Undersulfated Chondroitin Sulfate Research Articles

Association between Human Plasma Chondroitin Sulfate Isomers and Carotid Atherosclerotic Plaques.

Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS). The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P < 0.01) in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.

Acidic Glycosaminoglycans in Marine Teleost Larvae: Evidence for a Relationship Between Composition and Negative Charge Density in Elopomorph Leptocephali

Glycosaminoglycan (GAG) composition was determined in larvae of 10 species of true eels (subdivision Elopomorpha; order Anguilliformes; families Chlopsidae, Congridae, Moringuidae, Muraenidae and Ophichthidae), two species from the subdivision Clupeomorpha (order Clupeiformes; families Engraulidae and Clupeidae) and one species from the subdivision Euteleostei (order Aulopiformes; family Synodontidae). Using a combination of specific GAG-degrading enzymes, cellulose acetate electrophoresis with known GAG standards and relative degree of staining with Alcian Blue, the predominant GAG in anguilliform leptocephali was identified as undersulfated chondroitin sulfate (uCS); the only exception was Bathycongrus macrurus (Congridae), where heparan sulfate was tentatively identified together with uCS. In representatives of the Clupeomorpha and Euteleostei, however, the predominant GAG was chondroitin sulfate; uCS was not detected. These results, combined with those from previous studies, provide data on GAG composition in 21 species from three of the four currently recognized elopomorph orders (Albuliformes, Elopiformes and Anguilliformes). The combined data suggest that although several different body matrix GAGs have evolved in different groups of leptocephali, selection has favored GAGs (mainly uCS and keratan sulfate) with relatively low average negative charge densities (average of about one negative charge per disaccharide repeat of the acidic GAG chain). The presence of normally sulfated CS (average per disaccharide negative charge density of 2) as the major GAG in the three species of non-elopomorph larvae further suggests that some aspect of the unique developmental pattern in elopomorph leptocephali may place constraints on the maximum negative charge density permitted in the body matrix GAGs. The presence of GAGs with low negative charge density is interpreted as an additional elopomorph synapomorphy, offering further support for the view that the Elopomorpha represents a monophyletic group of fishes. Possible scenarios for the evolution of GAGs in leptocephali are presented.

Characterization and distribution of undersulfated chondroitin sulfate and chondroitin in leptocephalous larvae of elopomorph fishes

Undersulfated chondroitin sulfate (i.e., chondroitin sulfate with sulfate: hexosamine molar ratios ranging from 0.06-0.17) has been identified as a principal glycosaminoglycan (GAG) in whole-body extracts of leptocephalous larvae of four species of marine teleost fishes, representing two orders and three families. These include the eels (Anguilliformes)Ariosoma balearicum (Congridae),Rhechias dubia (Congridae) and Ophichthus sp. (Ophichthidae) and the ladyfish (Elopiformes: Elopidae: Elops saurus). A second GAG found in relatively large amounts in A. balearicum has been identified as chondroitin. Chondroitin and undersulfated chondroitin sulfate formed distinctive precipitates during extraction and were easily separated. Undersulfated chondroitin sulfate also was extracted and purified from early metamorphosing bonefish (Albuliformes: Albulidae: Albula sp.) leptocephali, where it is a minor GAG component, and was shown to be similar in composition to the compounds obtained from eels and ladyfish.

Glycosaminoglycans of solid and ascites forms of the P1798 murine lymphosarcoma

Total glycosaminoglycan levels are similar from subcutaneous, ascites and mesenteric forms of the P1798 lymphosarcoma. However, P1798 cells implanted subcutaneously produce a solid tumor rich in hyaluronic acid with lesser amounts of chondroitin 4-/6-sulfates as well as an undersulfated species of chondroitin sulfate, while cells implanted intraperitoneally produce ascites tumors which generate hyaluronic acid, almost exclusively. Mice bearing advanced ascites tumors develop solid mesenteric tumors which exhibit chondroitin 4-/6-sulfates and under-sulfated chondroitin sulfate in addition to hyaluronic acid.

Biochemical studies on Lowe's syndrome.

