A tandem mass spectrometric approach to determination of chondroitin/dermatan sulfate oligosaccharide glycoforms

Dermatan sulfate (DS) chains are variants of chondroitin sulfate (CS) that are expressed in mammalian extracellular matrices and are particularly prevalent in skin. DS has been implicated in varied biological processes including wound repair, infection, cardiovascular disease, tumorigenesis, and fibrosis. The biological activities of DS have been attributed to its high content of IdoA(alpha1-3)GalNAc4S(beta1-4) disaccharide units. Mature CS/DS chains consist of blocks with high and low GlcA/IdoA ratios, and sulfation may occur at the 4- and/or 6-position of GalNAc and 2-position of IdoA. Traditional methods for the analysis of CS/DS chains involve differential digestion with specific chondroitinases followed by steps of chromatographic isolation of the products and di-saccharide analysis on the individual fraction. This work reports the use of tandem mass spectrometry to determine the patterns of sulfation and epimerization of CS/DS oligosaccharides in a single step. The approach is first validated and then applied to a series of skin DS samples and to decorins from three different tissues. DS samples ranged from 74 to 99% of CSB-like repeats, using this approach. Decorin samples ranged from 30% CSB-like repeats for those samples from articular cartilage to 75% for those from sclera. These values agree with known levels of glucuronyl C5-epimerase in these tissues.     

Analysis of novel over‐ and under‐sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSⁿ). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di‐ and hexasaccharides were analyzed by ESI MSⁿ. MS² on bisulfated 4,5‐Δ‐HexAGalNAc revealed an additional sulfate ester group at 4,5‐Δ‐HexA. MS² data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5‐Δ‐HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS¹ mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSⁿ performed on ions that, according to mass calculation, correspond to pentasulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂], trisulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂] with IdoA‐derived 4,5‐Δ‐HexA at the nonreducing end, tetrasulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] and monosulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] with GlcA‐derived 4,5‐Δ‐HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over‐, regular, and under‐sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.     

Acidolysis-based component mapping of glycosaminoglycans by reversed-phase high-performance liquid chromatography with off-line electrospray ionization–tandem mass spectrometry: Evidence and tags to distinguish different glycosaminoglycans

Diverse monosaccharide analysis methods have been established for a long time, but few methods are available for a complete monosaccharide analysis of glycosaminoglycans (GAGs) and certain acidolysis-resistant components derived from GAGs. In this report, a reversed-phase high-performance liquid chromatography (RP–HPLC) method with pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization was established for a complete monosaccharide analysis of GAGs. Good separation of glucosamine/mannosamine (GlcN/ManN) and glucuronic acid/iduronic acid (GlcA/IdoA) was achieved. This method can also be applied to analyze the acidolysis-resistant disaccharides derived from GAGs, and the sequences of these disaccharides were confirmed by electrospray ionization–collision-induced dissociation–tandem mass spectrometry (ESI–CID–MS/MS). These unique disaccharides could be used as markers to distinguish heparin/heparan sulfate (HP/HS), chondroitin sulfate/dermatan sulfate (CS/DS), and hyaluronic acid (HA).     

Minimum substrate requirements of endoglycosidase activities toward dermatan sulfate by electrospray ionization-tandem mass spectrometry

The catabolism of dermatan sulfate (DS) commences with endohydrolysis of the polysaccharide to oligosaccharides by proposed endo-beta-N-acetylhexosaminidase and endohexuronidase activities. To investigate the substrate specificities of these activities, we developed an assay to measure specific products of their action upon oligosaccharide substrates. Tetra- to tetradecasaccharides, rich in glucuronic acid (GlcA) or iduronic acid (IdoA), were obtained from chondroitinase ABC digests of chondroitin sulfate (CS)-A and DS, respectively, separated by gel-filtration chromatography and characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Endo-beta-N-acetylhexosaminidase and endohexuronidase cleavage of these oligosaccharides was then assessed by incubating with cell homogenate (source of endoglycosidase activity) and measuring di- to octasaccharide products derived from the nonreducing end of the substrate by ESI-MS/MS. We found that both activities preferentially degraded the GlcA-rich substrate, with minor activity toward the IdoA-rich substrate and that a minimum of four and five monosaccharides were required on the reducing side of the target glycosidic linkage for endo-beta-N-acetylhexosaminidase and endohexuronidase cleavage, respectively. Thus, the minimum-sized substrates were a hexasaccharide for endo-beta-N-acetylhexosaminidase and an octasaccharide for endohexuronidase. We observed that endo-beta-N-acetylhexosaminidase sequentially removed tetrasaccharides from the nonreducing end of oligosaccharides when unrestricted by substrate length, whereas endohexuronidase activity was random and comparatively low. The activities displayed acidic pH optima and were shown by subcellular fractionation to reside in lysosomes and late endosomes. We suggest that these activities represent the known Hyal-1 and endo-beta-glucuronidase enzymes and that these enzymes act in concert to degrade GlcA-rich domains of DS but are less active toward regions containing IdoA.     

