Related Glycosaminoglycans Research Articles

Multiplex genome editing of mammalian cells for producing recombinant heparin

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.

Glycosaminoglycan‐Induced Emissive J‐Aggregate Formation in a meso‐Ester BODIPY Dye

AbstractThe analyte‐directed formation of emissive J‐aggregates of a meso‐ester BODIPY dye is explored in the design of fluorescence “light‐up” probes. The heparin‐specific pyridinium‐functionalized meso‐ester BODIPY dye C10‐Py+ self‐assembles in aqueous buffer into spherical aggregated nanostructures with spectroscopic features indistinguishable from the monomeric dye (signal off). In the presence of heparin, the aggregated assemblies rearrange and associate with heparin through charge‐pairing interactions, triggering the spontaneous formation of emissive J‐type aggregates (signal on). The heparin‐induced J‐aggregate formation of C10‐Py+ provides an excellent “turn‐on” signal with large spectral shifts. The response is highly selective for heparin over other closely related glycosaminoglycan (GAG) analogues, sensitive, fast, and operationally simple. Assays for heparin in human serum, and for the adulterant OSCS in heparin are demonstrated, confirming the reliability and robustness of the J‐aggregation approach using meso‐ester BODIPY dyes in the design of aggregation‐induced emission (AIE)‐based fluorogenic probes.

New Applications of Heparin and Other Glycosaminoglycans.

Heparin, the widely used pharmaceutical anticoagulant, has been in clinical use for well over half a century. Its introduction reduced clotting risks substantially and subsequent developments, including the introduction of low-molecular-weight heparin, made possible many major surgical interventions that today make heparin an indispensable drug. There has been a recent burgeoning of interest in heparin and related glycosaminoglycan (GAG) polysaccharides, such as chondroitin sulfates, heparan sulfate, and hyaluronate, as potential agents in various applications. This ability arises mainly from the ability of GAGs to interact with, and alter the activity of, a wide range of proteins. Here, we review new developments (since 2010) in the application of heparin and related GAGs across diverse fields ranging from thrombosis and neurodegenerative disorders to microbiology and biotechnology.

¹H and (15)N NMR Analyses on Heparin, Heparan Sulfates and Related Monosaccharides Concerning the Chemical Exchange Regime of the N-Sulfo-Glucosamine Sulfamate Proton.

Heparin and heparan sulfate are structurally related glycosaminoglycans (GAGs). Both GAGs present, although in different concentrations, N-sulfo-glucosamine (GlcNS) as one of their various composing units. The conditional fast exchange property of the GlcNS sulfamate proton in these GAGs has been pointed as the main barrier to its signal detection via NMR experiments, especially 1H-15N HSQC. Here, a series of NMR spectra is collected on heparin, heparan sulfate and related monosaccharides. The N-acetyl glucosamine-linked uronic acid types of these GAGs were properly assigned in the 1H-15N HSQC spectra. Dynamic nuclear polarization (DNP) was employed in order to facilitate 1D spectral acquisition of the sulfamate 15N signal of free GlcNS. Analyses on the multiplet pattern of scalar couplings of GlcNS 15N has helped to understand the chemical properties of the sulfamate proton in solution. The singlet peak observed for GlcNS happens due to fast chemical exchange of the GlcNS sulfamate proton in solution. Analyses on kinetics of alpha-beta anomeric mutarotation via 1H NMR spectra have been performed in GlcNS as well as other glucose-based monosaccharides. 1D 1H and 2D 1H-15N HSQC spectra recorded at low temperature for free GlcNS dissolved in a proton-rich solution showed signals from all exchangeable protons, including those belonging to the sulfamate group. This work suits well to the current grand celebration of one-century-anniversary of the discovery of heparin.

Role of EXT1 and Glycosaminoglycans in the Early Stage of Filovirus Entry.

Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients. Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.

Structure and function of a fungal adhesin that binds heparin and mimics thrombospondin-1 by blocking T cell activation and effector function.

Blastomyces adhesin-1 (BAD-1) is a 120-kD surface protein on B. dermatitidis yeast. We show here that BAD-1 contains 41 tandem repeats and that deleting even half of them impairs fungal pathogenicity. According to NMR, the repeats form tightly folded 17-amino acid loops constrained by a disulfide bond linking conserved cysteines. Each loop contains a highly conserved WxxWxxW motif found in thrombospondin-1 (TSP-1) type 1 heparin-binding repeats. BAD-1 binds heparin specifically and saturably, and is competitively inhibited by soluble heparin, but not related glycosaminoglycans. According to SPR analysis, the affinity of BAD-1 for heparin is 33 nM±14 nM. Putative heparin-binding motifs are found both at the N-terminus and within each tandem repeat loop. Like TSP-1, BAD-1 blocks activation of T cells in a manner requiring the heparan sulfate-modified surface molecule CD47, and impairs effector functions. The tandem repeats of BAD-1 thus confer pathogenicity, harbor motifs that bind heparin, and suppress T-cell activation via a CD47-dependent mechanism, mimicking mammalian TSP-1.