Lowe's syndrome (oculo-cerebro-renal syndrome) has been studied biochemically. The disease was characterized by proteinuria, sialic aciduria and the excretion of undersulfated chondroitin sulfate A due mainly to malfunction of renal tubules. However, cultured skin fibroblasts from patients were found also to produce markedly undersulfated glycosaminoglycans. The undersulfation was caused by depressed sulfation rather than by increased desulfation. Subsequent studies have revealed that degradation of active sulfate (adenosine 3'-phosphate 5'-phosphosulfate, PAPS) was markedly elevated in the cells from patients whereas PAPS biosynthesis or sulfate transfer of sulfate from PAPS to glycosaminoglycan acceptors were normal. The enzyme involved in PAPS degradation was then identified as a nucleotide pyrophosphatase which is capable of degrading various nucleotides. The level of the enzyme activity in patients' cells was about ten times higher than that in normal cells and the level in heterozygotes were intermediate between patients and normal individuals. It was suggested that Lowe's syndrome is caused by elevation of biosynthesis of a nucleotide pyrophosphatase having a capacity to degrade PAPS due to a defect in regulating the enzyme synthesis.

Undersulfated proteoglycans are induced by the ionophore monensin: Study of possible mechanisms

We have reported that the monovalent ionophore monensin causes undersulfated chondroitin sulfate biosynthesis in cultured chondrocytes. In order to clarify the mechanism of this diminished sulfation, we have measured the rate of incorporation of sulfate into chondrocytes and assayed the cellular ATP levels. We have also measured sulfatase activity, the incorporation of 35SO 4 into 3′-phosphoadenosine 5′-phospho[ 35S]sulfate and endogenous sulfotransferase activity in the cell-free extracts. We find that: (1) The incorporation of 35SO 4 into the free sulfate pool in chondrocytes was not inhibited by monensin. (2) The ATP levels of monensin-treated chondrocytes were the same as control cells. (3) There was no sulfatase activity in both control and monensin-treated chondrocytes. (4) Enzymatic analyses revealed that 35SO 4 incorporation into 3′-phosphoadenosine 5′-phospho[ 35S]sulfate and subsequent sulfotransferase activity were not inhibited in the presence of monensin. At present the most tenable hypothesis to account for monensin causing undersulfated chondroitin sulfate synthesis is that the ionophore impairs the access of proteoglycans to the sulfotransferases in the luminal walls of the Golgi structures.

Spondyloepiphyseal dysplasia, chondroitin sulfate type: A possible defect of paps — Chondroitin sulfate sulfotransferase in humans

Four patients with an unusual form of spondyloepiphyseal dysplasia excreted in the urine undersulfated chondroitin 6-sulfate (Biochem. Med. 7 , 415–423, 1973). The sera of these patients show a low activity of PAPS — chondroitin sulfate sulfotransferase, while the undersulfated chondroitin sulfate present in their urine is a much better acceptor of 35SO 4 than standard chondroitin sulfate when they are incubated with [ 35S ]PAPS and normal sulfotransferases. These results suggest that in these patients the skeletal lesions are secondary to a defect in the synthesis of chondroitin sulfate involving specifically the sulfotransferase activity.

Glycosaminoglycan metabolism of cultured cornea cells derived from bovine and human stroma and from bovine epithelium

The glycosaminoglycan metabolism of cultured epithelial and stroma cells from bovine cornea and of human stroma cells was investigated in comparative study. The following results were obtained: 1. (1) All cell types investigated distributed newly synthesized glycosaminoglycans into extracellular, pericellular and intracellular compartments. Epithelial cells, however, produced lower amounts of sulfated glycosaminoglycans and secreted a smaller proportion of them into the culture medium. 2. (2) Epithelial cells exhibited a more rapid turnover of intracellular glycosaminoglycans than the stroma cell lines. All cell types internalized [ 35S]proteoglycans to a similar extent, but epithelial cells degraded them more rapidly. 3. (3) The three cell types studied synthesized hyaluronic acid, undersulfated chondroitin sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratin sulfate. A characteristic distribution pattern of sulfated glycosaminoglycans was observed with respect to the origin of the cells as well as with regard to topographically distinct compartments. Heparan sulfate was found to be the main constituent of pericellular glycosaminoglycans. Bovine stroma cells secreted predominantly chondroitin-4-sulfate, human stroma cells heparan sulfate and epithelial cells dermatan sulfate into the culture medium. Intracellular glycosaminoglycans of bovine stroma cells consisted mainly of chondroitin-6-sulfate, whereas in human cells dermatan sulfate and in epithelial cells dermatan sulfate and chondroitin-6-sulfate were present in high proportions. Human stroma cells produced more hyaluronic acid and undersulfated chondroitin-sulfate than the other cell types.

