Glycosaminoglycan Oligosaccharides Research Articles

Synthesis and interaction with growth factors of sulfated oligosaccharides containing an anomeric fluorinated tail

Compounds that mimic the biological properties of glycosaminoglycans (GAGs) and can be more easily prepared than the native GAG oligosaccharides are highly demanded. Here, we present the synthesis of sulfated oligosaccharides displaying a perfluorinated aliphatic tag at the reducing end as GAG mimetics. The preparation of these molecules was greatly facilitated by the presence of the fluorinated tail since the reaction intermediates were isolated by simple fluorous solid-phase extraction. Fluorescence polarization competition assays indicated that the synthesized oligosaccharides interacted with two heparin-binding growth factors, midkine (MK) and FGF-2, showing higher binding affinities than the natural oligosaccharides, and can be therefore considered as useful GAG mimetics. Moreover, NMR experiments showed that the 3D structure of these compounds is similar to that of the native sequences, in terms of sugar ring and glycosidic linkage conformations. Finally, we also demonstrated that these derivatives are able to block the MK-stimulating effect on NIH3T3 cells growth.

Efficient platform for synthesizing comprehensive heparan sulfate oligosaccharide libraries for decoding glycosaminoglycan-protein interactions.

Glycosaminoglycans (GAGs) are abundant, ubiquitous carbohydrates in biology, yet their structural complexity has limited an understanding of their biological roles and structure-function relationships. Synthetic access to large collections of well defined, structurally diverse GAG oligosaccharides would provide critical insights into this important class of biomolecules and represent a major advance in glycoscience. Here we report a new platform for synthesizing large heparan sulfate (HS) oligosaccharide libraries displaying comprehensive arrays of sulfation patterns. Library synthesis is made possible by improving the overall synthetic efficiency through universal building blocks derived from natural heparin and a traceless fluorous tagging method for rapid purification with minimal manual manipulation. Using this approach, we generated a complete library of 64 HS oligosaccharides displaying all possible 2-O-, 6-O- and N-sulfation sequences in the tetrasaccharide GlcN-IdoA-GlcN-IdoA. These diverse structures provide an unprecedented view into the sulfation code of GAGs and identify sequences for modulating the activities of important growth factors and chemokines.

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics.

Oligosaccharide Blocks PAR1 (Proteinase-Activated Receptor 1)-PAR4-Mediated Platelet Activation by Binding to Thrombin Exosite II and Impairs Thrombosis.

Inappropriate activation and aggregation of platelets can lead to arterial thrombosis. Thrombin is the most potent platelet agonist that activates human platelets via two PARs (proteinase-activated receptors), PAR1 and PAR4. The aim is to study the activity and mechanism of an oligosaccharide HS-11 (the undecasaccharide, derived from sea cucumber Holothuria fuscopunctata) in inhibiting thrombin-mediated platelet activation and aggregation and to evaluate its antithrombotic activity. Platelet activation was analyzed by detecting CD62P/P-selectin expression using flow cytometry. The HS-11-thrombin interaction and the binding site were studied by biolayer interferometry. Intracellular Ca2+ mobilization of platelets was measured by FLIPR Tetra System using Fluo-4 AM (Fluo-4 acetoxymethyl). Platelet aggregation, thrombus formation, and bleeding Assay were assessed. An oligosaccharide HS-11, depolymerized from fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata blocks the interaction of thrombin with PAR1 and PAR4 complex by directly binding to thrombin exosite II, and completely inhibits platelet signal transduction, including intracellular Ca2+ mobilization and protein phosphorylation. Furthermore, HS-11 potently inhibits thrombin-PARs-mediated platelet aggregation and reduces thrombus formation in a model of ex vivo thrombosis. The study firstly report that the fucosylated glycosaminoglycan oligosaccharide has antiplatelet activity by binding to thrombin exosite II, and demonstrates that thrombin exosite II plays an important role in the simultaneous activation of PAR1 and PAR4, which may be a potential antithrombotic target for effective treatment of arterial thrombosis.

A Cluster Sequencing Strategy To Determine the Consensus Affinity Domains in Heparin for Its Binding to Specific Proteins.

