Host heparan sulfate promotes ACE2 super-cluster assembly and enhances SARS-CoV-2-associated syncytium formation.

SARS-CoV-2 infection causes spike-dependent fusion of infected cells with ACE2 positive neighboring cells, generating multi-nuclear syncytia that are often associated with severe COVID. To better elucidate the mechanism of spike-induced syncytium formation, we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical stimulator for spike-induced cell-cell fusion. We show that HS binds spike and promotes spike-induced ACE2 clustering, forming synapse-like cell-cell contacts that facilitate fusion pore formation between ACE2-expresing and spike-transfected human cells. Chemical or genetic inhibition of HS mitigates ACE2 clustering, and thus, syncytium formation, whereas in a cell-free system comprising purified HS and lipid-anchored ACE2, HS stimulates ACE2 clustering directly in the presence of spike. Furthermore, HS-stimulated syncytium formation and receptor clustering require a conserved ACE2 linker distal from the spike-binding site. Importantly, the cell fusion-boosting function of HS can be targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice. Thus, HS, as a host factor exploited by SARS-CoV-2 to facilitate receptor clustering and a stimulator of infection-associated syncytium formation, may be a promising therapeutic target for severe COVID.

Increased 3-O-sulfated heparan sulfate in Alzheimer's disease brain is associated with genetic risk gene HS3ST1.

HS3ST1 is a genetic risk gene associated with Alzheimer's disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3-O-sulfated HS displayed sevenfold increase in the AD group (n = 14, P < 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3-O-sulfated HS is made by 3-O-sulfotransferase isoform 1 (3-OST-1), which is encoded by the HS3ST1 gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3-O-sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3-O-sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression of HS3ST1 gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.

Heparan sulfate promotes ACE2 super-cluster assembly to enhance SARS-CoV-2-associated syncytium formation.

The mechanism of syncytium formation, caused by spike-induced cell-cell fusion in severe COVID-19, is largely unclear. Here we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical host factor exploited by SARS-CoV-2 to enhance spike’s fusogenic activity. HS binds spike to facilitate ACE2 clustering, generating synapse-like cell-cell contacts to promote fusion pore formation. ACE2 clustering, and thus, syncytium formation is significantly mitigated by chemical or genetic elimination of cell surface HS, while in a cell-free system consisting of purified HS, spike, and lipid-anchored ACE2, HS directly induces ACE2 clustering. Importantly, the interaction of HS with spike allosterically enables a conserved ACE2 linker in receptor clustering, which concentrates spike at the fusion site to overcome fusion-associated activity loss. This fusion-boosting mechanism can be effectively targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice.

A 6-O-endosulfatase activity assay based on synthetic heparan sulfate oligomers.

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the postsynthetic editing of heparan sulfate (HS), which regulates many important biological processes. The activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well-characterized synthetic substrates for the probing of its catalytic activity. We introduce synthetic HS oligosaccharides to fill this gap, and we use our recombinant Sulf-2 protein to show that a paranitrophenol (pNP)-labeled synthetic oligosaccharide allows a reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange high-pressure liquid chromatography and quantified by UV absorbance. This simple assay allows the detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that the N-glycosylation of the Sulf-2 enzyme affects the activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan.

Using heparan sulfate octadecasaccharide (18-mer) as a multi-target agent to protect against sepsis.

Sepsis is a lethal syndrome manifested by an unregulated, overwhelming inflammation from the host in response to infection. Here, we exploit the use of a synthetic heparan sulfate octadecasaccharide (18-mer) to protect against sepsis. The 18-mer not only inhibits the pro-inflammatory activity of extracellular histone H3 and high mobility group box 1 (HMGB1), but also elicits the anti-inflammatory effect from apolipoprotein A-I (ApoA-I). We demonstrate that the 18-mer protects against sepsis-related injury and improves survival in cecal ligation and puncture mice and reduces inflammation in an endotoxemia mouse model. The 18-mer neutralizes the cytotoxic histone-3 (H3) through direct interaction with the protein. Furthermore, the 18-mer enlists the actions of ApoA-I to dissociate the complex of HMGB1 and lipopolysaccharide, a toxic complex contributing to cell death and tissue damage in sepsis. Our study provides strong evidence that the 18-mer mitigates inflammatory damage in sepsis by targeting numerous mediators, setting it apart from other potential therapies with a single target.

Chemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras.

Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of H/C 12-mer 5 is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccharides using natural biosynthetic enzymes, offering a new class of glycan molecules for biological research.

Analysis of 3-O-Sulfated Heparan Sulfate Using Isotopically Labeled Oligosaccharide Calibrants.

The 3-O-sulfated glucosamine in heparan sulfate (HS) is a low-abundance structural component, but it is a key saccharide unit for the biological activities of HS. A method to determine the level of 3-O-sulfated HS is lacking. Here, we describe a LC-MS/MS based method to analyze the structural motifs. We determined the levels of 3-O-sulfated structural motifs from pharmaceutical heparin manufactured from bovine, porcine, and ovine. We discovered that saccharide chains carrying 3-O-sulfation from enoxaparin, an FDA-approved low-molecular weight heparin, displayed a slower clearance rate than non-3-O-sulfated sugar chains in a mouse model. Lastly, we detected the 3-O-sulfated HS from human brain. Furthermore, we found that a specific 3-O-sulfated structural motif, tetra-1, is elevated in the brain HS from Alzheimer's disease patients (n = 5, p = 0.0020). Our method offers a practical solution to measure 3-O-sulfated HS from biological sources with the sensitivity and quantitative capability.

Deciphering the substrate recognition mechanisms of the heparan sulfate 3-O-sulfotransferase-3.

The sulfation at the 3-OH position of a glucosamine saccharide is a rare modification, but is critically important for the biological activities of heparan sulfate polysaccharides. Heparan sulfate 3-O-sulfotransferase (3-OST), the enzyme responsible for completing this modification, is present in seven different isoforms in humans. Individual isoforms display substrate selectivity to uniquely sulfated saccharide sequences present in heparan sulfate polysaccharides. Here, we report two ternary crystal structures of heparan sulfate 3-OST isoform 3 (3-OST-3) with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates: non 6-O-sulfated octasaccharide (8-mer 1) and 6-O-sulfated octasaccharide (8-mer 3). The 8-mer 1 is a known favorable substrate for 3-OST-3, whereas the 8-mer 3 is an unfavorable one. Unlike the 8-mer 1, we discovered that the 8-mer 3 displays two binding orientations to the enzyme: productive binding and non-productive binding. Results from the enzyme activity studies demonstrate that 8-mer 3 can contribute to either substrate or product inhibition, possibly attributed to a non-productive binding mode. Our results suggest that heparan sulfate substrates interact with the 3-OST-3 enzyme in more than one orientation, which may regulate the activity of the enzyme. Our findings also suggest that different binding orientations between polysaccharides and their protein binding partners could influence biological outcomes.

Synthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4.

Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.

Construction of heparan sulfate microarray for investigating the binding of specific saccharide sequences to proteins.

Heparan sulfate (HS) is a heterogeneous, extracellular glycan that interacts with proteins and other molecules affecting many biological processes. The specific binding motifs of HS interactions are of interest, but have not been extensively characterized. Glycan microarrays are valuable tools that can be used to probe the interactions between glycans and their ligands while relying on relatively small amounts of samples. Recently, chemoenzymatic synthesis of HS has been employed to produce specific HS structures that can otherwise be difficult to produce. In this study, a microarray of diverse chemoenzymatically synthesized HS structures was developed and HS interactions were characterized. Fluorescently labeled antithrombin III (AT) and fibroblast growth factor-2 (FGF2) were screened against 95 different HS structures under three different printing concentrations to confirm the utility of this microarray. Specific sulfation patterns were found to be important for binding to these proteins and results are consistent with previous specificity studies. Furthermore, the binding affinities (KD,surf) of AT and FGF2 to multiple HS structures were determined using a microarray technique and is consistent with previous reports. Lastly, the 95-compound HS microarray was used to determine the distinct binding profiles for interleukin 12 and platelet factor 4. This technique is ideal for rapid expansion and will be pivotal to the high-throughput characterization of biologically important structure/function relationships.

Structural and substrate specificity analysis of 3-O-sulfotransferase isoform 5 to synthesize heparan sulfate.

