Dermatan Sulfate Disaccharide Research Articles

A highly active and stable chondroitin B lyase from Pedobacter schmidteae: Cloning, expression, and characterization

Chondroitin B lyase is a typical enzyme that catalyzes dermatan sulfate (DS) to produce oligosaccharides mainly 4,5-unsaturated dermatan sulfate disaccharides. It has been widely used in fields of medicine, functional food, and cosmetics because of its high selectivity. Here, a novel chondroitin B lyase from Pedobacter schmidteae (PsChonB) was cloned and heterologously expressed in Escherichia coli. The recombinant chondroitin B lyase had the highest activity in Tris-HCl buffer (100 mM, pH 8.0) at 40 °C. Characterization of the enzyme showed high stability of PsChonB, and its half-lives were 693 and 7949 min at 40 and 25 °C, and there was no significant decrease in activity after 30 days of storage at 4 °C. The specific activity, Km, Kcat, and Vmax of PsChonB were 395 U mg−1, 0.36 mg mL−1, 240.66 s−1, and 406.72 U mg−1. Molecular docking analysis of the enzyme indicated that the conserved residues Arg364, Arg363, His334, Arg318, Arg271, and Glu245 play vital roles in substrate binding. Compared with the previously reported chondroitin B lyases, PsChonB had the advantages of high activity and stability which indicated that it could be favorable for industrial production, clinic trials, and analytical the structures of glycosaminoglycans.

Systematic investigation of the skin inChst14−/−mice: A model for skin fragility in musculocontractural Ehlers–Danlos syndrome caused byCHST14variants (mcEDS-CHST14)

Loss-of-function variants in CHST14 cause a dermatan 4-O-sulfotransferase deficiency named musculocontractural Ehlers-Danlos syndrome-CHST14 (mcEDS-CHST14), resulting in complete depletion of the dermatan sulfate moiety of decorin glycosaminoglycan (GAG) chains, which is replaced by chondroitin sulfate. Recently, we uncovered structural alteration of GAG chains in the skin of patients with mcEDS-CHST14. Here, we conducted the first systematic investigation of Chst14 gene-deleted homozygote (Chst14-/-) mice. We used skin samples of wild-type (Chst14+/+) and Chst14-/- mice. Mechanical fragility of the skin was measured with a tensile test. Pathology was observed using light microscopy, decorin immunohistochemistry and electron microscopy (EM) including cupromeronic blue (CB) staining. Quantification of chondroitin sulfate and dermatan sulfate was performed using enzymatic digestion followed by anion-exchange HPLC. In Chst14-/- mice, skin tensile strength was significantly decreased compared with that in Chst14+/+ mice. EM showed that collagen fibrils were oriented in various directions to form disorganized collagen fibers in the reticular layer. Through EM-based CB staining, rod-shaped linear GAG chains were found to be attached at one end to collagen fibrils and protruded outside of the fibrils, in contrast to them being round and wrapping the collagen fibrils in Chst14+/+ mice. A very low level of dermatan sulfate disaccharides was detected in the skin of Chst14-/- mice by anion-exchange chromatography. Chst14-/- mice, exhibiting similar abnormalities in the GAG structure of decorin and collagen networks in the skin, could be a reasonable model for skin fragility of patients with mcEDS-CHST14, shedding light on the role of dermatan sulfate in maintaining skin strength.

Synthesis and anti-inflammatory activity of gold-nanoparticle bearing a dermatan sulfate disaccharide analog

A dermatan sulfate (DS) repeating disaccharide analog, α-l-idopyranosiduronate-(1→3)-2-amino-2-deoxy-4,6-di-O-sulfo-β-d-galactopyranoside, has been convergently synthesized and successfully applied to prepare the GAG-functionalized gold glyconanoparticle. This new material exhibited good anti-inflammatory activity which was comparable to that of the drug ibuprofen incarrageenan-induced paw edema in a rat model.

Toward the construction of dermatan sulfate (DS) partial disaccharide library: efficient synthesis of building blocks, common intermediate, and ligand conjugate of type-B DS disaccharide

Dermatan sulfate (DS) is composed of a repeating disaccharide unit containing iduronic acid (IdoA) and N-acetylgalactosamine (GalNAc). In the divergent synthesis of DS disaccharide, it is important to prepare the IdoA moiety with a diverse set of protecting groups. IdoA was efficiently obtained from glucose in 20 steps with some modifications following the method reported by van Boeckel et al. A disaccharide building block for constructing the DS disaccharide was synthesized by the glycosylation of the designed IdoA moiety with GalNAc. The disaccharide building block was used in the synthesis of DS-B disaccharide and its ligand conjugate.

