Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE)

Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal Deltadisaccharides including DeltaDiHA, DeltaDi0S, DeltaDi2S, DeltaDi4S, DeltaDi6S, DeltaDi2,4S, DeltaDi2,6S, DeltaDi4,6S, and DeltaDi2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, Di0S, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4, 6S-galNAc.     

Structural analysis of the linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1.

Swarm rat chondrosarcoma cell cultures were metabolically labeled with [35S]sulfate, [3H]glucose, or [3H]glucosamine. Chondroitin sulfate chains were isolated from purified aggrecan using alkaline borohydride treatment and Superose 6 chromatography. Various linkage region oligosaccharide alditols were derived from these chains using sequential chondroitinase digestions (ABC lyase followed by ACII lyase). They were then further processed by mercuric acetate treatment, which removed the 4,5-unsaturated uronosyl residue from the nonreducing end of the linkage, and then beta-galactosidase digestion which liberated the 2 galactose residues from the xylitol reducing terminus. Alkaline phosphatase digestions were performed to verify the presence of phosphate esters. All linkage region structures were isolated and identified using a combination of Progel-TSK G2500 and CarboPac PA1 chromatography steps in conjunction with monosaccharide analyses. This study revealed that chondroitin sulfate chains from aggrecan synthesized by rat chondrosarcoma cells in vitro have the following properties: 1) three out of every four of their linkage regions carry a phosphate ester on xylose, 2) nearly three out of every five chains begin the repeating disaccharide region with an unsulfated first disaccharide unit, 3) nearly twice as many nonphosphorylated chains have a sulfated first disaccharide than their phosphorylated counterparts, and 4) the vast majority of these chains do not contain sulfated galactose in their linkage regions. This report also describes a borohydride reduction procedure to confer alkali stability to the 3-substituted, unsaturated disaccharides derived from chondroitinase digests of chondroitin sulfate. Furthermore, a CarboPac PA1 method is demonstrated that separates these reduced disaccharides with exceptional resolution.     

Ion exchange HPLC microanalysis of chondroitin sulfate: quantitative derivatization of chondroitin lyase digestion products with 2-aminopyridine

Sulfated glycosaminoglycans such as chondroitin sulfate arecomposed of three structural domains, a linkage oligosaccharide,connecting the chain to the core protein, a variably sulfateddisaccharide repeat structure within the chain and a nonreducingterminal, and these domains may confer specific functions onparticular chain populations. We report here a new and highlysensitive method for the detection and quantitation of all nonreducingterminal residues and internal disaccharides obtained by chondroltinaseABC or ACII digestion of aggrecan chondroitin sulfate. The procedureinvolves a quantitative reductive amination of the reducingends of sulfated mono- and disaccharide chondroitinase productswith 2-aminopyridine and boranedimethylamine. All derivatizedsaccharides can be separated and quantitated by fluorescencein a single chromatographic step on an AS4A anion exchange column,eluted with a gradient (0–500 mM) of sodium trifluoroacetate.The reproducibility and stability of the derivatisation, togetherwith the sensitivity of the chromatography system, allowed forroutine quantitation in the range of 3–500 pmol of reducinggroup (corresponding to about 1.5–250 ng of disaccharideor 0.75–125 ng of monosaccharide). Moreover, the fluorescenceyield (fluorescence area units per pmol of reducing group) wasvirtually identical for all saccharides analyzed. Applicationof this method to an analysis of aggrecan purified from calfepiphyseal cartilage and from rat chondrosarcoma chondrocytecultures allowed a precise identification and quantitation ofthe internal disaccharides and the nonreducing terminal structures,together with an estimation of the number average molecularweight of CS chains in these aggrecan preparations. aggrecan chondroitin sulfate glycosaminoglycans proteoglycans     

Chimeric glycosaminoglycan oligosaccharides synthesized by enzymatic reconstruction and their use in substrate specificity determination of Streptococcus hyaluronidase

