are considered hot spots for biomedical intervention. The progress in biophysical techniques allowing access to an increasing molecular and structural understanding of these processes has led to the development of effective therapeutics. Indeed, strategies aimed at designing glycomimetics able to block specific lectin-carbohydrate interactions, carbohydrate-based vaccines mimicking self- and non-self-antigens as well as the exploitation of the therapeutic potential of glycosylated antibodies are being pursued. In this mini-review the most prominent contributions concerning recurrent diseases are highlighted, including bacterial and viral infections, cancer or immune-related pathologies, which certainly show the great promise of carbohydrates in drug discovery.

Environmental Effects Determine the Structure of Potential ß-Amino Acid Based Foldamers.

This work shows that hybrid peptides formed by alternating trans-2-aminocyclopentanecarboxylic acid (trans-ACPC) and trans-2-aminocyclohexanecarboxylic acid (trans-ACHC) do not fold in the solvents typically used in the study of their homo-oligomers. Only when the peptides are assayed in SDS micelles are the predicted helical structures obtained. This indicates that the environment could play an equally important role (as the backbone stereochemistry) in determining their fold, possibly by providing a sequestered environment.

Acetylated Trivalent Mannobioses: Chemical Modification, Structural Elucidation, and Biological Evaluation.

People suffering from allergies can be treated with repeated injections of increasing amounts of a specific allergen. This type of specific immunotherapy is currently the only way to treat the underlying pathological immune response associated with an allergy. The approach can afford long-lasting protection, but the process takes 3-5 years, can produce allergic reactions, and in severe cases treatment is often aborted due to anaphylaxis. However, treatment can be optimized with the use of specific adjuvants that modify the immune response, its duration, and that increase the production of the correct type of antibodies. In the pursuit of such adjuvants, two new trivalent acetylated ß-(1→2)-linked mannobioses based on a previously discovered lead molecule were prepared. The new molecules, along with the previously developed lead, were investigated by rigorous NMR and molecular modeling experiments in order to elucidate their behavior and preferred conformations in solution. Furthermore, the molecules were subjected to a biological investigation in which their immunostimulatory properties were evaluated by assessing their effect on the production of TH 2-type cytokine interleukin-4 (IL-4) and Treg pro-inflammatory cytokine tumor necrosis factor (TNF). Treatment of peripheral mononuclear blood cell cultures from patients suffering from birch allergy with birch allergen Bet v induced a strong IL-4 response, whereas the same treatment together with the trivalent acetylated mannobioses caused significant suppression of the induced IL-4.

Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides.

A shift to short-chain glycans is an observed change in mucin-type O-glycosylation in premalignant and malignant epithelia. Given the evidence that human galectin-3 can interact with mucins and also weakly with free tumor-associated Thomsen-Friedenreich (TF) antigen (CD176), the study of its interaction with MUC1 (glyco)peptides is of biomedical relevance. Glycosylated MUC1 fragments that carry the TF antigen attached through either Thr or Ser side chains were synthesized using standard Fmoc-based automated solid-phase peptide chemistry. The dissociation constants (Kd) for interaction of galectin-3 and the glycosylated MUC1 fragments measured by isothermal titration calorimetry decreased up to 10 times in comparison to that of the free TF disaccharide. No binding was observed for the nonglycosylated control version of the MUC1 peptide. The most notable feature of the binding of MUC1 glycopeptides to galectin-3 was a shift from a favorable enthalpy to an entropy-driven binding process. The comparatively diminished enthalpy contribution to the free energy (ΔG) was compensated by a considerable gain in the entropic term. (1)H-(15)N heteronuclear single-quantum coherence spectroscopy nuclear magnetic resonance data reveal contact at the canonical site mainly by the glycan moiety of the MUC1 glycopeptide. Ligand-dependent differences in binding affinities were also confirmed by a novel assay for screening of low-affinity glycan-lectin interactions based on AlphaScreen technology. Another key finding is that the glycosylated MUC1 peptides exhibited activity in a concentration-dependent manner in cell-based assays revealing selectivity among human galectins. Thus, the presentation of this tumor-associated carbohydrate ligand by the natural peptide scaffold enhances its affinity, highlighting the significance of model studies of human lectins with synthetic glycopeptides.

The crystal structure and small-angle X-ray analysis of CsdL/TcdA reveal a new tRNA binding motif in the MoeB/E1 superfamily.

Cyclic N6-threonylcarbamoyladenosine ('cyclic t6A', ct(6)A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct(6)A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct(6)A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t6A to form ct(6)A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K+ and Na+ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t6A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct(6)A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.

Algal lectin binding to core (α1-6) fucosylated N-glycans: structural basis for specificity and production of recombinant protein.