Analysis of oversulfation in biglycan chondroitin/dermatan sulfate oligosaccharides by chip-based nanoelectrospray ionization multistage mass spectrometry

Biglycan (BGN) is a small proteoglycan that consists of a protein core containing leucine-rich repeat regions and two glycosaminoglycan (GAG) chains of either chondroitin sulfate (CS) or dermatan sulfate (DS) type. The development of novel, highly efficient analytical methods for structural identification of BGN-derived CS/DS motifs, possibly implicated in biological events, is currently the focus of research. In this work, an improved analytical method based on fully automated chip-nanoelectrospray ionization (nanoESI) in conjunction with high-capacity ion trap (HCT) multistage mass spectrometry (MS) by collision-induced dissociation (CID) was for the first time applied to BGN CS/DS oligosaccharide analysis. The CS/DS chains were released from transfected 293 BGN by β-elimination. The chain was digested with AC I lyase, and the resulting mixture was purified and subsequently separated by size exclusion chromatography (SEC). Di- and tetrasaccharide fractions were pooled and characterized in detail using the developed chip-nanoESI protocol. The chip-nanoESI MS profile in the negative ion mode revealed the presence of under-, regularly, and oversulfated species in both di- and tetrasaccharide fractions. CID MS(2)-MS(3) yielded sequence patterns consistent with unusual oversulfated 4,5-Δ-GlcA(2S)-GalNAc(4S) and 4,5-Δ-GlcA(2S)-GalNAc(6S)-IdoA(2S)-GalNAc(6S) motifs.     

Chondroitin sulfate/dermatan sulfate sulfatases from mammals and bacteria

Sulfatases that specifically catalyze the hydrolysis of the sulfate groups on chondroitin sulfate (CS)/dermatan sulfate (DS) poly- and oligosaccharides belong to the formylglycine-dependent family of sulfatases and have been widely found in various mammalian and bacterial organisms. However, only a few types of CS/DS sulfatase have been identified so far. Recently, several novel CS/DS sulfatases have been cloned and characterized. Advanced studies have provided significant insight into the biological function and mechanism of action of CS/DS sulfatases. Moreover, further studies will provide powerful tools for structural and functional studies of CS/DS as well as related applications. This article reviews the recent progress in CS/DS sulfatase research and is expected to initiate further research in this field.     

Determination of iduronic acid and glucuronic acid in sulfated chondroitin/dermatan hybrid chains by <Superscript>1</Superscript>H-nuclear magnetic resonance spectroscopy

The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial eliminase. ¹H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious overlap of signals in the ¹H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) ¹H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D ¹H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains. The contributions of Fuchuan Li and Shuhei Yamada should be considered equal.     

Orbitrap mass spectrometry characterization of hybrid chondroitin/dermatan sulfate hexasaccharide domains expressed in brain

In the central nervous system, chondroitin/dermatan sulfate (CS/DS) glycosaminoglycans (GAGs) modulate neurotrophic effects and glial cell maturation during brain development. Previous reports revealed that GAG composition could be responsible for CS/DS activities in brain. In this work, for the structural characterization of DS- and CS-rich domains in hybrid GAG chains extracted from neural tissue, we have developed an advanced approach based on high-resolution mass spectrometry (MS) using nanoelectrospray ionization Orbitrap in the negative ion mode. Our high-resolution MS and multistage MS approach was developed and applied to hexasaccharides obtained from 4- and 14-week-old mouse brains by GAG digestion with chondroitin B and in parallel with AC I lyase. The expression of DS- and CS-rich domains in the two tissues was assessed comparatively. The analyses indicated an age-related structural variability of the CS/DS motifs. The older brain was found to contain more structures and a higher sulfation of DS-rich regions, whereas the younger brain was found to be characterized by a higher sulfation of CS-rich regions. By multistage MS using collision-induced dissociation, we also demonstrated the incidence in mouse brain of an atypical [4,5-Δ-GlcAGalNAc(IdoAGalNAc)₂], presenting a bisulfated CS disaccharide formed by 3-O-sulfate-4,5-Δ-GlcA and 6-O-sulfate-GalNAc moieties.     