A method was developed for the reconstruction of glycosaminoglycan (GAG) oligosaccharides using the transglycosylation reaction of an endo-beta-N-acetylhexosaminidase, testicular hyaluronidase, under optimal conditions. Repetition of the transglycosylation using suitable combinations of various GAGs as acceptors and donors made it possible to custom-synthesize GAG oligosaccharides. Thus we prepared a library of chimeric GAG oligosaccharides with hybrid structures composed of disaccharide units such as GlcA-GlcNAc (from hyaluronic acid), GlcA-GalNAc (from chondroitin), GlcA-GalNAc4S (from chondroitin 4-sulfate), GlcA-GalNAc6S (from chondroitin 6-sulfate), IdoA-GalNAc (from desulfated dermatan sulfate), and GlcA-GalNAc4,6-diS (from chondroitin sulfate E). The specificity of the hyaluronidase from Streptococcus dysgalactiae (hyaluronidase SD) was then investigated using these chimeric GAG oligosaccharides as model substrates. The results indicate that the specificity of hyaluronidase SD is determined by the following restrictions at the nonreducing terminal side of the cleavage site: (i) at least one disaccharide unit (GlcA-GlcNAc) is necessary for the enzymatic action of hyaluronidase SD; (ii) cleavage is inhibited by sulfation of the N-acetylgalactosamine; (iii) hyaluronidase SD releases GlcA-GalNAc and IdoA-GalNAc units as well as GlcA-GlcNAc. At the reducing terminal side of the cleavage site, the sulfated residues on the N-acetylgalactosamines in the disaccharide units were found to have no influence on the cleavage. Additionally, we found that hyaluronidase SD can specifically and endolytically cleave the internal unsulfated regions of chondroitin sulfate chains. This demonstration indicates that custom-synthesized GAG oligosaccharides will open a new avenue in GAG glycotechnology.     

Glycosaminoglycan composition of the large freshwater mollusc bivalve Anodonta anodonta

In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (DeltaUA2S(1-->4)-alpha-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (-->4)-beta-D-GlcA(1-->4)-alpha-D-GlcN2S3S6S(1-->) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs.     

Synthesis of chondroitin sulfate E hexasaccharide in the repeating region by an effective elongation strategy toward longer chondroitin oligosaccharide

We synthesized chondroitin and its sulfate E hexasaccharides (1 and 2) composed of the trimer of the repeating disaccharide, beta-D-GalNAc(+/-4,6-di-O-SO(3)Na)-(1-->4)-beta-D-GlcA, by employing an efficient synthetic strategy for longer chondroitin oligosaccharide. Successful elongation with the beta-D-GalNAc-(1-->4)-beta-D-GlcA unit instead of the corresponding disaccharide possessing an azide group avoided problematic reduction of the multiple azide groups on the hexasaccharide.     

Difference between N-acetylgalactosamine 4-sulfate 6-O-sulfotransferases from human serum and squid cartilage in specificity toward the terminal and interior portion of chondroitin sulfate

In the preceding paper (Inoue, H., Otsu, K., Yoneda, M., Kimata, K., Suzuki, S., and Nakanishi, Y. (1986) J. Biol. Chem. 261, 4460-4469), we reported the purification from human serum of an N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase fraction which was able to transfer sulfate predominantly to position 6 of the nonreducing terminal N-acetylgalactosamine 4-sulfate unit of chondroitin sulfate. We now show that the activity toward the terminal was co-purified with a minor activity toward the interior counterpart by sequential chromatography on heparin-Sepharose CL-6B, Matrex Blue B, hydroxyapatite, and Sephacryl S-300, and that the two activities were equally heatlabile. The enzyme purified 5000-fold from human serum was devoid of the sulfotransferase activities toward chondroitin, heparan sulfate, and keratan sulfate, but showed a strong terminal sulfotransferase activity toward dermatan sulfate (pig skin); over 97% of the sulfate residues incorporated were at position 6 of the nonreducing N-acetylgalactosamine 4,6-bissulfate end groups linked to the L-iduronic acid group. Although the enzyme introduces sulfate predominantly into the nonreducing terminal of chondroitin sulfate at physiological pH (approximately equal to 7.0) and Ca2+ concentration (approximately 2-3 mM), the activity toward the interior portion relative to that toward the terminal was increased by either lowering pH or elevating Ca2+ concentration, perhaps owing to changes in the conformation or ionic state of the acceptor molecule. Comparison between the human serum enzyme and the N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (formerly designated "E6-sulfotransferase") from squid cartilage indicated that the latter is distinct from the former in introducing sulfate predominantly into the interior portion of chondroitin sulfate. It appears that the role of the squid sulfotransferase is to synthesize so-called chondroitin sulfate E where over 50% of the interior hexosamine units are 4,6-bis-sulfated.     

