Effect of 6-O-sulfonate hexosamine residue on anticoagulant activity of fully O-sulfonated glycosaminoglycans

Intact and fully O[emsp4 ]-sulfonated glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, hyaluronan, heparan sulfate and heparin were chemically de-O-sulfonated on their hexosamine C-6 position (6-O[emsp4 ]-desulfonation) using N,O[emsp4 ]-bis(trimethylsilyl) acetamide. ¹H NMR spectroscopy and chemical compositional analysis showed that the chemical de-O[emsp4 ]-sulfonation at C-6 position of hexosamine residues in both intact and fully O[emsp4 ]-sulfonated GAGs was completely achieved. Since GAGs and their derivatives are often used as anticoagulant agents, their anti-amidolytic activities were determined. While most of anticoagulant activity of fully O[emsp4 ]-sulfonated GAGs (FGAGs) and heparin disappeared following chemical 6-O[emsp4 ]-desulfonation, the activity of 6-O-desulfonated fully O[emsp4 ]-sulfonated dermatan sulfate (De6FDS) remained. This observation suggests the importance of the position of O-sulfonate groups for anti-coagulant activity.     

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.     

Analysis of the structure and neuritogenic activity of chondroitin sulfate/dermatan sulfate hybrid chains from porcine fetal membranes

The amniotic membrane (AM) is the innermost layer of fetal membranes and possesses various biological activities. Although the mechanism underlying these biological activities remains unclear, unique components seem to be involved. AM contains various extracellular matrix components such as type I collagen, laminin, fibronectin, hyaluronan, and proteoglycans bearing chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycan side chains. Since CS/DS have been implicated in various biological processes, we hypothesized that CS/DS in AM may play a major role in the biological activities of AM. Therefore, the structure and bioactivity of the CS/DS chains from porcine fetal membranes (FM-CS/DS) were investigated. A compositional analysis using various chondroitinases revealed that the characteristic DS domain comprised of iduronic acid-containing disaccharide units is embedded in FM-CS/DS, along with predominant disaccharide units, GlcA-GalNAc, GlcA-GalNAc(4-O-sulfate), and GlcA-GalNAc(6-O-sulfate), where GlcA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. The average molecular mass of FM-CS/DS chains was unusually large and estimated to be 250 – 300 kDa. The FM-CS/DS chains showed neurite outgrowth-promoting activity, which was eliminated by digestion with chondroitinase ABC of the CS/DS chains. This activity was suppressed by antibodies against growth factors including pleiotrophin, midkine, and fibroblast growth factor-2, suggesting the involvement of these growth factors in the neurite outgrowth-promoting activity. The binding of these growth factors to FM-CS/DS was also demonstrated by surface plasmon resonance spectroscopy.     

Variation of acharan sulfate and monosaccharide composition and analysis of neutral <Emphasis Type="Italic">N</Emphasis>-glycans in African giant snail (<Emphasis Type="Italic">Achatina fulica</Emphasis>)

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide →4)-α-d-GlcNpAc (1→4)-α-l-IdoAp2S(1→, analyzed by SAX (strong-anion exchange)–HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex_2–4-HexNAc₂), high mannose (Hex_5–9-HexNAc₂), and complex (Hex₃-HexNAc_2–10) types. None showed core fucosylation.     

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.     

Patterns of uronosyl epimerization and 4-/6-0-sulphation in chondroitin/dermatan sulphate from decorin and biglycan of various bovine tissues