Glycosaminoglycans (GAGs) have high negative charge and are biologically and pharmaceutically important because their high charge promotes a strong interaction with many proteins. Due to the inherent heterogeneity of GAGs, multiple oligosaccharides, containing certain common domains, often can interact with clusters of basic amino acid residues on a target protein. The specificity of many GAG-protein interactions remains undiscovered since there is insufficient structural information on the interacting GAGs. Herein, we establish a cluster sequencing strategy to simultaneously deduce all major sequences of the affinity GAG oligosaccharides, leading to a definition of the consensus sequence they share that corresponds to the specific binding domain for the target protein. As a proof of concept, antithrombin III-binding oligosaccharides were examined, resulting in a heptasaccharide domain containing the well-established anticoagulant pentasaccharide sequence. Repeating this approach, a new pentasaccharide domain was discovered corresponding to the heparin motif responsible for binding interferon-γ (IFNγ). Our strategy is fundamentally important for the discovery of saccharide sequences needed in the development of novel GAG-based therapeutics.

Comprehensive structural assignment of glycosaminoglycan oligo- and polysaccharides by protein nanopore

Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues. Size discrimination of tetra- to icosasaccharides from heparin, chondroitin sulfate and dermatan sulfate was investigated and we show that different contents and distributions of sulfate groups can be detected. Remarkably, differences in α/β anomerization and 1,4/1,3 osidic linkages can also be detected in heparosan and hyaluronic acid, as well as the subtle difference between the glucuronic/iduronic epimers in chondroitin and dermatan sulfate. Although, at this stage, discrimination of each of the constituent units of GAGs is not yet achieved at the single-molecule level, the resolution reached in this study is an essential step toward this ultimate goal.

De Novo Sequencing of Heparin /Heparan Sulfate Oligosaccharides by Chemical Derivatization and LC-MS /MS.

The biological function of glycosaminoglycan (GAG) oligosaccharides is dictated in part by the pattern of modifications (sulfation, acetylation/deacetylation, and epimerization of uronic acids) occurring in oligosaccharide regions of the polysaccharide. The sequencing of the pattern of modifications of glycosaminoglycan (GAG) oligosaccharides is highly challenging due to the heterogeneity of most naturally occurring GAGs. While liquid chromatography coupled with mass spectrometry (LC-MS) is widely used to determine GAG oligosaccharide composition, the high lability of sulfates in the gas phase makes structural interrogation by tandem mass spectrometry (MS/MS) unlikely to yield useful sequence information. Here we describe a method for the chemical derivatization of GAG oligosaccharides that replaces sulfate groups in a site-specific manner. The resulting derivatized GAG oligosaccharides can be chromatographically separated with high efficiency using C18 reversed-phase chromatography and sequenced using standard LC-MS/MS methods.

Fluorous-Tag-Assisted Synthesis of GAG-Like Oligosaccharides.

The classic, solution-phase synthesis of glycosaminoglycan (GAG) oligosaccharides is hampered by the numerous, time-consuming chromatographic purifications required for the isolation of the glycosylation products after each coupling step between sugar building blocks. Here, we present a detailed experimental procedure for a glycosylation reaction involving a glycosyl acceptor unit that is equipped with a perfluorinated tag. The presence of this fluorous tail allows the quick purification of the desired glycosylation product by performing a simple fluorous solid-phase extraction (F-SPE). The described fluorous-tag-assisted glycosylation strategy greatly facilitates the assembly of building blocks, speeding up the preparation of biologically relevant GAG-like oligomers.

Insights into structure, affinity, specificity, and function of GAG-protein interactions through the chemoenzymatic preparation of defined sulfated oligohyaluronans.

High amounts of glycosaminoglycans (GAG) such as hyaluronan (HA) occur in connective tissues. There is nowadays increasing evidence that a "sulfation code" exists which mediates numerous GAG functions. High molecular weight and inhomogeneity of GAG, however, aggravated detailed studies. Thus, synthetic oligosaccharides were urgently required. We will review here chemoenzymatic and analytic strategies to provide defined sulfated and anomerically modified GAG oligosaccharides of the HA type. Representative studies of protein/GAG interactions by (bio)chemical and biophysical methods are reported yielding novel insights into GAG-protein binding. Finally, the biological conclusions and in vivo applications of defined sulfated GAG oligosaccharides will be discussed.

