Trisulfated Disaccharide Research Articles

Immune-mediated Neuropathy with Tri-sulfated Heparin Disaccharide or Fibroblast Growth Factor Receptor 3 Antibodies in Pediatric Patients: A Case Series

Immune-mediated Neuropathy with Tri-sulfated Heparin Disaccharide or Fibroblast Growth Factor Receptor 3 Antibodies in Pediatric Patients: A Case Series

Comparing FGFR-3 and TS-HDS Seropositive Small Fiber Neuropathy: Unique Patient Features, Symptoms, Laboratory, and Nerve Conduction Study Findings.

Small fiber neuropathy presents a significant diagnostic and therapeutic challenge. To solve this challenge, efforts have been made to identify autoantibodies associated with this condition. Previous literature has often considered tri-sulfated heparin disaccharide (TS-HDS) and fibroblast growth factor receptor 3 (FGFR3) as a singular seropositive group and/or focused primarily on symptomatic associations. One hundred seventy-two small fiber neuropathy patients with a Washington University Sensory Neuropathy panel were selected for TS-HDS seropositivity, FGFR-3 seropositivity, and seronegative controls. Data were collected to on the demographic, symptomatic, and laboratory profiles of each subgroup. Percent female (P = 0.0043), frequency of neuropathic pain symptoms (P = 0.0074), and erythrocyte sedimentation rate (P = 0.0293), vitamin D (P < 0.0001), and vitamin B12 (P = 0.0033) differed between the groups. Skin biopsy was more frequently normal within both the FGFR-3 and the TS-HDS cohort (P = 0.0253). TS-HDS and FGFR-3 display a distinct phenotype from both controls and one another. Immunoglobulin M (IgM) against FGFR-3 and IgM against TS-HDS may be individually valuable markers for the development of distinct clinical phenotypes.

Abstract P2136: TpTe Interval Behavior And Mortality In Wistar Rats Submitted To Cardiac Ischemia And Reperfusion Treated With The Heparin Fragment Trisulfated Disaccharide

Introduction: The Tp-Te ekg interval reflects ventricular transmural dispersion, being used for prediction of ventricular arrhythmia and sudden death. The heparin fragment called Trisulfated Disaccharide (TD), which has no effect on blood coagulation and acts on the sodium-calcium exchanger accelerating the output of calcium from the cell, decreases cytoplasmic calcium concentrations in situations of calcium overload due to ischemia and reperfusion (I-R). Objective: To evaluate the behavior of the Tp-Te interval and mortality due to cardiac arest in Wistar rats submitted to surgicaly induced I-R under treatment with TD at reperfusion. Materials and Method: Wistar rats (n=8) with average weight of 300g were anesthetized and under assisted breathing were electrocardiographically monitored and submitted to surgical cardiac ischemia by ligation of the anterior descending artery for 10 min, with reperfusion thereafter. The Tp-Te interval was analyzed before ischemia, at 10 min after ischemia induction and at 10 min after reperfusion, The incidence of arrhythmias was determined throughout the recording period. In the study group intravenous (0.2 mg/kg) TD was injected concomitantly with the reperfusion. In the control group (n=8), the same procedure was performed, without the TD infusion. Results: During ischemia there was a similar alteration of the Tp-Te interval in both groups when compared to the pre-ischemic phase, with an increase of about 90% in magnitude, as well as the presence of ventricular arrhythmias, without animal mortality. During reperfusion Tp-Te interval returned to normal in the treated group and there was no mortality. In the control group, the magnitude of the Tp-Te increase was maintained. The presence of ventricular arrhythmias was similar, but total atrioventricular block and/or sustained arrhythmia occurred in 50% of the animals, followed by animals death. Conclusion: Treatment with TD at the time of reperfusion normalized the Tp-Te interval 10 min after TD infusion as well as reduced mortality in wistar rats submitted to I-R, and may be useful in the protection of organs submitted to the same I-R conditions (transplants, surgical and hemodynamic procedures), where calcium overload is determinant in morbidity and mortality.