Dermatan sulphate is a co-polymer of two types of disac-chariderepeats: D-glucuronate-N-acetylgalactosamine and L-iduronate-N-acetylgalactosamlne.The former can be O-sulphated at C-4 or C-6 of the galactosamine,whereas the latter contains almost exclusively 4-O-sulphatedgalactosamine. A minor proportion of the L-iduronate may beO-sulphated at C-2. Chondroitin sulphate has no L-iduronate-containing repeats. We have used our recently developed methodsfor sequence analysis of galactos-aminoglycans to investigatethe structure of dermatan/ chondroitin sulphates of the proteoglycansdecorin and biglycan derived from various bovine tissues, likederails, sclera, tendon, aorta, cartilage and bone. The glycanchains, radioiodinated at the reducing end, were partially cleavedwith specific enzymes (chondroitin lyases), and subjected tohigh-resolution polyacrylamide gel electrophoresis, blottingand autoradiography to identify fragments extending from thelabelled reducing end to the point of cleavage. We used chondroitinB lyase to identify the location of L-iduronate, chondroitinAC-I lyase to locate D-glucuronate and chondroitin C lyase tocleave where D-glucuronate residues were succeeded by 6-O-sulphatedN-acetylgalactosamine. We could demonstrate tissue-specific,periodic and wave-like patterns of distribution for the twoepimeric uronic acids, as well as specific patterns of sulphationhi dermatan sulphates derived from either decorin or biglycan.For example, some dermatan sulphates contained D-glucuronate-richdomains that were always 6-sulphated (sclera] decorin), otherswere always 4-sulphated (decorin from bovine dermis, cartilageand bone; biglycan from aorta) or 6-sulphated near the linkageregion, but 4-sulphated hi more distal domains (decorin fromporcine dermis and bovine tendon). Decorin from bone and articularcartilage, as well as biglycan from articular and nasal cartilage,carried largely chondroitin sulphate chains, but also some dermatansulphate, whereas galactosaminoglycan chains derived from aggrecanof nasal cartilage were free of L-iduronate. Decorin and biglycanfrom the same tissue (articular cartilage or sclera) had similarglycan chains. The two side chains in a biglycan molecule areprobably also similar to one another. The portion of the glycanchains nearest to the core protein was substituted with chargedgroups to a variable degree, which may correlate with the structuralfeatures of the main chain. biglycan decorin     

Isolation and properties of two sialate-<Emphasis Type="Italic">O</Emphasis>-acetylesterases from horse liver with 4- and 9-<Emphasis Type="Italic">O</Emphasis>-acetyl specificities

Sialate-O-acetylesterase was purified almost 900-fold from particle-free supernatants of horse liver by gel filtration, ion-exchange chromatography and isoelectric focussing. The native enzyme on gel filtration exhibits a molecular weight of 54,000 Da. It was separated by isoelectric focussing into two forms with pI values of 4.8 and 5.7, respectively. The esterase with a lower pI hydrolyses only 9-O-acetyl groups from sialic acids (K_M 1.1 mM), while that with the higher pI esterifies both 4- and 9-O-acetylated monosaccharides at similar rates (K_M 0.3 M and 1.3 mM, respectively). Both forms are inactive with 7-O-acetylated N-acetylneuraminic acid. Enzyme assays were carried out at the pH optimum (pH 8.4–8.6) using free O-acetylated sialic acids followed by direct analysis of the reaction products by isocratic anion-exchange HPLC. Glycosidically bound sialic acids can also be de-O-acetylated. Horse liver esterase seems to be an essential enzyme for the catabolism of 4-O-acetylated sialoglycoconjugates, since sialidase from this tissue cannot act on 4-O-acetylated sialic acids.     

Permeabilization of <Emphasis Type="Italic">Microbacterium oxylans</Emphasis> shifts the conversion of puerarin from puerarin-7-<Emphasis Type="Italic">O</Emphasis>-glucoside to puerarin-7-<Emphasis Type="Italic">O</Emphasis>-fructoside

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 ± 31.9 μM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 ± 48.0 μM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, ¹³C NMR, ¹H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 ± 5.4 μM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 ± 27.7 μM puerarin-7-O-glucoside and 187.8 ± 29.5 μM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 ± 24.0 μM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product’s composition.     

The effect of substitution of the <Emphasis Type="Italic">N</Emphasis>-acetyl groups of <Emphasis Type="Italic">N</Emphasis>-acetylgalactosamine residues in chondroitin sulfate on its degradation by chondroitinase ABC

Chondroitinase ABC is a lyase that degrades chondroitin sulfate, dermatan sulfate and hyaluronic acid into disaccharides. The purpose of this study was to determine the ability of chondroitinase ABC to degrade chondroitin sulfate in which the N-acetyl groups are substituted with different acyl groups. The bovine tracheal chondroitin sulfate A (bCSA) was N-deacetylated by hydrazinolysis, and the free amino groups derivatized into N-formyl, N-propionyl, N-butyryl, N-hexanoyl or N-benzoyl amides. Treatment of the N-acyl or N-benzoyl derivatives of bCSA with chondroitinase ABC and analysis of the products showed that the N-formyl, N-hexanoyl and N-benzoyl derivatives are completely resistant to the enzyme. In contrast, the N-propionyl or N-butyryl derivatives were degraded into disaccharides with slower kinetics compared to that of unmodified bCSA. The rate of degradation of bCSA derivatives by the enzyme was found to be in the order of N-acetyl>N-propionyl>>N-butyryl bCSA. These results have important implications for understanding the interaction of N-acetyl groups of glycosaminoglycans with chondroitinase ABC.     

