Influenza A virus carries hundreds of trimeric hemagglutinin (HA) proteins on its viral envelope that interact with various sialylated glycans on a host cell. This interaction represents a multivalent binding event that is present in all the current receptor binding assays, including those employing viruses or precomplexed HA trimers. To study the nature of such multivalent binding events, we fused a superfolder green fluorescent protein (sfGFP) to the C-terminus of trimeric HA to allow for direct visualization of HA-receptor interactions without the need for additional fluorescent antibodies. The multivalent binding of the HA-sfGFP proteins was studied using glycan arrays and tissue staining. The HA-sfGFP with human-type receptor specificity was able to bind to a glycan array as the free trimer. In contrast, the HA-sfGFP with avian-type receptor specificity required multimerization by antibodies before binding to glycans on the glycan array could be observed. Interestingly, multimerization was not required for binding to tissues. The array data may be explained by the possible bivalent binding mode of a single human-specific HA trimer to complex branched N-glycans, which is not possible for the avian-specific HA due to geometrical constrains of the binding sites. The fact that this specificity pattern changes upon interaction with a cell surface probably represents the enhanced amount of glycan orientations and variable densities versus those on the glycan array.

O-GlcNAcylation is an essential post-translational modification that occurs on nuclear and cytoplasmic proteins, regulating their function in response to cellular stress and altered nutrient availability. O-GlcNAc transferase (OGT) is the enzyme that catalyzes this reaction and represents a potential therapeutic target, whose biological role is still not fully understood. To support this research field, a series of cell-permeable, low-nanomolar OGT inhibitors were recently reported. In this study, we resynthesized the most potent OGT inhibitor of the library, OSMI-4, and we used it to investigate OGT inhibition in different human cell lines. The compound features an ethyl ester moiety that is supposed to be cleaved by carboxylesterases to generate its active metabolite. Our LC-HRMS analysis of the cell lysates shows that this is not always the case and that, even in the cell lines where hydrolysis does not occur, OGT activity is inhibited.

Pseudomonas aeruginosa is a widespread opportunistic pathogen that is capable of colonizing various human tissues and is resistant to many antibiotics. LecA is a galactose binding tetrameric lectin involved in adhesion, infection and biofilm formation. This study reports on the binding characteristics of mono- and divalent (chelating) ligands to LecA using different techniques. These techniques include affinity capillary electrophoresis, bio-layer interferometry, native mass spectrometry and a thermal shift assay. Aspects of focus include: affinity, selectivity, binding kinetics and residence time. The affinity of a divalent ligand was determined to be in the low-nanomolar range for all of the used techniques and with a ligand residence time of approximately 7 h, while no strong binding was seen to related lectin tetramers. Each of the used techniques provides a unique and complementary insight into the chelation based binding mode of the divalent ligand to the LecA tetramer.

Scedosporium apiospermum is an emerging opportunistic fungal pathogen responsible for life-threatening infections in humans. Host-pathogen interactions often implicate lectins that have become therapeutic targets for the development of carbohydrate mimics for antiadhesive therapy. Here, we present the first report on the identification and characterization of a lectin from S. apiospermum named SapL1. SapL1 was found using bioinformatics as a homolog to the conidial surface lectin FleA from Aspergillus fumigatus known to play a role in the adhesion to host glycoconjugates present in human lung epithelium. In our strategy to obtain recombinant SapL1, we discovered the importance of osmolytes to achieve its expression in soluble form in bacteria. Analysis of glycan arrays indicates specificity for fucosylated oligosaccharides as expected. Submicromolar affinity was measured for fucose using isothermal titration calorimetry. We solved SapL1 crystal structure in complex with α-methyl-L-fucoside and analyzed its structural basis for fucose binding. We finally demonstrated that SapL1 binds to bronchial epithelial cells in a fucose-dependent manner. The information gathered here will contribute to the design and development of glycodrugs targeting SapL1.

O-GlcNAcylation is an essential post-translational modification installed by the enzyme O-β-N-acetyl-d-glucosaminyl transferase (OGT). Modulating this enzyme would be extremely valuable to better understand its role in the development of serious human pathologies, such as diabetes and cancer. However, the limited availability of potent and selective inhibitors hinders the validation of this potential therapeutic target. To explore new chemotypes that target the active site of OGT, we performed virtual screening of a large library of commercially available compounds with drug-like properties. We purchased samples of the most promising virtual hits and used enzyme assays to identify authentic leads. Structure-activity relationships of the best identified OGT inhibitor were explored by generating a small library of derivatives. Our best hit displays a novel uridine mimetic scaffold and inhibited the recombinant enzyme with an IC50 value of 7 µM. The current hit represents an excellent starting point for designing and developing a new set of OGT inhibitors that may prove useful for exploring the biology of OGT.