Urinary excretion of a large amount of bound sialic acid and of undersulfated chondroitin sulfate a by patients with the lowe syndrome

Urine samples from two patients with the Lowe syndrome were analyzed for sialic acid and mucopolysaccharides. The sialic acid content, relative to the creatinine content, was 4–5 times higher in these patients' urine than in normal urine. Most of the sialic acid was found in unidentified glycoproteins of high molecular weight, but the levels of sialyllactose and free sialic acid were also elevated about 2 fold. A most remarkable finding was the excretion of undersulfated chondroitin sulfate A without other mucopolysaccharides normally occurring in urine. It is suggested that a disorder in sulfation of mucopolysaccharides is etiologically implicated in the Lowe syndrome.

Heterogeneity of rat rib chondroitin sulfate and susceptibility to rat gastric chondrosulfatase

Cartilage chondroitin sulfate isolated directly from rat rib or from in vitro culture of rat rib constitutes a population of glycosaminoglycans which is heterogeneous with respect to size, degree of sulfation and content of N-acetylgalactosamine 4-sulfate. Fractions elute from Dowex-1 in order of increasing molecular size and degree of sulfation up to a certain limit. Unsulfated disaccharides and disulfated disaccharides are present in both the undersulfated chondroitin sulfate fractions and in the average or more representative chondroitin sulfate. A small content of disaccharide 6-sulfate is present in all fractions and appears to be an integral part of the chondroitin 4-sulfate molecules. Rat gastric chondrosulfatase hydrolyzes sulfate preferentially from the larger chondroitin 4-sulfate molecules, and the sulfate is removed primarily from the disaccharide 4-sulfate units.

Molecular weight-dependent distribution of acidic glycosaminoglycans in human plasma

Acidic glycosaminoglycans (AGAG) in human plasma were studied by enzymatic and electrophoretic procedures after fractionation by gel filtration on Sephadex G-100. Galactosaminoglycans were the predominant acidic glycosaminoglycans in the small molecular weight fraction, but glucosaminoglycans were predominant in the macromolecular weight fraction. The major constituents of plasma AGAG were estimated as undersulfated chondroitin sulfate isomers followed by a lesser amount of hyaluronic acid characterized by susceptibility to specific chondroitinases and streptomyces hyaluronidase. In addition, the presence of small amounts of heparan sulfate and oversulfated chondroitin sulfate in human plasma was indicated.

Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan.

Mouse 3T3 cells and their Simian Virus 40-transformed derivatives (3T3SV) were used to assess the relationship of transfromation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan (GAG). Glucosamine- and galactosamine-containing GAG were labeled equivalently by [3H=A1-glucose regardless of culture type, allowing incorporation into the various GAG to be compared under all conditions studied. Three components of each culture type were examined: the cells, which contain the bulk of newly synthesized GAG and are enriched in chondroitin sulfate and heparan sulfate; cell surface materials released by trypsin, which contain predominantly hyaluronic acid; and the media , which contain predominantly hyaluronic acid and undersulfated chondroitin sulfate. Increased cell density and viral transformation reduce incorporation into GAG relative to the incorporation into other polysaccharides. Transformation, however, does not substantially alter the type or distribution of newly synthesized GAG; the relative amounts and cellular distributions were very similar in 3T3 and 3T3SV cultures growing at similar rates at low densities. On the other hand, increased cell density as well as density-dependent growth inhibition modified the type and distribution of newly synthesized GAG. At high cell densities both cell types showed reduced incorporation into hyaluronate and an increase in cellular GAG due to enhanced labeling of chondroitin sulfate and heparan sulfate. These changes were more marked in confluent 3T3 cultures which also differed in showing substantially more GAG label in the medium and in chondroitin-6-sulfate and heparan sulfate at the cell surface. Since cell density and possibly density-dependent inhibition of growth but not viral transformation are major factors controlling the cellular distribution and type of newly synthesized GAG, differences due to GAG's in the culture behavior of normal and transformed cells may occur only at high cell density. The density-induced GAG alterations most likely involved are increased condroitin-6-sulfate and heparan sulfate and decreased hyaluronic acid at the cell surface.