Iduronic Acid in Chondroitin/Dermatan Sulfate Affects Directional Migration of Aortic Smooth Muscle Cells

Aortic smooth muscle cells produce chondroitin/dermatan sulfate (CS/DS) proteoglycans that regulate extracellular matrix organization and cell behavior in normal and pathological conditions. A unique feature of CS/DS proteoglycans is the presence of iduronic acid (IdoA), catalyzed by two DS epimerases. Functional ablation of DS-epi1, the main epimerase in these cells, resulted in a major reduction of IdoA both on cell surface and in secreted CS/DS proteoglycans. Downregulation of IdoA led to delayed ability to re-populate wounded areas due to loss of directional persistence of migration. DS-epi1−/− aortic smooth muscle cells, however, had not lost the general property of migration showing even increased speed of movement compared to wild type cells. Where the cell membrane adheres to the substratum, stress fibers were denser whereas focal adhesion sites were fewer. Total cellular expression of focal adhesion kinase (FAK) and phospho-FAK (pFAK) was decreased in mutant cells compared to control cells. As many pathological conditions are dependent on migration, modulation of IdoA content may point to therapeutic strategies for diseases such as cancer and atherosclerosis.     

Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library

The variation in the sulfation profile of chondroitin sulfate (CS)/dermatan sulfate (DS) chains regulates central nervous system development in vertebrates. Notably, the disulfated disaccharide D-unit, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate), correlates with the promotion of neurite outgrowth through the DSD-1 epitope that is embedded in the CS moiety of the proteoglycan DSD-1-PG/phosphacan. Monoclonal antibody (mAb) 473HD inhibits the DSD-1-dependent neuritogenesis and also recognizes shark cartilage CS-D, which is characterized by the prominent D-unit and is also recognized by two other mAbs, CS-56 and MO-225. We investigate the oligosaccharide epitope structures of these CS-D-reactive mAbs by ELISA and oligosaccharide microarrays using lipid-derivatized CS oligosaccharides. CS-56 and MO-225 recognized the octa- and larger oligosaccharides, though the latter also bound one unique hexasaccharide D-A-D, where A denotes the disaccharide A-unit GlcUA-GalNAc(4-O-sulfate). The octasaccharides reactive with CS-56 and MO-225 shared a core A-D tetrasaccharide, whereas the neighboring structural elements located on the reducing and/or nonreducing sides of the A-D gave a differential preference additionally to the recognition sequence for each antibody. In contrast, 473HD reacted with multiple hexa- and larger oligosaccharides, which also contained A-D or D-A tetrasaccharide sequences. Consistent with the distinct specificity of 473HD as compared with CS-56 and MO-225, the 473HD epitope displayed a different expression pattern in peripheral mouse organs as revealed by immunohistology, extending the previously reported CNS-restricted expression. The epitope of 473HD, but not of CS-56 or MO-225, was eliminated from DSD-1-PG by digestion with chondroitinase B, suggesting the close association of L-iduronic acid with the 473HD epitope. Despite such supplemental information, the integral epitope remains to be isolated for identification and comprehensive analytical characterisation. Thus novel information on the sugar sequences containing the A-D tetrasaccharide core was obtained for the epitopes of these three useful mAbs.     

Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a beta-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: (alpha/alpha)(5) for CS (chondroitin lyases AC) and beta-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located approximately 12 A from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.     

N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.     

Structure of Pleiotrophin- and Hepatocyte Growth Factor-binding Sulfated Hexasaccharide Determined by Biochemical and Computational Approaches

Endogenous pleiotrophin and hepatocyte growth factor (HGF) mediate the neurite outgrowth-promoting activity of chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains isolated from embryonic pig brain. CS/DS hybrid chains isolated from shark skin have a different disaccharide composition, but also display these activities. In this study, pleiotrophin- and HGF-binding domains in shark skin CS/DS were investigated. A high affinity CS/DS fraction was isolated using a pleiotrophin-immobilized column. It showed marked neurite outgrowth- promoting activity and strong inhibitory activity against the binding of pleiotrophin to immobilized CS/DS chains from embryonic pig brain. The inhibitory activity was abolished by chondroitinase ABC or B, and partially reduced by chondroitinase AC-I. A pentasulfated hexasaccharide with a novel structure was isolated from the chondroitinase AC-I digest using pleiotrophin affinity and anion exchange chromatographies. It displayed a potent inhibitory effect on the binding of HGF to immobilized shark skin CS/DS chains, suggesting that the pleiotrophin- and HGF-binding domains at least partially overlap in the CS/DS chains involved in the neuritogenic activity. Computational chemistry using molecular modeling and calculations of the electrostatic potential of the hexasaccharide and two pleiotrophin-binding octasaccharides previously isolated from CS/DS hybrid chains of embryonic pig brain identified an electronegative zone potentially involved in the molecular recognition of the oligosaccharides by pleiotrophin. Homology modeling of pleiotrophin based on a related midkine protein structure predicted the binding pocket of pleiotrophin for the oligosaccharides and provided new insights into the molecular mechanism of the interactions between the oligosaccharides and pleiotrophin.     

