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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.

Heparan sulfate (HS), a sulfated linear carbohydrate that decorates the cell surface and extracellular matrix, is ubiquitously distributed throughout the animal kingdom and represents a key regulator of biological processes and a largely untapped reservoir of potential therapeutic targets. The temporal and spatial variations in the HS structure underpin the concept of "heparanome" and a complex network of HS binding proteins. However, despite its widespread biological roles, the determination of direct structure-to-function correlations is impaired by HS chemical heterogeneity. Attempts to correlate substitution patterns (mostly at the level of sulfation) with a given biological activity have been made. Nonetheless, these do not generally consider higher-level conformational effects at the carbohydrate level. Here, the use of NMR chemical shift analysis, NOEs, and spin-spin coupling constants sheds new light on how different sulfation patterns affect the polysaccharide backbone geometry. Furthermore, the substitution of native O-glycosidic linkages to hydrolytically more stable S-glycosidic forms leads to observable conformational changes in model saccharides, suggesting that alternative chemical spaces can be accessed and explored using such mimetics. Employing a series of systematically modified heparin oligosaccharides (as a proxy for HS) and chemically synthesized O- and S-glycoside analogues, the chemical space occupied by such compounds is explored and described.

Supplementary 1: Table 1 - Sequences of shRNA used in the study. Supplementary 2: Table 2 - Sequences of primers used in the study. Supplementary 3: mRNA level of SULFs after shRNA knockdown. Supplementary 4: mRNA expression of HS biosynthetic enzymes

Two years since the outbreak of the novel coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS-CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side effect. One alternative, with structural similarities to heparin, is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated, and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective in inhibiting cell infection than low-molecular-weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.

Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

Heparan sulfate proteoglycans (HSPG) play a critical role in the interaction of tumor cells and their microenvironment. HSPG activity is dictated by sulfation patterns controlled by sulfotransferases, which add sulfate groups, and sulfatases (Sulf), which remove 6-O-sulfates. Here, we report altered expression of these enzymes in human neuroblastoma cells with higher levels of Sulf-2 expression, a specific feature of MYCN-amplified cells (MYCN-A cells) that represent a particularly aggressive subclass. Sulf-2 overexpression in neuroblastoma cells lacking MYCN amplification (MYCN-NA cells) increased their in vitro survival. Mechanistic investigations revealed evidence of a link between Sulf-2 expression and MYCN pathogenicity in vitro and in vivo. Analysis of Sulf-2 protein expression in 65 human neuroblastoma tumors demonstrated a higher level of Sulf-2 expression in MYCN-A tumors than in MYCN-NA tumors. In two different patient cohorts, we confirmed the association in expression patterns of Sulf-2 and MYCN and determined that Sulf-2 overexpression predicted poor outcomes in a nonindependent manner with MYCN. Our findings define Sulf-2 as a novel positive regulator of neuroblastoma pathogenicity that contributes to MYCN oncogenicity. Cancer Res; 74(21); 5999–6009. ©2014 AACR.

Analysis of enzyme expression in other non -neuroblastoma cell lines.

Negative control for figure 2.

Sequences of primers used for RT-PCR.

ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the ‘open’ conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.

Leukocyte recruitment from the vasculature into tissues is a crucial component of the immune system, but is also key to inflammatory disease. Chemokines are central to this process but have yet to be therapeutically targeted during inflammation, due to a lack of mechanistic understanding. Specifically, CXCL4 (PF4) has no established receptor that explains its function. Here we use biophysical, in vitro and in vivo techniques to determine the mechanism underlying CXCL4 mediated leukocyte recruitment. We demonstrate that CXCL4 binds to glycosaminoglycan (GAG) sugars within the endothelial extracellular matrix resulting in increased vascular permeability and non-specific recruitment of a range of leukocytes. Furthermore, GAG sulphation confers selectivity onto chemokine localisation. These findings represent a new understanding of chemokine biology, providing novel mechanisms for future therapeutic targeting.One sentence summaryCXCL4 binds to extracellular matrix proteoglycans resulting in increased vascular permeability and recruitment of a wide range of different leukocytes via a non-canonical mechanism.