Recognition of heparan sulfate by clinical strains of dengue virus serotype 1 using recombinant subviral particles

Dengue is the most important arthropod-borne viral disease in humans, with an estimated 3.6 billion people at risk for infection and more than 200 million infections per year. Identification of the cellular receptors for dengue virus (DV), the causative agent of dengue, is important toward understanding the pathogenesis of the disease. Heparan sulfate (HS) has been characterized as a DV receptor in multiple model systems, however the physiological relevance of these findings has been questioned by observations that flaviviruses, including DV, can undergo cell culture adaptation changes resulting in increased binding to HS. It thus remains unclear whether HS is utilized by clinical, non-cell culture-adapted strains of DV. To address this question, herein we describe a set of methodologies using recombinant subviral particles (RSPs) to determine the utilization of HS by clinical strains of DV serotype 1 (DV1). RSPs of clinically isolated strains with low cell culture passage histories were used to study HS interaction. Biochemically characterized RSPs showed dose-dependent binding to immobilized heparin, which could be competed by heparin and HS but not structurally related glycosaminoglycans chondroitin sulfate A and hyaluronic acid. The relevance of heparin and HS biochemical interactions was demonstrated by competition of RSP and DV binding to cells with soluble heparin and HS. Our results demonstrate that clinical strains of DV1 can specifically interact with heparin and HS. Together, these data support the possibility that HS on cell surfaces is utilized in the DV-human infection process.

Heparin Characterization: Challenges and Solutions

Although heparin is an important and widely prescribed pharmaceutical anticoagulant, its high degree of sequence microheterogeneity and size polydispersity make molecular-level characterization challenging. Unlike nucleic acids and proteins that are biosynthesized through template-driven assembly processes, heparin and the related glycosaminoglycan heparan sulfate are actively remodeled during biosynthesis through a series of enzymatic reactions that lead to variable levels of O- and N-sulfonation and uronic acid epimers. As summarized in this review, heparin sequence information is determined through a bottom-up approach that relies on depolymerization reactions, size- and charge-based separations, and sensitive mass spectrometric and nuclear magnetic resonance experiments to determine the structural identity of component oligosaccharides. The structure-elucidation process, along with its challenges and opportunities for future analytical improvements, is reviewed and illustrated for a heparin-derived hexasaccharide.

Low Anticoagulant Heparin Disrupts Plasmodium falciparum Rosettes in Fresh Clinical Isolates

The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria.

NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used 1H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the 1H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion. FigureDOSY spectra of intact (blue) and digested (red) heparin illustrating the differences in their diffusion coefficients.

Characterization of uniformly and atom-specifically 13C-labeled heparin and heparan sulfate polysaccharide precursors using 13C NMR spectroscopy and ESI mass spectrometry

The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin and heparan sulfate oligosaccharide or disaccharide fragments using simple 1D 1H NMR spectroscopy is often complicated by the extensive signal overlap. 13C NMR signals, on the other hand, overlap less and therefore, 13C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific 13C-labeled heparin and heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly 13C- and 15N-labeled precursors were also produced and characterized by 13C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of 13C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% 13C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome–proteome interactions.

The C. elegans hyaluronidase: A developmentally significant enzyme with chondroitin-degrading activity at both acidic and neutral pH

Mammalian hyaluronidases degrade hyaluronan and some structurally related glycosaminoglycans. We generated a deletion mutant in the Caenorhabditis elegans orthologue of mammalian hyaluronidase, hya-1. Mutant animals are viable and grossly normal, but exhibit defects in vulval morphogenesis and egg-laying and showed increased staining with alcian blue, consistent with an accumulation of glycosaminoglycan. A hya-1::GFP reporter was expressed in a restricted pattern in somatic tissues of the animal with strongest expression in the intestine, the PLM sensory neurons and the vulva. Total protein extracts from wild-type animals exhibited chondroitin-degrading but not hyaluronan-degrading activity. Chondroitinase activities were observed at both neutral and acidic pH conditions while both neutral and acidic activities were absent in extracts from hya-1 mutant strains. We also evaluated the function of oga-1, which encodes the C. elegans orthologue of MGEA-5, a protein with hyaluronan-degrading activity in vitro. oga-1 is expressed in muscles, vulval cells and the scavenger-like coelomocytes. An oga-1 mutant strain exhibited egg-laying and vulval defects similar to those of hya-1; chondroitinase activity was unaffected in this mutant.

