Inhibition of heparan sulfate and chondroitin sulfate proteoglycan biosynthesis

Proteoglycans (PGs) are composed of a protein moiety and a complex glycosaminoglycan (GAG) polysaccharide moiety. GAG chains are responsible for various biological activities. GAG chains are covalently attached to serine residues of the core protein. The first step in PG biosynthesis is xylosylation of certain serine residues of the core protein. A specific linker tetrasaccharide is then assembled and serves as an acceptor for elongation of GAG chains. If the production of endogenous GAG chains is selectively inhibited, one could determine the role of these endogenous molecules in physiological and developmental functions in a spatiotemporal manner. Biosynthesis of PGs is often blocked with the aid of nonspecific agents such as chlorate, a bleaching agent, and brefeldin A, a fungal metabolite, to elucidate the biological roles of GAG chains. Unfortunately, these agents are highly lethal to model organisms. Xylosides are known to prime GAG chains. Therefore, we hypothesized that modified xylose analogs may able to inhibit the biosynthesis of PGs. To test this, we synthesized a library of novel 4-deoxy-4-fluoroxylosides with various aglycones using click chemistry and examined each for its ability to inhibit heparan sulfate and chondroitin sulfate using Chinese hamster ovary cells as a model cellular system.     

Chondroitin sulfate/dermatan sulfate sulfatases from mammals and bacteria

Sulfatases that specifically catalyze the hydrolysis of the sulfate groups on chondroitin sulfate (CS)/dermatan sulfate (DS) poly- and oligosaccharides belong to the formylglycine-dependent family of sulfatases and have been widely found in various mammalian and bacterial organisms. However, only a few types of CS/DS sulfatase have been identified so far. Recently, several novel CS/DS sulfatases have been cloned and characterized. Advanced studies have provided significant insight into the biological function and mechanism of action of CS/DS sulfatases. Moreover, further studies will provide powerful tools for structural and functional studies of CS/DS as well as related applications. This article reviews the recent progress in CS/DS sulfatase research and is expected to initiate further research in this field.     

Isolation of the major chondroitin sulfate/dermatan sulfate and heparan sulfate proteoglycans from embryonic chicken retina

A technique is presented for the preparation of three major proteoglycans from 14-day embryonic chicken retinas following their culture overnight with [35S]sulfate and either [3H]glucosamine or [3H]serine. Homogenization of the tissue in saline permitted extraction of heterogeneous soluble proteoglycans separately from most of the heparan sulfate proteoglycans. The latter were extracted from the 140,000g pellet with 0.5% Triton X-100 in 8 M urea. The medium plus the saline and urea-detergent extracts were separated from low-molecular-weight contaminants, and fractionated into two peaks of radioactivity on Sephacryl S-300 in saline with 3 M urea and 0.5% Triton X-100. The proteoglycans were isolated directly from these fractions on DEAE-Sephacel, and subjected to ultrafiltration concentration and then further purification on cesium chloride density gradient centrifugation in 4 M guanidine hydrochloride. A further step involving cetylpyridinium chloride precipitation was examined, but it resulted in essentially no further purification. The fractionations separated a large chondroitin sulfate/dermatan sulfate proteoglycan from the culture medium that was excluded from S-300 and of low buoyant density; a large heparan sulfate proteoglycan from the urea-detergent extract that was also excluded from S-300 and of low buoyant density; and two smaller and possibly related heparan sulfate proteoglycans. One was found in the medium and showed low to intermediate buoyant density; the other was isolated from the urea-detergent extract and showed a significantly higher buoyant density, associated with a lower protein content. The saline extract contained both of the two larger proteoglycans and only minor amounts of the smaller molecules.     

Dextran sulfate and heparin sulfate inhibit platelet-activating factor-induced pulmonary edema.