Immune-Mediated Small Fiber Neuropathy With Trisulfated Heparin Disaccharide, Fibroblast Growth Factor Receptor 3, or Plexin D1 Antibodies: Presentation and Treatment With Intravenous Immunoglobulin.

Up to 50% of small fiber neuropathy (SFN) cases are idiopathic, but novel antibodies to Trisulfated Heparin Disaccharide (TS-HDS) and fibroblast growth factor receptor 3 (FGFR-3) have been implicated in half of these cases; the role of anti-Plexin D1 is less clear. We aimed to clarify presentation and management of these patients. An 18-month retrospective analysis revealed 54 cases of cryptogenic SFN who had testing for the 3 autoantibodies. Demographics, clinical features, epidermal nerve fiber density, and Quantitative Sudomotor Axon Reflex Test results were analyzed. Intravenous immunoglobulin (IVIG) treatment response was assessed. In total, 44.4% of patients had antibodies (62.5% TS-HDS, 29.2% FGFR-3, and 20.8% Plexin D1). Male patients were more likely to be FGFR-3 positive (P = 0.014). Facial involvement was more common in seropositive patients (P = 0.034), and patients with a higher Utah Early Neuropathy Scale score had a higher TS-HDS titer (P = 0.0469), but other clinical features were not significantly different. Seropositive patients trended toward a higher SFN screening list score (P = 0.16), abnormal Quantitative Sudomotor Axon Reflex Test (P = 0.052), and prior erroneous diagnosis (P = 0.19). In patients who completed IVIG, examinations and questionnaires improved and mean epidermal nerve fiber density increased by 297%. TS-HDS, FGFR-3, and Plexin D1 antibodies are present in a high proportion of cryptogenic SFN cases with more facial involvement, and greater disease severity is associated with higher antibody titers. They are often misdiagnosed but may respond subjectively and objectively to IVIG.

Abstract P1095: Analysis Of Pro-arrhythmic Effects Of The Trisulfated Disaccharide Heparin Fragment, A Potential Antiarrhythmic Drug.

Background: The heparin fragment called trisulfated disaccharide (TD) acts on the sodium-calcium exchanger by removing excess calcium from cytoplasm with antiarrhythmic action demonstrated in animal models in the reversal of supraventricular and ventricular arrhythmia. However, it is known that drugs with antiarrhythmic potential can induce arrhythmic phenomena, limiting and interrupting the study of new potentially antiarrhythmic drugs. Objective: To evaluate the arrhythmogenic potential of TD in relation to the stimulation of hERG channels in human cardiomyocytes. Material and Methods: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in culture were plated onto a fibronectin coated 48 well microelectrode array plate at a density of 25,000 cells per well in iCell® maintenance medium. The plate was incubated for 7 days until a stable beating phenotype was achieved, and the cells were ready for drug treatment. They were dosed with 0.2% DMSO vehicle, E-4031 (0.05μM), or cisapride (0.05μM) as controls (arrhythmogenic drugs). They were also dosed with TD at four concentrations in triplicate. The activity of the cells was recorded prior to dosing and at 1 hour using the Axion Biosystems Maestro MEA system. Beat period (s), fast Na+ slope (V/s), fast Na+ amplitude (mV) and field potential duration (FPD) were evaluated. Results: E-4031 treatment resulted in a significant increase in beat period length (decrease in beat rate) and a decrease in the Na slope and amplitude. There was a very large increase in the FPD. Cisapride showed a very similar response. Both compounds caused an increase in the ratio of the FPD to the beat period length. When the cardiomyocytes were incubated with the different doses of TD, there was a slight increase in the beat period length (decrease in the beat rate), which was observed regarding all the doses. There was an increase in the FPD, which is consistent with the change in beat rates suggesting that the change is purely related to beating and not indicative of any hERG effect or other responses. The ratio of the FPD to the beat period length was the same. There was a slight decrease in the sodium slope and amplitude, but they were not significant. Therefore, the only significant change observed was the beat rate. Conclusion: TD when exposed to human cardiomyocytes did not present an arrhythmogenic potential in relation to the hERG channels, and this is a desirable behavior for an antiarrhythmic drug.

