Epitope-specific changes in chondroitin sulfate/dermatan sulfate proteoglycans as markers in the lymphopoietic and granulopoietic compartments of developing bursae of Fabricius

The types and distributions of chondroitin sulfate proteoglycans within developing chick bursae of Fabricius were determined by indirect immunocytochemical analyses using mAb specific for chondroitin/dermatan sulfate epitopes. Analyses obtained from the use of well characterized mAb known to specifically identify chondroitin 4- and dermatan sulfates (antibody 2B6) and chondroitin 6-sulfate (antibody 3B3) were compared with those obtained from two additional mAb raised against chick chondroitin sulfates proteoglycans derived from hemopoietic tissue. The results indicate that chondroitin sulfate compositions of the adjacent lymphopoietic and granulopoietic compartments differ. Chondroitin 6-sulfate, notably absent from lymphopoietic regions, is a major chondroitin sulfate species in granulopoietic regions of day 13 bursae. Moreover, chondroitin 6-sulfate disappears from the granulopoietic compartment in a time course that corresponds to the decline in granulopoietic activity. Simultaneously, there is an apparent increase in chondroitin sulfates associated with developing medullary regions of lymphoid follicles. The content of chondroitin 4-/dermatan sulfates and, most significantly, of chondroitin/dermatan sulfates identified by antibodies raised against chick proteoglycans, increases within developing follicles. As a consequence, by day 18 of incubation, immunostained follicles become clearly demarcated from the connective tissue of the tunica propria. This study provides evidence that chondroitin sulfates are constituents of both lymphopoietic and granulopoietic microenvironments and that subtle changes occur within these proteoglycan structures during bursal development. These developmental changes in chondroitin sulfate compositions are consistent with these molecules playing a functional role in hemopoiesis.     

A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.     

Solvolytic depolymerization of chondroitin and dermatan sulfates

It is essential to establish a library of glycosaminoglycan oligosaccharides from the chondroitin and dermatan sulfates to investigate their biological functions and structure-activity relationships (SARs). There are several approaches to obtain oligosaccharides using chemical and enzymatic degradation procedures; however, purification of each resulting oligosaccharide is complicated because of the diversity of sulfonation patterns present in these oligosaccharides. We have developed a new method for the solvolytic degradation for chondroitin and dermatan sulfates to obtain an oligosaccharide mixture that can be easily purified into chondro/dermato oligosaccharides for characterization by both ¹H NMR and MALDI-TOFMS. These oligosaccharides have a methyl-esterified uronate residue and a methyl 2-acetamido-2-deoxy-d-galactofuranoside at the nonreducing and reducing ends, respectively. All other internal repeating disaccharide units were desulfonated, but maintained their core carbohydrate structures.     

Fractionation and isolation of heparan sulfates using poly-L-lysine-Sepharose

A simple procedure for the isolation of heparan sulfates from pig lung using a poly-L-lysine-Sepharose column is described. Glycosaminoglycans are absorbed on poly-L-lysine-Sepharose at pH 7.5 and eluted with an NaCl linear gradient in the following order: hyaluronic acid (0.32 M NaCl), chondroitin (0.36 M NaCl), keratan sulfate (0.80 M NaCl), chondroitin 4-sulfate (0.86 M NaCl), chondroitin 6-sulfate (0.95 M NaCl), dermatan sulfate (0.91 M NaCl), heparan sulfate (1.2 M NaCl), and heparin (1.35 M NaCl). Based on these observations, isolation of heparan sulfate from pig lung crude heparan sulfate fractions which contain chondroitin sulfates and dermatan sulfate was attempted, using this chromatographic technique.     

Pretreatment procedure for the microdetermination of chondroitin sulfate in plasma and urine

A new, simple, and rapid pretreatment method for the determination of chondroitin sulfate, dermatan sulfate, and hyaluronan from urine and blood plasma samples has been developed. Plasma proteins were first converted into small peptides by digestion using a nonspecific protease, actinase E, and the resulting small peptides were removed by centrifugal filtration. The retained, residual crude glycosaminoglycans, including chondroitin/dermatan sulfates and hyaluronan, were converted into unsaturated disaccharides through the action of chondroitin sulfate lyses. Next, these disaccharides were recovered and purified using centrifugal filtration together with DeltaDi-UA2S, added as an internal standard. The filtered disaccharide mixture was analyzed by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a fluorogenic reagent. This method was applied to a pharmacokinetic study of chondroitin sulfate administered intravenously to mice. The half-life of the administered chondroitin sulfates, having molecular masses from 6 to 50 kDa, varied depending on their molecular sizes. This new method should be useful for studies on the metabolic fate of exogenously administered glycosaminoglycans in small experimental animals.     

Determination of hexuronic acids in glycosaminoglycans by automated ion-exchange chromatography.

