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PubMed1995

Identity of dermatan and chondroitin sequences in dermatan sulfate chains determined by using fragmentation with chondroitinases and ion-pair high-performance liquid chromatography.

The structure of a dermatan sulfate fraction from porcine skin has been analyzed by using various chondro/dermatolyases (chondoitinases ABC, AC, and B) and a highly sensitive HPLC method. Chondroitinase AC degrades the chondroitin sulfate portions of the chain and releases the dermatan sulfate fragments as delta-saccharides, whereas chondroitinase B releases the chondroitin sulfate portions as delta-saccharides by degrading the dermatan sulfate sections of the chain. The variously sized, sulfate delta-saccharides have been completely separated by ion-pair reversed-phase HPLC and the type of repeating disaccharide units in each fragment was determined by digestion with chondroitinase ABC and anion-suppression HPLC or reversed-polarity HPCE. The combined use of the various chondro/dermatolyases and the HPLC/HPCE analyses has enabled us to elucidate the disaccharide sequence of portions of dermatan sulfate and quantitate the linkage region binding the galactosaminoglycans to the protein backbone. The findings suggest that the dermatan sulfate chain can be defined as a copolymer that contains short chondroitin sulfate and long dermatan sulfate regions which are distributed periodically and nonrandomly. Two glucuronic acid-containing repeats were identified--one repeat, located next to the -Gal-Gal-Xyl linkage trisaccharide, contains three glucuronic acid residues, and the other repeat contains two monosulfated disaccharides. A third non-random sequence consists of two chondroitin disaccharides separated by one non-sulfated dermatan-type disaccharide. Each of these repeats is followed by iduronic acid clusters composed of sequences with more than four sulfated disaccharides. Nonsulfated iduronic acid-containing disaccharides were identified in the nonreducing terminal in a cluster of three disaccharides, followed by a sulfated glucuronic acid-containing disaccharide. By the proposed method, the linkage region that contains fragments produced by each one of the three chondroitinases can be easily determined and the presence of phosphorylated xylose in the linkage trisaccharide is also readily identified. The dermatan sulfate fraction from pig skin that was studied contains no phosphorylated xylose, but a significant proportion is present in rat chondrosarcoma proteoglycans.

Animals↗
PubMed1995

Qualitative and quantitative studies of heparin and chondroitin sulfates in normal human plasma.

Heparin was extracted and purified from normal human plasma, and full characterization of its structure and physico-chemical properties was achieved for the first time. Plasma was submitted to exhaustive proteolytic treatment with papain, trypsin, chymotrypsin, collagenase and pepsin, anion-exchange chromatography and precipitation with organic solvents. By this procedure, we recovered heparin (about 0.7 mg/100 ml of plasma) and chondroitin sulfate (about 0.1 mg/100 ml of plasma). Chondroitin sulfate has a peak molecular mass of about 15,630, and it is composed of about 60% nonsulfated disaccharide, 3.5% disaccharide 6-monosulfate and about 40% disaccharide 4-monosulfate, with a sulfate-to-carboxyl ratio of 0.41. Heparin, identified by agarose-gel electrophoresis, is constituted by about 40% slow-moving component and about 60% fast-moving species. This glycosaminoglycan had a peak molecular mass of about 7000, and was identified as 'typical' heparin by its constituent disaccharide composition. About 70% of disaccharides were identified as trisulfated disaccharide, and about 18% as disulfated disaccharides, 3% as monosulfated disaccharides and 10% as nonsulfated disaccharide. Heparin extracted from normal human plasma has a high sulfate-to-carboxyl ratio (2.47) and in vitro anticoagulant activity of about 70 I.U. A more quantitative and statistical analysis performed on 10 ml of plasma obtained from 10 human healthy volunteers revealed a heparin level of 0.54 +/- 0.17 mg/100 ml plasma (mean +/- standard deviation) with a coefficient of variation of about +/- 32%. These findings demonstrate for the first time the presence of heparin molecules in normal human plasma and confirm the importance of adequate extraction processes to purify a molecule that strongly interacts with plasma protein components. This is discussed in light of other authors that described a polysaccharide molecule named heparan sulfate in human plasma.