Halophilic Properties of Metal Binding Protein Characterized by High Histidine Content from Chromohalobacter salexigens DSM3043

Periplasmic metal binding protein characterized by high histidine content was cloned from moderate halophile, Chromohalobacter salexigens. The protein, termed histidine-rich metal binding protein (HP), was expressed in and purified from E. coli as a native form. HP bound to Ni- and Cu-loaded chelate columns with high affinity, and Co- and Zn-columns with moderate affinity. Although the secondary structure was not grossly altered by the addition of 0.2–2.0 M NaCl, the thermal transition pattern was considerably shifted to higher temperature with increasing salt concentration: melting temperature was raised by ~20 °C at 2.0 M NaCl over the melting temperature at 0.2 M NaCl. HP showed reversible refolding from thermal melting in 0.2–1.15 M NaCl, while it formed irreversible aggregates upon thermal melting at 2 M NaCl. Addition of 0.01–0.1 mM NiSO₄ stabilized HP against thermal melting with high reversibility, while addition above 0.5 mM resulted in irreversible melting due to aggregation.     

Sequence analysis ofp-hydroxyphenyl-O-β-d-xyloside initiated and radio-iodinated dermatan sulfate from skin fibroblasts

To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated withp-hydroxyphenyl--d-xylopyranoside and [³H]galactose, and free [³H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After¹²⁵I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on theN-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-Å, Havsmark B, Silverberg I (1990)Biochem J 269:381–8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.Abbreviations GAG glycosaminoglycans - CS chondroitin sulfate - DS dermatan sulfate - Ser serine - Xyl d-xylose - Gal d-galactose - GlcA d-glucuronic acid - IdoA l-iduronic acid - GalNAc N-acetyl-d-galactosamine - GlcNAc N-acetyl-d-glucosamine - HexA 4-deoxy-l-threo-hex-4-enopyranosyluronic acid - HO-Phe p-hydroxyphenyl group - HO-Phe-Xyl p-hydroxyphenyl-O--d-xylopyranoside - O₂N-Phe-Xyl p-nitrophenyl--d-xylopyranoside - OSO₃ ester sulfate - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - FPLC fast performance liquid chromatography - LC standard liquid chromatography     

Determination and structural characterisation of dermatan sulfate in the presence of other galactosaminoglycans

Chondroitin sulfate and dermatan sulfate are galactosaminoglycans that have similar size and charge density thus making difficult their separation and accurate determination from tissue preparations. A procedure was developed, which was based on the specific action of chondroitinase B, that allowed the determination of dermatan sulfate content in a mixture of chondroitin sulfate/dermatan sulfate, its molecular mass (M_r), and iduronic acid content and distribution throughout the chain. According to this procedure, the galactosaminoglycan sample was treated with chondroitinase B and its profile, upon gel chromatography on Sepharose CL-6B, was compared to that of the initial sample. The differences in uronic acid content of the fractions of the gel chromatographies were plotted and a secondary profile was constructed, which corresponded to the elution profile of intact dermatan sulfate in the sample. From this profile, the size distribution of dermatan sulfate was obtained and its M_r was calculated. In addition, the accurate content of dermatan sulfate in the sample was determined. The digest contained oligosaccharides of variable size that were separated on BioGel P-10. From the separated oligosaccharides the distribution of iduronic acid throughout the dermatan sulfate chains was determined. The procedure was applied to the determination and partial characterisation of dermatan sulfate from sheep nasal cartilage, in which it is reported for the first time that it contains a significant proportion of dermatan sulfate chains of low iduronic acid content.     

Isolation and partial characterization of proteoglycans from sheep lung parenchyma.