This report describes a procedure for analyzing glucuronic and iduronic acids using the Technicon automated sugar chromatography system. Glueronic and iduronic acids of standard samples of glycosaminoglycans have been analyzed after hydrolysis by formic acid. The method has been applied to quantitate uronic acids in chondroitin sulfates and dermatan sulfate mixtures obtained by Dowex 1 Cl^? column fractionation of glycosaminoglycans from aortas of different animal species. The results are in good agreement with those obtained by the gas-liquid chromatographic technique.     

Isolation and characterization of an induced chondroitinase ABC from Flavobacterium heparium

During the investigation of alternative methods for the large sclae preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast tot he chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashiom, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chontroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of Δ-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30°C and at pH 6.0–7.5. Only 15% of the activity was observed at 37°C, indicating that the enzyme is very sensitive to thermal denaturation. It is stronly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed.     

Hyaluronic acid and chondroitin sulfate unsaturated disaccharides analysis by high-performance liquid chromatography and fluorimetric detection with dansylhydrazine

A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.     

Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high-performance liquid chromatography

Various under-sulfated, monosulfated, and over-sulfated chondroitin sulfate and dermatan sulfate isomers were analyzed in terms of disaccharide units before or after desulfation with chondrosulfatases in addition to digestion with chondroitinases. The unsaturated disaccharides were separable by a high-performance liquid chromatography (HPLC) method using a resin made from a sulfonized styrene-divinylbenzene copolymer. The retention times of the parent sulfated unsaturated disaccharides and newly generated unsaturated mono- or nonsulfated disaccharides were reproducible. On desulfation of the parent sulfated unsaturated disaccharides with chondrosulfatases, almost all ΔDi-S showed the same retention times as those of standard ΔDi-S from known components. Following digestion of ΔDi-diS_B with chondro-4-sulfatase as well as ΔDi-diS_D or ΔDi-diS_G with chondro-6-sulfatase, three ΔDi-monoS with the same retention time were detected with the HPLC method. These newly generated ΔDi-monoS₂ showed that the structure is N-acetyl-d-galactosamine, uronic acid 2-sulfate.     

Major chondroitin sulfate proteoglycans identified in L6J1 myoblast culture

The major proteoglycans from L6J1 rat myoblast culture were identified. The proteoglycans were isolated from different constituents of cell culture: culture medium, extracellular matrix (ECM), and myoblasts. To identify their core proteins, the proteoglycans were treated with enzymes specifically digesting chondroitin/dermatan sulfates or chondroitin sulfates. Subsequent electrophoresis and mass spectrometry revealed versican, collagen XII, and inter-α-trypsin inhibitor classified as chondroitin sulfate proteoglycans and biglycan known to be chondroitin/dermatan sulfate proteoglycan. Versican was identified in ECM and the other proteoglycans in the culture medium. Such difference in localization is likely to be a consequence of different biological functions. Versican, collagen XII, and biglycan are synthesized by myoblasts and inter-α-trypsin inhibitor originates from fetal bovine serum (a culture medium component).     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans II. Aggregated complexes-evidence for long-range order.

Aggregated complexes of acridine orange with dermatan and chondroitin sulfates have been studied in aqueous solution by absorption and circular dichroism spectroscopy. Aggregation was found to be favored at high-dye and glycosaminoglycan concentrations, and in solutions where anionic sites of the glycosaminoglycan are effectively complexed with dye. The aggregates can be removed from solution by centrifugation at 27,000 × g for 1 hr or by filtration through a membrane containing pores of 0.1 μm diameter. The aggregated complexes exhibit large-magnitude-ellipticity circular dichroism bands. In addition, the circular dichroism spectrum observed for a solution containing aggregated acridine orange/chondroitin 4-sulfate complexes is nearly a mirror image of that obtained for aggregated acridine orange/dermatan sulfate complexes. Cooperative alterations (sharp transitions) in the circular dichroism ellipticities of the aggregates occur at elevated temperatures, and result in spectroscopically distinct aggregates upon cooling. The circular dichroism properties and temperature effects are attributed to a supramolecular ordering of acridine orange/glycosaminoglycan complexes within the aggregates, which can be reorganized to a more stable form at high temperatures. Mixed aggregates, containing two different glycosaminoglycans, can be formed. The circular dichroism properties of the mixed aggregates also indicate the existence of long-range order in the arrangement of the complexes. Mixed aggregates containing dermatan sulfate and either chondroitin 4-sulfate or chondroitin 6-sulfate resemble pure dermatan sulfate aggregates in circular dichroism characteristics.     

A new electrophoretic method for the separation of disaccharides obtained by digestion of chondroitin sulfates and dermatan sulfate with chondroitinases

A new rapid and simple method has been developed for the separation of disaccharides obtained by chondroitinase digestion of chondroitin sulfates and dermatan sulfate using electrophoresis on cellulose acetate plates (Titan III cellulose acetate plates). Three disaccharides are completely separated by electrophoresis in barium acetate or calcium acetate in a short time, and less than 50 μg of glycosaminoglycan samples can be analyzed within 2 h.     

