Chondroitin Sulfate Oligosaccharides Research Articles

Inhibitory effect of chondroitin sulfate oligosaccharides on bovine testicular hyaluronidase.

Hyaluronan and chondroitin sulfates are prominent components of the extracellular matrices of animal tissues; however, their functions in relation to their oligosaccharide structures have not yet been fully elucidated. The oligosaccharides of hyaluronan and chondroitin sulfate were prepared and used to investigate their effects on the hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase when hyaluronan was used as a substrate. Hydrolysis and transglycosylation activities were assessed in independent reaction systems by analyzing the products by HPLC. The hydrolysis and transglycosylation reactions of bovine testicular hyaluronidase were dose-dependently inhibited by chondroitin sulfate oligosaccharides, but not by hyaluronan or chondroitin oligosaccharides. A kinetic analysis of the hydrolysis reaction using hyaluronan octasaccharide revealed that the inhibition mode by chondroitin sulfate oligosaccharides was competitive.

Novel chondroitin sulfate oligosaccharide motifs as biomarkers: insights into their involvement in brain development.

Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates. They are the linear polysaccharide side chains of proteoglycans that consist of various sulfated repeating disaccharides, [–4GlcUAβ1–3GalNAcβ1–]n, in which GlcUA and GalNAc represent d-glucuronic acid and N-acetyl-d-galactosamine, respectively. CS chains containing disaccharide designated D-units, GlcUA(2-O-sulfate)–GalNAc(6-O-sulfate), play a role in brain development by interacting with the neurotrophic factor, pleiotrophin, which generates signaling. A series of octasaccharide sequences containing at least one D-unit, were previously isolated from embryonic pig brains using a pleiotrophin-immobilized affinity column. These similar yet distinct sugar sequences were termed “wobble oligosaccharide motifs” on account of their structural flexibility. Oligosaccharides containing D-units were recently isolated from shark fin cartilage to obtain more detailed structural information on the CS chains containing D-units that are involved in brain development. Seven novel hexasaccharide sequences, in addition to three previously reported sequences, were isolated after exhaustive digestion of the CS chains with the chondroitin lyase AC-I, which cannot act on galactosaminidic linkages adjacent to the D-units. Of the epitopes involved in cerebellar development in mammals, the novel epitope sequences ∆A-D-A, ∆A-D-D, and ∆A-B-D were identified in binding studies using an anti-CS monoclonal antibody, with the symbol ∆ referring to the 4,5-unsaturated bond of GlcUA residues generated by the lyase, while A, B, and C represented GlcUA-GalNAc(4-O-sulfate), GlcUA(2-O-sulfate)-GalNAc(4-O-sulfate), and GlcUA-GalNAc(6-O-sulfate), respectively. The significance of multiple CS oligosaccharide sequences in the brain and electrostatic potential distribution at their molecular surface during ligand binding have been discussed.KeywordsChondroitin sulfateDermatan sulfateSugar sequencesSugar biomarkersBrain development

Structural analysis of isomeric chondroitin sulfate oligosaccharides using regioselective 6-O-desulfation method and tandem mass spectrometry

A strategy based on a regioselective 6-O-desulfation reaction and negative ion electrospray ionization tandem mass spectrometry (ESI-MS(n)) was developed for the structural delineation of isomeric chondroitin sulfate oligosaccharides. Product ions resulting from the glycosidic cleavage provided information about the number of sulfate groups in each sugar residue. After the regioselective 6-O-desulfation reaction, the number of sulfate groups on each residue was obtained using a tandem mass spectrometry analysis of the reaction product. The sulfation pattern could be obtained based on the product ions of analytes before and after the desulfation reaction. The strategy was demonstrated using a series of tetrasaccharides prepared from shark cartilage chondroitin sulfate D. Among the 12 identified tetrasaccharides, six structures had not been reported before.

