Sulfated Glycans Research Articles

Removable Dyes—The Missing Link for In-Depth N-Glycan Analysis via Multi-Method Approaches

As the roles of glycans in health and disease continue to be unraveled, it is becoming apparent that glycans’ immense complexity cannot be ignored. To fully delineate glycan structures, we developed an integrative approach combining a set of cost-effective, widespread, and easy-to-handle analytical methods. The key feature of our workflow is the exploitation of a removable fluorescent label—exemplified by 9-fluorenylmethyl chloroformate (Fmoc)—to bridge the gap between diverse glycoanalytical methods, especially multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Through the detailed structural analysis of selected, dauntingly complex N-glycans from chicken ovalbumin, horse serum, and bovine transferrin, we illustrate the capabilities of the presented strategy. Moreover, this approach “visualizes” N-glycans that have been difficult to identify thus far—such as the sulfated glycans on human immunoglobulin A—including minute changes in glycan structures, potentially providing useful new targets for biomarker discovery.

Glycoblotting enables seamless and straightforward workflow for MALDI-TOF/MS-based sulphoglycomics of N- and O-glycans.

SulfatedN-andO-glycans exist in trace levels which are challenging to detect, especially when abundant neutral and sialylated glycans are present. Current matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based sulfoglycomics approaches effectively utilize permethylation to discriminate sulfated glycans from sialyl-glycans. And a charge-based separation to isolate the sulfated glycans from the rest of the permethylated neutral and sialyl-glycans. However, these approaches suffer from concomitant sample losses during cleanup steps. Herein, we describe Glycoblotting as a straightforward complementary method with seamless glycan purification, enrichment, methylation, and labeling on a single platform to address sulfated glycan enrichment, sialic acid methylation, and sample loss. Glycoblottings' on-bead chemoselective ligation of reducing sugars with hydrazide showed excellent recovery of sulfated glycans, allowing the detection of more sulfated glycan species. On-bead methyl esterification of sialic acid using 3-methyl-1-p-tolyltriazene (MTT) effectively discriminates sulfated glycans from sialyl-glycans. Furthermore, we have shown that using MTT as a methylating agent allowed us to simultaneously detect and differentiate sulfate from phosphate groups in isobaricN-glycan species. We believe that Glycoblotting will contribute significantly to the MALDI-TOF MS-based Sulphoglycomics workflow.

The Sea Cucumber Thyonella gemmata Contains a Low Anticoagulant Sulfated Fucan with High Anti-SARS-CoV-2 Actions against Wild-Type and Delta Variants.

In this work, we isolated two new sulfated glycans from the body wall of the sea cucumber Thyonella gemmata: one fucosylated chondroitin sulfate (TgFucCS) (17.5 ± 3.5% kDa) and one sulfated fucan (TgSF) (383.3 ± 2.1% kDa). NMR results showed the TgFucCS backbone composed of [→3)-β-N-acetylgalactosamine-(1→4)-β-glucuronic acid-(1→] with 70% 4-sulfated and 30% 4,6-disulfated GalNAc units and one-third of the GlcA units decorated at the C3 position with branching α-fucose (Fuc) units either 4-sulfated (65%) or 2,4-disulfated (35%) and the TgSF structure composed of a tetrasaccharide repeating unit of [→3)-α-Fuc2,4S-(1→2)-α-Fuc4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→]n. Inhibitory properties of TgFucCS and TgSF were investigated using SARS-CoV-2 pseudovirus coated with S-proteins of the wild-type (Wuhan-Hu-1) or the delta (B.1.617.2) strains and in four different anticoagulant assays, comparatively with unfractionated heparin. Molecular binding to coagulation (co)-factors and S-proteins was investigated by competitive surface plasmon resonance spectroscopy. Among the two sulfated glycans tested, TgSF showed significant anti-SARS-CoV-2 activity against both strains together with low anticoagulant properties, indicating a good candidate for future studies in drug development.

Oxidative Release of O-Glycans under Neutral Conditions for Analysis of Glycoconjugates Having Base-Sensitive Substituents.

Protein O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of O-glycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on β-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, well-defined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of O-glycans suitable for glycomics. Treatment of O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by β-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of O-glycans from bovine submaxillary mucin, which identified mono- and di-O-acetylated sialoglycans as well as previously undetected tri-O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative O-glycan release under neutral conditions.

The αTSR Domain of Plasmodium Circumsporozoite Protein Bound Heparan Sulfates and Elicited High Titers of Sporozoite Binding Antibody After Displayed by Nanoparticles.