Ion mobility separates molecules in the gas-phase based on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a TOF mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking MS (XL-MS). Cross-linking reagents covalently link amino acids in proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50-70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

The aim of this study was to get insight into the internalization and transport of PEGylat-ed mixed micelles loaded by vitamin K, as mediated by Scavenger Receptor B1 (SR-B1) that is abundantly expressed by intestinal epithelium cells as well as by differentiated Caco-2 cells. Inhibition of SR-B1 reduced endocytosis and transport of vitamin-K-loaded 0%, 30% and 50% PEGylated mixed micelles and decreased colocalization of the micelles with SR-B1. Confocal fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, and surface plasmon resonance (SPR) were used to study the interaction between the mixed micelles of different compositions (varying vitamin K loading and PEG content) and SR-B1. Interaction of PEGylated micelles was independent of the vitamin K content, indicating that the PEG shell prevented vitamin K exposure at the surface of the micelles and binding with the receptor and that the PEG took over the micelles' ability to bind to the receptor. Molecular docking calculations corroborated the dual binding of both vita-min K and PEG with the binding domain of SR-B1. In conclusion, the improved colloidal stability of PEGylated mixed micelles did not compromise their cellular uptake and transport due to the affinity of PEG for SR-B1. SR-B1 is able to interact with PEGylated nanoparticles and mediates their subsequent internalization and transport.

Clostridioides difficile infection is expected to become the most common healthcare-associated infection worldwide. C. difficile-induced pathogenicity is significantly attributed to its enterotoxin, TcdA, which primarily targets Rho-GTPases involved in regulating cytoskeletal and tight junction (TJ) dynamics, thus leading to cytoskeleton breakdown and ultimately increased intestinal permeability. This study investigated whether two non-digestible oligosaccharides (NDOs), alginate (AOS) and chitosan (COS) oligosaccharides, possess antipathogenic and barrier-protective properties against C. difficile bacteria and TcdA toxin, respectively. Both NDOs significantly reduced C. difficile growth, while cell cytotoxicity assays demonstrated that neither COS nor AOS significantly attenuated the TcdA-induced cell death 24 h post-exposure. The challenge of Caco-2 monolayers with increasing TcdA concentrations increased paracellular permeability, as measured by TEER and LY flux assays. In this experimental setup, COS completely abolished, and AOS mitigated, the deleterious effects of TcdA on the monolayer's integrity. These events were not accompanied by alterations in ZO-1 and occludin protein levels; however, immunofluorescence microscopy revealed that both AOS and COS prevented the TcdA-induced occludin mislocalization. Finally, both NDOs accelerated TJ reassembly upon a calcium-switch assay. Overall, this study established the antipathogenic and barrier-protective capacity of AOS and COS against C. difficile and its toxin, TcdA, while revealing their ability to promote TJ reassembly in Caco-2 cells.

Galectin inhibitors are urgently needed to understand the mode of action and druggability of different galectins, but potent and selective agents still evade researchers. Small-sized inhibitors based on thiodigalactoside (TDG) have shown their potential while modifications at their C3 position indicated a strategy to improve selectivity and potency. Considering the role of galectins as glycoprotein traffic police, involved in multivalent bridging interactions, we aimed to create multivalent versions of the potent TDG inhibitors. We herein present for the first time the multivalent attachment of a TDG derivative using bovine serum albumin (BSA) as the scaffold. An efficient synthetic method is presented to obtain a novel type of neoglycosylated proteins loaded with different numbers of TDG moieties. A polyethylene glycol (PEG)-spacer is introduced between the TDG and the protein scaffold maintaining appropriate accessibility for an adequate galectin interaction. The novel conjugates were evaluated in galectin binding and inhibition studies in vitro. The conjugate with a moderate density of 19 conjugated TDGs was identified as one of the most potent multivalent Gal-3 inhibitors so far, with a clear demonstration of the benefit of a multivalent ligand presentation. The described method may facilitate the development of specific galectin inhibitors and their application in biomedical research.