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) involved in chondroitin sulfate initiation: Impact of sulfation on activity and specificity

Glycosaminoglycan (GAG) assembly initiates through the formation of a linkage tetrasaccharide region serving as a primer for both chondroitin sulfate (CS) and heparan sulfate (HS) chain polymerization. A possible role for sulfation of the linkage structure and of the constitutive disaccharide unit of CS chains in the regulation of CS-GAG chain synthesis has been suggested. To investigate this, we determined whether sulfate substitution of galactose (Gal) residues of the linkage region or of N-acetylgalactosamine (GalNAc) of the disaccharide unit influences activity and specificity of chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1), a key glycosyltransferase of CS biosynthesis. We synthesized a series of sulfated and unsulfated analogs of the linkage oligosaccharide and of the constitutive unit of CS and tested these molecules as potential acceptor substrates for the recombinant human CSGalNAcT-1. We show here that sulfation at C4 or C6 of the Gal residues markedly influences CSGalNAcT-1 initiation activity and catalytic efficiency. Kinetic analysis indicates that CSGalNAcT-1 exhibited 3.6-, 1.6-, and 2.2-fold higher enzymatic efficiency due to lower K(m) values toward monosulfated trisaccharides substituted at C4 or C6 position of Gal1, and at C6 of Gal2, respectively, compared with the unsulfated oligosaccharide. This highlights the critical influence of Gal substitution on both CSGalNAcT-1 activity and specifity. No GalNAcT activity was detected toward sulfated and unsulfated analogs of the CS constitutive disaccharide (GlcA-β1,3-GalNAc), indicating that CSGalNAcT-1 was involved in initiation but not in elongation of CS chains. Our results strongly suggest that sulfation of the linkage region acts as a regulatory signal in CS chain initiation.     

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide was isolated from this mucopolysaccharide by the action of the chondroitinase C, indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.     

Structures of Streptococcus pneumoniae hyaluronate lyase in complex with chondroitin and chondroitin sulfate disaccharides. Insights into specificity and mechanism of action

Streptococcus pneumoniae hyaluronate lyase is a surface enzyme of this Gram-positive bacterium. The enzyme degrades hyaluronan and chondroitin/chondroitin sulfates by cleaving the beta1,4-glycosidic linkage between the glycan units of these polymeric substrates. This degradation helps spreading of this bacterial organism throughout the host tissues and facilitates the disease process caused by pneumococci. The mechanism of this degradative process is based on beta-elimination, is termed proton acceptance and donation, and involves selected residues of a well defined catalytic site of the enzyme. The degradation of hyaluronan alone is thought to proceed through a processive mode of action. The structures of complexes between the enzyme and chondroitin as well as chondroitin sulfate disaccharides allowed for the first detailed insights into these interactions and the mechanism of action on chondroitins. This degradation of chondroitin/chondroitin sulfates is nonprocessive and is selective for the chondroitin sulfates only with certain sulfation patterns. Chondroitin sulfation at the 4-position on the nonreducing site of the linkage to be cleaved or 2-sulfation prevent degradation due to steric clashes with the enzyme. Evolutionary studies suggest that hyaluronate lyases evolved from chondroitin lyases and still retained chondroitin/chondroitin sulfate degradation abilities while being specialized in the degradation of hyaluronan. The more efficient processive degradation mechanism has come to be preferred for the unsulfated substrate hyaluronan.     