Synthesis of Chondroitin Sulfate Oligosaccharides Using N‐(Tetrachlorophthaloyl)‐ and N‐(Trifluoroacetyl)galactosamine Building Blocks

AbstractWe have explored synthetic routes for the preparation of chondroitin sulfate (CS) oligosaccharides based on the use of N‐tetrachlorophthaloyl‐ (N‐TCP) and N‐trifluoroacetyl‐substituted (N‐TFA) galactosamine building blocks. Using N‐TCP units, we carried out the total synthesis of two CS disaccharides, demonstrating the compatibility of TCP protection with the final deprotection/sulfation steps. However, an attempted 2 + 2 coupling of N‐TCP‐containing disaccharides for the synthesis of CS tetrasaccharides failed. In contrast, a synthetic route using N‐TFA galactosamine units efficiently led to biologically relevant CS‐like oligosaccharides. The N‐TFA groups could easily be removed at the end of the synthesis, and microwave irradiation greatly facilitated the sulfation reactions. The utility of this approach is illustrated with the total synthesis of two CS‐like tetrasaccharides with different sulfate distribution patterns. Finally, we used a fluorescence polarization assay to estimate the relative abilities of the synthesized compounds to inhibit the interaction between FGF‐2 and heparin.

Mass preparation of oligosaccharides by the hydrolysis of chondroitin sulfate polysaccharides with a subcritical water microreaction system

The biological functions of chondroitin sulfate (CS) are executed by the interaction of specific oligosaccharide sequences in the polysaccharide chain with effective proteins. Thus, CS oligosaccharides are expected to have pharmacological applications. Furthermore, the demand for CS in health food supplements and medication is growing. However, the absorbency of CS polysaccharides in the digestive system is very low. Since the activity of orally administered CS is expected to increase by depolymerization, industrial production of CS oligosaccharides is required. In this study, hydrolysis with subcritical and super-critical water was applied to the depolymerization of CS for the first time, and hydrolytic conditions for oligosaccharide production were examined. CS oligosaccharides principally containing an N-acetyl-d-galactosamine residue at their reducing ends were successfully obtained. No significant desulfation was found in CS oligosaccharides prepared under optimized conditions. The production of CS oligosaccharides by this method will have a strong influence on the CS-related materials market.

Synthesis of chondroitin/dermatan sulfate-like oligosaccharides and evaluation of their protein affinity by fluorescence polarization

Here, we present a novel approach for the chemical synthesis of chondroitin and dermatan sulfate oligosaccharides. A key point of this strategy is the preparation and use of an N-trifluoroacetyl galactosamine building block containing a 4,6-O-di-tert-butylsilylene group. Glycosylation reactions proceeded in good yields (74-91%) with our protecting group distribution. Using this approach, we have synthesized, for the first time, a chondroitin/dermatan sulfate-like tetrasaccharide that contains both types of uronic acids, D-glucuronic and L-iduronic acid. Moreover, we have employed a fluorescence polarization competition assay to evaluate the interactions between the synthesized oligosaccharides and FGF-2 (basic fibroblast growth factor). Our results show that this method, using standard instrumentation and minimal sample consumption, is a powerful tool for the rapid analysis of the glycosaminoglycan affinity for proteins in solution.

ChemInform Abstract: Synthetic and Semi‐Synthetic Chondroitin Sulfate Oligosaccharides, Polysaccharides, and Glycomimetics

AbstractReview: 81 refs.

Synthetic and semi-synthetic chondroitin sulfate oligosaccharides, polysaccharides, and glycomimetics

Chondroitin sulfate (CS) is a sulfated polysaccharide involved in a myriad of biological processes. Due to the variable sulfation pattern of CS polymer chains, the need to study in detail structure–activity relationships regarding CS biomedical features has provoked much interest in obtaining synthetic CS species. This paper reviews two decades of synthetic and semi-synthetic CS oligosaccharides, polysaccharides, and glycomimetics obtained by chemical, chemoenzymatic, enzymatic, and microbiological-chemical strategies.