Prebiotic galacto-oligosaccharides (GOS) were shown to support mucosal immune development by enhancing regulatory-type Th1 immune polarization induced by synthetic CpG oligodeoxynucleotides (TLR9 agonist mimicking a bacterial DNA trigger). Epithelial-derived galectin-9 was associated with these immunomodulatory effects. We aimed to identify the most active fractions within GOS based on the degree of polymerization (DP), and to study the immunomodulatory capacities of DP3-sized β-3'galactosyllactose (β-3'GL) using a transwell co-culture model of human intestinal epithelial cells (IEC) and activated peripheral blood mononuclear cells (PBMC). IEC were apically exposed to different DP fractions of GOS or β-3'GL in the presence of CpG, and basolaterally co-cultured with αCD3/CD28-activated PBMC, washed, and incubated in fresh medium for IEC-derived galectin analysis. Only DP3-5 in the presence of CpG enhanced galectin-9 secretion. DP3-sized β-3'GL promoted a regulatory-type Th1 response by increasing IFNγ and IL-10 or galectin-9 concentrations as compared to CpG alone. In addition, IEC-derived galectin-3, -4, and -9 secretion was increased by β-3'GL when combined with CpG. Therefore, the GOS DP3-5 and most effectively DP3-sized β-3'GL supported the immunomodulatory properties induced by CpG by enhancing epithelial-derived galectin secretion, which, in turn, could support mucosal immunity.

SARS-CoV-2 viruses engage ACE2 as a functional receptor with their spike protein. The S1 domain of the spike protein contains a C-terminal receptor binding domain (RBD) and an N-terminal domain (NTD). The NTD of other coronaviruses includes a glycan binding cleft. However, for the SARS-CoV-2 NTD, protein-glycan binding was only observed weakly for sialic acids with highly sensitive methods. Amino acid changes in the NTD of variants of concern (VoC) show antigenic pressure, which can be an indication of NTD-mediated receptor binding. Trimeric NTD proteins of SARS-CoV-2, alpha, beta, delta, and omicron did not reveal a receptor binding capability. Unexpectedly, the SARS-CoV-2 beta subvariant strain (501Y.V2-1) NTD binding to Vero E6 cells was sensitive to sialidase pretreatment. Glycan microarray analyses identified a putative 9-O-acetylated sialic acid as a ligand, which was confirmed by catch-and-release ESI-MS, STD-NMR analyses, and a graphene-based electrochemical sensor. The beta (501Y.V2-1) variant attained an enhanced glycan binding modality in the NTD with specificity toward 9-O-acetylated structures, suggesting a dual-receptor functionality of the SARS-CoV-2 S1 domain, which was quickly selected against. These results indicate that SARS-CoV-2 can probe additional evolutionary space, allowing binding to glycan receptors on the surface of target cells.

beta-lactoglobulin (BLG) is one of the major cow's milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity.

Divalent inhibitors of the neuraminidase enzyme (NA) of the Influenza A virus were synthesized with vastly different spacers. The spacers varied from 14 to 56 atoms and were relatively rigid by way of the building blocks and their connection by CuAAC. As the ligand for these constructs, a Δ4-β-d-glucoronide was used, which can be prepared form N-acetyl glucosamine. This ligand showed good NA inhibitory potency but with room for improvement by multivalency enhancement. The synthesized compounds showed modest potency enhancement in NA activity assays but a sizeable potency increase in a 4-day cytopathic effect assay. The demonstration that the compounds can also inhibit hemagglutinin in addition to NA may be the cause of the enhancement.

Streptococcus suis is a Gram-positive bacterium, which causes sepsis and meningitis in pigs and humans. This review examines the role of known S. suis virulence factors in adhesion and S. suis carbohydrate-based adhesion mechanisms, as well as the inhibition of S. suis adhesion by anti-adhesion compounds in in vitro assays. Carbohydrate-binding specificities of S. suis have been identified, and these studies have shown that many strains recognize Galα1-4Gal-containing oligosaccharides present in host glycolipids. In the era of increasing antibiotic resistance, new means to treat infections are needed. Since microbial adhesion to carbohydrates is important to establish disease, compounds blocking adhesion could be an alternative to antibiotics. The use of oligosaccharides as drugs is generally hampered by their relatively low affinity (micromolar) to compete with multivalent binding to host receptors. However, screening of a library of chemically modified Galα1-4Gal derivatives has identified compounds that inhibit S. suis adhesion in nanomolar range. Also, design of multivalent Galα1-4Gal-containing dendrimers has resulted in a significant increase of the inhibitory potency of the disaccharide. The S. suis adhesin binding to Galα1-4Gal-oligosaccharides, Streptococcal adhesin P (SadP), was recently identified. It has a Galα1-4Gal-binding N-terminal domain and a C-terminal LPNTG-motif for cell wall anchoring. The carbohydrate-binding domain has no homology to E. coli P fimbrial adhesin, which suggests that these Gram-positive and Gram-negative bacterial adhesins recognizing the same receptor have evolved by convergent evolution. SadP adhesin may represent a promising target for the design of anti-adhesion ligands for the prevention and treatment of S. suis infections.

