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.

Evidence for Multiple Binding Modes in the Initial Contact Between SARS-CoV-2 Spike S1 Protein and Cell Surface Glycans.

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.

Pentosan Polysulfate Inhibits Attachment and Infection by SARS-CoV-2 In Vitro: Insights into Structural Requirements for Binding.

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.

Saturated tetrasaccharide profile of enoxaparin. An additional piece to the heparin biosynthesis puzzle.

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

Heparin Inhibits Cellular Invasion by SARS-CoV-2: Structural Dependence of the Interaction of the Spike S1 Receptor-Binding Domain with Heparin.

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.

Structural Features of Heparan Sulfate from Multiple Osteochondromas and Chondrosarcomas.

Multiple osteochondromas (MO) is a hereditary disorder associated with benign cartilaginous tumors, known to be characterized by absence or highly reduced amount of heparan sulfate (HS) in the extracellular matrix of growth plate cartilage, which alters proper signaling networks leading to improper bone growth. Although recent studies demonstrated accumulation of HS in the cytoplasm of MO chondrocytes, nothing is known on the structural alterations which prevent HS from undergoing its physiologic pathway. In this work, osteochondroma (OC), peripheral chondrosarcoma, and healthy cartilaginous human samples were processed following a procedure previously set up to structurally characterize and compare HS from pathologic and physiologic conditions, and to examine the phenotypic differences that arise in the presence of either exostosin 1 or 2 (EXT1 or EXT2) mutations. Our data suggest that HS chains from OCs are prevalently below 10 kDa and slightly more sulfated than healthy ones, whereas HS chains from peripheral chondrosarcomas (PCSs) are mostly higher than 10 kDa and remarkably more sulfated than all the other samples. Although deeper investigation is still necessary, the approach here applied pointed out, for the first time, structural differences among OC, PCS, and healthy HS chains extracted from human cartilaginous excisions, and could help in understanding how the structural features of HS are modulated in the presence of pathological situations also involving different tissues.

Fine structural characterization of sulodexide.

Sulodexide is a heparinoid which combines the properties of its components heparin and dermatan sulfate and is used not only for the prophylaxis and treatment of thromboembolic diseases but also for the treatment of diabetic nephropathy. Despite many clinical studies have been conducted to investigate its activity and safety, no data are available on the fine chemical characterization of its components. In this work, the in-depth investigation on the structural features of both the whole mixture and the isolated components was accomplished, involving the analysis of molecular weight distribution and of their mono, di and oligosaccharide composition by HP-SEC/TDA, 2D-NMR and HPLC-MS techniques. Moreover, also the separation of fractions endowed of graded affinity to antithrombin was achieved followed again by detailed structural analysis. The combination of different techniques permits to profile in depth the structural features of such a drug and offers a useful tool for possible analysis of batch production.

Structural Characterization of the Low-Molecular-Weight Heparin Dalteparin by Combining Different Analytical Strategies.

A number of low molecular weight heparin (LMWH) products are available for clinical use and although all share a similar mechanism of action, they are classified as distinct drugs because of the different depolymerisation processes of the native heparin resulting in substantial pharmacokinetic and pharmacodynamics differences. While enoxaparin has been extensively investigated, little information is available regarding the LMWH dalteparin. The present study is focused on the detailed structural characterization of Fragmin® by LC-MS and NMR applied both to the whole drug and to its enzymatic products. For a more in-depth approach, size homogeneous octasaccharide and decasaccharide components together with their fractions endowed with high or no affinity toward antithrombin were also isolated and their structural profiles characterized. The combination of different analytical strategies here described represents a useful tool for the assessment of batch-to-batch structural variability and for comparative evaluation of structural features of biosimilar products.

Determination of the molecular weight of low-molecular-weight heparins by using high-pressure size exclusion chromatography on line with a triple detector array and conventional methods.