Depolymerized Holothurian Glycosaminoglycan Inhibits Plasma Thrombin Generation Via Interaction with the Factor IXa Heparin-Binding Exosite

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties in rodent thrombosis and dog hemodialysis models. DHG demonstrates significantly less bleeding in template or tail transection assays than therapeutically equivalent doses of heparins. Several potential in vitro mechanisms have been described for DHG, including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The relevant mechanism(s) for inhibition of tissue factor (TF) induced plasma thrombin generation by DHG were examined in HCII or mock-immunodepleted, and factor-deficient human plasmas, using selected recombinant factor IX(a) with mutations in the heparin-binding exosite. Plasma thrombin generation was detected by fluorogenic substrate cleavage in the presence of corn trypin inhibitor to block contact activation, and compared to a standard curve generated with α2-macroglobulin-thrombin complex. The dose-dependent decrease in velocity index, a parameter reflecting the rate of thrombin generation between lag phase and peak thrombin concentration, was used to compare DHG potency. When triggered by 0.2 pM TF, the EC50 for inhibition of thrombin generation by DHG was 0.16 ± 0.01 μM in both HCII-depleted and mock-depleted plasma, suggesting that DHG acts independently of HCII. When triggered by excess (4 pM) TF, plasma thrombin generation was independent of factors VIII and IX. Under these conditions, the EC50 for DHG inhibition of thrombin generation was increased 13-fold in mock-depleted plasma (2.02 ± 0.09 μM) and 28-fold in HCII-depleted plasma (4.31 ± 0.23 μM). These results suggest that components of the intrinsic tenase complex contribute to inhibition of plasma thrombin generation by DHG, and HCII contributes only at high tissue factor concentrations. In the presence of 0.2 pM TF, Western blotting under nonreducing conditions showed preservation of the prothrombin/meizothrombin band and delayed/reduced thrombin generation in the presence of 0.5 μM DHG, confirming that the inhibition involves reduced prothrombin activation rather than accelerated thrombin inhibition. When triggered by 0.2 pM TF in factor VIII-deficient plasma supplemented with 700 pM factor VIII or thrombin-activated factor VIIIa, the EC50 for inhibition by DHG was 0.41 ± 0.02 μM and 0.44 ± 0.05 μM, respectively. Similarly, the EC50 for DHG inhibition of thrombin generation in factor IX deficient plasma supplemented with 0.2 pM TF and 100% plasma-derived factor IX (90 nM), or 100 pM plasma-derived factor IXa alone, was 0.36 ± 0.01 μM and 0.34 ± 0.02 μM, respectively. Thus, activation of factors VIII and IX do not contribute significantly to the inhibition mechanism for DHG. The contribution of intrinsic tenase activity to DHG inhibition of plasma thrombin generation was assessed using recombinant factor IX(a) mutants with moderate (R170A) or marked (R233A) reductions in heparin affinity. Factor IX deficient plasma was supplemented with 0.2 pM TF and 100% recombinant factor IX, or 100 pM factor IXa, with increasing concentrations of DHG. Similar to plasma-derived factor IX(a), DHG demonstrated an EC50 of 0.38 ± 0.01 μM for inhibition of thrombin generation in the presence of factor IX(a) wild type (WT) zymogen or protease. In the presence of factor IX(a) R170A, the EC50 for DHG was 0.86 ± 0.06 μM and 1.02 ± 0.02 μM, respectively, a 2–3 fold increase relative to WT (P ≤ 0.01). For factor IX(a) R233A, the EC50 for DHG was 3.55 ± 0.47 μM for zymogen and 2.98 ± 0.64 μM for protease, an 8–9 fold increase relative to WT (P ≤ 0.01). Thus, mutations in the factor IXa heparin-binding exosite induced resistance to DHG inhibition of thrombin generation as follows: factor IX(a) R233A> R170A> WT. These findings are consistent with the common mechanism for intrinsic tenase inhibition demonstrated for heparin and DHG in purified systems, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition of plasma thrombin generation by DHG. This antithrombin-independent mechanism likely mediates the antithrombotic efficacy of DHG and related glycosaminoglycans, and may represent a novel therapeutic target with lower bleeding risk.