Composition of acidic glycosaminoglycans in human cerebral arteries

1. (1) The composition of acidic glycosaminoglycans (AGAG) in the pooled human cerebral arteries was investigated by electrophoretic characterization before and after digestion with chondroitinases. Constitution of the chondroitin sulfate (CS) family was determined qualitatively on the basis of the disaccharide subunits of the CS chains after depolymerization with the enzymes. 2. (2) The data obtained indicated that the AGAG in cerebral arteries consisted of, in order of amount, heparan sulfates, chondroitin-6-sulfate, dermatan sulfate, chondroitin-4-sulfate and hyaluronic acid. 3. (3) The existence of oversulfated CS and undersulfated CS in the cerebral AGAG was supported by the detection of unsaturated di-sulfated and non-sulfated disaccharides on paper chromatography. In addition, the presence of hyaluronic acid was indicated by electrophoretic and enzymatic separation. 4. (4) The distribution of the individual AGAG in cerebral arteries was also examined on the basis of molecular weight by gel filtration on a Sephadex G-100 column.

On acid glycosaminoglycans (mucopolysaccharides) in pleural effusion.

The concentration of acid glycosaminoglycans (mucopolysaccharides) in pleural fluid was found to be in the range of 4.0 to 60.3 mug per ml. By chromatographic analysis and by electrophoresis of the eluates, in combination with enzymatic digestion, it was found that pleural fluid contained protein-free hyaluronic acid, protein-bound hyaluronic acid, undersulfated chondroitin sulfate A(C), chondroitin sulfate A(C), protein-bound chondroitin sulfate A(C), and dermatan sulfate. The composition of acid glycosaminoglycans in pleural fluid seems to reflect the metabolism of the pleural cavity.

Isolation and preliminary characterization of glycosaminoglycans in human plasma

Plasma glycosaminoglycans were isolated, after pronase digestion by precipitation with cetylpyridinium chloride. The glycosaminoglycans in plasma were found exclusively in the protein fraction precipitated by trichloroacetic acid. The concentration of glycosaminoglycans in normal plasma was 168 to 272 μg per 100 ml. Electrophoresis of plasma glycosaminoglycans in barium acetate buffer on cellulose acetate revealed three fractions, all resistant to streptomyces hyaluronidase but susceptible to testicular hyaluronidase. Electrophoretic mobility of the major fraction was similar to dermatan sulfate and hyaluronic acid. Enzymatic assay with chondroitinases, however, indicated that the major constituent of plasma glycosaminoglycans was undersulfated chondroitin sulfate composed of equimolar amounts of unsaturated non-sulfated disaccharides and unsaturated 4-sulfated disaccharides at the disaccharide subunit levels. The other two fractions were identical with chondroitin sulfate A and C. The quantity of chondroitin sulfate C was least. Hyaluronic acid and over-sulfated chondroitin sulfate were not demonstrated in plasma.

Glycosaminoglycan synthesis by the early embryonic chick heart

Glycosaminoglycans of embryonic chick hearts labeled in situ were characterized by means of labeled precursor incorporation, electrophoretic mobility, sensitivity to testicular hyaluronidase, elution characteristics from CPC-cellulose columns, and hexosamine content. During the initial period of overt cardiac muscle differentiation (approximately stage 10) chondroitin sulfates are not detectable but an undersulfated component is present. Chondroitin sulfate synthesis appears shortly after overt muscle differentiation. Hyaluronate is present both during and after overt myocardial differentiation. Although epimerization of 3H-glucosamine-derived labeled UDP- N-acetyl- d-glucosamine occurs (determined by recovery of incorporated labeled galactosamine), label does not appear in chondroitin sulfate. 3H-Glucosamine is thus a relatively specific precursor for unsulfated glycosaminoglycans, a fact that we exploited in demonstrating their distribution radioautographically. Glycosaminoglycan synthesis was also examined in hearts labeled (a) in isolated organ culture, (b) in situ but exposed directly to the medium by removal of the splanchnopleure. In both cases fully sulfated chondroitin sulfate and chondroitin are not synthesized. Hearts make only hyaluronate and undersulfated chondroitin sulfate.