Loss of dermatan sulfate epimerase (DSE) function results in musculocontractural Ehlers–Danlos syndrome

The sulfated polysaccharide dermatan sulfate (DS) forms proteoglycans with a number of distinct core proteins. Iduronic acid-containing domains in DS have a key role in mediating the functions of DS proteoglycans. Two tissue-specific DS epimerases, encoded by DSE and DSEL, and a GalNAc-4-O-sulfotransferase encoded by CHST14 are necessary for the formation of these domains. CHST14 mutations were previously identified for patients with the musculocontractural type of Ehlers-Danlos syndrome (MCEDS). We now identified a homozygous DSE missense mutation (c.803C>T, p.S268L) by the positional candidate approach in a male child with MCEDS, who was born to consanguineous parents. Heterologous expression of mutant full-length and soluble recombinant DSE proteins showed a loss of activity towards partially desulfated DS. Patient-derived fibroblasts also showed a significant reduction in epimerase activity. The amount of DS disaccharides was markedly decreased in the conditioned medium and the cell fraction from cultured fibroblasts of the patient when compared with a healthy control subject, whereas no apparent difference was observed in the chondroitin sulfate (CS) chains from the conditioned media. However, the total amount of CS disaccharides in the cell fraction from the patient was increased ∼1.5-fold, indicating an increased synthesis or a reduced conversion of CS chains in the cell fraction. Stable transfection of patient fibroblasts with a DSE expression vector increased the amount of secreted DS disaccharides. DSE deficiency represents a specific defect of DS biosynthesis. We demonstrate locus heterogeneity in MCEDS and provide evidence for the importance of DS in human development and extracellular matrix maintenance.

Heparan sulfate and dermatan sulfate disaccharide levels for newborn screening in MPS I, MPS II and MPS III

Heparan sulfate and dermatan sulfate disaccharide levels for newborn screening in MPS I, MPS II and MPS III

Determination of sulfation pattern in brain glycosaminoglycans by chip-based electrospray ionization ion trap mass spectrometry

Chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans display variability of sulfation in their constituent disaccharide repeats during chain elongation. Since a large proportion of the extracellular matrix of the central nervous system (CNS) is composed of proteoglycans, CS/DS disaccharide degree and profile of sulfation play important roles in the functional diversity of neurons, brain development, and some of its pathological states. To investigate the sulfation pattern of CS/DS structures expressed in CNS, we introduced here a novel method based on an advanced system encompassing fully automated chip nanoelectrospray ionization (nanoESI) in the negative ion mode and high capacity ion trap multistage mass spectrometry (MS(2)-MS(3)) by collision-induced dissociation (CID). This method, introduced here for the first time in glycomics of brain glycosaminoglycans, was particularly applied to structural investigation of disaccharides obtained by beta-elimination and digestion with chondroitin B and AC I lyase of hybrid CS/DS chains from wild-type mouse brain. Screening in the chip-MS mode of DS disaccharide fraction resulting after depolymerization with chondroitin B lyase revealed molecular ions assigned to monosulfated disaccharide species having a composition of 4,5-Delta-[IdoA-GalNAc]. By optimized CID MS(2)-MS(3), fragment ions supporting the localization of sulfate ester group at C4 within GalNAc were produced. Chip ESI MS profiling of CS disaccharide fraction obtained by depolymerization of the same CS/DS chain using chondroitin AC I lyase indicated the occurrence of mono- and bisulfated 4,5-Delta-[GlcA-GalNAc]. The site of oversulfation was determined by MS(2)-MS(3), which provided sequence patterns consistent with a rare GlcA-3-sulfate-GalNAc-6-sulfate structural motif. Figure Mouse brain GlcA-3-sulfate-GalNAc-6-sulfate structural motif.