Neuroprotective effect of heparin Trisulfated disaccharide on ischemic stroke.

Cells undergoing hypoxia experience intense cytoplasmic calcium (Ca2+) overload. High concentrations of intracellular calcium ([Ca2+]i) can trigger cell death in the neural tissue, a hallmark of stroke. Neural Ca2+ homeostasis involves regulation by the Na+/Ca2+ exchanger (NCX). Previous data published by our group showed that a product of the enzymatic depolymerization of heparin by heparinase, the unsaturated trisulfated disaccharide (TD; ΔU, 2S-GlcNS, 6S), can accelerate Na+/Ca2+ exchange via NCX, in hepatocytes and aorta vascular smooth muscle cells. Thus, the objective of this work was to verify whether TD could act as a neuroprotective agent able to prevent neuronal cell death by reducing [Ca2+]i. Pretreatment of N2a cells with TD reduced [Ca2+]i rise induced by thapsigargin and increased cell viability under [Ca2+]I overload conditions and in hypoxia. Using a murine model of stroke, we observed that pretreatment with TD decreased cerebral infarct volume and cell death. However, when mice received KB-R7943, an NCX blocker, the neuroprotective effect of TD was abolished, strongly suggesting that this neuroprotection requires a functional NCX to happen. Thus, we propose TD-NCX as a new therapeutic axis for the prevention of neuronal death induced by [Ca2+]i overload.

Functional Cellular Anti-Tumor Mechanisms are Augmented by Genetic Proteoglycan Targeting

While recent research points to the importance of glycans in cancer immunity, knowledge on functional mechanisms is lacking. In lung carcinoma among other tumors, anti-tumor immunity is suppressed; and while some recent therapies boost T-cell mediated immunity by targeting immune-checkpoint pathways, robust responses are uncommon. Augmenting tumor antigen-specific immune responses by endogenous dendritic cells (DCs) is appealing from a specificity standpoint, but challenging. Here, we show that restricting a heparan sulfate (HS) loss-of-function mutation in the HS sulfating enzyme Ndst1 to predominantly conventional DCs (Ndst1f/f CD11cCre+ mutation) results in marked inhibition of Lewis lung carcinoma growth along with increased tumor-associated CD8+ T cells. In mice deficient in a major DC HS proteoglycan (syndecan-4), splenic CD8+ T cells showed increased anti-tumor cytotoxic responses relative to controls. Studies examining Ndst1f/f CD11cCre + mutants revealed that mutation was associated with an increase in anti-tumor cytolysis using either splenic CD8+ T cells or tumor-infiltrating (TIL) CD8+ T cells purified ex-vivo, and tested in pooled effector-to-target cytolytic assays against tumor cells from respective animals. On glycan compositional analysis, HS purified from Ndst1f/f CD11cCre + mutant DCs had reduced overall sulfation, including reduced sulfation of a tri-sulfated disaccharide species that was intriguingly abundant on wildtype DC HS. Interestingly, antigen presentation in the context of major histocompatibility complex class-I (MHC-I) was enhanced in mutant DCs, with more striking effects in the setting of HS under-sulfation, pointing to a likely regulatory role by sulfated glycans at the antigen/MHC-I – T-cell interface; and possibly future opportunities to improve antigen-specific T cell responses by immunologic targeting of HS proteoglycans in cancer.

Cryptogenic small-fiber neuropathies: Serum autoantibody binding to trisulfated heparan disaccharide and fibroblast growth factor receptor-3.

Causes of small-fiber peripheral neuropathies (SFN) are often undefined. In this study we investigated associations of serum autoantibodies, immunoglobulin G (IgG) vs fibroblast growth factor receptor-3 (FGFR-3), and immunoglobulin M (IgM) vs trisulfated heparan disaccharide (TS-HDS) in cryptogenic SFN. One hundred fifty-five patients with biopsy-proven SFN and no identified cause for their neuropathy were blindly tested for serum IgM vs TS-HDS and IgG vs FGFR-3. Forty-eight percent of SFN patients had serum antibodies, 37% with IgM vs TS-HDS and 15% with IgG vs FGFR-3. TS-HDS antibodies were more frequent in SFN patients than in controls (P = .0012). Both antibodies were more common in females, and with non-length-dependent nerve pathology. Nintey-two percent of patients with acute-onset SFN had serum IgM vs TS-HDS. Autoantibodies directed against TS-HDS and FGFR-3 suggest an immune disorder in otherwise idiopathic SFN. Serum IgM vs TS-HDS may be a marker for SFN with an acute onset.