Malaria is a devastating infectious illness caused by protozoan Plasmodium parasites. The circumsporozoite protein (CSP) on Plasmodium sporozoites binds heparan sulfate proteoglycan (HSPG) receptors for liver invasion, a critical step for prophylactic and therapeutic interventions. In this study, we characterized the αTSR domain that covers region III and the thrombospondin type-I repeat (TSR) of the CSP using various biochemical, glycobiological, bioengineering, and immunological approaches. We found for the first time that the αTSR bound heparan sulfate (HS) glycans through support by a fused protein, indicating that the αTSR is a key functional domain and thus a vaccine target. When the αTSR was fused to the S domain of norovirus VP1, the fusion protein self-assembled into uniform S60-αTSR nanoparticles. Three-dimensional structure reconstruction revealed that each nanoparticle consists of an S60 nanoparticle core and 60 surface displayed αTSR antigens. The nanoparticle displayed αTSRs retained the binding function to HS glycans, indicating that they maintained authentic conformations. Both tagged and tag-free S60-αTSR nanoparticles were produced via the Escherichia coli system at high yield by scalable approaches. They are highly immunogenic in mice, eliciting high titers of αTSR-specific antibody that bound specifically to the CSPs of Plasmodium falciparum sporozoites at high titer. Our data demonstrated that the αTSR is an important functional domain of the CSP. The S60-αTSR nanoparticle displaying multiple αTSR antigens is a promising vaccine candidate potentially against attachment and infection of Plasmodium parasites.

Structural requirements of Holothuria floridana fucosylated chondroitin sulfate oligosaccharides in anti-SARS-CoV-2 and anticoagulant activities.

Fucosylated chondroitin sulfate (FucCS) is a unique glycosaminoglycan found primarily in sea cucumbers. This marine sulfated glycan is composed of a chondroitin sulfate backbone decorated with fucosyl branches attached to the glucuronic acid. FucCS exhibits potential biological actions including inhibition of blood clotting and severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection. These biological effects have been attributed to certain structural features, including molecular weight (MW), and/or those related to fucosylation, such as degrees of fucosyl branches, sulfation patterns and contents. In a previous work, we were able to generate oligosaccharides of the FucCS from Pentacta pygmaea (PpFucCS) with reduced anticoagulant effect but still retaining significant anti-SARS-CoV-2 activity against the delta strain. In this work, we extended our study to the FucCS extracted from the species Holothuria floridana (HfFucCS). The oligosaccharides were prepared by free-radical depolymerization of the HfFucCS via copper-based Fenton reaction. One-dimensional 1H nuclear magnetic resonance spectra were employed in structural analysis. Activated partial thromboplastin time and assays using protease (factors Xa and IIa) and serine protease inhibitors (antithrombin, and heparin cofactor II) in the presence of the sulfated carbohydrates were used to monitor anticoagulation. Anti-SARS-CoV-2 effects were measured using the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 wild-type and delta variant spike (S)-proteins. Furthermore, the cytotoxicity of native HfFucCS and its oligosaccharides was also assessed. Like for PpFucCS, we were able to generate a HfFucCS oligosaccharide fraction devoid of high anticoagulant effect but still retaining considerable anti-SARS-CoV-2 actions against both variants. However, compared to the oligosaccharide fraction derived from PpFucCS, the average MW of the shortest active HfFucCS oligosaccharide fraction was significantly lower. This finding suggests that the specific structural feature in HfFucCS, the branching 3,4-di-sulfated fucoses together with the backbone 4,6-di-sulfated N-acetylgalactosamines, is relevant for the anti-SARS-CoV-2 activity of FucCS molecules.

SPR Sensor-Based Analysis of the Inhibition of Marine Sulfated Glycans on Interactions between Monkeypox Virus Proteins and Glycosaminoglycans.