Structural determination of five novel tetrasaccharides containing 3-O-sulfated D-glucuronic acid and two rare oligosaccharides containing a beta-D-glucose branch isolated from squid cartilage chondroitin sulfate E

Oversulfated chondroitin sulfate E (CS-E) derived from squid cartilage exhibits intriguing biological activities, which appear to reflect the biological activities of mammalian CS chains containing the so-called E disaccharide unit [GlcAbeta1-3GalNAc(4,6-O-disulfate)]. Previously, we isolated novel tetra- and hexasaccharides containing a rare GlcA(3-O-sulfate) at the nonreducing end after digestion of squid cartilage CS-E with testicular hyaluronidase. In this study, squid cartilage CS-E was extensively digested with chondroitinase AC-II, which yielded five highly sulfated novel tetrasaccharides and two odd-numbered oligosaccharides (tri- and pentasaccharides) containing D-Glc. Their structures were determined by fast atom bombardment mass spectrometry and (1)H NMR spectroscopy. The results revealed an internal GlcA(3-O-sulfate) residue for all the novel tetrasaccharide sequences, which rendered the oligosaccharides resistant to the enzyme. The results suggest that GlcA(3-O-sulfate) units are not clustered but rather interspersed in the CS-E polysaccahride chains, being preferentially located in the highly sulfated sequences. The predominant structure on the nearest nonreducing side of a GlcA(3-O-sulfate) residue was GalNAc(4-O-sulfate) (80%), whereas that on the reducing side was GalNAc(4,6-O-disulfate) (59%). The structural variety in the vicinity of the GlcA(3-O-sulfate) residue might represent the substrate specificity of the unidentified chondroitin GlcA 3-O-sulfotransferase. The results also revealed a trisaccharide and a pentasaccahride sequence, both of which contained a beta-d-Glc branch at the C6 position of the constituent GalNAc residue. Approximately 5 mol % of all disaccharide units were substituted by Glc in the CS-E preparation used.     

A unique nonreducing terminal modification of chondroitin sulfate by N-acetylgalactosamine 4-sulfate 6-o-sulfotransferase

N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 6 of N-acetylgalactosamine 4-sulfate (GalNAc(4SO4)). We previously identified human GalNAc4S-6ST cDNA and showed that the recombinant GalNAc4S-6ST could transfer sulfate efficiently to the nonreducing terminal GalNAc(4SO4) residues. We here present evidence that GalNAc4S-6ST should be involved in a unique nonreducing terminal modification of chondroitin sulfate A (CSA). From the nonreducing terminal of CS-A, a GlcA-containing oligosaccharide (Oligo I) that could serve as an acceptor for GalNAc4S-6ST was obtained after chondroitinase ACII digestion. Oligo I was found to be GalNAc(4SO4)-GlcA(2SO4)-GalNAc(6SO4) because GalNAc(4SO4) and deltaHexA(2SO4)-GalNAc(6SO4) were formed after chondroitinase ABC digestion. When Oligo I was used as the acceptor for GalNAc4S-6ST, sulfate was transferred to position 6 of GalNAc(4SO4) located at the nonreducing end of Oligo I. Oligo I was much better acceptor for GalNAc4S-6ST than GalNAc(4SO4)-GlcAGalNAc(6SO4). An oligosaccharide (Oligo II) whose structure is identical to that of the sulfated Oligo I was obtained from CS-A after chondroitinase ACII digestion, indicating that the terminal modification occurs under the physiological conditions. When CS-A was incubated with [35S]PAPS and GalNAc4S-6ST and the 35S-labeled product was digested with chondroitinase ACII, a 35S-labeled trisaccharide (Oligo III) containing [35S]GalNAc(4,6-SO4) residue at the nonreducing end was obtained. Oligo III behaved identically with the sulfated Oligos I and II. These results suggest that GalNAc4S-6ST may be involved in the terminal modification of CS-A, through which a highly sulfated nonreducing terminal sequence is generated.     