Building Protein-Protein and Protein-Glycosaminoglycan Interaction Networks Using MatrixDB, the Extracellular Matrix Interaction Database.

The interaction database MatrixDB reports protein-protein and protein-glycosaminoglycan interactions in human, mammalian, and model organisms, involving at least one extracellular matrix (ECM) constituent, namely full-length proteins, ECM multimeric proteins considered as stable complexes, proteoglycans, glycosaminoglycans (GAGs), and bioactive fragments called matricryptins, which are released upon limited proteolysis of ECM proteins. The current version of MatrixDB (as of October 2020) contains 106,543 experimentally supported interactions, with all types of biomolecules combined. MatrixDB is the only database focusing on the curation of ECM protein and GAG interactions. The iNavigator integrated in MatrixDB allows users to build interaction networks online and to filter them according to expression data, quantitative proteomics data, or interaction detection methods. MatrixDB belongs to the International Molecular Exchange (IMEx) consortium, and uses its curation rules to capture interaction data, which are available in standardized exchange formats according to the Human Proteome Organization-Proteomics Standards Initiative (HUPO-PSI). © 2021 Wiley Periodicals LLC. Basic Protocol 1: Browse MatrixDB Basic Protocol 2: Create a list of biomolecules of interest to build interaction networks Basic Protocol 3: Build and export interaction networks of selected biomolecules using the iNavigator Basic Protocol 4: Build specific interaction networks using the iNavigator widgets Basic Protocol 5: Generate 3D models of glycosaminoglycan oligosaccharides using the GAG Builder tool.

Pleiotrophin interacts with glycosaminoglycans in a highly flexible and adaptable manner.

Pleiotrophin (PTN) is a potent mitogenic cytokine whose activities are controlled by its interactions with glycosaminoglycan (GAG). We examined the specificity of PTN for several types of GAG oligosaccharides. Our data indicate that the interaction of PTN with GAGs is dependent on the sulfation density of GAGs. Surprisingly, an acidic peptide also had similar interactions with PTN as GAGs. This shows that the interaction of PTN with anionic polymers is flexible and adaptable and that the charge density is the main determinant of the interaction. In addition, we show that PTN can compensate for the loss of its termini in interactions with heparin oligosaccharides, allowing it to maintain its affinity for GAGs in the absence of the termini. Taken together, these data provide valuable insight into the interactions of PTN with its proteoglycan receptors.

Ischemic stroke disrupts the endothelial glycocalyx through activation of proHPSE via acrolein exposure

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC–MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

Peracylation Coupled with Tandem Mass Spectrometry for Structural Sequencing of Sulfated Glycosaminoglycan Mixtures without Depolymerization.

The structures of glycosaminoglycans (GAGs), especially the patterns of modification, are crucial to modulate interactions with various protein targets. It is very challenging to determine the fine structures using liquid chromatography-mass spectrometry (LC-MS) due in large part to the gas-phase sulfate losses upon collisional activation. Previously, our group reported a method for fine structure analysis that required permethylation of the GAG oligosaccharide. However, uncontrolled depolymerization during the permethylation process due to esterification of uronic acid lowers the reliability of the method to resolve structures of GAGs, especially for larger oligosaccharides. Here, we describe a simplified derivatization method using propionylation and desulfation. The oligosaccharides have all hydroxyl and amine groups protected with propionyl groups and then have sulfate groups removed to generate unprotected hydroxyl and amine groups at all sites that were previously sulfated. This derivatized oligosaccharide generates informative fragments during collision-induced dissociation that resolve the original sulfation patterns. This method is demonstrated to enable accurate determination of sulfation patterns of even the highly sulfated pentasaccharide fondaparinux by MS2 and MS3. Using a mixture of dp6 from porcine heparin, we demonstrate that this method allows for structural characterization of complex mixtures, including clear chromatographic separation and sequencing of structural isomers, all at high yields without evidence of depolymerization. This represents a marked improvement in the reliability to structurally characterize GAG oligosaccharides over permethylation-based derivatization schemes.