Analysis of glycosaminoglycan-derived oligosaccharides using reversed-phase ion-pairing and ion-exchange chromatography with suppressed conductivity detection

Oligosaccharides prepared from glycosaminoglycans (GAGs) including heparin, heparan sulfate, chondroitin sulfates, dermatan sulfate, and keratan sulfate were analyzed using reverse-phase ion-pairing HPLC and ion-exchange HPLC with suppressed conductivity detection. The results were compared with those obtained by strong anion-exchange HPLC using uv detection. These oligosaccharides were first prepared by enzymatically depolymerizing the GAGs with enzymes including heparin lyase (EC 4.2.2.7), heparan sulfate lyase (EC 4.2.2.8), chondroitin ABC lyase (EC 4.2.2.4), and keratan sulfate hydrolase (EC 3.2.1.103). Analysis was then performed without derivitization under isocratic conditions with a limit of sensitivity in the picomole range. Preliminary studies suggest that this approach may be particularly useful in examining oligosaccharides having no uv chromophore such as those prepared from keratan sulfate.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

An automated version of the periodate-thiobarbituric acid assay for analysis of delta-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acids and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.     

Rapid and sensitive determination of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography

The separation and quantitative analysis of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography (HPLC) are described. The substituted unsaturated disaccharides which result from digestion of chondroitin sulfates with chondroitinase are quickly separated on polar adsorbents such as silica gel. The UV absorption properties of these unsaturated disaccharides permit UV measurement with detection limits of approximately 100 ng. Their separation by HPLC facilitates the use of enzymatic methods for the determination of chondroitin sulfates A, B and C.

The potential of this method in clinical application is demonstrated by quantitative assays of glycosaminoglycans from a normal urine and urine from a patient with Hunter syndrome. The results are consistent with amount of isomeric chondroitin sulfates found in comparable urines by others.     

Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

Profiling of the eye aqueous humor in exfoliation syndrome by high-performance liquid chromatographic analysis of hyaluronan and galactosaminoglycans

The concentrations of hyaluronan and galactosaminoglycans - i.e., chondroitin sulfate and dermatan sulfate - were measured in the aqueous humor of the eye from patients with exfoliation syndrome and from healthy persons. The glycosaminoglycans/proteoglycans were almost completely precipitated (>97%) with ethanol in the presence of dextran as carrier and, following enzymic digestion, hyaluronan and galactosaminoglycans, were quantitatively converted to Δ^4,5-disaccharides. Non-degraded heparan sulfate and proteins/glycoproteins were removed by ultrafiltration using a Centricon 3 membrane. Separation and determination of hyaluronan- and galactosaminoglycan-derived Δ-disaccharides were performed by ion-suppression HPLC. For an accurate analysis in triplicate, as little as 50 μl of aqueous humor is required. Application of this method to the analysis of samples from six patients with exfoliation syndrome and three healthy persons showed that hyaluronan levels in patients (6.65–16.15 μg ml⁻¹) were significantly higher (3–8 times) than in healthy persons (2.0–2.24 μg ml⁻¹). There was no significant alteration in the galactosaminoglycan concentration. The obtained data open a new area in the deeper understanding of the exfoliation syndrome pathophysiology and in establishing a highly sensitivity and accurate HPLC method for its diagnosis and patient's follow-up.     

Quantitative analysis of glycosaminoglycans,chondroitin/dermatan sulfate,hyaluronic acid,heparan sulfate,and keratan sulfate by liquid chromatography–electrospray ionization–tandem mass spectrometry

We developed a method using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive “GAGomic” analysis of biological tissues.     

Sulfate composition of glycosaminoglycans determined by infrared spectroscopy

Anhydrous sodium sulfate (Na2SO4) was analyzed at varying concentrations by infrared (ir) spectroscopy. A standard curve was obtained from a linear plot of sulfate (SO2-(4] concentration vs the weight of the ir band area of S = O stretching. Standard chondroitin 4-sulfate, chondroitin 6-sulfate, heparan sulfate, heparin, keratan sulfates, and various dermatan sulfates isolated from human and rat skins were also studied by ir spectroscopy. The spectrum of every glycosaminoglycan (GAG) displayed an ir band around 1230 cm-1 which originated from S = O stretching of sulfate esters. Therefore, the weight of the latter band was employed to quantify sulfate, by using the standard curve indicated above. Sulfate was also estimated quantitatively by the gelatin/BaCl2 method of K.S. Dodgson and R.G. Price (Biochem. J. 1962, 84, 106-110). The sulfate composition determined by ir spectroscopy ranged from 8.5 to 22.1% (w/w), and agreed closely with the values obtained chemically. In the ir spectroscopy method, sulfate was determined using the polymer forms of the GAGs. After analysis, these heteropolysaccharides were recovered unaffected in a yield greater than 95%. The data show that the infrared spectroscopy technique, in addition to being sensitive and reliable, is much more economical than the chemical procedures currently employed to quantify GAG sulfate.