Sulfation of chondroitin sulfate in human articular cartilage. The effect of age, topographical position, and zone of cartilage on tissue composition.

The chondroitin ABC lyase digestion products of normal human femoral condyle articular cartilage and of purified aggrecan were analyzed for their mono- and nonsulfated disaccharide composition. Changes in the total tissue chemistry were most pronounced during the period from birth to 20 years of age, when the -[GlcAbeta,3GalNAc6]- disaccharide content increased from approximately 50% to 85% of the total disaccharide content and there was a concomitant decrease in the content of the 4-sulfated disaccharide. In general, the disaccharide content of the deeper layers of immature cartilage were richer in the 4-sulfated residue than the upper regions of the tissue. As the tissue aged and decreased in thickness, the disaccharide composition became more evenly 6-sulfated. The newly synthesized chondroitin sulfate chains had a similar composition to the endogenous chains and also underwent the same age and zonal changes. The monoclonal antisera 3B3(+) and 2B6(+) were used to immunolocalize the unsaturated 6- and 4-sulfated residues generated at the reducing termini of the chondroitin sulfate chains by digestion with chondroitin ABC lyase, and these analyses indicated that the sulfation pattern at this position did not necessarily reflect the internal disaccharide composition of the chains. In summary, the sulfation pattern of chondroitin sulfate disaccharides from human normal articular cartilage varies with the age of the specimen, the position (topography) on the joint surface, and the zone of cartilage analyzed. Furthermore, these changes in composition are a consequence of both extracellular, post-translational processing of the core protein of aggrecan and changes in the sulfotransferase activity of the chondrocyte.     

Characterization of a low-sulfated chondroitin sulfate from the body of Viviparus ater (mollusca gastropoda). Modification of its structure by lead pollution

A chondroitin sulfate was purified from the body of Viviparus ater (Mollusca gastropoda) and analyzed for molecular mass, constituent disaccharides, and structure by 1H NMR and 1H 2D NMR. A quite unique glycosaminoglycan species was isolated having a high molecular mass (greater than 45,000) and low charge density, about 0.60, due to the presence of 42% non-sulfated disaccharide, 5% 6-sulfated disaccharide, 48% 4-sulfated disaccharide, and 5% 4,6-disulfated disaccharide. Specimens of Mollusca were also submitted to lead exposure for different times, and the effect on chondroitin sulfate structure was studied. After 96 h treatment a strong decrease in chondroitin sulfate content was observed with a significant modification of its structure producing a more desulfated polymer, in particular in position 4 of the galactosamine unit. Simultaneously, the amount of unsaturated non-sulfated disaccharide increased with an overall decrease of the charge density.     

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.     

Glycosaminoglycan conformation: do aqueous molecular dynamics simulations agree with x-ray fiber diffraction?

Glycosaminoglycan-protein interactions are biologically important and require an appreciation of glycan molecular shape in solution, which is presently unavailable. In previous studies we found strong similarity between aqueous molecular dynamics (MD) simulations and published x-ray diffraction refinements of hyaluronan. We have applied a similar approach here to chondroitin and dermatan, attempting to clarify some of the issues raised by the x-ray diffraction literature relating to chondroitin and dermatan sulfate. We predict that chondroitin has the same beta(1-->4) linkage conformation as hyaluronan, and that their average beta(1-->3) conformations differ. This is explained by changes in hydrogen-bonding across this linkage, resulting from its axial hydroxyl, causing a different sampling of left-handed helices in chondroitin (2.5- to 3.5-fold) as compared with hyaluronan (3.0- to 4.0-fold). Few right-handed helices, which lack intramolecular hydrogen-bonds, were sampled during our MD simulations. Thus, we propose that the 8-fold helix observed in chondroitin-6-sulfate, represented in the literature as an 8(3) helix (right-handed), though it has never been refined, is more likely to be 8(5) (left-handed) helix. Molecular dynamics simulations implied that (4)C(1) and (2)S(O), but not (1)C(4), forms of iduronate could be used in refinements of dermatan x-ray fiber diffraction patterns. Current models of 8-fold dermatan sulfate chains containing (4)C(1) iduronate refine to right-handed helices, which possess no intramolecular hydrogen-bonds. However, MD simulations predict that models containing (2)S(O) iduronate could provide better (8(5) helix) starting structures for refinement. Thus, the 8-fold dermatan sulfate refinement (8(3) helix) could be in error.     