Carbohydrate Sequence↗
PubMed1999

Structural requirements of carbohydrates to bind Agaricus bisporus lectin.

Galbeta1-3GalNAc (T-disaccharide) and related molecules were assayed to describe the structural requirements of carbohydrates to bind Agaricus bisporus lectin (ABL). Results provide insight into the most relevant regions of T-disaccharide involved in the binding of ABL. It was found that monosaccharides bind ABL weakly indicating a more extended carbohydrate-binding site as compared to those involvedin the T-disaccharide specific lectins such as jacalin and peanut agglutinin. Lacto-N-biose (Galbeta1-3GlcNAc) unlike T-disaccharide, is unable to inhibit the ABL interaction, thus showing the great importance of the position of the axial C-4 hydroxyl group of GalNAc in T-disaccharide. This finding could explain the inhibitory ability of Galbeta1-6GlcNAc and lactose because C-4 and C-3 hydroxyl groups of reducing Glc, respectively, occupy a similar position as reported by conformational analysis. From the comparison of different glycolipids bearing terminal T-disaccharide bound to different linkages, it can be seen than ABL binding is even more impaired by an adjacent C-6 residual position than by the anomeric influence of T-disaccharide. Furthermore, the addition of beta-GlcNAc to the terminal T-disaccharide in C-3 position of Gal does not affect the ABL binding whereas if an anionic group such as glucuronic acid is added to C-3, the binding is partially affected. These findings demonstrate that ABL holds a particular binding nature different from that of other T-disaccharide specific lectins.

Binding Sites↗
PubMed1979

Age-dependent constitution of chondroitin sulfate isomers in cartilage proteoglycans under associative conditions.

Associated proteoglycans were prepared with guanidine-HCl from bovine articular cartilage of various ages. They were purified and fractionated by equilibrium centrifugation in cesium chloride (CsCl) density gradients. The compositions of chondroitin sulfate (CS) isomers in associated proteoglycans of articular cartilages of three ages were compared based on the relative amounts of disaccharide units. The results indicated that the proportions of 4-sulfated disaccharide units comprised around 2/3, 1/3, and 1/6 of the total CS in the associated proteoglycans of calf, 18-month-old cow, and 8-year-old cow, respectively. In contrast, the proportions of 6-sulfated disaccharide units in the proteoglycans were in the reverse order; they comprised nearly 1/3, 1/2, and 2/3 of the total CS, respectively, at the three ages. Thus, with increasing age, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units decreased significantly. With the decrease of CsCl density in the gradients, the proportion of 4-sulfated disaccharide units to total CS as well as that of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased in the associated proteoglycans of all ages. The increased ratios of 4-sulfated to 6-sulfated disaccharide units with decreasing CsCl density were significant among the individual proteoglycans: 1.84-2.36 in calf, 0.40-0.89 in 18-month-old cow, and 0.16-0.28 in 8-year-old cow.

Aging↗
PubMed1988

Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade.

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (beta 1-6)-D-Glucosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (beta 1-6)-D-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity. Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.

Animals↗
PubMed1990

Synthesis of chondroitin sulfate D and heparin proteoglycans in murine lymph node-derived mast cells. The dependence on fibroblasts.