LC-MSn Analysis of Isomeric Chondroitin Sulfate Oligosaccharides Using a Chemical Derivatization Strategy

Improved methods for structural analyses of glycosaminoglycans (GAGs) are required to understand their functional roles in various biological processes. Major challenges in structural characterization of complex GAG oligosaccharides using liquid chromatography-mass spectrometry (LC-MS) include the accurate determination of the patterns of sulfation due to gas-phase losses of the sulfate groups upon collisional activation and inefficient on-line separation of positional sulfation isomers prior to MS/MS analyses. Here, a sequential chemical derivatization procedure including permethylation, desulfation, and acetylation was demonstrated to enable both on-line LC separation of isomeric mixtures of chondroitin sulfate (CS) oligosaccharides and accurate determination of sites of sulfation by MS(n). The derivatized oligosaccharides have sulfate groups replaced with acetyl groups, which are sufficiently stable to survive MS(n) fragmentation and reflect the original sulfation patterns. A standard reversed-phase LC-MS system with a capillary C18 column was used for separation, and MS(n) experiments using collision-induced dissociation (CID) were performed. Our results indicate that the combination of this derivatization strategy and MS(n) methodology enables accurate identification of the sulfation isomers of CS hexasaccharides with either saturated or unsaturated nonreducing ends. Moreover, derivatized CS hexasaccharide isomer mixtures become separable by LC-MS method due to different positions of acetyl modifications.

Extraction, Isolation and Analysis of Chondroitin Sulfate Glycosaminoglycans

Glycosaminoglycans (GAGs) including chondroitin sulfate (CS) and chondroitin sulfate/dermatan sulfate (CS/DS) copolymers are anionic straight chain polysaccharides. They are galactosamine containing GAGs (galactosaminoglycans) having wide range of applications in pharmaceutical, cosmetic and food industries. This article reviews techniques to isolate and characterize these galactosaminoglycans from animal and poultry tissues. Patent based information is also discussed. Cartilaginous tissues are the major source of CS consisting entirely of D-glucuronosyl-N-acetylgalactosamine repeating disaccharide units, in which the galactosamine is sulfated at C4 or C6. In contrast, most galactosaminoglycans in non-cartilaginous connective tissues (e.g. skin and tendon) are CS/DS copolymers comprised of varying proportions of D-glucuronosyl-N-acetylgalactosamine and L-iduronosyl-N-acetylgalactosamine. Tissues are digested with proteinase (e.g. papain) to liberate GAGs, which are fractionated to isolate and purify galactosaminoglycans. Common techniques used for fractionation of GAGs include: precipitation with different concentrations of ethanol; solubilization of GAG precipitated as GAG-quarternary ammonium compound complexes with different concentrations of NaCl; anion exchange chromatography and gel filtration chromatography. Purified galactosaminoglycans are examined by various methods including chondroitinase digestion, high performance liquid chromatography and electrophoresis. Histological methods are used to localize galactosaminoglycans in tissues. The patent information on the CS hydrolase and ultraviolet irradiation may be useful for the preparation of CS oligosaccharide.

Ultraperformance liquid chromatography with electrospray ionization ion trap mass spectrometry for chondroitin disaccharide analysis

Chondroitin sulfate (CS) has an important role in cell division, in the central nervous system, and in joint-related pathologies such as osteoarthritis. Due to the complex chemical structure and biological importance of CS, simple, sensitive, high resolution, and robust analytical methods are needed for the analysis of CS disaccharides and oligosaccharides. An ion-pairing, reversed-phase, ultraperformance liquid chromatography (IPRP-UPLC) separation, coupled to electrospray ionization mass spectrometry with an ion trap mass analyzer, was applied for the analyses of CS-derived disaccharides. UPLC separation technology uses small particle diameter, short column length, and elevated column temperature to obtain high resolution and sensitivity. Hexylamine (15 mM) was selected as the optimal ion-pairing reagent.