The evaluation of weight average molecular weight (Mw) and molecular weight distribution represents one of the most controversial aspects concerning the characterization of low molecular weight heparins (LMWHs). As the most commonly used method for the measurement of such parameters is high performance size exclusion chromatography (HP-SEC), the soundness of results mainly depends on the appropriate calibration of the chromatographic columns used. With the aim of meeting the requirement of proper Mw standards for LMWHs, in the present work the determination of molecular weight parameters (Mw and Mn) by HP-SEC combined with a triple detector array (TDA) was performed. The HP-SEC/TDA technique permits the evaluation of polymeric samples by exploiting the combined and simultaneous action of three on-line detectors: light scattering detectors (LALLS/RALLS); refractometer and viscometer. Three commercial LMWH samples, enoxaparin, tinzaparin and dalteparin, a γ-ray depolymerized heparin (γ-Hep) and its chromatographic fractions, and a synthetic pentasaccharide were analysed by HP-SEC/TDA. The same samples were analysed also with a conventional HP-SEC method employing refractive index (RI) and UV detectors and two different chromatographic column set, silica gel and polymeric gel columns. In both chromatographic systems, two different calibration curves were built up by using (i) γ-Hep chromatographic fractions and the corresponding Mw parameters obtained via HP-SEC/TDA; (ii) the whole γ-Hep preparation with broad Mw dispersion and the corresponding cumulative distribution function calculated via HP-SEC/TDA. In addition, also a chromatographic column calibration according to European Pharmacopoeia indication was built up. By comparing all the obtained results, some important differences among Mw and size distribution values of the three LMWHs were found with the five different calibration methods and with HP-SEC/TDA method. In particular, the detection of the lower molecular weight components turned out to be the most critical aspect. Whereas HP-SEC/TDA may underestimate species under 2 KDa when present in low concentration, other methods appeared to emphasize their content.

A heparin-like glycosaminoglycan from shrimp containing high levels of 3-O-sulfated D-glucosamine groups in an unusual trisaccharide sequence.

The detailed characterization of a novel heparin-like glycosaminoglycan purified from the viscera (heads) of the shrimp Litopenaeus vannamei is reported. Structural analysis performed by mono- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed it to be rich in both glucuronic acid and N,6-sulfated glucosamine residues. The key peculiarities were its high 3-O-sulfated glucosamine content compared to mammalian heparins; a residue which is usually associated with the antithrombin (AT) binding site, and the location of these residues within 2-O-sulfated iduronate and glucuronate-containing sequences (I2S-A(∗)-G), a situation not found in mammalian heparin. It also exhibited higher molecular weight (∼36kDa) than conventional heparin (∼16kDa) but, negligible anticoagulant activity (∼5IU/mg compared to heparin ∼190IU/mg) and stabilization of AT, which has been linked directly to anticoagulation activity. A high affinity fraction, eluting at a similar salt concentration (0.75-1.5M NaCl) from an antithrombin affinity column, to the high affinity fraction of heparin, also showed only weak thermal stabilization of AT (+∼2°C). These structural peculiarities may help elucidate more clearly the relationship between structure and function of sulfated polysaccharides, and provide useful model compounds with which to better understand interactions of biological significance.

Low-molecular-weight heparins: differential characterization/physical characterization.

Low-molecular-weight heparins (LMWHs), derived from unfractionated heparin (UFH) through different depolymerization processes, have advantages with respect to the parent heparin in terms of pharmacokinetics, convenience of administration, and reduced side effects. Each LMWH can be considered as an independent drug with its own activity profile, placing significance on their biophysical characterization, which will also enable a better understanding of their structure-function relationship. Several chemical and physical methods, some involving sample modification, are now available and are reviewed.

Heparin-like heparan sulfate from rabbit cartilage.

Glycosaminoglycans were extracted from both young rabbit growth plate (GRP) and articular (ART) cartilage tissues and enzymatically treated to selectively eliminate chondroitin sulfates and hyaluronic acid. The procedure avoided any fractionation step that could enrich the extract with over- or under-sulfated species. Isolated heparan sulfate (HS) was characterized by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy to quantify their specific structural features and/or by mass spectrometry to establish the disaccharide composition. Both GRP and ART HSs, despite differing in their yield (GRP at least 100 times greater than ART), exhibited a surprisingly high degree of sulfation. Quantitative two-dimensional heteronuclear single-quantum coherence-NMR analysis of GRP HS revealed unusually high N-sulfated glucosamine and 2-O-sulfated iduronic acid contents, similar to heparin. The unique pentasaccharide sequence of the binding site for antithrombin was also detected in a significant amount. High-performance liquid chromatography mass spectrometry analysis of the enzymatic digests with a cocktail of heparin lyases of both cartilaginous HSs confirmed the NMR results. As well as the discovery of an unusual HS structure in the two different types of rabbit cartilage, the feasibility of the analytical method adopted here has been demonstrated within this study. Such a method can be used to isolate and analyze HS from both normal and pathologic tissues. Characterization of healthy and pathological HS structures will contribute to improve the understanding of diseases related to malfunctions of HS biosynthesis and/or metabolism.

Structural features of low-molecular-weight heparins affecting their affinity to antithrombin.