We tested the hypothesis that dextran sulfate and heparin sulfate inhibit platelet-activating factor- (PAF) induced pulmonary edema in the isolated perfused guinea pig lung via a charge-dependent mechanism. Dextran sulfate prevented the changes in pulmonary capillary pressure (Ppc, 7.8 +/- 0.9 vs. 14.0 +/- 0.7 cmH2O), lung weight gain (dW, +0.48 +/- 0.29 vs. +8.41 +/- 2.07 g), and pulmonary edema formation or wet-to-dry weight ratio [(W-D)/D, 6.5 +/- 0.3 vs. 13.2 +/- 2.6] occurring 60 min after PAF infusion (10(-11) M) into an isolated lung. The unsulfated form of dextran had no protective effect [Ppc, dW, and (W-D)/D, 11.9 +/- 1.4 cmH2O, +5.33 +/- 2.18 g, and 11.2 +/- 3.2, respectively]. The unrelated anionic compound, heparin sulfate, also inhibited the PAF response [Ppc, dW, and (W-D)/D, 7.0 +/- 0.5 cmH2O, +0.61 +/- 0.32 g, and 6.1 +/- 0.2, respectively], whereas the partially desulfated form of heparin was not effective in inhibiting PAF-induced edema [Ppc, dW, and (W-D)/D, 15.1 +/- 0.7 cmH2O, +6.07 +/- 1.58 g, and 10.0 +/- 1.2, respectively]. When the metachromatic dye crystal violet was used as an indicator of charge interactions, the sulfated compounds interacted with PAF in vitro. The data indicate that PAF-induced pulmonary edema is inhibited by sulfated polysaccharides, possibly via a charge interaction between negatively charged compounds and PAF.     

Chondroitin sulfate and heparan sulfate proteoglycans of PC12 pheochromocytoma cells

We have isolated and characterized the cell-associated and secreted proteoglycans synthesized by a clonal line of rat adrenal medullary PC12 pheochromocytoma cells, which have been extensively employed for the study of a wide variety of neurobiological processes. Chondroitin sulfate accounts for 70-80% of the [35S] sulfate-labeled proteoglycans present in PC12 cells and secreted into the medium. Two major chondroitin sulfate proteoglycans were detected with molecular sizes of 45,000-100,000 and 120,000-190,000, comprising 14- and 105-kDa core proteins and one or two chondroitin sulfate chains with an average molecular size of 34 kDa. In contrast to the chondroitin sulfate proteoglycans, one major heparan sulfate proteoglycan accounts for most of the remaining 20-30% of the [35S] sulfate-labeled proteoglycans present in the PC12 cells and medium. It has a molecular size of 95,000-170,000, comprising a 65-kDa core protein and two to six 16-kDa heparan sulfate chains. Both the chondroitin sulfate and heparan sulfate proteoglycans also contain O-glycosidically linked oligosaccharides (25-28% of the total oligosaccharides) and predominantly tri- and tetraantennary N-glycosidic oligosaccharides. Proteoglycans produced by the original clone of PC12 cells were compared with those of two other PC12 cell lines (B2 and F3) that differ from the original clone in morphology, adhesive properties, and response to nerve growth factor. Although the F3 cells (a mutant line derived from B2 and reported to lack a cell surface heparan sulfate proteoglycan) do not contain a large molecular size heparan sulfate proteoglycan species, there was no significant difference between the B2 and F3 cells in the percentage of total heparan sulfate released by mild trypsinization, and both the B2 and F3 cells synthesized cell-associated and secreted chondroitin sulfate and heparan sulfate proteoglycans having properties very similar to those of the original PC12 cell line but with a reversed ratio (35:65) of chondroitin sulfate to heparan sulfate.     

Mutational study of heparan sulfate 2-O-sulfotransferase and chondroitin sulfate 2-O-sulfotransferase

Heparan sulfate (HS) and chondroitin sulfate (CS) are highly sulfated polysaccharides with a wide range of biological functions. Heparan sulfate 2-O-sulfotransferase (HS-2OST) transfers the sulfo group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the 2-OH position of the hexauronic acid that is adjacent to N-sulfated glucosamine, whereas chondroitin sulfate 2-O-sulfotransferase (CS-2OST) transfers the sulfo group to the hexauronic acid that is adjacent to N-acetylated galactosamine. Here we report a systematic mutagenesis study of HS-2OST and CS-2OST based on their structural homology to estrogen sulfotransferase and HS 3-O-sulfotransferase isoform 3 (3-OST3), for which crystal structures exist. We have identified six residues possibly involved in binding to PAPS. HS-2OST carrying mutations of these residues lacks sulfotransferase activity and the ability to bind 3'-phosphoadenosine 5'-phosphate, a PAPS analogue, as determined by isothermal titration calorimetry. Similar residues involved in binding to PAPS were also identified in CS-2OST. Additional residues that participate in carbohydrate substrate binding were also identified in both enzymes. Mutations at these residues led to the loss of sulfotransferase activity but maintained the ability to bind to phosphoadenosine 5'-phosphate. The catalytic function of HS-2OST appears to involve two histidine residues (His140 and His142), whereas only one histidine (His168) of CS 2-OST is likely to be critical. This unique feature of HS 2-OST catalytic residues directed us to characterize the Drosophila heparan sulfate 2-O-sulfotransferase. The results from this study provide insight into the differences and similarities various residues play in the biological roles of the HS-2OST and CS-2OST enzymes.     

Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate

Recent glycobiology studies have suggested fundamental biological functions for chondroitin, chondroitin sulfate and dermatan sulfate, which are widely distributed as glycosaminoglycan sidechains of proteoglycans in the extracellular matrix and at cell surfaces. They have been implicated in the signaling functions of various heparin-binding growth factors and chemokines, and play critical roles in the development of the central nervous system. They also function as receptors for various pathogens. These functions are closely associated with the sulfation patterns of the glycosaminoglycan chains. Surprisingly, nonsulfated chondroitin is indispensable in the morphogenesis and cell division of Caenorhabditis elegans, as revealed by RNA interference experiments of the recently cloned chondroitin synthase gene and by the analysis of mutants of squashed vulva genes.     

Heparan sulfate and chondroitin sulfate proteoglycans inhibit E-selectin binding to endothelial cells

E-selectin is a cell adhesion molecule involved in the initial rolling and adhesion of leukocytes to the endothelium during inflammation. In addition, in vitro studies have suggested that an interaction between E-selectin and binding sites such as sialyl Lewis X-containing oligosaccharides on endothelial cells may be important for angiogenesis. In order to investigate the binding of E-selectin to endothelial cells, we developed an ELISA assay using chimeric E-selectin-Ig molecules and endothelial cells fixed on poly-L-lysine coated plates. Our results indicate that E-selectin-Ig binds to both bovine capillary endothelial cells and human dermal microvascular endothelial cells in a calcium-dependent and saturable manner. The binding is inhibited markedly by heparin and by syndecan-1 ectodomain, and moderately by chondroitin sulfate, but not by sialyl Lewis X-containing oligosaccharides. These results suggest that heparan sulfate and chondroitin sulfate proteoglycans on endothelial cells are potential ligands for E-selectin.     

Estrone sulfate and dehydroepiandrosterone sulfate concentrations in normal subjects and men with cirrhosis.

Circulation levels of estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHAS) have been measured in plasma using a radioimmunoassay for estrone and dehydroepiandrosterone following extraction and hydrolysis of the sulfate. The mean +/- SE concentrations of E1S and DHAS in normal men were 458 +/- 25 pg/ml and 1.45 +/- 0.19 micrograms/ml, respectively. In normal women the values for days 5-7 of the cycle were 880 +/- 117 pg/ml and 1.25 +/- 0.12 micrograms/ml which were not different than the values for days 20-22 of 1195 +/- 176 pg/ml and 1.58 +/- 0.29 micrograms/ml. The mean values in post-menopausal women were 250 +/- 33 pg/ml and 0.47 +/- 0.07 micrograms/ml, both lower than the values in young women. In a group of cirrhotic men the mean values were 325 +/- 55 pg/ml and 0.38 +/- 0.12 micrograms/ml, both significantly lower than the normal values. This suggests a defect in sulfurylation in men with hepatic cirrhosis.     

Quinine sulfate and bacterial invasion

Background

As many patients who receive antimalarial drugs for treatment of noninfectious, inflammatory diseases are also immunosuppressed and might have a concomitant bacterial infection, we studied the effectiveness of these drugs against bacterial infections, to find out whether they could protect against (and even treat) such conditions and obviate the need for an additional antibiotic drug.

Methods

Effect of QS on bacterial growth: Escherichia coli (E. coli) HB101 pRI203 were cultured overnight at 37°C in TSB and inoculated (approx 1 × 10⁷ cells /ml) in MEM in the presence of QS at various concentrations (0, 50 and 100 μM).The effect of QS at concentration of 50 and 100 μM on the entry process of E. coli HB101 pRI203 into HeLa cells was studied under different experimental conditions: 1. QS was incubated with 3 × 10⁵ HeLa cells for 60 min at 37°C prior to infection. 2. QS was added to HeLa cell monolayers during the infection period.

Results

QS showed no antibacterial activity after 24 h of incubation.The invasive efficiency of the bacteria was significantly inhibited at a dose-dependent manner, when QS was added to HeLa cells for 60 min at 37°C prior to infection (condition 1), and to a lesser extent when added during the period of infection (condition 2).

Conclusions

Although the antimalarials are generally regarded as being inactive against most extracellular bacterial species, our results indicate that QS significantly inhibited the internalization/invasion efficacy of E. coli in the host cells.