Proteoglycans synthesized in cultured mast cells derived from horse serum-immunized lymph node cells were analyzed. Treatment of the 35S-proteoglycans extracted from these cells with either chondroitinase ABC or AC resulted in 95% +/- 7% and 84% +/- 7%, respectively (mean +/- S.E., n = 3), of the radioactivity associated with disaccharides eluting in the included volume of PD-10. The 35S-proteoglycans were not hydrolyzed by nitrous acid elimination treatment. The chondroitinase ABC-generated disaccharides were analyzed by aminocyano high performance liquid chromatography. 35S-Disaccharides eluted in a major peak at a retention time of 8.1 min, corresponding to the disaccharide of chondroitin 4-sulfate proteoglycan (delta Di-4S), and a second peak at 12 min, corresponding to the disaccharide of chondroitin sulfate D proteoglycan (delta Di-diSD). Further treatment with chondro-4-sulfatase did not affect the retention time of the disaccharide corresponding to delta Di-diSD whereas this peak disappeared after the digested proteoglycan was treated either by chondro-6-sulfatase or by both sulfatases. Therefore, this disaccharide was identified as chondroitin sulfate D. Quantification of the radiolabeled disaccharides showed that delta Di-diSD contributed 20% +/- 2% (n = 3) of the total sulfated disaccharides of the chondroitin sulfate of these cultured cells. The role of fibroblasts in inducing the shift of chondroitin sulfate D into heparin proteoglycan in these mast cells was also investigated by using three types of monolayers: mouse embryonic skin fibroblasts (MESF), rat embryonic skin fibroblasts (RESF), and 3T3 fibroblasts. 35S-Proteoglycans that were extracted from the lymph node-derived mast cells cultured for 30 days on MESF and on 3T3 fibroblast monolayers were 93% +/- 4% and 30% +/- 7% (n = 3) susceptible to nitrous acid elimination, respectively. No degradation by nitrous acid was observed in 35S-proteoglycans extracted from cells cultured on RESF monolayer. Since the MESF was found to be the most potent monolayer in the induction of heparin synthesis, the kinetics of changes in the synthesis of proteoglycan types were determined in lymph node-derived mast cells cultured on MESF for up to 30 days. It was found that the synthesis of chondroitin sulfate gradually declined whereas that of heparin starting between 4 and 7 days after plating gradually increased. From the 17th day on, only the synthesis of heparin was detected.

Animals↗
PubMed1990

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.

Carbohydrate Sequence↗
PubMed1981

Selective depolymerisation of heparin to produce radio-labelled substrates for sulfamidase, 2-acetamido-2-deoxy-alpha-D-glucosidase, acetyl-CoA:2-amino-2-deoxy-alpha-D-glucoside N-acetyltransferase, and 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase.

Heparin was carboxyl-reduced with NaBT4, and degraded under conditions of acid hydrolysis that selectively cleaved the 2-0-sulfo-L-idopyranosidic linkages. The resulting, radiolabelled-disaccharides and -tetrasaccharides were isolated by gel chromatography, and then fractionated by ion-exchange chromatography, paper chromatography, and paper electrophoresis. Of the nine disaccharides isolated and identified, eight were probably derived from the major repeating-disaccharide unit in heparin (2-deoxy-2-sulfoamino-D-glucosyl 6-sulfate leads to L-idosyluronic acid 2-sulfate). Sodium borotritide reduction and/or HNO2 deamination of these eight disaccharide fractions indicated four to contain L-idopyranose residues and the other four to contain 1,6-anhydro-L-idopyranose residues as terminal units. The latter, terminal unit probably represents a minor component formed during the acid hydrolysis. On the basis of N-acetylation, N-sulfation, and HNO2-deamination studies, and the known positions and configurations of the glycosidic and sulfate linkages in heparin, four disaccharides were identified as 0-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-(1 leads to 4)-L-[6-3H]idopyranose, 0-(2-amino-2-deoxy-alpha-D-glycopyranosyl)-(1 leads to 4)-L-[6-3H]idopyranose 2-sulfate, and 0-(2-amino-2-deoxy-alpha-D-glucopyranosyl 6-sulfate]-(1 leads to 4)-L-[6-3H]idopyranose 2-sulfate. A similar set of four disaccharides contained 1,6-anhydro-L-[6-3H]idopyranose residues in place of the L-[6-3H]idopyranose residues. The other disaccharide was tentatively identified as 0-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1 leads to 4)-L-[6-3H]idopyranose, the isolation of which suggests the presence of an IdA(OSO-3)-GlcNAc-IdA(OSO-3) sequence in the heparin preparation, which accounts for at least 1% of its total sequence. The tetrasaccharides were fractionated, on the basis of their sulfate content, into at least five species by ion-exchange chromatography or by paper electrophoresis. These were fractionated further into species with and without carboxyl groups, and with L-idopyranose or 1,6-anhydro-L-idopyranose residues as terminal units. Tentative structures for some of these tetrasaccharides are proposed. Disaccharide and tetrasaccharide species were evaluated before and after N-acetylation or N-sulfation, as substrates for sulfamidase, acetyl-CoA: 2-amino-2-deoxy-alpha-D-glucoside N-acetyl-transferase, 2-acetamido-2-deoxy-alpha-D-glucosidase, or 2-acetamido-2-deoxy-D-glucose 6-sulfate sulfatase in human-skin fibroblasts.