Sulfated carbohydrate metabolism is a fundamental process, which occurs in all domains of life. Carbohydrate sulfatases are enzymes that remove sulfate groups from carbohydrates and are essential to the depolymerisation of complex polysaccharides. Despite their biological importance, carbohydrate sulfatases are poorly studied and challenges remain in accurately assessing the activity, specificity and kinetic parameters. Most notably, separation of desulfated products from sulfated substrates is currently a time-consuming process. In this paper, we describe the development of rapid capillary electrophoresis coupled to substrate fluorescence detection as a high-throughput and facile means of analysing carbohydrate sulfatase activity. The approach has utility for the determination of both kinetic and inhibition parameters and is based on existing microfluidic technology coupled to a new synthetic fluorescent 6S-GlcNAc carbohydrate substrate. Furthermore, we compare this technique in terms of both time and resources, to high performance anion exchange chromatography and NMR-based methods, which are the two current ‘gold standards’ for enzymatic carbohydrate sulfation analysis. Our study clearly demonstrates the advantages of mobility shift assays for the quantification of near real-time carbohydrate desulfation by purified sulfatases, and could support the search for small molecule inhibitors of these disease-associated enzymes.One sentence summarySulfatases remove sulfate groups from biomolecules; in this study we report a rapid and robust capillary electrophoresis assay for the quantification of carbohydrate desulfation.

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2-O or 6-O sulfate groups than on N-sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the ‘open’ conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evident multiplicity of binding modes also suggests that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required

Complex glycans are ubiquitous in Nature and essential to life; glycosaminoglycans surround all mammalian cells and are key players in development and cell signalling, while plant cell walls provide structural rigidity, even during phases of growth, and plant cells account for the vast majority of carbon fixation on earth. Despite their diverse roles, however, only a fraction of the potential chemical space hypothetically available to complex glycans has been explored. New regions of this chemical space can, nevertheless, be accessed by generating new ones which do not occur in Nature or, by modifying naturally-occurring polysaccharide structures. Collectively, we term these new polysaccharides (NPs). Two synthetic routes to NPs are described here; the de novo route, directly from monosaccharide starting materials and the functionalization route, involving glycosylation of existing polysaccharides. The reaction in both cases involves a simple condensation step under microwave heating, catalysed by benign organic acids. The approach is illustrated by the generation of structures with biological activities ranging from cell signalling and inhibition of bacterial growth to mimicking carbohydrate antigens from pathogenic microorganisms. The method is as applicable to fine chemicals as it is to industrial waste, for example, biotechnologically-derived D-allulose (D-psicose) or biofermentation waste, and enables rapid access to extensive chemical space, encompassing both existing biological structures and those previously unattainable. Accessing this chemical space unlocks new functionalities and applications for complex glycans in the biological, medical, biotechnological and materials science arenas.

The cytotoxic mode of action of four antimicrobial peptides (AMPs) (gomesin, tachyplesin, protegrin, and polyphemusin) against a HeLa cell tumor model is discussed. A study of cell death by AMP stimulation revealed some similarities, including annexin-V externalization, reduction of mitochondrial potential, insensitivity against inhibitors of cell death, and membrane permeabilization. Evaluation of signaling proteins and gene expression that control cell death revealed wide variation in the responses to AMPs. However, the ability to cross cell membranes emerged as an important characteristic of AMP-dependent cell death, where endocytosis mediated by dynamin is a common mechanism. Furthermore, the affinity between AMPs and glycosaminoglycans (GAGs) and GAG participation in the cytotoxicity of AMPs were verified. The results show that, despite their primary and secondary structure homology, these peptides present different modes of action, but endocytosis and GAG participation are an important and common mechanism of cytotoxicity for β-hairpin peptides.

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19.