Heparin and structurally related polymers attenuate eotaxin‐1 (CCL11) release from human airway smooth muscle

The glycosaminoglycan heparin has anti-inflammatory activity and is exclusively found in mast cells, which are localized within airway smooth muscle (ASM) bundles of asthmatic airways. Interleukin (IL)-13 induces the production of multiple inflammatory mediators from ASM including the eosinophil chemoattractant chemokine, eotaxin-1. Heparin and related glycosaminoglycan polymers having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule linked to anti-inflammatory activity. Cultured human ASM cells were stimulated with interleukin (IL)-13 in the absence or presence of heparin and related polymers. Eotaxin-1 was quantified using chemokine antibody arrays and ELISA. Unfractionated heparin attenuated IL-13-dependent eotaxin-1 production and this effect was reproduced with low molecular weight heparins (3 and 6 kDa), demonstrating a minimum activity fragment of at least 3 kDa. N-desulphated, 20% re-N-acetylated heparin (anticoagulant) was ineffective against IL-13-dependent eotaxin-1 production compared with 90% re-N-acetylated (anticoagulant) or O-desulphated (non-anticoagulant) heparin, suggesting a requirement for N-sulphation independent of anticoagulant activity. Other sulphated molecules with variable anionic charge and molecular weight exceeding 3 kDa (dextran sulphate, fucoidan, chondroitin sulphate B) inhibited IL-13-stimulated eotaxin-1 release to varying degrees. However, non-sulphated dextran had no effect. Inhibition of IL-13-dependent eotaxin-1 release by heparin involved but did not depend upon sulphation, though loss of N-sulphation reduced the attenuating activity, which could be restored by N-acetylation. This anti-inflammatory effect was also partially dependent on anionic charge, but independent of molecular size above 3 kDa and the anticoagulant action of heparin.

Heparin‐binding proteins of human seminal plasma: purification and characterization

Human seminal plasma (HuSP) contains several proteins that bind heparin and related glycosaminoglycans. Heparin binding proteins (HBPs) from seminal plasma have been shown to participate in modulation of capacitation or acrosome reaction and thus have been correlated with fertility in some species. However, these have not been studied in detail in human. The objective of this study was to purify major HBPs from HuSP in order to characterize these proteins. HBPs were isolated by affinity-chromatography on Heparin-Sepharose column, purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and Size-exclusion chromatography and checked for purity on sodium-dodecyl PAGE (SDS-PAGE). Identification of HBPs was done by matrix-assisted laser desorption-ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Here we report the purification and identification of seven HBPs in seminal fluid. The major HBPs are lactoferrin and its fragments, semenogelin I fragments, semenogelin II, prostate specific antigen, homolog of bovine seminal plasma-proteins (BSP), zinc finger protein (Znf 169) and fibronectin fragments. In this study we are reporting for the first time the purification and identification of BSP-homolog and Znf 169 from HuSP and classified them as HBPs. Here we report the purification of seven clinically important proteins from human seminal fluid through heparin affinity chromatography and RP-HPLC, in limited steps with higher yield.

Platelet GPIIb/IIIa antagonist, XV459, in heparin‐induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a serious, immune-related complication of heparin therapy. One of the most severe manifestations of HIT is the development of thromboembolic events, which is based on platelet activation and aggregation caused by HIT-associated antibodies. Therapeutic options for patients with HIT are limited despite advancement toward the development of alternative (nonheparin) anticoagulants, such as direct thrombin inhibitors and indirect anti-factor Xa agents. Platelet GPIIb/IIIa receptor antagonists have been shown to be the final common pathway for platelet aggregation regardless of the use of activator or anticoagulant. In this study, the ability of a novel platelet GPIIb/IIIa antagonist, a free acid form of roxifiban (XV459), to block platelet activation/aggregation in response to highly characterized heparin-PF4 antibody-positive plasma/heparin was examined using light transmittance aggregometry, serotonin release, and (125)I-fibrinogen binding assays to human platelets. XV459 at 20 nM maximally inhibited (P < 0.001) the platelet-activation/aggregation responses as mediated by the HIT antibody-positive plasma (in the presence of therapeutic heparin concentrations). Compared with controls, both HIT antibodies/heparin and TEAC (a mixture of thrombin [0.1 IU/ml], epinephrine [1 microg/ml], arachidonate [0.1 mM], and collagen [10 microml]) resulted in significantly higher levels of fibrinogen binding to human platelets (5-7-fold increase; P < 0.001). Concentration-dependent profiles of XV459 on the mean percent inhibition of (125)I-fibrinogen binding in the presence of HIT antibodies and TEAC were achieved ( approximately 50% inhibition at 10 nM XV459). The platelet GPIIb/IIIa receptor antagonist (XV459) might be of potential benefit in the management of thrombotic thrombocytopenia produced by heparin and/or related glycosaminoglycans.