Acetylglucosaminidase↗
PubMed1999

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.

Adolescent↗
PubMed1994

Fractionation of heparin, dermatan sulfate, and chondroitin sulfate by sequential precipitation: a method to purify a single glycosaminoglycan species from a mixture.

Purified heparin, dermatan sulfate, and chondroitin sulfate in mixtures were fractionated by sequential precipitation with increasing volumes of acetone and analyzed by agarose-gel electrophoresis and for M(r), charge density, constituent disaccharides, and anticoagulant activity (for heparin). Purified glycosaminoglycans are generally utilized for pharmaceutical purposes and show physicochemical properties of glycosaminoglycans used as drugs. Heparin is the first glycosaminoglycan to precipitate at low percentages of acetone. The relative amount of slow moving and fast moving components, the M(r) and charge density, and the disaccharide pattern of fractionated heparin depend on the percentage of solvent. The activated partial thromboplastin time activity of fractions composed of heparin decreases with the charge density and M(r). Dermatan sulfate is precipitated by acetone over a narrow range (0.6-0.7 vol, 37-41%), and one of these fractions is constituted by 100% of this polysaccharide. These species of dermatan sulfate have different percentages of constituent disaccharides compared to the native polysaccharide. Nonsulfated disaccharide and disaccharide-6-sulfate are enriched. The dermatan sulfate species precipitated by acetone are also enriched in disaccharide-4,6-disulfated. Chondroitin sulfate is the most soluble glycosaminoglycan in mixed acetone/water solvent. It begins to precipitate at 0.8 vol (44%) of acetone. Different species of chondroitin sulfate can be recovered by precipitation at different percentages of solvent, and they show a decrease in M(r) and charge density depending on the percentage of acetone. The chondroitin sulfate species fractionated are also enriched in disulfated disaccharides compared to native polysaccharide. A different distribution of the three disulfated disaccharides can be pointed out in the fractionated chondroitin sulfate. Sequential precipitation performed by carefully increasing acetone percentages can help obtain purified species of glycosaminoglycan with desired properties from a mixture and tissue extracts, and achieve savings in time.

Animals↗
PubMed2000

Length distribution of the peptidoglycan chains in the sacculus of Escherichia coli.

The stress-bearing fabric of bacteria is made of peptidoglycan. This crosslinked fabric is formed from disaccharide pentapeptide units that are transported through the cytoplasmic membrane and then polymerized in two directions: (i) to form oligoglycan chains; and (ii) to cross link these chains by tail-to-tail bonds from the muropeptides to the protruding peptides of other chains. The distribution of the glycan chain lengths is reminiscent of the "most probable distribution of polymer chemistry. Of course, the process is more complex than solely the random addition of units to growing chains. The complexity precludes mathematical analysis, but computer modeling of the Monte Carlo type is capable of including a range of possibilities. At each time point a specified number of disaccharides are singly added to the muramic acid residue ends of existing chains chosen at random. The transfer is in exchange for the cleavage of pyrophosphate bactoprenol that transported the disaccharide pentapeptide through the membrane. The progam then selects, again at random, which chain to cleave and between which two disaccharides of the chain the cleavage event is to occur. The cleavage generates an N -acetyl 1,6 anhydro-muramic acid end and a non-reducing N -acetyl glucosamine end. The simulation can be modified so that the program does not cleave off a disaccharide next to either end of the chain. Comparisons are shown with the experimental results of Obermann & H]oltje (1994. Microbiology140, 79-87.) They obtained their data by taking the results with normal growing cells and subtracting the similar data from minicells to estimate the chain length distribution in the cylinder part of the cell. In its most basic form the computer simulation has only one fitted parameter, K, which is the number of disaccharides added to the murein for every internal cleavage event. In this form the fitting to the experimental results is poor. One possible reason for this is that the tension on the chains, and therefore the probability of being cleaved by autolysins varies with orientation of the chain on the cylinder surface. It is well known that the tension in the cylindrical wall is twice as large in the circumferential direction as in the axial one, so one class would consist of those chains aligned longitudinally, subject to lower stress, and would have a higher energy of activation for autolysis than chains aligned circumferentially. A good fit is obtained on the assumption that there are only two classes of chains; one more likely to be cleaved than the other. The key point is that only two processes: adding of disaccharide pentapeptides at random to glycan chains and cleavage between the disaccharides at random, together with the assumption that the wall is less easily hydrolysed in the axial direction is sufficient to account for the experimental distribution.