Determination of Keratan Sulfate and Chondroitin /Dermatan Sulfate Disaccharides during Chick Corneal Development by Electrospray Tandem Mass Spectrometry

In cornea, keratan sulfate (KS) and chondroitin/dermatan sulfate (CS/DS) are the two major glycosaminoglycans (GAGs). In this study, electrospray ionization tandem mass spectrometry (ESI-MS/MS) was employed to quantify changes in KS and CS/DS sulfated disaccharides in the developing chick cornea. The concentration of KS monosulfated disaccharide (MSD) Gal-â-1,4-GlcNAc(6S) in embryonic Day 8 (E8) cornea equals that at E20, falls to its lowest level by E10, rises to a second peak by E14, falls to a second low by E18, peaks again by E20, and remains high in adult corneas. A similar concentration profile is observed for KS disulfated disaccharide (DSD) Gal(6S)-â-1,4-GlcNAc(6S), and thus also for total sulfated KS disaccharides. The molar percent of DSD is higher than that of MSD from E8-E18, equivalent at E20, and less than that of MSD in adult corneas. In contrast, total concentration of CS/DS Ddi-4S plus Ddi-6S decreases as development progresses and is lowest in adult corneas. Concentration and molar percent of Ädi-6S is highest at E8, then decreases through development as the concentration and molar percent of Ddi-4S increases from E8 and exceeds that of Ddi-6S after E14. New rapid, direct chemical analysis of extracellular matrix components obtained from sections from embryonic and adult chick corneas reveals heretofore undetected changes in sulfation characteristics of KS and CS/DS disaccharides during corneal development.

Structural and Sequence Motifs in Dermatan Sulfate for Promoting Fibroblast Growth Factor-2 (FGF-2) and FGF-7 Activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.

Chondroitin sulfate proteoglycans of bovine cornea: structural characterization and assessment for the adherence of Plasmodium falciparum-infected erythrocytes

The structures of the bovine corneal chondroitin sulfate (CS) chains and the nature of core proteins to which these chains are attached have not been studied in detail. In this study, we show that structurally diverse CS chains are present in bovine cornea and that they are mainly linked to decorin core protein. DEAE-Sephacel chromatography fractionated the corneal chondroitin sulfate proteoglycans (CSPGs) into three distinct fractions, CSPG-I, CSPG-II, and CSPG-III. These CSPGs markedly differ in their CS and dermatan sulfate (DS) contents, and in particular the CS structure—the overall sulfate content and 4- to 6-sulfate ratio. In general, the CS chains of the corneal CSPGs have low to moderate levels (15–64%) of sulfated disaccharides and 0–30% DS content. Structural analysis indicated that the DS disaccharide units in the CS chains are segregated as large blocks. We have also assessed the suitability of the corneal CSPGs as an alternative to placental CSPG or the widely used bovine tracheal chondroitin sulfate A (CSA) for studying the structural interactions involved in the adherence of Plasmodium falciparum-infected red blood cells (IRBCs) to chondroitin 4-sulfate. The data demonstrate that the corneal CSPGs efficiently bind IRBCs, and that the binding strength is either comparable or significantly higher than the placental CSPG. In contrast, the IRBC binding strength of bovine tracheal CSA is markedly lower than the human placental and bovine corneal CSPGs. Thus, our data demonstrate that the bovine corneal CSPG but not tracheal CSA is suitable for studying structural interactions involved in IRBC-C4S binding.

The Structure of Chondroitin B Lyase Complexed with Glycosaminoglycan Oligosaccharides Unravels a Calcium-dependent Catalytic Machinery

Chondroitinase B from Pedobacter heparinus is the only known enzyme strictly specific for dermatan sulfate and is a widely used enzymatic tool for the structural characterization of glycosaminoglycans. This beta-helical polysaccharide lyase belongs to family PL-6 and cleaves the beta(1,4) linkage of dermatan sulfate in a random manner, yielding 4,5-unsaturated dermatan sulfate disaccharides as the product. The previously reported structure of its complex with a dermatan sulfate disaccharide product identified the -1 and -2 subsites of the catalytic groove. We present here the structure of chondroitinase B complexed with several dermatan sulfate and chondroitin sulfate oligosaccharides. In particular, the soaking of chondroitinase B crystals with a dermatan sulfate hexasaccharide results in a complex with two dermatan sulfate disaccharide reaction products, enabling the identification of the +2 and +1 subsites. Unexpectedly, this structure revealed the presence of a calcium ion coordinated by sequence-conserved acidic residues and by the carboxyl group of the l-iduronic acid at the +1 subsite. Kinetic and site-directed mutagenesis experiments have subsequently demonstrated that chondroitinase B absolutely requires calcium for its activity, indicating that the protein-Ca(2+)-oligosaccharide complex is functionally relevant. Modeling of an intact tetrasaccharide in the active site of chondroitinase B provided a better understanding of substrate specificity and the role of Ca(2+) in enzymatic activity. Given these results, we propose that the Ca(2+) ion neutralizes the carboxyl moiety of the l-iduronic acid at the cleavage site, whereas the conserved residues Lys-250 and Arg-271 act as Brønsted base and acid, respectively, in the lytic degradation of dermatan sulfate by chondroitinase B.