Recombinant Expression, Purification, and Biochemical Characterization of Chondroitinase ABC II from Proteus vulgaris

Chondroitin lyases (or chondroitinases) are a family of enzymes that depolymerize chondroitin sulfate (CS) and dermatan sulfate (DS) galactosaminoglycans, which have gained prominence as important players in central nervous system biology. Two distinct chondroitinase ABC enzymes, cABCI and cABCII, were identified in Proteus vulgaris. Recently, cABCI was cloned, recombinantly expressed, and extensively characterized structurally and biochemically. This study focuses on recombinant expression, purification, biochemical characterization, and understanding the structure-function relationship of cABCII. The biochemical parameters for optimal activity and kinetic parameters associated with processing of various CS and DS substrates were determined. The profile of products formed by action of cABCII on different substrates was compared with product profile of cABCI. A homology-based structural model of cABCII and its complexes with CS oligosaccharides was constructed. This structural model provided molecular insights into the experimentally observed differences in the product profile of cABCII as compared with that of cABCI. The critical active site residues involved in the catalytic activity of cABCII identified based on the structural model were validated using site-directed mutagenesis and kinetic characterization of the mutants. The development of such a contaminant-free cABCII enzyme provides additional tools to decode the biologically important structure-function relationship of CS and DS galactosaminoglycans and offers novel therapeutic strategies for recovery after central nervous system injury.

コンドロイチン硫酸オリゴ糖の構造解析における重酸素標識の利用

Numerous studies have been reported on structural analysis of chondroitin sulfate (CS) oligosaccharides using multiple-stage mass spectrometry (MSn). However, their sequence determination is highly complex because of the simply repeating backbone structure. This study attempts to overcome this issue using an 18O-labeling method. The usefulness of the 18O-labeling method in the MSn analysis of CS oligosaccharide is shown in this paper.

Software Tool for the Structural Determination of Glycosaminoglycans by Mass Spectrometry

Structural elucidation of glycosaminoglycans (GAGs) is one of the major challenges in biochemical analysis. This is mainly because of the diversity of GAG sulfation and N-acetylation patterns and variations in uronate isomers. ESI-MS and recently MALDI-MS methodologies are important strategies for investigating the molecular structure of GAGs. However, the interpretation of MS data produced by these strategies must take into account a large number of variables (including the number of monosaccharide residues, acetylations, sulfate groups, multiple charges, and exchanges between different cations). We have developed a bioinformatics tool to assist this complex interpretation task. The software is based on GlycoWorkbench, a tool for semiautomatic interpretation of glycan MS data. The tool generates the sugar backbones in all their variants (GAG family, composition, acetylation positions, and number of sulfates) and automatically matches them with the selected MS peaks. The backbones corresponding to a given peak are validated against the selected MS/MS peaks by generating all possible fragmentations. Native chondroitin sulfate and heparin oligosaccharides as well as chemically modified heparin oligomers have been successfully analyzed by MALDI- and ESI-MS and MS/MS, and the results of the semiautomated annotation of these mass spectra are presented here.

Two Related but Distinct Chondroitin Sulfate Mimetope Octasaccharide Sequences Recognized by Monoclonal Antibody WF6

Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, DeltaD-C-C-C and DeltaC-C-A-D (where A represents GlcUAbeta1-3GalNAc(4-O-sulfate), C is GlcUAbeta1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)beta1-3GalNAc(6-O-sulfate), DeltaCis Delta(4,5)HexUAalpha1-3GalNAc(6-O-sulfate), and DeltaDis Delta(4,5)HexUA(2-O-sulfate)alpha1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, DeltaC-A-D-C and DeltaC-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (DeltaD-C-C-C and DeltaC-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody.