Greater than 90% of the proteoglycans of sheep lung parenchyma, as measured by uronic acid, were solubilized employing a sequential procedure with guanidine hydrochloride, dithiothreitol and Triton X-100. The amounts solubilized were 68.7%, 16.2% and 5.9%, respectively. The guanidine hydrochloride extract was chromatographed using DEAE-cellulose in urea and eluted with increasing concentrations of NaCl. A major fraction (containing a 6.5-fold enrichment of uronic acid) was obtained with 0.5 M NaCl and further purified by Sepharose Cl-6B chromatography in guanidine hydrochloride. To demonstrate the presence of protein-linked glycosaminoglycans, the void volume peak containing protein and uronic acid was digested with papain and rechromatographed. Evidence for the presence of proteoglycans was obtained by observing an almost complete loss of uronic acid in the void volume and the appearance of a uronic acid peak in the included volume, migrating in the same area as single-chain glycosaminoglycans. Electrophoretic migration and disappearance of bands in electrophoresis after digestion with specific mucopolysaccharide lyases indicated that the small amount of uronic acid remaining in the void volume was hyaluronic acid whereas the included volume contained hyaluronic acid, heparan sulfate, chondroitin sulfates and/or dermatan sulfate.     

Anatomical changes induced by increasing NaCl salinity in three fodder shrubs, <Emphasis Type="Italic">Nitraria retusa</Emphasis>, <Emphasis Type="Italic">Atriplex halimus</Emphasis> and <Emphasis Type="Italic">Medicago arborea</Emphasis>

Nitraria retusa and Atriplex halimus (xero-halophytes) plants were grown in the range 0–800 mM NaCl while Medicago arborea (glycophyte) in 0–300 mM NaCl. Plants were harvested after 120 days of salt-treatment. The present study was designed to study the effect of salinity on root, stem and leaf anatomy, water relationship, and plant growth in greenhouse conditions. Salinity induced anatomical changes in the roots, stems and leaves. The cuticle and epidermis of N. retusa and A. halimus stems were unaffected by salinity. However, root anatomical parameters (root cross section area, cortex thickness and stele to root area ratio), and stem anatomical parameters (stem cross section area and cortex area) were promoted at 100–200 mM NaCl. Indicating that low to moderate salinity had a stimulating effect on root and stem growth of these xero-halophytic species. At higher salinities, root and stem structures were altered significantly, and their percentages of reduction were higher in A. halimus than in N. retusa whereas, in M. arborea, they were strongly altered as salinity rose. NaCl (100–300 mM) reduced leaf water content by 21.2–56.2% and specific leaf area by 51–88.1%, while increased leaf anatomical parameters in M. arborea (e.g. increased thickness of upper and lower epidermis, palisade and spongy mesophyll, entire lamina, and increased palisade to spongy mesophyll ratio). Similar results were evidenced in A. halimus leaves with salinity exceeding 100 mM NaCl. Leaves of N. retusa were thinner in salt-stressed plants while epidermis thickness and water content was unaffected by salinity. The size of xylem vessel was unchanged under salinity in the leaf’s main vein of the three species while we have increased number in M. arborea leaf main vein in the range of 200–300 mM NaCl. A longer distance between leaf vascular bundle, a reduced size and increased number of xylem vessel especially in stem than in root vascular system was evidenced in M. arborea treated plants and only at (400–800 mM) in the xero-halophytic species. The effects of NaCl toxicity on leaf, stem and root ultrastructure are discussed in relation to the degree of salt resistance of these three species. Our results suggest that both N. retusa and A. halimus show high tolerance to salinity while M. arborea was considered as a salt tolerant species.     

Structure and Antiviral Activity of the Galactofucan Sulfates Extracted from Undaria Pinnatifida (Phaeophyta)