Expression of sulfotransferases involved in the biosynthesis of chondroitin sulfate E in the bone marrow derived mast cells

Bone marrow-derived mast cells (BMMCs) contain chondroitin sulfate (CS)-E comprised of GlcA-GalNAc(4SO4) units and GlcA-GalNAc(4,6-SO4) units. GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of CS. On the basis of the specificity of GalNAc4S-6ST, it is thought that CS-E is synthesized in BMMC through the sequential sulfation by chondroitin 4-sulfotransferase (C4ST)-1 and GalNAc4S-6ST. In this paper, we investigated whether GalNAc4S-6ST and C4ST-1 are actually expressed in BMMCs in which CS-E is actively synthesized. As the bone marrow cells differentiate to BMMCs, level of C4ST-1 and GalNAc4S-6ST messages increased, whereas chondroitin 6-sulfotransferase (C6ST)-1 message decreased. In the extract of BMMCs, activity of GalNAc4S-6ST and C4ST but not C6ST were detected. The recombinant mouse GalNAc4S-6ST transferred sulfate to both nonreducing terminal and internal GalNAc(4SO4) residues; the activity toward nonreducing terminal GalNAc(4SO4) was increased with increasing pH. When CS-E synthesized by BMMCs was metabolically labeled with 35SO4 in the presence of bafilomycin A, chloroquine or NH4Cl, the proportion of the nonreducing terminal GalNAc(4,6-SO4) was increased compared with the control, suggesting that GalNAc4S-6ST in BMMC may elaborate CS-E in the intracellular compartment with relatively low pH where sulfation of the internal GalNAc(4SO4) by GalNAc4S-6ST preferentially occurs.     

Urinary excretion of sulphated N-acetylhexosamines in patients with various mucopolysaccharidoses.

Sulphated N-acetylhexosamines have been isolated from human urine and tentatively identified as N-acetylglucosamine 6-sulphate (GlcNAc6S), N-acetylgalactosamine 6-sulphate (GalNAc6S), N-acetylgalactosamine 4-sulphate (GalNAc4S) and N-acetylgalactosamine 4,6-disulphate (GalNAc4,6diS). Urine from mucopolysaccharidosis-Type-IIID, -IVA and -VI patients compared with that from normal individuals contains elevated levels of GlcNAc6S (380-fold), GalNAc6S (180-fold) and GalNAc4S (420-fold) respectively. Urine from mucopolysaccharidosis-Type-VI patients also contain more than 600 times the normal level of GalNAc4,6diS. Urine from a mucolipidosis-Type-II and a multiple-sulphatase-deficient patient, and, in general, all mucopolysaccharidosis patients studied, contain at least 5-10-fold elevations of sulphated N-acetylhexosamines over the levels detected in urine from normal controls and a alpha-mannosidosis patient. Urine from patients with clinically mild phenotypes contains less sulphated N-acetylhexosamines than isolated from urine of clinically severe mucopolysaccharidosis patients. The source of the four sulphated N-acetylhexosamines is not known. However, incubation of a series of oligosaccharide substrates, derived from keratan sulphate and chondroitin 6-sulphate and containing non-reducing-end beta-linked 6-sulphated N-acetylhexosamine residues, with homogenates of cultured human skin fibroblasts has indirectly been shown to release GlcNA6S and GalNAc6S respectively. Release of GalNAc4S could not be demonstrated in similar incubations of oligosaccharide substrates derived from chondroitin 4-sulphate and containing non-reducing-end beta-linked GalNAc4S residues. We propose that some, if not all, of the sulphated N-acetylhexosamine present in human urine is derived from the action of beta-N-acetylhexosaminidase on sulphated GlcNAc or GalNAc residues at the non-reducing end of keratan sulphate, dermatan sulphate or chondroitin sulphate.     