Structural Basis for Binding of Plasmodium falciparum Erythrocyte Membrane Protein 1 to Chondroitin Sulfate and Placental Tissue and the Influence of Protein Polymorphisms on Binding Specificity

Chondroitin sulfate (CS) A is a key receptor for adhesion of Plasmodium falciparum-infected erythrocytes (IEs) in the placenta and can also mediate adhesion to microvascular endothelial cells. IEs that adhere to CSA express var2csa-type genes, which encode specific variants of the IE surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1). We report direct binding of native PfEMP1, isolated from IEs and encoded by var2csa, to immobilized CSA. Binding of PfEMP1 was dependent on 4-O-sulfated disaccharides and glucuronic acid rather than iduronic acid, consistent with the specificity of intact IEs. Using immobilized CS oligosaccharides as neoglycolipid probes, the minimum chain length for direct binding of PfEMP1 was eight monosaccharide units. Similarly for IE adhesion to placental tissue there was a requirement for 4-O-sulfated GalNAc and glucuronic acid mixed with non-sulfated disaccharides; 6-O-sulfation interfered with the interaction between placental CSA and IEs. The minimum chain length for maximal inhibition of adhesion was 10 monosaccharide residues. Partially 4-O-sulfated CS oligosaccharides (45-55% sulfation) were highly effective inhibitors of placental adhesion (IC(50), 0.15 microg/ml) and may have potential for therapeutic development. We used defined P. falciparum isolates expressing different variants of var2csa in adhesion assays and found that there were isolate-specific differences in the preferred structural motifs for adhesion to CSA that correlated with polymorphisms in PfEMP1 encoded by var2csa-type genes. This may influence sites of IE sequestration or parasite virulence. These findings have significant implications for understanding the pathogenesis and biology of malaria, particularly during pregnancy, and the development of targeted interventions.

Reduced sulfation of chondroitin sulfate in thyroglobulin derived from human papillary thyroid carcinomas

The presence of a chondroitin sulfate (CS) chain on human thyroglobulin (Tg) distinguishes it from Tg of other species; the role played by this chain in normal thyroid function is unclear. In the present study, we determined the structure of the CS oligosaccharides in human thyroid-derived Tg. Q-Sepharose anion exchange column chromatography of thyroid extracts indicated that the negative charge of human Tg was primarily due to the presence of the CS chain. Interestingly, the Tg of papillary carcinomas was less negatively charged, suggesting that its CS side chain was less sulfated. Structural analysis of the CS in Tg revealed that its most abundant disaccharide is the DeltaDi-0S unit (50.2 +/- 18.3%), which is not sulfated. The DeltaDi-0S, DeltaDi-6S (31.7 +/- 13.7%) and DeltaDi-diSD (12.8 +/- 4.3%) units comprise more than 90% of the disaccharides in normal Tg. However, the DeltaDi-6S (0.0-21.2%) and DeltaDi-diSD (0.0-7.7%) units were significantly reduced in Tg extracted from papillary thyroid carcinomas, whereas DeltaDi-0S (86.0 +/- 21.3%) was increased. These results suggest that the Tg in papillary carcinomas has a less sulfated CS side chain and, by virtue of that fact, is less negatively charged. What role this change in carcinoma cells has in their transformation and spread remains to be determined.

Activation of phospholipase C pathways by a synthetic chondroitin sulfate‐E tetrasaccharide promotes neurite outgrowth of dopaminergic neurons