Two years since the outbreak of the novel coronavirus SARS-CoV-2 pandemic, there remain few clinically effective drugs to complement vaccines. One is the anticoagulant, heparin, which in 2004 was found able to inhibit invasion of SARS CoV (CoV-1) and which has been employed during the current pandemic to prevent thromboembolic complications and moderate potentially damaging inflammation. Heparin has also been shown experimentally to inhibit SARS-CoV-2 attachment and infection in susceptible cells. At high therapeutic doses however, heparin increases the risk of bleeding and prolonged use can cause heparin-induced thrombocytopenia, a serious side-effect. One alternative, with structural similarities to heparin is the plant-derived, semi-synthetic polysaccharide, pentosan polysulfate (PPS). PPS is an established drug for the oral treatment of interstitial cystitis, is well-tolerated and exhibits weaker anticoagulant effects than heparin. In an established Vero cell model, PPS and its fractions of varying molecular weights, inhibited invasion by SARS-CoV-2. Intact PPS and its size-defined fractions were characterized by molecular weight distribution and chemical structure using NMR spectroscopy and LC-MS, then employed to explore the structural basis of interactions with SARS-CoV-2 spike protein receptor-binding domain (S1 RBD) and the inhibition of Vero cell invasion. PPS was as effective as unfractionated heparin, but more effective at inhibiting cell infection than low molecular weight heparin (on a weight/volume basis). Isothermal titration calorimetry and viral plaque-forming assays demonstrated size-dependent binding to S1 RBD and inhibition of Vero cell invasion, suggesting the potential application of PPS as a novel inhibitor of SARS-CoV-2 infection.

During the manufacture of pharmaceutical heparin, a range of treatments are applied to sanitize, decolourise and reduce the pyrogenic properties of the samples. The structural effects of bleaching, an oxidative process, are examined. Among

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV–visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

International audience; Humans have co-evolved with a dense community of microbial symbionts that 34 inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that 35 separates these microbes from the intestinal epithelium. Some gut bacteria are able to 36 utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, 37 it remains unclear which enzymes initiate the degradation of the highly complex O-38 glycans found in mucins. In the colon, these glycans are heavily sulfated, but sulfatases active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-linked glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans and even possess the ability to cleave additional linkages not yet known to occur in host glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated Oglycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases like inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

Galectins are involved in the regulation of divergent physiological and pathological processes and are increasingly recognized to play important roles in a number of diseases. However, a simple and effective way in assessing galectin-ligand interactions is lacking. Our examination of the sequence of all 12 human galectin members reveals the presence of one or more tryptophan residues in the carbohydrate-recognition domains of each galectin. This led us to investigate the possibility that alteration of the galectin intrinsic tryptophan fluorescence could be used in determining the strength of galectin-ligand interactions. One representative member from each of the three subtype galectins, galectin-2 (proto-), galectin-3 (chimera-) and galectin-4 (tandem repeat-type), was selected and analysed for galectin interaction with three ligands of different affinities: galactose, lactose and N-acetyl-lactosamine using tryptophan fluorescence spectroscopy (TFS) and, as a comparison, isothermal titration calorimetry (ITC). Good agreement between TFS and ITC measurements were revealed in ligand bindings of all galectin members. Moreover, TFS detected very weak galectin binding where ITC could not reliably do so. The reliability of TFS in determining galectin-ligand interactions was further validated by analysis of galectin-3 interaction with a semisynthetic ligand, F3. Thus, TFS can be used as a simple, sensitive and reliable way to determine galectin-ligand interactions and also as a drug-discovery platform in developing galectin-targeted therapeutic drugs.

Fibroblast growth factors (FGFs) regulate embryonic development and homeostasis, including tissue and organ repair and specific aspects of metabolism. The basic FGF and acidic FGF, now known as FGF2 and FGF1, are widely used protein drugs for tissue repair. However, they are susceptible to denaturation at ambient temperatures and during long-time storage, which will reduce their biological activity. The interaction of FGFs with the sulfated domains of heparan sulfate and heparin is essential for their cellular signaling and stability. Therefore, we analyzed the interactions of FGF1 and FGF2 with four sulfated polysaccharides: heparin, dextran sulfate (DXS), λ-carrageenan, and chondroitin sulfate. The results of thermal stability and cell proliferation assays demonstrate that heparin, DXS, and λ-carrageenan bound to both FGFs and protected them from denaturation. Our results suggest heparin, DXS, and λ-carrageenan are potential formulation materials that bind and stabilize FGFs, and which may also potentiate their activity and control their delivery.