     

Organization of glycosaminoglycan sulfation in the biosynthesis of proteochondroitin sulfate and proteodermatan sulfate

Although the intermediates for sulfation of proteochondroitin and proteodermatan have been known for several decades, organizational aspects of this formation have not been clearly defined. Work in several laboratories, including our own, have indicated a pattern which strongly suggests that sulfation ordinarily takes place together with glycosaminoglycan polymerization in the same Golgi sites, and with close relationship to aspects of polymer elongation, polymer modification and polymer termination. the organization of sulfation together with polymerization may be a major factor controlling the location, type, and degree of sulfation, which in turn may direct specific functions of these proteoglycans.     

Chondrocytes respond to an altered heparan sulfate composition with distinct changes of heparan sulfate structure and increased levels of chondroitin sulfate

Heparan sulfate (HS) regulates the activity of many signaling molecules critical for the development of endochondral bones. Even so, mice with a genetically altered HS metabolism display a relatively mild skeletal phenotype compared to the defects observed in other tissues and organs pointing to a reduced HS dependency of growth-factor signaling in chondrocytes. To understand this difference, we have investigated the glycosaminoglycan (GAG) composition in two mouse lines that produce either reduced levels of HS (Ext1^gt/gt mice) or HS lacking 2-O-sulfation (Hs2st1^−/− mice). Analysis by RPIP-HPLC revealed an increased level of sulfated disaccarides not affected by the mutation in both mouse lines indicating that chondrocytes attempt to restore a critical level of sulfation. In addition, in both mutant lines we also detected significantly elevated levels of CS. Size exclusion chromatography further demonstrated that Ext1^gt/gt mutants produce more but shorter CS chains, while the CS chains produced by (Hs2st1^−/− mice) mutants are of similar length to that of wild type littermates indicating that chondrocytes produce more rather than longer CS chains. Expression analysis revealed an upregulation of aggrecan, which likely carries most of the additionally produced CS. Together the results of this study demonstrate for the first time that not only a reduced HS synthesis but also an altered HS structure leads to increased levels of CS in mammalian tissues. Furthermore, as chondrocytes produce 100-fold more CS than HS the increased CS levels point to an active, precursor-independent mechanism that senses the quality of HS in a vast excess of CS. Interestingly, reducing the level of cell surface CS by chondroitinase treatment leads to reduced Bmp2 induced Smad1/5/9 phosphorylation. In addition, Erk phosphorylation is increased independent of Fgf18 treatment indicating that both, HS and CS, affect growth factor signaling in chondrocytes in distinct manners.     

A high-performance liquid chromatography for constituent disaccharides of chondroitin sulfate and dermatan sulfate isomers

An improved high-performance liquid chromatography for unsaturated disaccharides prepared from chondroitin sulfate and dermatan sulfate isomers was developed using an ion-exchange resin made from a sulfonized styrene-divinylbenzene copolymer. By this newly devised method, it was found that the retention times of representative unsaturated disaccharides are very unique and appear in the following order: unsaturated 6-sulfated, nonsulfated, and 4-sulfated disaccharides. The content of the individual unsaturated disaccharides could be measured at similar sensitivities with ultraviolet absorbance. Sensitive and unique retention times as well as good resolution were found for various unsaturated disulfated disaccharides. The new microassay method by HPLC can be used to determine chondroitin sulfate and dermatan sulfate isomers in amounts as small as 100 ng to 8 micrograms. The practicality of this method was verified by application to the separation and quantitation of chondroitin sulfate and dermatan sulfate isomers from human coronary arteries.     

Residual keratan sulfate in chondroitin sulfate formulations for oral administration

Chondroitin sulfate is a biomedical glycosaminoglycan (GAG) mostly used as a dietary supplement. We undertook analysis on some formulations of chondroitin sulfates available for oral administration. The analysis was based on agarose-gel electrophoresis, strong anion-exchange chromatography, digestibility with specific GAG lyases, uronic acid content, NMR spectroscopy, and size-exclusion chromatography. Keratan sulfate was detected in batches from shark cartilage, averaging ～16% of the total GAG. Keratan sulfate is an inert material, and hazardous effects due to its presence in these formulations are unlikely to occur. However, its unexpected high percentage compromises the desired amounts of the real ingredient specified on the label claims, and forewarns the pharmacopeias to update their monographs. The techniques they recommended, especially cellulose acetate electrophoresis, are inefficient in detecting keratan sulfate in chondroitin sulfate formulations. In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Analysis based on strong anion-exchange chromatography is shown to be more reliable than the methods presently suggested by standard pharmacopeias.