Extraction of glycosaminoglycans from chicken eggshell

The objectives of this study were to analyze glycosaminoglycan (GAG) and mineral composition in the chicken eggshell. Eggshells were decalcified with acetic acid, and GAG was extracted from the decalcified shell by digestion with papain. The eggshell contained an average of 0.024% of its dry weight as uronic acid, a carbohydrate moiety of GAG. The eggshell GAG consisted of approximately 48% hyaluronic acid and and 52% galactosaminoglycan. In the latter, chondroitin sulfate-dermatan sulfate copolymers were the major galactosaminoglycans with dermatan sulfate disaccharide as a relatively minor component. The inorganic material recovered after decalcification accounted for approximately 140% of dry weight of the eggshell and contained 24.11% calcium, 0.04% phosphorous, and 0.23% magnesium, with an undetectable amount of nitrogen.

Crystal structure of chondroitinase B from Flavobacterium heparinum and its complex with a disaccharide product at 1.7 Å resolution

Glycosaminoglycans (GAGs) are a family of acidic heteropolysaccharides, including such molecules as chondroitin sulfate, dermatan sulfate, heparin and keratan sulfate. Cleavage of the O-glycosidic bond within GAGs can be accomplished by hydrolases as well as lyases, yielding disaccharide and oligosaccharide products. We have determined the crystal structure of chondroitinase B, a glycosaminoglycan lyase from Flavobacterium heparinum, as well as its complex with a dermatan sulfate disaccharide product, both at 1.7 Å resolution. Chondroitinase B adopts the right-handed parallel β-helix fold, found originally in pectate lyase and subsequently in several polysaccharide lyases and hydrolases. Sequence homology between chondroitinase B and a mannuronate lyase from Pseudomonas sp. suggests this protein also adopts the β-helix fold. Binding of the disaccharide product occurs within a positively charged cleft formed by loops extending from the surface of the β-helix. Amino acid residues responsible for recognition of the disaccharide, as well as potential catalytic residues, have been identified. Two arginine residues, Arg318 and Arg364, are found to interact with the sulfate group attached to O-4 of N-acetylgalactosamine. Cleavage of dermatan sulfate likely occurs at the reducing end of the disaccharide, with Glu333 possibly acting as the general base.

Alterations of glycosaminoglycans during patellar tendon autograft healing after posterior cruciate ligament replacement. A biochemical study in a sheep model.

In each of 30 skeletally mature sheep, the posterior cruciate ligament was replaced in one knee by a free patellar tendon autograft using the central third of the ipsilateral patellar tendon. The healing autograft was compared with the contralateral posterior cruciate ligament and the patellar tendons and posterior cruciate ligaments of nonoperated animals. The content of glycosaminoglycans, chondroitin sulfate disaccharides, and dermatan sulfate disaccharides was assessed biochemically at six periods during the 2 years after surgery. The total glycosaminoglycans and chondroitin sulfate disaccharides in the native posterior cruciate ligament was threefold that in the native patellar tendon. In contrast, the amount of dermatan sulfate disaccharides was similar in both the native tendon and native ligament. In the autograft, glycosaminoglycans and chondroitin sulfate disaccharides increased significantly to about 144% and 172%, respectively, of the contralateral posterior cruciate ligament at Week 104. The dermatan sulfate disaccharides in the autograft also showed a significant increase up to Week 26, followed by a remarkable but not significant decrease until the end of the study. In the contralateral posterior cruciate ligament, the dermatan sulfate disaccharides increased significantly between Weeks 52 and 104. Thus, the amount of dermatan sulfate disaccharides was similar in both the autograft and the contralateral posterior cruciate ligament after 2 years. This study suggests that the patellar tendon autograft did not completely assume the biochemical properties of the posterior cruciate ligament.