Heparan sulfate 3-O-sulfotransferase (3-OST) transfers a sulfo group to the 3-OH position of a glucosamine saccharide unit to form 3-O-sulfated heparan sulfate. 3-O-sulfation is known to be critically important for bestowing anticoagulant activity and other biological functions of heparan sulfate. Here, we report two ternary crystal structures of 3-OST-5 with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates. We also used 3-OST-5 to synthesize six 3-O-sulfated 8-mers. Results from the structural analysis of the six 3-O-sulfated 8-mers revealed the substrate specificity of 3-OST-5. The enzyme prefers to sulfate a 6-O-sulfo glucosamine saccharide that is surrounded by glucuronic acid over a 6-O-sulfo glucosamine saccharide that is surrounded by 2-O-sulfated iduronic acid. 3-OST-5 modified 8-mers display a broad range of anti-factor Xa activity, depending on the structure of the 8-mer. We also discovered that the substrate specificity of 3-OST-5 is not governed solely by the side chains from amino acid residues in the active site. The conformational flexibility of the 2-O-sulfated iduronic acid in the saccharide substrates also contributes to the substrate specificity. These findings advance our understanding for how to control the biosynthesis of 3-O-sulfated heparan sulfate with desired biological activities.

Using engineered 6-O-sulfotransferase to improve the synthesis of anticoagulant heparin.

Heparan sulfate (HS) and heparin are sulfated polysaccharides exhibiting diverse physiological functions. HS 6-O-sulfotransferase (6-OST) is a HS biosynthetic enzyme that transfers a sulfo group to the 6-OH position of glucosamine to synthesize HS with desired biological activities. Chemoenzymatic synthesis is a widely adopted method to obtain HS oligosaccharides to support biological studies. However, this method is unable to synthesize all possible structures due to the specificity of natural enzymes. Here, we report the use of an engineered 6-OST to achieve fine control of the 6-O-sulfation. Unlike wild type enzyme, the engineered 6-OST only sulfates the non-reducing end glucosamine residue. Utilizing the engineered enzyme and wild type enzyme, we successfully completed the synthesis of five hexasaccharides and one octasaccharide differing in 6-O-sulfation patterns. We also identified a hexasaccharide construct as a new anticoagulant drug candidate. Our results demonstrate the feasibility of using an engineered HS biosynthetic enzyme to prepare HS-based therapeutics.

Quantitative analysis of heparan sulfate using isotopically labeled calibrants.

Heparan sulfate is a sulfated polysaccharide that displays essential physiological functions. Here, we report a LC-MS/MS-based method for quantitatively determining the individual disaccharide composition and total amount of heparan sulfate. Using eight 13C-labeled disaccharide calibrants and one 13C-labeled polysaccharide calibrant, we complete the analysis in one-pot process. The method is both sensitive and has the throughput to analyze heparan sulfate from mouse tissues and plasma.

Design of anti-inflammatory heparan sulfate to protect against acetaminophen-induced acute liver failure.

Acetaminophen/paracetamol (APAP) overdose is the leading cause of drug-induced acute liver failure (ALF) in the United States and Europe. The progression of the disease is attributed to sterile inflammation induced by the release of high mobility group box 1 (HMGB1) and the interaction with receptor for advanced glycation end products (RAGE). A specific, effective, and safe approach to neutralize the proinflammatory activity of HMGB1 is highly desirable. Here, we found that a heparan sulfate (HS) octadecasaccharide (18-mer-HP or hepatoprotective 18-mer) displays potent hepatoprotection by targeting the HMGB1/RAGE axis. Endogenous HS proteoglycan, syndecan-1, is shed in response to APAP overdose in mice and humans. Furthermore, purified syndecan-1, but not syndecan-1 core protein, binds to HMGB1, suggesting that HMGB1 binds to HS polysaccharide side chains of syndecan-1. Last, we compared the protection effect between 18-mer-HP and N-acetyl cysteine, which is the standard of care to treat APAP overdose. We demonstrated that 18-mer-HP administered 3 hours after a lethal dose of APAP is fully protective; however, the treatment of N-acetyl cysteine loses protection. Therefore, 18-mer-HP may offer a potential therapeutic advantage over N-acetyl cysteine for late-presenting patients. Synthetic HS provides a potential approach for the treatment of APAP-induced ALF.

Synthesis of 3-O-Sulfated Disaccharide and Tetrasaccharide Standards for Compositional Analysis of Heparan Sulfate.