Circulating heparin oligosaccharides rapidly target the hippocampus in sepsis, potentially impacting cognitive functions

Sepsis induces heparanase-mediated degradation of the endothelial glycocalyx, a heparan sulfate-enriched endovascular layer critical to vascular homeostasis, releasing highly sulfated domains of heparan sulfate into the circulation. These domains are oligosaccharides rich in heparin-like trisulfated disaccharide repeating units. Using a chemoenzymatic approach, an undecasaccharide containing a uniformly 13C-labeled internal 2-sulfoiduronic acid residue was synthesized on a p-nitrophenylglucuronide acceptor. Selective periodate cleavage afforded a heparin nonasaccharide having a natural structure. This 13C-labeled nonasaccharide was intravenously administered to septic (induced by cecal ligation and puncture, a model of polymicrobial peritonitis-induced sepsis) and nonseptic (sham) mice. Selected tissues and biological fluids from the mice were harvested at various time points over 4 hours, and the 13C-labeled nonasaccharide was recovered and digested with heparin lyases. The resulting 13C-labeled trisulfated disaccharide was quantified, without interference from endogenous mouse heparan sulfate/heparin, using liquid chromatography-mass spectrometry with sensitive and selective multiple reaction monitoring. The 13C-labeled heparin nonasaccharide appeared immediately in the blood and was rapidly cleared through the urine. Plasma nonasaccharide clearance was only slightly prolonged in septic mice (t1/2 ∼ 90 minutes). In septic mice, the nonasaccharide penetrated into the hippocampus but not the cortex of the brain; no hippocampal or cortical brain penetration occurred in sham mice. The results of this study suggest that circulating heparan sulfates are rapidly cleared from the plasma during sepsis and selectively penetrate the hippocampus, where they may have functional consequences.

Prevalence of Axonal sensory neuropathy with IgM binding to Trisulfated heparin disaccharide (TS-HDS) in patients with Fibromyalgia (P2.2-100)

Prevalence of Axonal sensory neuropathy with IgM binding to Trisulfated heparin disaccharide (TS-HDS) in patients with Fibromyalgia (P2.2-100)

Prevalence of Axonal Sensory Neuropathy With IgM Binding to Trisulfated Heparin Disaccharide in Patients With Fibromyalgia.

To assess the intraepidermal nerve fiber density in patients diagnosed with fibromyalgia (FM) and to evaluate the role of IgM binding to trisulfated heparin disaccharide (TS-HDS) in these patients. FM is a poorly understood pain disorder with several proposed pathophysiologic mechanisms. It is characterized by widespread pain, fatigue, and sleep abnormalities. Small fiber neuropathy (SFN) has been proposed as an underlying mechanism, and patients with FM have been shown to have a reduction in the intraepidermal nerve fiber density. An underlying inflammatory process that could be a result of autoimmune phenomena has also been suggested. Non-length-dependent SFN (NLDSFN) has been shown to have a higher incidence of autoimmune disease. Twenty-two patients with established diagnosis of FM underwent skin biopsy at 2 sites; 10 cm above the lateral malleolus and 10 cm above the patella. Serum IgM binding to TS-HDS was assayed using an ELISA method. A total of 5/22 patients had positive TS-HDS antibodies; of these, 4 had NLDSFN (P = 0.0393). Comparison with a control group at Washington University showed no significant difference in percentage with TS-HDS antibodies (P = 0.41). When compared with Washington University database of skin biopsy, there was a trend for an increased percentage of NLDSFN in patients with FM (P = 0.06). This study further supports the hypothesis that a subgroup of patients with FM has SFN. We suggest a correlation between the presence of NLDSFN and TS-HDS antibodies.