Development of a functional bioassay for arylsulfatase B using the natural substrates of the enzyme

A functional bioassay has been developed for measuring the intracellular activity of recombinant human arylsulfatase B (rhASB) on its natural glycosaminoglycan (GAG) substrates, dermatan sulfate (DS), and chondroitin sulfate (CS) when the enzyme is taken up into cultured ASB-deficient human fibroblasts (GM00519). The enzyme ASB is a lysosomal exohydrolase, cleaving sulfate from the N-acetylgalactosamine-4-sulfate (GalNAc-4S) residue at the nonreducing terminal of GAG structures. ASB-deficient cells accumulate DS and CS, which may be partially hydrolyzed by other lysosomal hydrolases, with the reactions stopping if a GalNAc-4S residue is reached on the nonreducing end of the oligosaccharide. When rhASB is added to the culture medium, the enzyme is taken up and translocates to the lysosomes and the intracellular DS and CS are depleted, demonstrating that the uptake of rhASB is able to restore lysosomal function in an in vitro cell-based assay. The accumulation and depletion of DS and CS are measured by digesting the residual intracellular DS and CS content with chondroitin ABC lyase and monitoring a characteristic disaccharide digestion product by laser-induced fluorescence–capillary zone electrophoresis (LIF–CZE). In the proposed assay format, GM00519 cells are cultured 5 weeks postconfluence to accumulate DS/CS, followed by incubation with rhASB (1–20 pM) for 5 days, and the CS/DS depletion profiles are compared between samples. The assay measures depletion of DS/CS independently of their molecular size or processing state; in this approach, all DS- and CS-like substances accumulating in the absence of ASB activity are considered to be natural substrates of the enzyme.     

Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo-beta-galactosidase digestion products

Many tissues contain glycoproteins and proteoglycans, which are substituted with N-or O-linked keratan sulfate, a glycosaminoglycan in which the lactosamine (-galbeta1,4glcNAc-) disaccharide backbone is variably modified by sulfation, fucosylation, and sialylation. We report here a rapid, sensitive, and quantitative procedure for obtaining a complete disaccharide compositional analyses for keratan sulfates after FACE separation of products generated by hydrolysis of the glycosaminoglycans with B. fragillis keratanase II and E. freundii endo-beta-galactosidase. Seven digestion end products are separable in a single electrophoretic step using Monosaccharide composition gels. These are: the unsulfated disaccharide, glcNAcbeta1,3gal, the fucosylated trisaccharide, galbeta1,2[fucalpha1,3]glcNAc6S, the mono- and disulfated disaccharides, galbeta1,4glcNAc6S or gal6Sbeta1,4glcNAc6S from the chain interior, and the sialylated mono- and disulfated trisaccharides neuAalpha2,3galbeta1,4glcNAc6S or neuAalpha2,3gal6Sbeta1,4glcNAc6S from the nonreducing terminus. FACE analyses also revealed the presence of a contaminant beta-galactosidase activity in keratanase II enzyme preparations which cleaves the disaccharide, galbeta1,4glcNAc6S to its constituent monosaccharides, gal and glcNAc6S. It was particularly prominent at enzyme concentrations > 2 mU per nmole substrate glcNH(2) or after prolonged digestion times (> 12 h), and was not inhibitable by thiogalactosides or N-acetyl-lactosamine. As these monosaccharide products would not be detectable using the commonly described analytical methods for KS hydrolase products, such as (1)H-NMR and HPLC analyses, our data illustrate that the FACE procedure represents an improved approach for accurate compositional microanalyses of corneal and skeletal keratan sulfates, especially applicable to experimentation involving small amounts (1-2 microg) of this glycosaminoglycan.