We determined the specificity of BTL, a lectin from the red marine alga Bryothamnion triquetrum, toward fucosylated oligosaccharides. BTL showed a strict specificity for the core α1,6-fucosylation, which is an important marker for cancerogenesis and quality control of therapeutical antibodies. The double fucosylation α1,6 and α1,3 was also recognized, but the binding was totally abolished in the sole presence of the α1,3-fucosylation. A more detailed analysis of the specificity of BTL showed a preference for bi- and tri-antennary nonbisected N-glycans. Sialylation or fucosylation at the nonreducing end of N-glycans did not affect the recognition by the lectin. BTL displayed a strong affinity for a core α1,6-fucosylated octasaccharide with a Kd of 12 µM by titration microcalorimetry. The structural characterization of the interaction between BTL and the octasaccharide was obtained by STD-NMR. It demonstrated an extended epitope for recognition that includes the fucose residue, the distal GlcNAc and one mannose residue. Recombinant rBTL was obtained in Escherichia coli and characterized. Its binding properties for carbohydrates were studied using hemagglutination tests and glycan array analysis. rBTL was able to agglutinate rabbit erythrocytes with strong hemagglutination activity only after treatment with papain and trypsin, indicating that its ligands were not directly accessible at the cell surface. The hemagglutinating properties of rBTL confirm the correct folding and functional state of the protein. The results show BTL as a potent candidate for cancer diagnosis and as a reagent for the preparation and quality control of antibodies lacking core α1,6-fucosylated N-glycans.

Targeting matrix metalloproteinases: design of a bifunctional inhibitor for presentation by tumour-associated galectins.

A new strategy to exploit galectin presence to target matrix metalloproteinases (MMPs) is presented. A bifunctional conjugate with lactose and an inhibitor for MMPs is able to bind MMP and Gal-3 simultaneously. This compound might allow the lectin to attract the MMP inhibitor to the tumour site and to block protumoural activities of the lectin at the same time.

Studies related to Norway spruce galactoglucomannans: chemical synthesis, conformation analysis, NMR spectroscopic characterization, and molecular recognition of model compounds.

Galactoglucomannan (GGM) is a polysaccharide mainly consisting of mannose, glucose, and galactose. GGM is the most abundant hemicellulose in the Norway spruce (Picea abies), but is also found in the cell wall of flax seeds, tobacco plants, and kiwifruit. Although several applications for GGM polysaccharides have been developed in pulp and paper manufacturing and the food and medical industries, attempts to synthesize and study distinct fragments of this polysaccharide have not been reported previously. Herein, the synthesis of one of the core trisaccharide units of GGM together with a less-abundant tetrasaccharide fragment is described. In addition, detailed NMR spectroscopic characterization of the model compounds, comparison of the spectral data with natural GGM, investigation of the acetyl-group migration phenomena that takes place in the polysaccharide by using small model compounds, and a binding study between the tetrasaccharide model fragment and a galactose-binding protein (the toxin viscumin) are reported.

Molecular recognition of the Thomsen-Friedenreich antigen-threonine conjugate by adhesion/growth regulatory galectin-3: nuclear magnetic resonance studies and molecular dynamics simulations.

Nuclear magnetic resonance (NMR) spectroscopy and molecular modeling methods have been strategically combined to elucidate the molecular recognition features of the binding of threonine O-linked Thomsen-Friedenreich (TF) antigen to chimera-type avian galectin-3 (CG-3). Saturation transfer difference (STD) NMR experiments revealed the highest intensities for the H4 protons of both the ß-D-Galp and α-D-GalpNAc moieties, with 100 and 71% of relative STD, respectively. The methyl protons of the threonine residue exhibited a small STD effect, <15%, indicating that the interaction of the amino acid with the protein is rather transient. Two-dimensional transferred nuclear Overhauser effect spectroscopy NMR experiments and molecular modeling suggested some differences in conformer populations between the free and bound states. A dynamic binding mode for the TF antigen-CG-3 complex consisting of two poses has been deduced. In one pose, intermolecular interactions were formed between the terminal threonine residue and the receptor. In the second pose, intermolecular interactions involved the internal GalpNAc. The difference in the trend of some shifts in the heteronuclear single-quantum coherence titration spectra indicates some disparities in the binding interactions of CG-3 with lactose and TF antigen. The results obtained from this model of the avian orthologue of human galectin-3 will allow detailed interspecies comparison to give sequence deviations in phylogeny a structural and functional meaning. Moreover, the results indicate that the peptide scaffold presenting TF antigen could be relevant for binding and thus provides a possible route for the design of galectin-3 inhibitors with improved affinity and selectivity.

Insights into the dynamics and molecular recognition features of glycopeptides by protein receptors: the 3D solution structure of hevein bound to the trisaccharide core of N-glycoproteins.

Protein-carbohydrate interactions are at the heart of a variety of essential molecular recognition events. Hevein, a model lectin related to the superantigen family, recognizes the trisaccharide core of N-glycoproteins (1). A combined approach of NMR spectroscopy and molecular modeling has permitted us to demonstrate that an Asn-linked Man(GlcNAc)(2) (2) is bound with even higher affinity than (GlcNAc)(3). The molecular recognition process entails conformational selection of only one of the possibilities existing for chitooligosaccharides. The deduced 3D structure of the hevein/2 complex permits the extension of polypeptide chains from the Asn moiety of 2, as well as glycosylation at Man O-3 and Man O-6 of the terminal sugar. Given the ubiquity of the Man(GlcNAc)(2) core in all mammalian N-glycoproteins, the basic recognition mode presented herein might be extended to a variety of systems with biomedical importance.

New cytotoxic cembranes from the sea pen Gyrophyllum sibogae.

Two new cembrane-type diterpenoids have been isolated from the 2-propanol extract of the sea pen Gyrophyllum sibogae collected in South Africa: 7,8-dihydroflabellatene A (1) and 7,8-dihydroflabellatene B (2). Their structures were determined on the basis of detailed spectroscopic analysis and by single-crystal X-ray analysis of the major metabolite 1, which showed strong in vitro cytotoxicity against a panel of 13 tumor cell lines.