Cell Wall Skeleton↗
PubMed1983

Selective depolymerisation of dermatan sulfate: production of radiolabelled substrates for alpha-L-iduronidase, sulfoiduronate sulfatase, and beta-D-glucuronidase.

Radiolabelled disaccharide substrates for alpha-L-iduronidase, beta-D-glucuronidase, and sulfoiduronate sulfatase have been prepared from dermatan sulfate by application in sequence of N-deacetylation, deaminative cleavage, and reduction with NaBT4. The yield of disaccharides was approximately 87% of the total oligosaccharide fraction. Five disaccharides were isolated and tentatively identified. The major disaccharide, O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA-anT4S), represented approximately 75% of the total disaccharide fraction. The other disaccharides were O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA2S-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (GlcA-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 6-sulfate (GlcA-anT6S), and O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol (IdoA-anT), which represented approximately 4.5, 11.2, 1.0, and 1.8%, respectively, of the total disaccharide fraction. When incubated with cultured skin-fibroblasts from normal controls, IdoA-anT4S was shown to be a sensitive substrate for alpha-L-iduronidase to produce 2,5-anhydro-D-talitol 4-sulfate (anT4S). Activity toward IdoA-anT4S was not observed with fibroblast homogenates from alpha-L-iduronidase-deficient patients (Mucopolysaccharidosis Type I). Similarly, normal-fibroblast homogenates degraded GlcA-anT6S to anT6S, and GlcA-anT4S to anT4S, at a rate 6 to 8 times greater than found for fibroblasts from beta-D-glucuronidase-deficient patients (Mucopolysaccharidosis Type VII). IdoA-anT4S was hydrolysed by alpha-L-iduronidase at a rate 365 times greater than that for IdoA-anT. Sulfation of the anhydro-D-[1-3H]talitol residues is an important structural determinant in the mechanism of action of alpha-L-iduronidase on disaccharide substrates. IdoA2S-anT4S was degraded to IdoA-anT4S and then to anT4S by normal-fibroblast homogenates, whereas fibroblasts from alpha-L-iduronidase-deficient and sulfoiduronate sulfatase-deficient (Mucopolysaccharidosis Type II) patients produced considerably decreased levels of anT4s and IdoA-anT4S (and anT4S), respectively.

Chondroitin↗
PubMedDate not supplied

Alterations of glycosaminoglycans during patellar tendon autograft healing after posterior cruciate ligament replacement. A biochemical study in a sheep model.

In each of 30 skeletally mature sheep, the posterior cruciate ligament was replaced in one knee by a free patellar tendon autograft using the central third of the ipsilateral patellar tendon. The healing autograft was compared with the contralateral posterior cruciate ligament and the patellar tendons and posterior cruciate ligaments of nonoperated animals. The content of glycosaminoglycans, chondroitin sulfate disaccharides, and dermatan sulfate disaccharides was assessed biochemically at six periods during the 2 years after surgery. The total glycosaminoglycans and chondroitin sulfate disaccharides in the native posterior cruciate ligament was threefold that in the native patellar tendon. In contrast, the amount of dermatan sulfate disaccharides was similar in both the native tendon and native ligament. In the autograft, glycosaminoglycans and chondroitin sulfate disaccharides increased significantly to about 144% and 172%, respectively, of the contralateral posterior cruciate ligament at Week 104. The dermatan sulfate disaccharides in the autograft also showed a significant increase up to Week 26, followed by a remarkable but not significant decrease until the end of the study. In the contralateral posterior cruciate ligament, the dermatan sulfate disaccharides increased significantly between Weeks 52 and 104. Thus, the amount of dermatan sulfate disaccharides was similar in both the autograft and the contralateral posterior cruciate ligament after 2 years. This study suggests that the patellar tendon autograft did not completely assume the biochemical properties of the posterior cruciate ligament.