Enzyme immobilization offers a robust tool to scale up the production of longer, diverse, natural glycosaminoglycan oligosaccharides.

Although structurally diverse, longer glycosaminoglycan (GAG) oligosaccharides are critical to understand human biology, few are available. The major bottleneck has been the predominant production of oligosaccharides, primarily disaccharides, upon enzymatic depolymerization of GAGs. In this work, we employ enzyme immobilization to prepare hexasaccharide and longer sequences of chondroitin sulfate in good yields with reasonable homogeneity. Immobilized chondroitinase ABC displayed good efficiency, robust operational pH range, broad thermal stability, high recycle ability and excellent distribution of products in comparison to the free enzyme. Diverse sequences could be chromatographically resolved into well-defined peaks and characterized using LC-MS. Enzyme immobilization technology could enable easier access to diverse longer GAG sequences.

IR action spectroscopy of glycosaminoglycan oligosaccharides

Glycosaminoglycans (GAGs) are a physio- and pharmacologically highly relevant class of complex saccharides, possessing a linear sequence and strongly acidic character. Their repetitive linear core makes them seem structurally simple at first glance, yet differences in sulfation and epimerization lead to an enormous structural diversity with only a few GAGs having been successfully characterized to date. Recent infrared action spectroscopic experiments on sulfated mono- and disaccharide ions show great promise. Here, we assess the potential of two types of gas-phase action spectroscopy approaches in the range from 1000 to 1800 cm−1 for the structural analysis of complex GAG oligosaccharides. Synthetic tetra- and pentasaccharides were chosen as model compounds for this benchmark study. Utilizing infrared multiple photon dissociation action spectroscopy at room temperature, diagnostic bands are largely unresolved. In contrast, cryogenic infrared action spectroscopy of ions trapped in helium nanodroplets yields resolved infrared spectra with diagnostic features for monosaccharide composition and sulfation pattern. The analysis of GAGs could therefore significantly benefit from expanding the conventional MS-based toolkit with gas-phase cryogenic IR spectroscopy.Graphical abstract

Chemoenzymatic Synthesis of Glycosaminoglycans.

Glycosaminoglycans (GAGs) are a family of structurally complex heteropolysaccharides composed of alternating hexosamine and uronic acid or galatose residue that include hyaluronan, chondroitin sulfate and dermatan sulfate, heparin and heparan sulfate, and keratan sulfate. GAGs display a range of critical biological functions, including regulating cell-cell interactions and cell proliferation, inhibiting enzymes, and activating growth factor receptors during various metabolic processes. Indeed, heparin is a widely used GAG-based anticoagulant drug. Unfortunately, naturally derived GAGs are highly heterogeneous, limiting studies of their structure-activity relationships and even resulting in safety concerns. For example, the heparin contamination crisis in 2007 reportedly killed more than a hundred people in the United States. Unfortunately, the chemical synthesis of GAGs, or their oligosaccharides, based on repetitive steps of protection, activation, coupling, and deprotection, is incredibly challenging. Recent advances in chemoenzymatic synthesis integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions, mimicking the biosynthetic pathway of GAGs, and represent a promising strategy to solve many of these synthetic challenges. In this critical Account, we examine the recent progress made, in our laboratory and by others, in the chemoenzymatic synthesis of GAGs, focusing on heparan sulfate and heparin, a class of GAGs with profound physiological and pharmacological importance. A major challenge for the penetration of the heparin market by homogeneous heparin products is their cost-effective large-scale synthesis. In the past decade, we and our collaborators have systematically explored the key factors that impact this process, including better enzyme expression, improved biocatalysts using protein engineering and immobilization, low cost production of enzyme cofactors, optimization of the order of enzymatic transformations, as well as development of efficient technologies, such as using ultraviolet absorbing or fluorous tags, to detect and purify synthetic intermediates. These improvements have successfully resulted in multigram-scale synthesis of low-molecular-weight heparins (LMWHs), with some showing excellent anticoagulant activity and even resulting in more effective protamine reversal than commercial, animal-sourced LMWH drugs. Sophisticated structural analysis is another challenge for marketing heparins, since impurities and contaminants can be present that are difficult to distinguish from heparin drug products. The availability of the diverse library of structurally defined heparin oligosaccharides has facilitated the systematic analytical studies undertaken by our group, resulting in important information for characterizing diverse heparin products, safeguarding their quality. Recently, a series of chemically modified nucleotide sugars have been investigated in our laboratory and have been accepted by synthases to obtain novel GAGs and GAG oligosaccharides. These includefluoride and azido regioselectivelyfunctionalizedsugarsand stable isotope-enriched GAGs and GAG oligosaccharides, critical for better understanding the biological roles of these important biopolymers. We speculate that the repertoire of unnatural acceptors and nucleotide sugar donors will soon be expanded to afford many new GAG analogues with new biological and pharmacological properties including improved specificity and metabolic stability.