Modern developments in mass spectrometry of chondroitin and dermatan sulfate glycosaminoglycans

Chondroitin sulfate (CS) and dermatan sulfate (DS) are special types of glycosaminoglycan (GAG) oligosaccharides able to regulate vital biological functions that depend on precise motifs of their constituent hexose sequences and the extent and location of their sulfation. As a result, the need for better understanding of CS/DS biological role called for the elaboration and application of straightforward strategies for their composition and structure elucidation. Due to its high sensitivity, reproducibility, and the possibility to rapidly generate data on fine CS/DS structure determinants, mass spectrometry (MS) based on either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) brought a major progress in the field. Here, modern developments in MS of CS/DS GAGs are gathered in a critical review covering the past 5 years. The first section is dedicated to protocols for CS/DS extraction from parent proteoglycan, digestion, and purification that are among critical prerequisites of a successful MS experiment. The second part highlights several MALDI MS aspects, the requirements, and applications of this ionization method to CS/DS investigation. An ample chapter is devoted to ESI MS strategies, which employ either capillary- or advanced chip-based sample infusion in combination with multistage MS (MS ⁿ ) using either collision-induced (CID) or electron detachment dissociation (EDD). At last, the potential of two versatile separation techniques, capillary electrophoresis (CE), and liquid chromatography (LC) in off- and/or on-line coupling with ESI MS and MS ⁿ , is discussed, alongside an assessment of particular buffer/solvent conditions and instrumental parameters required for CS/DS mixture separation followed by on-line mass analysis of individual components.     

Characterization of glycosaminoglycans from human normal and scoliotic nasal cartilage with particular reference to dermatan sulfate.

The composition and the distribution of glycosaminoglycans (GAGs) present in normal human nasal cartilage (HNNC), were examined and compared with those in human scoliotic nasal cartilage (HSNC). In both tissues, hyaluronan (HA), keratan sulfate (KS) and the galactosaminoglycans (GalAGs)--chondroitin sulfate (CS) and dermatan sulfate (DS)--were identified. The overall GAG content in HSNC was approx. 30% higher than the HNNC. Particularly, a 114% increase in HA, and 46% and 86% in KS and DS, respectively, was recorded. CS was the main type of GAG in both tissues with no significant compositional difference. GalAG chains in HSNC exhibited an altered disaccharide composition which was associated with significant increases of non-sulfated and 6-sulfated disaccharides. DS, which was identified and quantitated for the first time in HNNC and HSNC, contained low amounts of iduronic acid (IdoA), 18% and 28% respectively. In contrast to other tissues, where IdoA residues are organized in long IdoA rich repeats, the IdoA residues of DS in human nasal cartilage seemed to be randomly distributed along the chain. DS chains in HSNC were of larger average molecular size than those from HNNC. These results clearly indicate the GAG content and pattern in both HNNC and HSNC and demonstrate that scoliosis of nasal septum cartilage is related to quantitative and structural modifications at the GAG level.     

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.     

Dermatan sulfate epimerase 2 is the predominant isozyme in the formation of the chondroitin sulfate/dermatan sulfate hybrid structure in postnatal developing mouse brain