Sulfated glycans from marine organisms are excellent sources of naturally occurring glycosaminoglycan (GAG) mimetics that demonstrate therapeutic activities, such as antiviral/microbial infection, anticoagulant, anticancer, and anti-inflammation activities. Many viruses use the heparan sulfate (HS) GAG on the surface of host cells as co-receptors for attachment and initiating cell entry. Therefore, virion-HS interactions have been targeted to develop broad-spectrum antiviral therapeutics. Here we report the potential anti-monkeypox virus (MPXV) activities of eight defined marine sulfated glycans, three fucosylated chondroitin sulfates, and three sulfated fucans extracted from the sea cucumber species Isostichopus badionotus, Holothuria floridana, and Pentacta pygmaea, and the sea urchin Lytechinus variegatus, as well as two chemically desulfated derivatives. The inhibitions of these marine sulfated glycans on MPXV A29 and A35 protein-heparin interactions were evaluated using surface plasmon resonance (SPR). These results demonstrated that the viral surface proteins of MPXV A29 and A35 bound to heparin, which is a highly sulfated HS, and sulfated glycans from sea cucumbers showed strong inhibition of MPXV A29 and A35 interactions. The study of molecular interactions between viral proteins and host cell GAGs is important in developing therapeutics for the prevention and treatment of MPXV.

Functions and specificity of bacterial carbohydrate sulfatases targeting host glycans.

Sulfated host glycans (mucin O-glycans and glycosaminoglycans [GAGs]) are critical nutrient sources and colonisation factors for Bacteroidetes of the human gut microbiota (HGM); a complex ecosystem comprising essential microorganisms that coevolved with humans to serve important roles in pathogen protection, immune signalling, and host nutrition. Carbohydrate sulfatases are essential enzymes to access sulfated host glycans and are capable of exquisite regio- and stereo-selective substrate recognition. In these enzymes, the common recognition features of each subfamily are correlated with their genomic and environmental context. The exo-acting carbohydrate sulfatases are attractive drug targets amenable to small-molecule screening and subsequent engineering, and their high specificity will help elucidate the role of glycan sulfation in health and disease. Inhibition of carbohydrate sulfatases provides potential routes to control Bacteroidetes growth and to explore the influence of host glycan metabolism by Bacteroidetes on the HGM ecosystem. The roles of carbohydrate sulfatases from the HGM organism Bacteroides thetaiotaomicron and the soil isolated Pedobacter heparinus (P. heparinus) in sulfated host glycan metabolism are examined and contrasted, and the structural features underpinning glycan recognition and specificity explored.

Inhibition of Cytomegalovirus by Pentacta pygmaea Fucosylated Chondroitin Sulfate Depends on Its Molecular Weight.

Many viruses attach to host cells by first interacting with cell surface proteoglycans containing heparan sulfate (HS) glycosaminoglycan chains and then by engaging with specific receptor, resulting in virus entry. In this project, HS-virus interactions were targeted by a new fucosylated chondroitin sulfate from the sea cucumber Pentacta pygmaea (PpFucCS) in order to block human cytomegalovirus (HCMV) entry into cells. Human foreskin fibroblasts were infected with HCMV in the presence of PpFucCS and its low molecular weight (LMW) fractions and the virus yield at five days post-infection was assessed. The virus attachment and entry into the cells were visualized by labeling the purified virus particles with a self-quenching fluorophore octadecyl rhodamine B (R18). The native PpFucCS exhibited potent inhibitory activity against HCMV specifically blocking virus entry into the cell and the inhibitory activities of the LMW PpFucCS derivatives were proportional to their chain lengths. PpFucCS and the derived oligosaccharides did not exhibit any significant cytotoxicity; moreover, they protected the infected cells from virus-induced lytic cell death. In conclusion, PpFucCS inhibits the entry of HCMV into cells and the high MW of this carbohydrate is a key structural element to achieve the maximal anti-viral effect. This new marine sulfated glycan can be developed into a potential prophylactic and therapeutic antiviral agent against HCMV infection.

Antiviral activity of marine sulfated glycans against pathogenic human coronaviruses

Great interest exists towards the discovery and development of broad-spectrum antivirals. This occurs due to the frequent emergence of new viruses which can also eventually lead to pandemics. A reasonable and efficient strategy to develop new broad-spectrum antivirals relies on targeting a common molecular player of various viruses. Heparan sulfate is a sulfated glycosaminoglycan present on the surface of cells which plays a key role as co-receptor in many virus infections. In previous work, marine sulfated glycans (MSGs) were identified as having antiviral activities. Their mechanism of action relies primarily on competitive inhibition of virion binding to heparan sulfate, preventing virus attachment to the cell surface prior to entry. In the current work we used pseudotyped lentivirus particles to investigate in a comparative fashion the inhibitory properties of five structurally defined MSGs against SARS-CoV-1, SARS-CoV-2, MERS-CoV, and influenza A virus (IAV). MSGs include the disaccharide-repeating sulfated galactan from the red alga Botryocladia occidentalis, the tetrasaccharide-repeating sulfated fucans from the sea urchin Lytechinus variegatus and from the sea cucumber Isostichopus badionotus, and the two marine fucosylated chondroitin sulfates from the sea cucumbers I. badionotus and Pentacta pygmaea. Results indicate specificity of action against SARS-CoV-1 and SARS-CoV-2. Curiously, the MSGs showed decreased inhibitory potencies against MERS-CoV and negligible action against IAV. Among the five MSGs, the two sulfated fucans here studied deserve further attention since they have the lowest anticoagulant effects but still present potent and selective antiviral properties.