The galactofucan sulfate extract (GFS) obtained from the brown seaweed Undaria pinnatifida by extraction with dilute acid is a potent inhibitor of the herpes viruses HSV-1, HSV-2 and HCMV, with IC₅₀ values determined in vitro of 1.1, 0.2 and 0.5 μgmL⁻¹, respectively. Fractionation of GFS by anion exchange chromatography gave three fractions which differed in their uronic acid and sulfate contents and in their antiviral activity, as well as in having somewhat reduced molecular weights compared to GFS. The low uronic acid/high sulfate fraction (F2M), obtained in 63% yield, had similar molar proportions of galactopyranosyl and fucopyranosyl residues, little associated protein and was equipotent with GFS (IC₅₀ values of 1.1, 0.1 and 0.5 μgmL⁻¹, respectively). The high uronic acid/low sulfate fraction (F1M), obtained in 18% yield, had a much lower proportion of galactopyranosyl residues and was less active (IC₅₀ values of 4.6, 1.0 and 4.0 μgmL⁻¹, respectively). The minor low uronic acid/high sulfate fraction (F4M) had a significant amount of associated protein and was also less active (IC₅₀ = 3.1, 1.0 and 2.0 μgmL⁻¹, respectively). The structure of the major fraction (F2M) was shown to be complex by glycosyl linkage analysis before and after solvolytic desulfation, with many component sugar residues being identified, although 3-linked fucopyranosyl 2,4-disulfate residues were a prominent feature.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

Occurrence of a dermatan sulfate isomer in sea urchin larvae

The dermatan sulfate isomer was isolated from the larvae of a sea urchin, Pseudocentrotus depressus. Its repeating unit was characterized as 2-acetamide-2-deoxy-3-O-(β-D-idopyranosyluronic acid)-4,6-O-disulfo-galactose. This report is the first one to show the occurrence of a dermatan sulfate in invertebrates.     

Chemoenzymatic preparation of dermatan sulfate oligosaccharides as arylsulfatase B and α-L-iduronidase substrates

Dermatan sulfate was partially depolymerized with chondroitin ABC lyase to obtain an oligosaccharide mixture from which an unsaturated disulfated tetrasaccharide was purified and characterized using nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Chemical removal of the unsaturated uronate residue with mercuric acetate, followed by de-4-O-sulfation with arylsulfatase B (N-acetylgalactosamine 4-sulfatase) and N- acetylhexo-saminidase catalyzed removal of the 2-acetamido-2-deoxy-D-galactospyranosyl residue at the non-reducing end afforded a monosulfated disaccharide of the structure -L-idopyranosyluronic acid (13)-,-D-2-acetamido-2-deoxy-4-O-sulfo galactopyranose. This monosulfated disaccharide serves as a substrate for mammalian -L-iduronidase as demonstrated using fluorophore assisted carbohydrate electrophoresis.     

Molecular Mechanism of Substrate Specificity for Heparan Sulfate 2-O-Sulfotransferase

Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.     

Effects of primer-concentration on uronosyl-epimerization and sulfation patterns in p-hydroxyphenyl-O-β-D-xylopyranoside-primed galactosaminoglycans produced by skin fibroblasts

By supplying skin fibroblasts with different concentrations of the galactosaminoglycan chain-primer p-hydroxyphenyl-O-β-D-xylopyranoside we have produced and recovered glycan-chains that were subsequently radio-iodinated in the hydroxyphenyl group and subjected to sequence analysis by using graded enzymic treatment followed by a combination of gel chromatography and electrophoresis. Fragments extending from the tagged reducing end to the cleavage-point were identified and quantified. Degradation by chondroitin B lyase of chains primed at 0.1 or 0.5 mM xyloside gave profiles indicating a periodic and wave-like distribution of iduronate-containing repeats, with high incidence around positions 2, 5 and onwards, whereas in chains produced at 1.0 mM xyloside the incidence of iduronate was similar in positions 1–4 and then declined. Degradation by chondroitin AC lyase indicated a high incidence of glucuronate in or near the linkage-region. There was a relatively uniform degree of sulfation in chains primed at low xyloside concentration, whereas chains primed at 1.0 mM xyloside gave very heterogeneous charge-patterns in all segments of the chain, including the linkage-region, giving the impression that adequate sulfation, probably at C-4 and at the first opportunity, is necessary to obtain an ordered and periodic epimerization pattern. Abbreviations:CS, chondroitin sulfate; DS, dermatan sulfate; GAG, glycosaminoglycan; Gal, D-galactose, GaINAc, N-acetyl-D-galactosamine; GlcUA, D-glucuronic acid; GlyUA, glycuronic acid; ΔGlyUA, 4,5-unsaturated glycuronic acid; IdoUA, L-iduronic acid; Xyl-Phe-OH, p-hydroxyphenyl-O-β-D-xylopyranoside; Xyl, D-xylose This revised version was published online in November 2006 with corrections to the Cover Date.