SULFATION CODE FOUND

A STUDY ON THE CARBOHYDRATE chondroitin sulfate (CS) reveals that it harbors a sulfation code: The carbohydrate encodes functional information sequence-specifically, similar to DNA and proteins. The findings have implications for understanding processes like neuronal development, viral invasion, and spinal cord injury. CS is found in joints and cartilage and is sold as an over-the-counter arthritis treatment. Many researchers assumed that the role of CS and related glycosaminoglycan polymers was primarily structural and gave them short shrift as a focus of study. Others suspected that glycosaminoglycans played a functional role as well, and variable sulfation was suggested as a way their bioactivity might be modulated. But a systematic functional understanding of glycosaminoglycan sulfation was hampered by the structural complexity and heterogeneity of these polysaccharides. Researchers had no way to study CS sulfation without synthesizing CS oligosaccharides having specific sulfation patterns, which is e...

Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: Role in the interactions with receptor activator of nuclear factor κB ligand (RANKL) and RANK

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor κB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity ( K D: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK–RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK–RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK–RANKL–OPG.

Cooperative Dimerization of Fibroblast Growth Factor 1 (FGF1) upon a Single Heparin Saccharide May Drive the Formation of 2:2:1 FGF1·FGFR2c·Heparin Ternary Complexes

The related glycosaminoglycans heparin and heparan sulfate are essential for the activity of the fibroblast growth factor (FGF) family as they form an integral part of the signaling complex at the cell surface. Using size-exclusion chromatography we have studied the capacities of a variety of heparin oligosaccharides to bind FGF1 and FGFR2c both separately and together in ternary complexes. In the absence of heparin, FGF1 had no detectable affinity for FGFR2c. However, 2:2:1 complexes formed spontaneously in solution between FGF1, FGFR2c, and heparin octasaccharide (dp8). The dp8 sample was the shortest chain length that bound FGFR2c, that dimerized FGF1, and that promoted a strong mitogenic response to FGF1 through FGFR2c. Heparin hexasaccharide and various selectively desulfated heparin dp12s failed to bind FGFR2c and could only interact with FGF1 monomerically. These saccharides formed 1:1:1 complexes with FGF1 and FGFR2c, which had no tendency to self-associate, suggesting that binding of two FGF1 molecules to the same saccharide chain is a prerequisite for subsequent FGFR2c dimerization. We found that FGF1 dimerization upon heparin was favored over monomeric interactions even when a large excess of saccharide was present. A cooperative mechanism of FGF1 dimerization could explain how 2:2:1 signaling complexes form at the cell surface, an environment rich in heparan sulfate.

Some structural determinants of the antiproliferative effect of heparin-like molecules on human airway smooth muscle.

Accumulation of airway smooth muscle (ASM) and its infiltration by mast cells are key pathological features of airway remodelling in asthma. Heparin, a major component of mast cell granules, inhibits ASM proliferation by an unknown mechanism. Here, unfractionated heparins and related glycosaminoglycans having structurally heterogeneous polysaccharide side chains that varied in molecular weight, sulphation and anionic charge were used to identify features of the heparin molecule that were required for its antiproliferative activity in cultured human ASM cells. Proliferation induced by 10% fetal bovine serum (FBS) was abrogated by two unfractionated commercial heparin preparations (Sigma and Multiparin) and this effect was reproduced with each of three low-molecular weight heparin preparations (3, 5 and 6 kDa, respectively), demonstrating that antiproliferative activity resided in at least a 3 kDa heparin fraction. N-desulphated 20% re-acetylated (N-de) heparin (anticoagulant) and O-desulphated heparin (O-de) (non-anticoagulant) fractions also inhibited FBS-dependent proliferation (rank potency: Sigma heparin > O-de > N-de) suggesting that the antiproliferative action of heparin involved N-sulphation but was independent of its anticoagulant activity. Other sulphated molecules with variable anionic charge (dextran sulphate, fucoidan, chondroitin sulphates A or B, heparan sulphate) inhibited proliferation to varying degrees, as did the non-sulphated molecules hyaluronic acid and poly-L-glutamic acid. However, nonsulphated dextran had no effect. In summary, attenuation of FBS-dependent proliferation of human ASM by heparin involves but does not depend upon sulphation, although loss of N-sulphation reduces antiproliferative activity. This antiproliferative effect is independent of anionic charge and the anticoagulant actions of heparin.