Capillary electrophoresis of heparin and dermatan sulfate unsaturated disaccharides with triethylamine and acetonitrile as electrolyte additives

Capillary electrophoresis at constant voltage with the addition of triethylamine as electrolyte to a running buffer containing borate using fused-silica capillaries permits the complete resolution in less than 30 min of 11 standard heparin and 8 standard dermatan sulfate disaccharides, which represent degradation products of heparin and dermatan sulfate by specific lyases. Triethylamine influences the migration time of disaccharides by reducing both their electrophoretic mobility towards the anode and the electroosmotic flow towards the cathode. A modulated combination of these effects together with borate-disaccharide complex formation is responsible for separation, especially in the case of isomers which differ in the position of the sulfate groups. The addition of acetonitrile did not introduce any favourable effect in the separation of disaccharide mixtures. Under these conditions different dermatan sulfates were analysed to assess the source of the preparations.

Purity of glycosaminoglycan-related compounds using capillary electrophoresis.

High performance capillary electrophoresis was used to determine impurities in glycosaminoglycans. The counterion of glycosaminoglycans was analyzed with indirect UV-detection using a 40 mM 4-aminopyridine buffer. Calcium, lithium, potassium and sodium could be resolved. A linear correlation between the area under the curve and the concentration of sodium (r2 = 0.98) and calcium (r2 = 0.99) was found. Using enzymatic depolymerization, chondroitin sulfates were cleaved to disaccharides. The resulting disaccharides, with the structure 4-deoxy-alpha-L-threo-hex-4-enopyranosyl uronic acid (delta UA) 2 x (1-->3)-D-GalNY6X (X = H, sulfate and Y = acetyl, sulfate) for dermatan sulfate, were detected selectively at 230 nm using capillary electrophoresis. Dermatan sulfate disaccharides were analyzed using a 50 cm long fused silica capillary (75 microns ID). The buffer used was 10 mM sodium tetraborate and 50 mM SDS, pH 8.8. The detection was at 230 nm. Using the main peak delta UA (1-->3)-D-GalNAc4S as standard, between 1 and 80% dermatan sulfate in heparin preparations were analyzed. The disaccharide showed a linear correlation of the peak area versus the concentration with a correlation coefficient r2 = 0.98. The methods are useful in characterizing the identity and concentration of the counterion of glycosaminoglycans after chondroitinase degradation.

Disaccharide compositional analysis of heparin and heparan sulfate using capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was used to separate eight commercial disaccharide standards of the structure ΔUA2X(1 → 4)- d-GlcNY6X (where ΔUA is 4-deoxy-α- l- threo-hex-4-enopyranosyluronic acid, GlcN is 2-deoxy-2-aminoglucopyranose, S is sulfate, Ac is acetate, X may be S, and Y is S or Ac). These eight disaccharides had been prepared from heparin, heparan sulfate, and derivatized heparins. A similar CZE method was recently reported for the analysis of eight chondroitin and dermatan sulfate disaccharides (A. Al-Hakim and R. J. Linhardt, Anal. Biochem. 195, 68–73, 1991). Two of the standard heparin/heparan sulfate disaccharides, having an identical charge of −2, ΔUA2S(1 → 4)- d-GlcNAc and ΔUA(1 → 4)- d-GlcNS, were not fully resolved using standard sodium borate/boric acid buffer. This buffer had proven effective in separating chondroitin/dermatan sulfate disaccharides of identical charge. Resolution of these two heparin/heparan sulfate disaccharides could be improved by extending the capillary length, preparing the buffer in 2H 2O, or eliminating boric acid. Baseline resolution was achieved in sodium dodecyl sulfate in the absence of buffer. The structure and purity of each of the eight new commercial heparin/heparan sulfate disaccharide standards were confirmed using fast-atom-bombardment mass spectrometry and high-field 1H-NMR spectroscopy. Heparin and heparan sulfate were then depolymerized using heparinase (EC 4.2.2.7), heparin lyase II (EC 4.2.2.-), heparinitase (EC 4.2.2.8), and a combination of all three enzymes. CZE analysis of the products formed provided a disaccharide composition of each glycosaminoglycan. As little as 50 fmol of disaccharide could be detected by ultraviolet absorbance.