3-O-Sulfation on the glucosamine sugar unit in heparan sulfate (HS) is linked to various biological functions, including the anticoagulant activity to treat thrombotic disorders in hospitals. The 3-O-sulfated glucosamine is biosynthesized by heparan sulfate glucosamine 3-sulfotransferases. Because of its biological significance, there is a need for 3-O-sulfated oligosaccharide standards to facilitate the compositional analysis of HS. These oligosaccharides must contain a Δ4,5-unsaturated uronic acid (ΔUA) residue at the nonreducing end, which is due to the depolymerization reaction catalyzed by heparin lyases used during the compositional analysis procedure. Here, we describe a protocol for the preparation of one 3-O-sulfated disaccharide (compound 4) and three 3-O-sulfated tetrasaccharides (compound 1-3) in a milligram scale. The synthesis of 3-O-sulfated disaccharide and tetrasaccharide standards was completed by degrading synthetic octasaccharides using heparin lyases. Further analysis revealed that 3-O-sulfated oligosaccharide standards are labile under basic conditions, confirming the findings from a previous study. The unwanted degradation was reduced by decreasing the pH in the presence of phosphate buffer. The 3-O-sulfated oligosaccharide standards are reagents to characterize 3-O-sulfation in HS derived from biological sources.

New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3'-phosphoadenosine 5'-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

Synthetic oligosaccharides can replace animal-sourced low-molecular weight heparins.

Low-molecular weight heparin (LMWH) is used clinically to treat clotting disorders. As an animal-sourced product, LMWH is a highly heterogeneous mixture, and its anticoagulant activity is not fully reversible by protamine. Furthermore, the reliability of the LMWH supply chain is a concern for regulatory agencies. We demonstrate the synthesis of heparin dodecasaccharides (12-mers) at the gram scale. In vitro experiments demonstrate that the anticoagulant activity of the 12-mers could be reversed using protamine. One of these, labeled as 12-mer-1, reduced the size of blood clots in the mouse model of deep vein thrombosis and attenuated circulating procoagulant markers in the mouse model of sickle cell disease. An ex vivo experiment demonstrates that the anticoagulant activity of 12-mer-1 could be reversed by protamine. 12-mer-1 was also examined in a nonhuman primate model to determine its pharmacodynamic parameters. A 7-day toxicity study in a rat model showed no toxic effects. The data suggest that a synthetic homogeneous oligosaccharide can replace animal-sourced LMWHs.

Chemoenzymatic synthesis of heparan sulfate and heparin oligosaccharides and NMR analysis: paving the way to a diverse library for glycobiologists.

Heparan sulfate (HS) is a member of the glycosaminoglycans (GAG) family that plays essential roles in biological processes from animal sources. Heparin, a highly sulfated form of HS, is widely used as anticoagulant drug worldwide. The high diversity and complexity of HS and heparin represent a roadblock for structural characterization and biological activity studies. Access to structurally defined oligosaccharides is critical for the successful development of HS and heparin structure-activity relationships. In this study, a library of 66 HS and heparin oligosaccharides covering different sulfation patterns and sizes was prepared through an efficient method of chemoenzymatic synthesis. A systematic nuclear magnetic resonance spectroscopy study was firstly undertaken for every oligosaccharide in the library. In addition to the availability of different oligosaccharides, this work also provides spectroscopic data helpful for characterizing more complicated polysaccharide structures providing a safeguard to ensure the quality of the drug heparin. This HS/heparin library will be useful for activity screening and facilitate future structure-activity relationship studies.

The TRPM1 channel in ON-bipolar cells is gated by both the α and the ßγ subunits of the G-protein Go.

Transmission from photoreceptors to ON bipolar cells in mammalian retina is mediated by a sign-inverting cascade. Upon binding glutamate, the metabotropic glutamate receptor mGluR6 activates the heterotrimeric G-protein Gαoß3γ13, and this leads to closure of the TRPM1 channel (melastatin). TRPM1 is thought to be constitutively open, but the mechanism that leads to its closure is unclear. We investigated this question in mouse rod bipolar cells by dialyzing reagents that modify the activity of either Gαo or Gßγ and then observing their effects on the basal holding current. After opening the TRPM1 channels with light, a constitutively active mutant of Gαo closed the channel, but wild-type Gαo did not. After closing the channels by dark adaptation, phosducin or inactive Gαo (both sequester Gßγ) opened the channel while the active mutant of Gαo did not. Co-immunoprecipitation showed that TRPM1 interacts with Gß3 and with the active and inactive forms of Gαo. Furthermore, bioluminescent energy transfer assays indicated that while Gαo interacts with both the N- and the C- termini of TRPM1, Gßγ interacts only with the N-terminus. Our physiological and biochemical results suggest that both Gαo and Gßγ bind TRPM1 channels and cooperate to close them.