Animals↗
PubMed2003

Anomalous structure of urinary glycosaminoglycans in patients with pseudoxanthoma elasticum.

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a hereditary connective tissue disease in which proteoglycans have altered properties. We investigated whether altered proteoglycan metabolism occurs in vivo and may be reflected in the urine of PXE individuals by analyzing the excreted polysaccharides. METHODS: We measured sulfated glycosaminoglycans in the urine of 10 PXE-affected patients, 12 healthy carriers, and 20 healthy controls by agarose gel electrophoresis. Chondroitin sulfate and heparan sulfate disaccharides were also quantified by treatment with specific lyases and separation of products by chromatography. RESULTS: Total polysaccharides were 34% lower in the urine of PXE-affected patients and 17% lower in healthy carriers than in the control group. Chondroitin sulfate was significantly (P <0.01) decreased, and heparan sulfate was significantly increased. The ratio of chondroitin sulfate to heparan sulfate was 2.7 for PXE-affected patients, 2.3 for healthy carriers, and 10.7 for controls. In PXE-affected individuals and carriers, chondroitin sulfate contained more 4-sulfated disaccharide, less 6-sulfated disaccharide, and decreased nonsulfated disaccharide. Heparan sulfate from PXE-affected individuals and healthy carriers produced significantly less N-sulfated disaccharide and more disaccharide sulfated at the C-6 position with no significant abnormality of the nonsulfated disaccharide percentage and sulfates:disaccharide ratio. CONCLUSIONS: The urinary data support the concept that the inherited defect of the ABCC6/MRP6 transporter in PXE alters metabolism of key polysaccharides. Structural analysis of urinary sulfated polyanions may be useful in the diagnosis of PXE.

Chondroitin Sulfates↗
PubMed1985

Far-ultraviolet circular dichroism and uronic acid components of anticoagulant deca-, dodeca-, tetradeca-, and octadecasaccharide heparin fractions.

The therapeutic anticoagulant action of heparin is mediated by the ability of a multifunctional octadecasaccharide region of the molecule to bind to and differentially alter the conformational integrity of antithrombin, and the sugar sequence of the primary binding domain is known. Low ultraviolet circular dichroism spectroscopy of heparin-derived anticoagulant octa-, deca-, dodeca-, tetradeca-, and octadecasaccharides has been useful in elucidating the nature of the sugars that are contained within the second functional domain of the octadecasaccharide. The difference between the spectra of the molar ellipticity of the above sequential oligosaccharides was taken to be the CD spectrum of the corresponding additional disaccharide unit(s). Optical models of the component disaccharides of heparin were derived from CD spectra of heparins having a high degree of sulfation, synthetic glycosides of N-acetylglucosamine, and glycosides of component uronic acids. These were sufficiently distinct in magnitude and spectral position to warrant interpretation of the experimental difference CD spectra. The uronic acids of the disaccharides between deca- and octamer, dodeca- and decamer, and tetradeca- and dodecamer were thereby ascribed to sulfated iduronate, unsulfated iduronate, and glucuronate residues, respectively, while those of the tetrasaccharide between the octadeca- and tetradecasaccharide were tentatively assigned to sulfated iduronate moieties. Interpretation of the difference CD spectra on the basis of the optical models was less certain in regard to the amino sugar components. It appears that the amino sugar derivative between the dodeca- and decamer was N-acetylglucosamine, while the other disaccharides of the octa- to octadecasaccharide probably contained the N-sulfated derivative. A speculative disaccharide sequence drawn from these data indicates that relatively less strongly anionic disaccharides, having nonsulfated uronic acid moieties and N-acetylglucosamine, were flanked by trisulfated disaccharide units, constituting a structural element similar to that which contains the primary binding domain of the anticoagulant.

Acetylglucosamine↗
PubMed1995

Nonreducing end structures of chondroitin sulfate chains on aggrecan isolated from Swarm rat chondrosarcoma cultures.