The vast microbial community that resides in the human colon, termed the human gut microbiota, performs important roles in maintaining host health. Sulfated host glycans comprise both a major nutrient source and important colonisation factors for this community. Carbohydrate sulfatases remove sulfate groups from glycans and are essential in many bacteria for the utilisation of sulfated host glycans. Additionally, carbohydrate sulfatases are also implicated in numerous host diseases, but remain some of the most understudied carbohydrate active enzymes to date, especially at the structural and molecular level. In this work, we analyse 7 carbohydrate sulfatases, spanning 4 subfamilies, from the human gut symbiont Bacteroides thetaiotaomicron, a major utiliser of sulfated host glycans, correlating structural and functional data with phylogenetic and environmental analyses. Together, these data begin to fill the knowledge gaps in how carbohydrate sulfatases orchestrate sulfated glycan metabolism within their environment.

The linear anionic class of polysaccharides, glycosaminoglycans (GAGs), are critical throughout the animal kingdom for developmental processes and the maintenance of healthy tissues. They are also of interest as a means of influencing biochemical processes. One member of the GAG family, heparin, is exploited globally as a major anticoagulant pharmaceutical and there is a growing interest in the potential of other GAGs for diverse applications ranging from skin care to the treatment of neurodegenerative conditions, and from the treatment and prevention of microbial infection to biotechnology. To realize the potential of GAGs, however, it is necessary to develop effective tools that are able to exploit the chemical manipulations to which GAGs are susceptible. Here, the current knowledge concerning the chemical modification of GAGs, one of the principal approaches for the study of the structure-function relationships in these molecules, is reviewed. Some additional methods that were applied successfully to the analysis and/or processing of other carbohydrates, but which could be suitable in GAG chemistry, are also discussed.

The vast microbial community that resides in the human colon, termed the human gut microbiota, performs important roles in maintaining host health. Sulfated host glycans comprise both a major nutrient source and important colonisation factors for this community. Carbohydrate sulfatases remove sulfate groups from glycans and are essential in many bacteria for the utilisation of sulfated host glycans. Additionally, carbohydrate sulfatases are also implicated in numerous host diseases, but remain some of the most understudied carbohydrate active enzymes to date, especially at the structural and molecular level. In this work, we analyse 7 carbohydrate sulfatases, spanning 4 subfamilies, from the human gut symbiont Bacteroides thetaiotaomicron, a major utiliser of sulfated host glycans, correlating structural and functional data with phylogenetic and environmental analyses. Together, these data begin to fill the knowledge gaps in how carbohydrate sulfatases orchestrate sulfated glycan metabolism within their environment.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward 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 while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

Heparin is a complex glycosaminoglycan, derived mainly from pig mucosa, used therapeutically for its anticoagulant activity. Yet, owing largely to the chain complexity, the progressive effects of environmental conditions on heparin structure have not been fully described. A systematic study of the influence of acidic hydrolysis on heparin chain length and substitution has therefore been conducted. Changes in the sulfation pattern, monitored via 2D NMR, revealed initial de-N-sulfation of the molecule (pH 1/ 40 °C) and unexpectedly identified the secondary sulfate of iduronate as more labile than the 6-O-sulfate of glucosamine residues under these conditions (pH 1/ 60 °C). Additionally, the loss of sulfate groups, rather than depolymerization, accounted for most of the reduction in molecular weight. This provides an alternative route to producing partially 2-O-de-sulfated heparin derivatives that avoids using conventional basic conditions and may be of value in the optimization of processes associated with the production of heparin pharmaceuticals.

Background and Purpose: Currently, there are no licensed vaccines and limited antivirals for the treatment of COVID-19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID-19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID-19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS-CoV-2, in vitro, is needed. Experimental Approach: Seven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS-CoV-2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined. Key Results: All the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150-fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor. Conclusion and Implications: This comparison of clinically relevant heparins shows that UFH has significantly stronger SARS-CoV-2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID-19.

BACKGROUND Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated. AIM To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis. METHODS In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated. RESULTS Extracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice. CONCLUSION Extracellular histones potentially enhance fibrogenesis via the TLR4–MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.

SummaryHumans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that separates these microbes from the intestinal epithelium. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate the degradation of the highly complex O-glycans found in mucins. In the colon, these glycans are heavily sulfated, but the specific sulfatases that are active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We have characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases such as inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.

International audience; Glycosaminoglycans (GAGs) are an important nutrient source for the gut microbiome. Here, the authors characterize the genetic loci that underpins glycosaminoglycan utilization in Bacteroides thetaiotaomicron; providing insights into the metabolism of GAGs by a predominant member of the gut microbiota.The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the 'sulfation problem' is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.