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

Analysis of Procollagen C-Proteinase Enhancer-1/Glycosaminoglycan Binding Sites and of the Potential Role of Calcium Ions in the Interaction.

In this study, we characterize the interactions between the extracellular matrix protein, procollagen C-proteinase enhancer-1 (PCPE-1), and glycosaminoglycans (GAGs), which are linear anionic periodic polysaccharides. We applied molecular modeling approaches to build a structural model of full-length PCPE-1, which is not experimentally available, to predict GAG binding poses for various GAG lengths, types and sulfation patterns, and to determine the effect of calcium ions on the binding. The computational data are analyzed and discussed in the context of the experimental results previously obtained using surface plasmon resonance binding assays. We also provide experimental data on PCPE-1/GAG interactions obtained using inhibition assays with GAG oligosaccharides ranging from disaccharides to octadecasaccharides. Our results predict the localization of GAG-binding sites at the amino acid residue level onto PCPE-1 and is the first attempt to describe the effects of ions on protein-GAG binding using modeling approaches. In addition, this study allows us to get deeper insights into the in silico methodology challenges and limitations when applied to GAG-protein interactions.

Structural analysis of urinary glycosaminoglycans from healthy human subjects.

Urinary glycosaminoglycans (GAGs) can reflect the health condition of a human being, and the GAGs composition can be directly related to various diseases. In order to effectively utilize such information, a detailed understanding of urinary GAGs in healthy individuals can provide insight into the levels and structures of human urinary GAGs. In this study, urinary GAGs were collected and purified from healthy males and females of adults and young adults. The total creatinine-normalized urinary GAG content, molecular weight distribution and disaccharide compositions were determined. Using capillary zone electrophoresis (CZE)-mass spectrometry (MS) and CZE-MS/MS relying on negative electron transfer dissociation, the major components of healthy human urinary GAGs were determined. The structures of 10 GAG oligosaccharides representing the majority of human urinary GAGs were determined.

Investigation of the Amide Proton Solvent Exchange Properties of Glycosaminoglycan Oligosaccharides.

One-dimensional 1H NMR experiments were conducted for aqueous solutions of glycosaminoglycan oligosaccharides to measure the amide proton temperature coefficients and activation energy barriers for solvent exchange and evaluate the effect of pH on the solvent exchange properties. A library of mono- and oligosaccharides was prepared by enzymatic depolymerization of amide-containing polysaccharides and by chemical modification of heparin and heparan sulfate saccharides including members that contain a 3- O-sulfated glucosamine residue. The systematic evaluation of this saccharide library facilitated assessment of the effects of structural characteristics, such as size, sulfation number and site, and glycosidic linkage, on amide proton solvent exchange rates. Charge repulsion by neighboring negatively charged sulfate and carboxylate groups was found to have a significant impact on the catalysis of amide proton solvent exchange by hydroxide. This observation leads to the conclusion that solvent exchange rates must be interpreted within the context of a given chemical environment. On their own, slow exchange rates do not conclusively establish the involvement of a labile proton in a hydrogen bond, and additional supporting experimental evidence such as reduced temperature coefficients is required.