Chondrocyte cultures derived from the Swarm rat chondrosarcoma were metabolically labeled with [35S]sulfate or [6-3H]GlcN. Radiolabeled aggrecan was purified from the cell layer and exhaustively digested with chondroitin ABC lyase. Digestion products were resolved into disaccharide and monosaccharide residues using Toyopearl HW40S chromatography. The separated saccharide pools were reduced with NaBH4 and applied onto a CarboPac PA1 column to resolve all of the internal disaccharide alditols (unsaturated) from the nonreducing end disaccharide (saturated) and monosaccharide alditols. Mercuric acetate treatment was used prior to carbohydrate analysis to identify unambiguously the saturated from the unsaturated disaccharides. The chondroitin sulfate (CS) chains from these aggrecan preparations contained: (a) an internal disaccharide composition of unsulfated (3-4 per chain), 4-sulfated (approximately 32 per chain), 6-sulfated (approximately 1 per 14 chains), and 4,6-sulfated disaccharides (approximately 1 per 6 chains) and (b) a nonreducing terminal composition of 4-sulfated GalNAc (approximately 4 out of every 7 chains), 4,6-disulfated GalNAc (approximately 2 out of every 7 chains), and GlcUA adjacent to a 4-sulfated GalNAc residue (approximately 1 out of every 7 chains). Thus, the vast majority of these CS chains terminated with a sulfated GalNAc residue. The presence of 4,6-disulfated GalNAc at nonreducing termini is 60-fold more abundant than 4,6-disulfated GalNAc in interior disaccharides. This observation is consistent with the suggestion that disulfation of terminal GalNAc residues is involved in chain termination.

Aggrecans↗
PubMed1988

Mechanism of expression of Thomsen-Friedenreich (T) antigen at the cell surface of a mammary adenocarcinoma.

The Thomsen-Friedenreich (T) antigen and its disaccharide component Gal beta 1,3GalNAc, which is recognized by the plant lectin peanut agglutinin (PNA), have been proposed as useful tumor markers because of their apparently specific occurrence in certain types of carcinomas. We have investigated the mechanism for the appearance of the disaccharide at the cell surface of ascites 13762 rat mammary adenocarcinoma cells using pulse-chase glucosamine labeling, proteolysis, and PNA precipitation of the cell-surface sialomucin ASGP-1. Glucosamine-labeled disaccharide appears at the cell surface in less than 10 min. Although the appearance of larger oligosaccharides continues to increase, the appearance of labeled disaccharide levels off within an hour. Analysis of intracellular vs. cell surface-labeled oligosaccharides showed that all disaccharide synthesized more than an hour before reaching the cell surface is converted to larger oligosaccharides. Thus, the presence of the disaccharide at the cell surface results from its synthesis late in the transit pathway of the sialomucin to the cell surface. We propose that the presence of T antigen at the surface of carcinoma cells results from an aberration of the pathway for O-linked glycosylation in these cells, probably caused by inappropriate localization of the enzymes involved in synthesis of the disaccharide.

Adenocarcinoma↗
PubMed1980

Constitutional variations of acidic glycosaminoglycans in normal and arthritic bovine articular cartilage proteoglycans at different ages.

Proteoglycans extracted from normal and arthritic bovine articular cartilage of various ages were fractionated and purified under associative and dissociative conditions. After proteolytic digestion, the composition of the acidic glycosaminoglycans (AGAG) in the proteoglycans was determined enzymatically by digestion with chondroitinase-AC II, chondroitinase-ABC, Streptomyces, hyaluronidase and keratanase. Under both associative and dissociative conditions, uniform distribution of chondroitin sulfate (CS) isomers from proteoglycans of different ages was observed: With increasing age, the relative proportion of 4-sulfated disaccharide units in total AGAG decreased, whereas that of 6-sulfated disaccharide units increased. The relative proportion of 4-sulfated disaccharide units in total CS and the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units were greater in arthritic cartilages than in normal cartilages of the same ages. At all three ages studied, the relative proportion of 4-sulfated disaccharide units in sequential fractions increased with the decrease of cesium chloride (CsC1) density, as the proportion of 6-sulfated disaccharide units decreased. The relative proportions of hyaluronic acid (HA) and keratan sulfate (KS) increased with age. The AGAG components of cartilage proteoglycans were distributed with a certain regularity in the fractions of CsCl density gradients, but underwent changes with increasing age and in arthritic process.

Aging↗