Distinct sulfated glycans expressed in intrahepatic cholangiocarcinoma: a potential target for new therapy.

Cholangiocytes exhibit morphological and functional heterogeneity, depending on their anatomical localization; however, like other ductal organs, their mucosal surface is covered with mucin, which functions to prevent the entry of foreign substances, lubricateand prevent clogging by bile. Recently, the authors discovered that distinct sulfated glycans recognized by a series of antisulfated glycan antibodies are expressed not only in normal intrahepatic bile ducts but also in intrahepatic cholangiocarcinoma (iCCA). In this review, the authors first describe the anatomy of bile ducts and the biochemical characteristics of bile-duct-associated mucins, and then describe differences in structure and expression patterns of these sulfated glycans in physiological and pathological conditions. Finally, potential therapeutic strategies foriCCAusing antisulfated glycan antibodies are discussed.

Structural and evolutionary insights into the multidomain galectin from the red abalone Haliotis rufescens with specificity for sulfated glycans

Galectins are an evolutionarily ancient family of lectins characterized by their affinity for β-galactosides and a conserved binding site in the carbohydrate recognition domain (CRD). These lectins are involved in multiple physiological functions, including the recognition of glycans on the surface of viruses and bacteria. This feature supports their role in innate immune responses in marine mollusks. Here, we identified and characterized a galectin, from the mollusk Haliotis rufescens (named HrGal), with four CRDs that belong to the tandem-repeat type. HrGal was purified by affinity chromatography in a galactose-agarose resin and exhibited a molecular mass of 64.11 kDa determined by MALDI-TOF mass spectrometry. The identity of HrGal was verified by sequencing, confirming that it is a 555 amino acid protein with a mass of 63.86 kDa. This protein corresponds to a galectin reported in GenBank with accession number AHX26603. HrGal is stable in the presence of urea, reducing agents, and ions such as Cu2+ and Zn2+. The recombinant galectin (rHrGal) was purified from inclusion bodies in the presence of these ions. A theoretical model obtained with the AlphaFold server exhibits four non-identical CRDs, with a β sandwich folding and the representative motifs for binding β-galactosides. This allows us to classify HrGal within the tandem repeat galectin family. On the basis of a phylogenetic analysis, we found that the mollusk sequences form a monophyletic group of tetradomain galectins unrelated to vertebrate galectins. HrGal showed specificity for galactosides and glucosides but only the sulfated sugars heparin and ι-carrageenan inhibited its hemagglutinating activity with a minimum inhibitory concentration of 4 mM and 6.25 X 10−5% respectively. The position of the sulfate groups seemed crucial for binding, both by carrageenans and heparin.

Computationally guided conversion of the specificity of E-selectin to mimic that of Siglec-8

Sulfated glycans have been found to be associated with various diseases and therefore have significant potential in molecular pathology as biomarkers. Although lectins are useful reagents for detecting glycans, there is a paucity of sulfate-recognizing lectins, and those that exist, such as from Maackia amurensis, display mixed specificities. Recombinant lectin engineering offers an emerging tool for creating novel glycan recognition by altering and/or enhancing endogenous specificities. The present study demonstrated the use of computational approaches in the engineering of a mutated form of E-selectin that displayed highly specific recognition of 6'-sulfo-sialyl Lewis X (6'-sulfo-sLex), with negligible binding to its endogenous nonsulfated ligand, sLex. This new specificity mimics that of the unrelated protein Siglec-8, for which 6'-sulfo-sLex is its preferred ligand. Molecular dynamics simulations and energy calculations predicted that two point mutations (E92A/E107A) would be required to stabilize binding to the sulfated oligosaccharide with E-selectin. In addition to eliminating putative repulsions between the negatively charged side chains and the sulfate moiety, the mutations also abolished favorable interactions with the endogenous ligand. Glycan microarray screening of the recombinantly expressed proteins confirmed the predicted specificity change but also identified the introduction of unexpected affinity for the unfucosylated form of 6'-sulfo-sLex (6'-sulfo-sLacNAc). Three key requirements were demonstrated in this case for engineering specificity for sulfated oligosaccharide: 1) removal of unfavorable interactions with the 6'-sulfate, 2) introduction of favorable interactions for the sulfate, and 3) removal of favorable interactions with the endogenous ligand.