Metabolic changes of urinary acidic glycosaminoglycans in Weber christian disease

1. (1) An augmented yield of acidic glycosaminoglycans, both digested with and resistant to chondroitinases, was observed in 24-hour urine of a patient with Weber Christian disease. 2. (2) The elevation of the excreted acidic glycosaminoglycans was reflected as an increase of acidic glycosaminoglycans eluted with lower concentrations of NaCl from Dowex 1-X2 columns. This finding correlated with the elevated proportion of the non-sulfated disaccharide subunit derived from undersulfated chondroitin sulfate isomers. 3. (3) Enzymatic assay of chondroitin sulfate isomers in the disease indicated that the disulfated disaccharide derived from oversulfated chondroitin sulfate was clearly found at the higher molecular weights by gel filtration on Sephadex G-100. The proportion of the 4-sulfated disaccharide to the 6-sulfated disaccharide was relatively greater in the disease in comparison to normal subjects. 4. (4) The analytical results from gel filtration indicated that depolymerization of urinary acidic glycosaminoglycans and glycopeptides occurs in the disease. The grade of the depolymerization of acidic glycosaminoglycans was associated with the degree of desulfation of chondroitin sulfates in terms of the disaccharide subunit level. 5. (5) Electrophoretic separation of the fractionated urinary acidic glycosaminoglycans on gel filtration revealed that heparan sulfates accounted for the major glucosaminoglycans with higher molecular weights, which were also eluted at lower NaCl concentrations from the Dowex column. On the other hand, chondroitin sulfates accounted for major galactosaminoglycans with lower molecular weights, which were eluted at the relatively higher NaCl concentrations on the Dowex column.

Enzymatic Analysis of the Disaccharide Subunits Derived from Urinary Chondroitin Sulfate Isomers in Mucopolysaccharidosis

Abstract The qualitative and quantitative analyses of urinary chondroitin sulfates in patients with Hurler's Hunter's and Maroteaux-Lamy's syndromes were carried out in comparison with those of a normal subject on the basis of disaccharide subunit levels after digestion of the chondroitin sulfates with chondroitinases and chondrosulfatases. The minor quantities of unsaturated 4-sulfated and 6-sulfated disaccharides were detected in the degraded production of urinary chondroitin sulfates in these patients after digestion with chondroitinase-AC. A striking increase of unsaturated 4-sulfated disaccharide derived from chondrotin sulfate B was shown after degradation with chondroitinase-ABC. The minorities of unsaturated non-sulfated and di-sulfated disaccharides were also detected as disaccharide chains of the isomers. The findings indicate that minor constituents of chondroitin sulfate A and C are present in addition to the majority of chondroitin sulfate B in the urinary chondroitin sulfate group in these syndromes. The presence of minor quantities of the disaccharide subunits of non- and/or undersulfated chondroitin sulfate and oversulfated chondroitin sulfate isomers in the syndromes.

Chondroitin sulfates in atherosclerotic human aorta

1. (1) Chondroitin sulfate isomers in the tunica intima of human aorta at different grades of atherosclerosis were studied by using enzymatic digestion methods with chondroitinases and chondrosulfatases. 2. (2) The total amounts of chondroitin sulfates tended to decrease with advanced atherosclerosis, but there was no obvious change in the distribution of chondroitin sulfate isomers in relation to atherosclerosis. 3. (3) Chondroitin or undersulfated chondroitin sulfate as well as oversulfated chondroitin sulfate were found in the disaccharide subunits as minor components of aortic chondroitin sulfates.

Chondroitin sulfate isomers in normal human urine

1. 1. Enzymic analysis of urinary chondroitin sulfate isomers in normal subjects was carried out on the basis of unsaturated disaccharide subunits produced by the digestion of urinary chondroitin sulfates with chondroitinases and chondrosulfatases. 2. 2. By paper chromatographic and electrophoretic separations, four unsaturated disaccharides were detected as the products of degradation of urinary chondroitin sulfate isomers with chondroitinases. 3. 3. Minor constituents of unsaturated nonsulfated disaccharide and unsaturated di-sulfated disaccharide were demonstrated in addition to the major components of unsaturated 4-sulfated and 6-sulfated disaccharides in normal human urine. This finding indicates that nosulfated and/or undersulfated chondroitin sulfate, as well as oversulfated chondroitin sulfate, are present in normal urine. The distribution of the unsaturated disaccharides fractioned by NaCl elution from Dowex columns reflected the diverse spectrum of urinary chondroitin sulfate isomers.