Heparin: An essential drug for modern medicine.

Heparin is a life-saving drug, which belongs to few clinically used drugs without defined molecular structures in modern medicine. Heparin is the mostly negatively charged biopolymer with a broad distributions in molecular weight, charge density, and biological activities. Heparin is mainly composed of repeating trisulfated disaccharide units, which is made by mast cells that are enriched in the intestines, lungs or livers of animals. Porcine intestines and bovine lungs are two mostly used sources for heparin isolation. Heparin is well known for its anticoagulant and antithrombotic pharmacological effects. The anticoagulant activity of heparin is attributable to a 3-O-sulfate and 6-O-sulfate containing pentasaccharide sequence or a minimum eight-repeating disaccharide units containing the pentasaccharide sequence that catalyzes the suicidal inactivation of factor Xa or thrombin by a serpin or serine protease inhibitor named antithrombin III, respectively. Thus, heparin is responsible for the simultaneous inhibition of both thrombin generation and thrombin activity in the blood circulation. Moreover, heparin has many pharmacological properties such as anti-inflammatory, anti-viral, anti-angiogenesis, anti-neoplastic, and anti-metastatic effects though high affinity interactions with a variety of proteases, protease inhibitors, chemokines, cytokines, growth factors, and their respective receptors. The one drug multiple molecular targeting properties make heparin a very special drug in that various clinical trials are still conducting worldwide even 100 years after its discovery. In this review, we will summarize the structure-function relationship and the molecular mechanisms of heparin. We will also provide an overview of different clinical and potential clinical applications of heparin.

The Accumulation of Heparan Sulfate S-Domains in Kidney Transthyretin Deposits Accelerates Fibril Formation and Promotes Cytotoxicity

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In invitro assays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain-dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils.

Heparan sulfate S-domains and extracellular sulfatases (Sulfs): their possible roles in protein aggregation diseases.

Highly sulfated domains of heparan sulfate (HS), also known as HS S-domains, consist of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)-]. The expression of HS S-domains at the cell surface is determined by two mechanisms: tightly regulated biosynthetic machinery and enzymatic remodeling by extracellular endoglucosamine 6-sulfatases, Sulf-1 and Sulf-2. Intracellular or extracellular deposits of misfolded and aggregated proteins are characteristic of protein aggregation diseases. Although proteins can aggregate alone, deposits of protein aggregates in vivo contain a number of proteinaceous and non-protein components. HS S-domains are one non-protein component of these aggregated deposits. HS S-domains are considered to be critical for signal transduction of several growth factors and several disease conditions, such as tumor progression, but their roles in protein aggregation diseases are not yet fully understood. This review summarizes the current understanding of the possible roles of HS S-domains and Sulfs in the formation and cytotoxicity of protein aggregates.

Heparan Sulfate Glycosaminoglycans in Glioblastoma Promote Tumor Invasion.

Glioblastoma (GBM) is the most common primary malignant brain tumor of adults and confers a poor prognosis due, in part, to diffuse invasion of tumor cells. Heparan sulfate (HS) glycosaminoglycans, present on the cell surface and in the extracellular matrix, regulate cell signaling pathways and cell-microenvironment interactions. In GBM, the expression of HS glycosaminoglycans and the enzymes that regulate their function are altered, but the actual HS content and structure are unknown. However, inhibition of HS glycosaminoglycan function is emerging as a promising therapeutic strategy for some cancers. In this study, we use liquid chromatography-mass spectrometry analysis to demonstrate differences in HS disaccharide content and structure across four patient-derived tumorsphere lines (GBM1, 5, 6, 43) and between two murine tumorsphere lines derived from murine GBM with enrichment of mesenchymal and proneural gene expression (mMES and mPN, respectively) markers. In GBM, the heterogeneous HS content and structure across patient-derived tumorsphere lines suggested diverse functions in the GBM tumor microenvironment. In GBM5 and mPN, elevated expression of sulfatase 2 (SULF2), an extracellular enzyme that alters ligand binding to HS, was associated with low trisulfated HS disaccharides, a substrate of SULF2. In contrast, other primary tumorsphere lines had elevated expression of the HS-modifying enzyme heparanase (HPSE). Using gene editing strategies to inhibit HPSE, a role for HPSE in promoting tumor cell adhesion and invasion was identified. These studies characterize the heterogeneity in HS glycosaminoglycan content and structure across GBM and reveal their role in tumor cell invasion.Implications: HS-interacting factors promote GBM invasion and are potential therapeutic targets. Mol Cancer Res; 15(11); 1623-33. ©2017 AACR.

Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization

Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI–MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined.

Trisulfate Disaccharide Decreases Calcium Overload and Protects Liver Injury Secondary to Liver Ischemia/Reperfusion.

BackgroundIschemia and reperfusion (I/R) causes tissue damage and intracellular calcium levels are a factor of cell death. Sodium calcium exchanger (NCX) regulates calcium extrusion and Trisulfated Disaccharide (TD) acts on NCX decreasing intracellular calcium through the inhibition of the exchange inhibitory peptide (XIP).ObjectivesThe aims of this research are to evaluate TD effects in liver injury secondary to I/R in animals and in vitro action on cytosolic calcium of hepatocytes cultures under calcium overload.MethodsWistar rats submitted to partial liver ischemia were divided in groups: Control: (n = 10): surgical manipulation with no liver ischemia; Saline: (n = 15): rats receiving IV saline before reperfusion; and TD: (n = 15): rats receiving IV TD before reperfusion. Four hours after reperfusion, serum levels of AST, ALT, TNF-α, IL-6, and IL-10 were measured. Liver tissue samples were collected for mitochondrial function and malondialdehyde (MDA) content. Pulmonary vascular permeability and histologic parameters of liver were determined. TD effect on cytosolic calcium was evaluated in BRL3A hepatic rat cell cultures stimulated by thapsigargin pre and after treatment with TD.ResultsAST, ALT, cytokines, liver MDA, mitochondrial dysfunction and hepatic histologic injury scores were less in TD group when compared to Saline Group (p<0.05) with no differences in pulmonary vascular permeability. In culture cells, TD diminished the intracellular calcium raise and prevented the calcium increase pre and after treatment with thapsigargin, respectively.ConclusionTD decreases liver cell damage, preserves mitochondrial function and increases hepatic tolerance to I/R injury by calcium extrusion in Ca2+ overload situations.

The Sulfs: expression, purification, and substrate specificity.

Sulf-1 and Sulf-2 are endo-acting extracellular sulfatases. The Sulfs liberate 6-O sulfate groups, mainly from N, 6-O, and 2-O trisulfated disaccharides of heparan sulfate (HS)/heparin chains. The Sulfs have been shown to modulate the interaction of a number of protein ligands including growth factors and morphogens with HS/heparin and thus regulate the signaling of these ligands. They also play important roles in development and are dysregulated in many cancers. The establishment of the expression of the Sulfs and methods of assaying them has been desirable to investigate these enzymes. In this chapter, methods to express and purify recombinant Sulfs and to analyze HS structures in an extracellular fraction of HSulf-transfected HEK293 cells are described. The application of these enzymes for ex vivo degradation of an anti-HS epitope accumulated in the brain of a neurodegenerative disease model mouse is also described.

Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

Sodium butyrate, a histone deacetylase inhibitor, has been used to improve transgene expression in Chinese hamster ovary (CHO) cells. The current study explores the impact of butyrate treatment on heparan sulfate (HS) biosynthesis and structural composition in a recombinant CHO-S cell line expressing enzymes in the heparin (HP)/(HS) biosynthetic pathway (Dual-10 stably expressing NDST2 and HS3st1). Flow cytometric analysis showed that antithrombin binding was increased in Dual-10 cells and basic fibroblast growth factor binding was decreased in response to sodium butyrate treatment. The results were in agreement with the AMAC-LCMS (2-aminoacridine-tagged HS/HP analysis by liquid chromatography mass spectrometry) data that showed that there was an increase in heparan sulfate tri-sulfated disaccharides and a decrease in N-sulfated disaccharides in the butyrate-treated cells. However, we could not detect any changes in the chondroitin sulfate pathway in Dual-10 cells treated with butyrate. The current study is the first to report the effect of butyrate on glycosaminoglycan profiles.