As part of a more extensive investigation on structural features of different low-molecular-weight heparins (LMWHs) that can affect their biological activities, Enoxaparin, Tinzaparin and Dalteparin were characterised with regards to the distribution of different chain length oligosaccharides as determined by size-exclusion (SE) chromatography, as well as their structure as defined by 2D-NMR spectra (HSQC). The three LMWHs were also fractionated into high affinity (HA) and no affinity (NA) pools with regards to their ability to bind antithrombin (AT). The HA fractions were further subfractionated and characterised. For the parent LMWHs and selected fractions, molecular weight parameters were measured using a SE chromatographic system with a triple detector (TDA) to obtain absolute molecular weights. The SE chromatograms clearly indicate that Enoxaparin is consistently richer in shorter oligosaccharides than Tinzaparin and Dalteparin. Besides providing the content of terminal groups and individual glucosamine and uronic acid residues with different sulfate substituents, the HSQC-NMR spectra permitted us to evaluate and correlate the content of the pentasaccharide, AT-binding sequence A-G-A*-I-A (AT-bs) through quantification of signals of the disaccharide sequence G-A*. Whereas the percent content of HA species is approximately the same for the three LMWHs, substantial differences were observed for the chain distribution of AT-bs as a function of length, with the AT-bs being preferentially contained in the longest chains of each LMWH. The above information will be useful in establishing structure-activity relationships currently under way. This study is therefore critical for establishing correlations between structural features of LMWHs and their AT-mediated anticoagulant activity.

The tainted heparin story: an update.

Recently, certain batches of heparin have been associated with an acute, rapid onset of serious side-effects indicative of allergic-type reactions. These reports generated significant concern regarding the possible presence of a dangerous contaminant within heparin and highlighted the need to re-assess the purity criteria of heparin preparations for clinical use. Given the nature of the array of all possible contaminants, traditional screening tests cannot safely differentiate between contaminated and uncontaminated heparin preparations. Mono- and bi-dimensional NMR spectroscopy are powerful techniques that are able to detect and quantify a wide variety of potential sulfated polysaccharide contaminants. As such, these techniques are powerful tools for the analysis and assessment of heparin preparations.

Focusing of low-molecular-mass heparins in polycationic polyacrylamide matrices.

A novel method for separation of low-molecular-mass heparins is reported here, on the basis of migrating the polyanionic heparins in a polycationic polyacrylamide gel, made by incorporating a gradient of positively charged monomers (the Immobilines used for creating immobilized pH gradients) into the neutral polyacrylamide backbone. Separations can be operated either in linear or nonlinear gradients of positive charges, thus modulating at whim the separation power. This allows the polydisperse heparins to reach a steady-state position along the migration path and condense (focus) in an environment inducing charge neutralization. It is shown that the separations obtained are a complex function of both size and charge distribution along the oligosaccharide chains. This novel methodology represents a marked improvement over existing techniques and appears to hold promise for applications in screening of commercial lots of heparins, also in view of possible presence of contaminants, such as those recently detected in imported heparins.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events.

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.

High-performance liquid chromatographic/mass spectrometric studies on the susceptibility of heparin species to cleavage by heparanase.

Heparanase is an endo-beta-D-glucuronidase that cleaves the heparan sulfate chains of heparan sulfate proteoglycans and is implicated in angiogenesis and metastasis. With the aim of establishing a simple and reliable method for studying the susceptibility of heparin/heparan sulfate oligosaccharides to be cleaved by heparanase, an on-line ion pair reversed-phase high-performance liquid chromatographic/electrospray ionization mass spectrometric method was set up. The method works in the micromolar range of concentration and does not require derivatization of the substrate or of the products. It is based on mass identification of oligosaccharide fragments generated by heparanase and their quantification with reference to an internal heparin disaccharide standard. Substrates were (1) the synthetic pentasaccharides GlcN (NS,6S) - GlcA - GlcN (NS,3S,6S) - IdoA (2S) - GlcN (NS,6S) - OMe (AGA*IA (M)) and GlcN (NS,6S) - GlcA - GlcN (NS,6S) - IdoA (2S) - GlcN (NS,6S) - OMe (AGAIA (M)), corresponding to the heparin/heparan sulfate active site for antithrombin, and to the same sequence devoid of the 3- O-sulfate group in the central glucosamine, respectively; and (2) two natural heparin octasaccharides containing the AGA*IA sequence in different locations along the chain. The two pentasaccharides exhibited a higher susceptibility to heparanase cleavage with respect to the octasaccharides. The commercial availability of AGA*IA (M) makes it an ideal substrate to determine the specific activity of heparanase preparations. The present method could also be used for rapid screening of potential heparanase inhibitors.