Shiga toxin is an AB5 toxin produced by Shigella species, while related toxins are produced by Shiga toxin-producing Escherichia coli (STEC). Infection by Shigella can lead to bloody diarrhea followed by the often fatal hemolytic uremic syndrome (HUS). In the present paper, we aimed for a simple and effective toxin inhibitor by comparing three classes of carbohydrate-based inhibitors: glycodendrimers, glycopolymers, and oligosaccharides. We observed a clear enhancement in potency for multivalent inhibitors, with the divalent and tetravalent compounds inhibiting in the millimolar and micromolar range, respectively. However, the polymeric inhibitor based on galabiose was the most potent in the series exhibiting nanomolar inhibition. Alginate and chitosan oligosaccharides also inhibit Shiga toxin and may be used as a prophylactic drug during shigella outbreaks.

O-GlcNAcylation is an important post-translational and metabolic process in cells that must be carefully regulated. O-GlcNAc transferase (OGT) is ubiquitously present in cells and is the only enzyme that catalyzes the transfer of O-GlcNAc to proteins. OGT is a promising target in various pathologies such as cancer, immune system diseases, or nervous impairment. In our previous work we identified the 2-oxo-1,2-dihydroquinoline-4-carboxamide derivatives as promising compounds by a fragment-based drug design approach. Herein, we report the extension of this first series with several new fragments. As the most potent fragment, we identified 3b with an IC50 value of 116.0 μM. If compared with the most potent inhibitor of the first series, F20 (IC50 = 117.6 μM), we can conclude that the new fragments did not improve OGT inhibition remarkably. Therefore, F20 was used as the basis for the design of a series of compounds with the elongation toward the O-GlcNAc binding pocket as the free carboxylate allows easy conjugation. Compound 6b with an IC50 value of 144.5 μM showed the most potent OGT inhibition among the elongated compounds, but it loses inhibition potency when compared to the UDP mimetic F20. We therefore assume that the binding of the compounds in the O-GlcNAc binding pocket is likely not crucial for OGT inhibition. Furthermore, evaluation of the compounds with two different assays revealed that some inhibitors most likely interfere with the commercially available UDP-Glo™ glycosyltransferase assay, leading to false positive results. This observation calls for caution, when evaluating UDP mimetic as OGT inhibitors with the UDP-Glo™ glycosyltransferase assay, as misinterpretations can occur.

Divalent ligands were prepared as inhibitors for the adhesion protein of the problematic Pseudomonas aeruginosa pathogen. Bridging two binding sites enables simultaneous binding of two galactose moieties, which strongly enhances binding. An alternating motif of glucose and triazole and aryl groups was shown to have the right mix of rigidity, solubility, and ease of synthesis. Spacers were varied with respect to the core unit as well as the aglycon portions in an attempt to optimize dynamics and enhance interactions with the protein. Affinities of the divalent ligands were measured by ITC, and Kd's as low as 12 nM were determined, notably for a compounds with either a rigid (phenyl) or flexible (butyl) unit at the core. Introducing a phenyl aglycon moiety next to the galactoside ligands on both termini did indeed lead to a higher enthalpy of binding, which was more than compensated by entropic costs. The results are discussed in terms of thermodynamics and theoretical calculations of the expected and observed multivalency effects.

The bacterial pathogens Streptococcus agalactiae (GBS) and Staphylococcus aureus (S. aureus) cause serious infections in humans and animals. The emergence of antibiotic-resistant isolates and bacterial biofilm formation entails the urge of novel treatment strategies. Recently, there is a profound scientific interest in the capabilities of non-digestible oligosaccharides as antimicrobial and anti-biofilm agents as well as adjuvants in antibiotic combination therapies. In this study, we investigated the potential of alginate oligosaccharides (AOS) and chitosan oligosaccharides (COS) as alternative for, or in combination with antibiotic treatment. AOS (2-16%) significantly decreased GBS V growth by determining the minimum inhibitory concentration. Both AOS (8 and 16%) and COS (2-16%) were able to prevent biofilm formation by S. aureus wood 46. A checkerboard biofilm formation assay demonstrated a synergistic effect of COS and clindamycin on the S. aureus biofilm formation, while AOS (2 and 4%) were found to sensitize GBS V to trimethoprim. In conclusion, AOS and COS affect the growth of GBS V and S. aureus wood 46 and can function as anti-biofilm agents. The promising effects of AOS and COS in combination with different antibiotics may offer new opportunities to combat antimicrobial resistance.

A novel miniaturized sensor for electrochemical detection that contains graphene- and gold nanoparticles was functionalized with proteins. Using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) it was possible to observe and quantify interactions of molecules with these proteins. The protein binders included carbohydrate ligands as small as carbohydrates up to COVID-19 spike protein variants engaged in protein-protein interactions. The system uses off-the-shelf sensors combined with an affordable potentiostat and yet is sensitive enough for small ligand binding.