Structure of Chondroitin Sulfate
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Keywords:
Iduronic acid
Uronic acid
Dermatan sulfate
Tetrasaccharide
Chondroitin
Galactosamine
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.
Dermatan sulfate
Chondroitin
Galactosamine
Disaccharide
Iduronic acid
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The presence of sulfated uronic acid residues in dermatan sulfate has been known for some time. In view of the copolymeric nature of pig skin dermatan sulfate, it was of interest to see whether the extra sulfate groups were attached to iduronic acid or glucuronic acid residues. Hyaluronidase‐degraded dermatan sulfate was fractionated on Dowex‐1 to yield fractions of varying sulfate content. It was observed that the degree of sulfation was invesely proportional to the glucuronic acid content. Oversulfated dermatan sulfate was subsequently treated with chondroitinase‐AC to yield a completely glucuronic acid‐free polymeric product still bearing all the extra sulfate groups. More direct evidence for the presence of sulfated induronic acid moieties was obtained by Smith degradation of oversulfated, chondroitinse‐AC treated dermatan sulfate. A fast‐moving component isolated by preparative electrophoresis had the molar ratio sulfate; hexosamine 1.8:1 and contained exclusively L‐iduronic acid.
Iduronic acid
Dermatan sulfate
Uronic acid
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Chondroitin/dermatan sulfate is a highly complex linear polysaccharide ubiquitously found in the extracellular matrix and at the cell surface. Several of its functions, such as binding to growth factors, are mediated by domains composed of alternating iduronic acid and 4-O-sulfated N-acetylgalactosamine residues, named 4-O-sulfated iduronic acid blocks. These domains are generated by the action of two DS-epimerases, which convert D-glucuronic acid into its epimer L-iduronic acid, in close connection with 4-O-sulfation. In this study, dermatan sulfate structure was evaluated after downregulating or increasing dermatan 4-O-sulfotransferase 1 (D4ST-1) expression. siRNA-mediated downregulation of D4ST-1 in primary human lung fibroblasts led to a drastic specific reduction of iduronic acid blocks. No change of epimerase activity was found, indicating that the influence of D4ST-1 on epimerization is not due to an altered expression level of the DS-epimerases. Analysis of the dermatan sulfate chains showed that D4ST-1 is essential for the biosynthesis of the disulfated structure iduronic acid-2-O-sulfate-N-acetylgalactosamine-4-O-sulfate, thus confirmed to be strictly connected with the iduronic acid blocks. Also the biologically important residue hexuronic acid-N-acetylgalactosamine-4,6-O-disulfate considerably decreased after D4ST-1 downregulation. In conclusion, D4ST-1 is a key enzyme and is indispensable in the formation of important functional domains in dermatan sulfate and cannot be compensated by other 4-O-sulfotransferases.
Iduronic acid
Dermatan sulfate
Sulfotransferase
Chondroitin
Epimer
Uronic acid
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Iduronic acid
Galactosamine
Uronic acid
Hexosamines
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Abstract The products obtained after digestion of dermatan sulfate with testicular hyaluronidase have been investigated. Stepwise precipitation with ethanol (18, 25, 40, and 50%) and gel filtration of the material soluble in 50% ethanol yielded a number of polysaccharide and oligosaccharide fractions, ranging in molecular weight from 500 (disaccharide) to 16,000. Analysis of a tetrasaccharide fraction which contained both d-glucuronic and l-iduronic acid demonstrated that it was a mixture of chondroitin 4-sulfate tetrasaccharide and a hybrid tetrasaccharide of the following structure: [see PDF for equation] An analogous, unsulfated tetrasaccharide was found in an acid hydrolysate of dermatan sulfate. Besides its oligosaccharide components, the hyaluronidase digest also contained glycopeptides, presumably derived from the carbohydrate-protein linkage region.
Tetrasaccharide
Dermatan sulfate
Iduronic acid
Oligosaccharide
Ethanol precipitation
Chondroitin
Disaccharide
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A second dermatan sulfate epimerase (DS-epi2) was identified as a homolog of the first epimerase (DS-epi1), which was previously described by our group. DS-epi2 is 1,222 amino acids long and has an approximately 700-amino acid N-terminal epimerase domain that is highly conserved between the two enzymes. In addition, the C-terminal portion is predicted to be an O-sulfotransferase domain. In this study we found that DS-epi2 has epimerase activity, which involves conversion of d-glucuronic acid to l-iduronic acid (EC 5.1.3.19), but no O-sulfotransferase activity was detected. In dermatan sulfate, iduronic acid residues are either clustered together in blocks or alternating with glucuronic acid, forming hybrid structures. By using a short interfering RNA approach, we found that DS-epi2 and DS-epi1 are both involved in the biosynthesis of the iduronic acid blocks in fibroblasts and that DS-epi2 can also synthesize the hybrid structures. Both iduronic acid-containing domains have been shown to bind to several growth factors, many of which have biological roles in brain development. DS-epi2 has been genetically linked to bipolar disorder, which suggests that the dermatan sulfate domains generated by a defective enzyme may be involved in the etiology of the disease.
Iduronic acid
Dermatan sulfate
Sulfotransferase
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Dermatan sulfate
Iduronic acid
Uronic acid
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Dermatan sulfate epimerase 2 (DS-epi2), together with its homolog DS-epi1, transform glucuronic acid into iduronic acid in DS polysaccharide chains. Iduronic acid gives DS increased chain flexibility and promotes protein binding. DS-epi2 is ubiquitously expressed and is the predominant epimerase in the brain. Here, we report the generation and initial characterization of DS-epi2 null mice. DS-epi2-deficient mice showed no anatomical, histological or morphological abnormalities. The body weights and lengths of mutated and wild-type littermates were indistinguishable. They were fertile and had a normal lifespan. Chondroitin sulfate (CS)/DS isolated from the newborn mutated mouse brains had a 38% reduction in iduronic acid compared with wild-type littermates, and compositional analysis revealed a decrease in 4-O-sulfate and an increase in 6-O-sulfate containing structures. Despite the reduction in iduronic acid, the adult DS-epi2−/− brain showed normal extracellular matrix features by immunohistological stainings. We conclude that DS-epi1 compensates in vivo for the loss of DS-epi2. These results extend previous findings of the functional redundancy of brain extracellular matrix components.
Dermatan sulfate
Iduronic acid
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Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.
Iduronic acid
Dermatan sulfate
Biglycan
Chondroitin
Fibrillogenesis
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Dermatan sulfate
Uronic acid
Chondroitin
Iduronic acid
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