Human Milk Glycosaminoglycans in Feces of Breastfed Newborns: Preliminary Structural Elucidation and Possible Biological Role

Much evidence has been obtained that several human milk glycans (glycoproteins, glycolipids, and especially oligosaccharides), behaving as cell surface receptor homologs, are able to inhibit binding of pathogens and thus protect the newborn against several enteric infections. Moreover, recent studies have focused attention on human milk complex sulfated polysaccharides, the glycosaminoglycans (GAGs), as well as their structure and their possible biological roles. However, no data are yet available regarding the metabolic fate of GAGs and their possible presence in the feces of breastfed newborns or regarding their composition and structure, which would be useful to shed light on their metabolism. Following written parental consent, morning fecal samples were obtained from 12 term breastfed newborns (six females, six males) between days 5 and 33 postpartum (mean– SD, 13.92– 7.89 days). Samples were immediately stored at 20 C and then lyophilized. GAGs were were extracted and purified, and disaccharide mapping was determined according to previous methodology to obtain GAG structural characterization. We determined the presence of chondroitin sulfate (CS) (approximately 67%) and dermatan sulfate (approximately 4%) for a total content of approximately 32 mg/100 g of feces, and the total amount of heparan sulfate (HS)/heparin (Hep) (approximately 29%) was approximately 13 mg/100 g of feces. Disaccharide evaluation of CS showed the presence of a large percentage of disaccharide sulfated in position 4 with a lower content of disaccharide sulfated in position 6 and of the nonsulfated disaccharide for a charge density value of approximately 0.81 ( T1c Table 1). The nonsulfated disaccharide 4,5-unsaturated uronic acidN-acetylglucosamine, typical of HS, was found to constitute the main species (approximately 28%) present in feces along with the three main monosulfated (approximately 23%), the three main disulfated (approximately 35%), and the trisulfated (approximately 14%)disaccharide typical ofHep (Table 1). Because of this peculiar disaccharide composition, the HS extractedfromhumannewbornfeceswasfoundtohaveacharge density value (approximately 1.40) similar to the polymer characterized in human milk showing a high content of disulfated and trisulfated disaccharides and a high charge density. Our results demonstrate the presence in the feces of breastfed newborns of a highly sulfated HS molecule rather similar in structure and disaccharide composition to the polyanion characterized in human milk. However, the CS structure was found to be different, in particular because of a higher content of 4-sulfated disaccharide and a lower contribution of the nonsulfated species for a generally high charge density, with respect to CS from human milk (0.81 vs. 0.36 of human milk). This primary structure of feces CS may be the result of the partial absorption of its chains, in particular those lacking in sulfate groups as demonstrated in previous studies. In fact, the partial (and preferential) absorption of CS chains/oligomers may be the result of its degradation by enzymes produced by intestinal bacteria, such as chondroitinases produced by Bacteroides strains from the human colon able to degrade CS of various structures exhibiting broad specificity. The presence of Bacteroides species from the human intestine suggests that the catabolism of human milk GAGs might partially take place in newborns. However, we still found the presence in newborns’ feces of CS and, in particular, HS/Hep structurally similar to those evaluated in human milk, demonstrating that a part of these polymers could be excreted as unmodified macromolecules. This is new information enabling us to understand more fully the role and the presence of these complex macromolecules in human milk and their relationship with the health of newborns. In fact, Newburg et al. have demonstrated that GAGs in human milk are able to inhibit the binding of the human immunodeficiency virus envelope glycoprotein gp120 to the cellular CD4 receptor. From these preliminary data, we can suppose that human milk GAGs, in particular HS/Hep, pass partially undigested through the entire digestive system of newborns, acting as possible soluble receptors able to inhibit the binding of different pathogens to the intestinal mucosa, thus protecting the infant from infections. However, although feces HS/Hep seems likely to be derived from ingested milk, the origin or potential modification