Interactions of marine sulfated glycans with antithrombin and platelet factor 4.

The molecular interactions of sulfated glycans, such as heparin, with antithrombin (AT) and platelet factor 4 (PF4) are essential for certain biological events such as anticoagulation and heparin induced thrombocytopenia (HIT). In this study, a library including 84 sulfated glycans (polymers and oligomers) extracted from marine algae along with several animal-originated polysaccharides were subjected to a structure-activity relationship (SAR) study regarding their specific molecular interactions with AT and PF4 using surface plasmon resonance. In this SAR study, multiple characteristics were considered including different algal species, different methods of extraction, molecular weight, monosaccharide composition, sulfate content and pattern and branching vs. linear chains. These factors were found to influence the binding affinity of the studied glycans with AT. Many polysaccharides showed stronger binding than the low molecular weight heparin (e.g., enoxaparin). Fourteen polysaccharides with strong AT-binding affinities were selected to further investigate their binding affinity with PF4. Eleven of these polysaccharides showed strong binding to PF4. It was observed that the types of monosaccharides, molecular weight and branching are not very essential particularly when these polysaccharides are oversulfated. The sulfation levels and sulfation patterns are, on the other hand, the primary contribution to strong AT and PF4 interaction.

A glycan-based approach to cell characterization and isolation: Hematopoiesis as a paradigm.

Cell surfaces display a wide array of molecules that confer identity. While flow cytometry and cluster of differentiation (CD) markers have revolutionized cell characterization and purification, functionally heterogeneous cellular subtypes remain unresolvable by the CD marker system alone. Using hematopoietic lineages as a paradigm, we leverage the extraordinary molecular diversity of heparan sulfate (HS) glycans to establish cellular "glycotypes" by utilizing a panel of anti-HS single-chain variable fragment antibodies (scFvs). Prospective sorting with anti-HS scFvs identifies functionally distinct glycotypes within heterogeneous pools of mouse and human hematopoietic progenitor cells and enables further stratification of immunophenotypically pure megakaryocyte-erythrocyte progenitors. This stratification correlates with expression of a heptad of HS-related genes that is reflective of the HS epitope recognized by specific anti-HS scFvs. While we show that HS glycotyping provides an orthogonal set of tools for resolution of hematopoietic lineages, we anticipate broad utility of this approach in defining and isolating novel, viable cell types across diverse tissues and species.

Specificity of viscumin revised. As probed with a printed glycan array

Viscumin, a lectin used in anti-cancer therapy, was originally considered as βGal recognizing protein; later, an ability to bind 6′-sialyl N-acetyllactosamine (6′SLN) terminated gangliosides was found. Here we probed viscumin with a printed glycan array (PGA) containing a large number of mammalian sulfated glycans, and found a strong binding to glycans with 6-O-SuGal moiety as lactose, N-acetyllactosamine (LN), di-N-acetyllactosamine (LacdiNAc), and even 6-O-SuGalNAcα (but not SiaTn). Also, the ability to bind some of αGal terminated glycans, including Galα1-3Galβ1-4GlcNAc, was observed. Unexpectedly, only weak interaction was detected with parent neutral β-galactosides including LN-LN-LN and branched (LN)2LN oligolactosamines; in the light of these data, one should not confidently classify viscumin as a β-galactoside-binding lectin. Carrying out PGA in the presence of neutral or sulfated/sialylated glycan, together with sequential elution from lactose-sepharose and consideration of the protein structure, lead to the conclusion that two glycan-binding sites of viscumin have different specificities, one of which prefers charged sulfated and sialylated moieties.

Complementary Role of GlcNAc6ST2 and GlcNAc6ST3 in Synthesis of CL40-Reactive Sialylated and Sulfated Glycans in the Mouse Pleural Mesothelium.