In dopaminergic neurons, chondroitin sulfate (CS) proteoglycans play important roles in neuronal development and regeneration. However, due to the complexity and heterogeneity of CS, the precise structure of CS with biological activity and the molecular mechanisms underlying its influence on dopaminergic neurons are poorly understood. In this study, we investigated the ability of synthetic CS oligosaccharides and natural polysaccharides to promote the neurite outgrowth of mesencephalic dopaminergic neurons and the signaling pathways activated by CS. CS-E polysaccharide, but not CS-A, -C or -D polysaccharide, facilitated the neurite outgrowth of dopaminergic neurons at CS concentrations within the physiological range. The stimulatory effect of CS-E polysaccharide on neurite outgrowth was completely abolished by its digestion into disaccharide units with chondroitinase ABC. Similarly to CS-E polysaccharide, a synthetic tetrasaccharide displaying only the CS-E sulfation motif stimulated the neurite outgrowth of dopaminergic neurons, whereas a CS-E disaccharide or unsulfated tetrasaccharide had no effect. Analysis of the molecular mechanisms revealed that the action of the CS-E tetrasaccharide was mediated through midkine-pleiotrophin/protein tyrosine phosphatase zeta and brain-derived neurotrophic factor/tyrosine kinase B receptor pathways, followed by activation of the two intracellular phospholipase C (PLC) signaling cascades: PLC/protein kinase C and PLC/inositol 1,4,5-triphosphate/inositol 1,4,5-triphosphate receptor signaling leading to intracellular Ca(2+) concentration-dependent activation of Ca(2+)/calmodulin-dependent kinase II and calcineurin. These results indicate that a specific sulfation motif, in particular the CS-E tetrasaccharide unit, represents a key structural determinant for activation of midkine, pleiotrophin and brain-derived neurotrophic factor-mediated signaling, and is required for the neuritogenic activity of CS in dopaminergic neurons.

Graphitized Carbon LC−MS Characterization of the Chondroitin Sulfate Oligosaccharides of Aggrecan

A novel in-gel endoglycosidase technique to study oligosaccharides with graphitized carbon LC-MS has revealed differences in the sulfation profile between the linkage and repeat regions of chondroitin sulfate on aggrecan. Bovine articular cartilage aggrecan was isolated in a composite agarose PAGE gel or diluted in ammonium acetate buffer and was digested overnight with chondroitinase ABC. Including a chemical release/reduction protocol after digestion, we could separate and detect three differentially sulfated chondroitin sulfate disaccharides of the repeat region (DeltaUA1-3GalNAc0/4/6S-ol) from the three differentially sulfated linkage region hexasaccharides (DeltaUA1-3GalNAc0/4/6Sbeta1-4GlcAbeta1-3Galbeta1-3Galbeta1-4Xylitol). Graphitized carbon LC-MS in the negative ion mode was able to resolve isomeric disaccharides and linkage region hexasaccharides. Specific MS2 and MS3 enabled us to confirm the sulfate location on all oligosaccharides by comparing their fragmentation with sulfated disaccharide standards. The presence of unsulfated, 6-sulfated, and 4-sulfated linkage regions was correlated with positive Western blot staining with the respective CS linkage region neoepitope antibodies (1B5, 3B3, 2B6) on digested aggrecan. Our strategy of examining linkage region and repeat region profiles is applicable to screening GAGs from various biological samples in order to detect differences between normal and disease states.

The Role of Mobile Protons in Negative Ion CID of Oligosaccharides

Carbohydrates of all classes consist of glycoform mixtures built on common core units. Determination of compositions and structures of such mixtures relies heavily on tandem mass spectrometric data. Analysis of native glycans is often necessary for samples available in very low quantities and for sulfated glycan classes. Negative tandem mass spectrometry (MS) provides useful product ion profiles for neutral oligosaccharides and is preferred for acidic classes. In previous work from this laboratory, site-specific influences of sialylation on product ion profiles in the negative mode were elucidated. The present results show how the interplay of two other acidic groups, uronic acids and sulfates, determines product ion patterns for chondroitin sulfate oligosaccharides. Unsulfated chondroitin oligosaccharides dissociate to form C-type ions almost exclusively. Chondroitin sulfate oligosaccharides produce abundant B- and Y-type ions from glycosidic bond cleavage with C- and Z-types in low abundances. These observations are explained in terms of competing proton transfer reactions that occur during the collisional heating process. Mechanisms for product ion formation are proposed based on tandem mass spectra and the abundances of product ions as a function of collision energy.