Chondroitin sulfate (CS) and dermatan sulfate (DS) are expressed in significant amounts in the brain and play important roles in the development of the central nervous system in mammals. CS and DS structures are often found in a single CS/DS hybrid chain. The l-iduronic acid (IdoA)-containing domain, which defines a DS-type domain, appears key to the biological functions of the CS/DS hybrid chain. In this study, to clarify the distribution of the DS-type structure in the brain during development, the expression patterns of DS epimerase 1 (DS-epi1) and DS-epi2, both of which convert d-glucuronic acid into IdoA, were investigated by in situ hybridization. DS-epi2 was ubiquitously expressed in the developing brain after birth, whereas the expression of DS-epi1 was faint and obscure at all developmental stages. Quantitative real-time polymerase chain reaction revealed the expression of DS-epi2 to be higher than that of DS-epi1 throughout development, suggesting that DS-epi2 but not DS-epi1 is mostly expressed in the brain and plays key roles in the epimerization of CS/DS during its biosynthesis. Moreover, an analysis of the disaccharides of CS/DS demonstrated significant amounts of IdoA-containing iD units [IdoA(2S)-GalNAc(6S)] and iB units [IdoA(2S)-GalNAc(4S)], where 2S, 4S and 6S stand for 2-O-, 4-O- and 6-O-sulfate, respectively, in every region of the brain examined. The proportion of these units in cerebellar CS/DS was greatly altered during postnatal development. These results suggest that the IdoA-containing structures in the developing brain are mainly produced by the actions of DS-epi2 and play crucial roles in postnatal development.     

An automated mass spectrometry-based screening method for analysis of sulfated glycosaminoglycans

Glycosaminoglycans (GAGs) are linear polysaccharides, consisting of repeated disaccharide units, attached to core proteins in all multicellular organisms. Chondroitin sulfate (CS) and dermatan sulfate (DS) constitute a subgroup of sulfated GAGs for which the degree of sulfation varies between species and tissues. One major goal in GAG characterization is to correlate structure to function. A common approach is to exhaustively degrade the GAG chains and thereafter determine the amount of component disaccharide units. In large-scale studies, there is a need for high-throughput screening methods since existing methods are either very time- or samples consuming. Here, we present a new strategy applying MALDI-TOF MS in positive ion mode for semi-qualitative and quantitative analysis of CS/DS derived disaccharide units. Only a few picomoles of sample are required per analysis and 10 samples can be analyzed in 25 min, which makes this approach an attractive alternative to many established assay methods. The total CS/DS concentration in 19 samples derived from Caenorhabditis elegans and mammalian tissues and cells was determined. The obtained results were well in accordance with concentrations determined by a standard liquid chromatography-based method, demonstrating the applicability of the method for samples from various biological matrices containing CS/DS of different sulfation degrees.     

Studies on the chemistry of arterial wall, XVII. Metabolic characteristics of different types of chondroitin sulfate-dermatan sulfate hybrids in arterial tissue.

1) Chondroitin sulfate and dermatan sulfate of bovine arterial tissue exist as copolymers with a varying degree of hybridization between chondroitin and dermatan sulfates. A fraction rich in dermatan sulfate hybridized with 20% chondroitin sulfate (termed DS-rich hybrid) and a fraction rich in chondroitin sulfate containing 17% DS as copolymer constituent (CS-rich hybrid) can be isolated by the subfractionation of the arterial tissue CS-DS preparation. 2) When arterial tissue segments were preincubated with [14C]glucosamine, 95% of the radioactivity incorporated into the glycosaminoglycans was found to be present in the galactosamine moiety of all of the CS-DS subfractions, whereas the relative proportion of 14C radioactivity incorporated into the galactosamine and uronic acid components was 51:49 following preincubation with [14C]glucose. In both experiments the specific radioactivity of the DS-rich hybrids was twice as high as that of the CS-rich hybrids. 3) Enzymatic degradation of the hybrid CS-DS subfractions by chondroitin AC and ABC lyases revealed that the specific radioactivity of the CS and DS disaccharide units released from the DS-rich hybrids was twice as high as those isolated from the CS-rich hybrids, but within each hybrid fraction the galactosamine moieties of the CS and DS units and their glucuronic and iduronic acid components exhibited equal specific radioactivities. 4) The results strongly support the assumption that distinct compartments exist for the formation of hybrid CS-DS proteoglycans with different proportions of CS and DS.     

Synthesis of proteoglycans and hyaluronic acid by long-term cultures of testicular cells from immature and pubertal rats

Long-term cultures of somatic testicular cells derived from immature and pubertal rats were used to study the synthesis of proteoglycans (PG) and hyaluronic acid (HA). Labelled PG and HA in the culture medium, membrane-associated and intracellular pools were characterized by gel filtration, ion exchange chromatography and selected enzymatic and chemical treatments. Somatic cells synthesize a PG containing both heparan and chondroitin/dermatan sulfate (CS/DS) chains and a PG containing only CS/DS chains. No major qualitative changes in the type of PG were observed in cells derived from immature and pubertal animals. However, significant age-dependent differences in the cell distribution pattern of PG and HA were determined. This may have implications in the regulation of spermatogenesis.