Sialyl 6-sulfo Lewis X (6-sulfo sLeX) and its derivative sialyl 6-sulfo N-acetyllactosamine (LacNAc) are sialylated and sulfated glycans of sialomucins found in the high endothelial venules (HEVs) of secondary lymphoid organs. A component of 6-sulfo sLeX present in the core 1-extended O-linked glycans detected by the MECA-79 antibody was previously shown to exist in the lymphoid aggregate vasculature and bronchial mucosa of allergic and asthmatic lungs. The components of 6-sulfo sLeX in pulmonary tissues under physiological conditions remain to be analyzed. The CL40 antibody recognizes 6-sulfo sLeX and sialyl 6-sulfo LacNAc in O-linked and N-linked glycans, with absolute requirements for both GlcNAc-6-sulfation and sialylation. Immunostaining of normal mouse lungs with CL40 was performed and analyzed. The contribution of GlcNAc-6-O-sulfotransferases (GlcNAc6STs) to the synthesis of the CL40 epitope in the lungs was also elucidated. Here, we show that the expression of the CL40 epitope was specifically detected in the mesothelin-positive mesothelium of the pulmonary pleura. Moreover, GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5), but not GlcNAc6ST1 (encoded by Chst2) or GlcNAc6ST4 (encoded by Chst7), are required for the synthesis of CL40-positive glycans in the lung mesothelium. Furthermore, neither GlcNAc6ST2 nor GlcNAc6ST3 is sufficient for in vivo expression of the CL40 epitope in the lung mesothelium, as demonstrated by GlcNAc6ST1/3/4 triple-knock-out and GlcNAc6ST1/2/4 triple-knock-out mice. These results indicate that CL40-positive sialylated and sulfated glycans are abundant in the pleural mesothelium and are synthesized complementarily by GlcNAc6ST2 and GlcNAc6ST3, under physiological conditions in mice.

Inhibition of SARS-CoV-2 wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) strains by marine sulfated glycans.

The Coronavirus disease pandemic has steered the global therapeutic research efforts toward the discovery of potential anti-severe acute respiratory syndrome coronavirus (SARS-CoV-2) molecules. The role of the viral spike glycoprotein (S-protein) has been clearly established in SARS-CoV-2 infection through its capacity to bind to the host cell surface heparan sulfate proteoglycan (HSPG) and angiotensin-converting enzyme-2. The antiviral strategies targeting these 2 virus receptors are currently under intense investigation. However, the rapid evolution of the SARS-CoV-2 genome has resulted in numerous mutations in the S-protein posing a significant challenge for the design of S-protein-targeted inhibitors. As an example, the 2 key mutations in the S-protein receptor-binding domain (RBD), L452R, and T478K in the SARS-CoV-2 Delta variant (B.1.617.2) confer tighter binding to the host epithelial cells. Marine sulfated glycans (MSGs) demonstrate excellent inhibitory activity against SARS-CoV-2 via competitive disruption of the S-protein RBD-HSPG interactions and thus have the potential to be developed into effective prophylactic and therapeutic molecules. In this study, 7 different MSGs were evaluated for their anti-SARS-CoV-2 activity in a virus entry assay utilizing a SARS-CoV-2 pseudovirus coated with S-protein of the wild-type (Wuhan-Hu-1) or the Delta (B.1.617.2) strain. Although all tested MSGs showed strong inhibitory activity against both strains, no correlations between MSG structural features and virus inhibition could be drawn. Nevertheless, the current study provides evidence for the maintenance of inhibitory activity of MSGs against evolving SARS-CoV-2 strains.

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have co-evolved with eukaryotic hosts. Bacteria metabolise sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognise their glycan substrate remain poorly understood. Here, we utilise structural biology to determine how sulfatases from the human gut microbiota recognise sulfated glycans. We reveal 7 new carbohydrate sulfatase structures span four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent the first structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity towards related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism, whilst revealing the roles carbohydrate sulfatases play in host-glycan catabolism.

GALAXY ver3: updated web application for glycosylation profiling based on 3D HPLC map.

High-performance liquid chromatography (HPLC) elution data provide a useful tool for quantitative glycosylation profiling, discriminating isomeric oligosaccharides. The web application Glycoanalysis by the Three Axes of MS and Chromatography (GALAXY), which is based on the three-dimensional HPLC map of N-linked oligosaccharides with pyridyl-2-amination developed by Dr. Noriko Takahashi, has been extensively used for N-glycosylation profiling at molecular, cellular, and tissue levels. Herein, we describe the updated GALAXY as version 3, which includes new HPLC data including those of glucuronylated and sulfated glycans, an improved graphical user interface using modern technologies, and linked to glycan information in GlyTouCan and the GlyCosmos Portal. This liaison will facilitate glycomic analyses of human and other organisms in conjunction with multiomics data.