Molecular weight determination of heparin and dermatan sulfate by size exclusion chromatography with a triple detector array.

The determination of molecular weight (M) and molecular weight distribution (MD) of heparins by a novel approach, consisting of a high performance size exclusion chromatography (HP-SEC) combined with a triple detector array (TDA) is described. HP-SEC/TDA permits the evaluation of MD of polymeric samples through a combined and simultaneous action of three on-line detectors, right-angle laser light scattering (RALLS), refractometer (RI), and viscometer. The method does not require any chromatographic column calibration, thus overcoming also the difficulty to obtain adequate reference standards. It permits the size determination also of small molecules, even when scattering dissimmetry is not observable. Unfractionated heparins, eight fractions of a size fractionated heparin, and dermatan sulfates were analyzed by HP-SEC/TDA. The M values found for the heparin fractions were used to build up a calibration curve of a conventional HP-SEC system: the results obtained analyzing unfractionated heparin samples with both HP-SEC/TDA and HP-SEC were in excellent agreement, suggesting the possibility to use the TDA data to generate standard samples with known MD and intrinsic viscosity [eta]. Moreover, HP-SEC/TDA can successfully be employed also for the determination of the Mark-Houwink a and k parameters.

Active conformations of glycosaminoglycans. NMR determination of the conformation of heparin sequences complexed with antithrombin and fibroblast growth factors in solution.

Binding to proteins usually induces perturbation of nuclear magnetic resonances of ligand molecules. Using sensitive nuclear magnetic resonance (NMR) spectroscopy techniques, these perturbations have been measured for heparin oligosaccharides in aqueous solution in the presence of proteins and the NMR data have been used to characterize the three-dimensional (3D) structure of the oligosaccharides in the bound state. The pentasaccharide corresponding to the active site of heparin/heparan sulfate for antithrombin (AT) adopts in the complex with the protein a conformation different from that in the absence of the protein. A notable difference involves the 2-O-sulfated iduronic acid (IdoA2S) residue, which is driven to adopt an exclusively skew-boat @affil2: 2S 0 form in the complex. In addition, complexing induces a change in the geometry around the glycosidic linkage between the nonreducing end glucosamine and the adjacent glucuronic acid residue as compared with the free state. NMR and molecular modeling data also indicate that the 2-O-sulfate group in the IdoA2S residue is not directly involved in binding to AT. This suggests that its role is mainly that of affecting the conformational equilibrium of this residue, leading to a 3D structure of pentasaccharide in the bound state that meets the stereochemical requirements of the receptor and results in high-affinity binding to the protein. On the other hand, NMR studies of heparin tetrasaccharides in the presence of fibroblast growth factors FGF-1 and FGF-2 indicate that FGF binding stabilizes the @affil1: 1C 4 conformation of the IdoA2S residue directly involved in binding. These studies also confirm the crucial role of the 6-O-sulfate group on at least one glucosamine residue in the formation of the complex with FGF-1 but not with FGF-2.

Minimal heparin/heparan sulfate sequences for binding to fibroblast growth factor-1.

The glycosaminoglycans heparin and heparan sulfate (HS) bind to fibroblast growth factor FGF1 and promote its dimerization, a proposed prerequisite for binding to a cellular receptor and triggering mitogenic signals. The problem of minimal structural requirements for heparin/HS sequences to bind FGF1 was approached by surface plasmon resonance (SPR), NMR spectroscopy, and MALDI mass spectrometry studies using the three synthetic tetrasaccharides GlcNSO(3)6OR-IdoA2SO(3)-GlcNSO(3)6OR'-IdoA2SO(3)OPr (AA, R = R' = SO(3); BA, R = H, R' = SO(3); BB, R = R' = H; Pr, propyl). AA and BA significantly interact with the protein, whereas BB is practically inactive. The NMR spectra show that, whereas the interaction of AA primarily involves the GlcNSO(3)6SO(3)IdoA2SO(3) disaccharide moiety at its nonreducing end, residues at both the nonreducing (NR) and reducing side (R) appear to be involved in the weaker complex of BA. Furthermore, MALDI experiments show that, in addition to 1:1 protein:tetrasaccharide complexes, AA and BA are able to form 2:1 complexes, indicating that heparin/HS-induced dimerization of FGF1 requires only one 6-OSO(3) group per tetrasaccharide.