Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica.

Acharan sulfate is a glycosaminoglycan (GAG), having the structure -->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose(1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3-5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.

Determination of lipid-bound sulfate by ion chromatography and its application to quantification of sulfolipids from kidneys of various mammalian species.

A variety of procedures have been developed for determining the sulfate ester content of various biomolecules. Ion chromatography (IC), that is, quantitation of ionic substances by ion conductimetry after separation by anion-exchange chromatography, has been increasingly utilized for the determination of inorganic sulfate in clinical and environmental samples. We adopted suppressed-mode IC to the determination of lipid- or glycolipid-bound sulfate released by acid hydrolysis and found that it has the advantage of increased precision for wide concentration ranges (30 pmol to approximately micromol) and lack of interference from other lipids. To minimize deterioration of the separation column, the lipophilic constituents in the acid hydrolysate were removed by a two-phase partition system of chloroform-methanol-water. The inorganic sulfate was quantitatively extracted into the aqueous phase by replacing water with an alkaline buffer. By this method, the concentration of sulfolipids was determined in the kidney of mammals with various body mass. Sulfolipids were more concentrated in the kidney of smaller animals, which have higher maximum urine concentrating activity per gram of body mass, supporting the hypothesis of the function of sulfolipids as an ion barrier on the luminal surface of renal tubules.

Quantitative changes in glycosaminoglycans in the lungs of rats exposed to diesel exhaust.

Exposure to diesel exhaust (DE) induces lesions in lung epithelium by generation of reactive oxygen species. Glycosaminoglycans (GAG), components of extracellar matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. We investigated how GAG are related to the recovery of lung tissue from injury. Using high-performance liquid chromatography analysis, we determined the amounts of GAG, such as chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) in the lungs of rats exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m(3) as suspended particulate matter, or to filtered air. The contents of CS and HA in the surroundings of the bronchi were significantly increased after exposure to DE. In addition, immunohistochemical staining showed that the number of 8-hydroxydeoxyguanosine-positive cells as a marker of cell damage, and proliferating cell nuclear antigen-positive cells also increased in the same areas in which the levels of GAG were elevated in the lungs of rats exposed to 3 mg/m(3) DE. These results suggest that CS and HA in the lung contribute to cell proliferation and remodeling in the process of recovery from injury caused by exposure to DE.

(1)H nuclear magnetic resonance spectroscopic analysis for determination of glucuronic and iduronic acids in dermatan sulfate, heparin, and heparan sulfate.

(1)H NMR spectroscopy has been established for the determination of uronate residues in glycosaminoglycans (GAGs) such as dermatan sulfate (DS), heparin (HP), and heparan sulfate (HS). Because of variation in the sulfonation positions in DS, HP, or HS, interpretation of spectra is difficult. Solvolysis was applied to remove O-sulfo groups from these GAG chains in dimethyl sulfoxide containing 10% methanol at 80 degrees C for 5 h. In the cases of HP and HS, N-sulfo groups on glucosamine residues were also removed under the same conditions. The resulting unsubstituted amino groups in HP and HS chains were re-N-acetylated using acetic anhydride to obtain homogeneous core structure with the exception of the variation of uronate residues. The contents of glucuronate and iduronate residues in the chemically modified DS, HP, and HS samples were analyzed by 600-MHz (1)H NMR spectroscopy. These methods were applied to compositional analysis of uronate residues in GAGs isolated from various sources.

Analytical method of chondroitin/dermatan sulfates using high performance liquid chromatography/turbo ionspray ionization mass spectrometry: application to analyses of the tumor tissue sections on glass slides.

We established a highly sensitive quantitative analytical method for chondroitin/dermatan sulfates by LC/MS method. By this method, the unsaturated disaccharides produced after the enzymatic digestion of chondroitin/dermatan sulfates can be determined in the amounts as low as 0.5 pmol levels. The use of tetrabutylammonium hydroxide as an ion-pair reagent for LC/MS allowed us to separate unsaturated 4-sulfated disaccharide and unsaturated 6-sulfated disaccharide. Furthermore, the peak areas of unsaturated disaccharides were increased almost 10 times by the postcolumn addition of acetonitrile. We applied this LC/MS method to the analyses of unsaturated disaccharides from chondroitin/dermatan sulfates in the tissues sections on glass slides, which were prepared from MethA tumor-bearing mice. This method brought about considerable reduction in the time distance from sample collection to preparation of analytical results.

Separation of chondroitin sulfate and hyaluronic acid fragments by centrifugal precipitation chromatography.

Centrifugal precipitation chromatography (CPC) was applied for the first time to the separation of fragments of chondroitin sulfate (ChS) and hyaluronic acid (HA). The separation was performed using a gradient elution system between ethanol and water since solubility of these biopolymers highly depends on the concentration of ethanol in aqueous solution. ChS and HA were each eluted into several peaks through a flow-through UV detector at 275 nm, despite they have almost no absorbance at this wavelength in an aqueous solution. The separation was also confirmed by redissolving the dried fraction in water and measuring the absorbance at 210 nm. These results suggest that the CPC system can detect small precipitates of these biopolymers by light scattering at 275 nm. The separated fragments of biopolymers are not easily characterized because no suitable analytical method is available for identification of these compounds. However, the overall results demonstrate that CPC may be a useful separation of biopolymers such as glycosaminoglycans which quantitatively produce precipitates in an organic solvent mixture.

1,25-Dihydroxyvitamin D(3) prevents the conversion of adipose tissue into fibrous tissue in skin exposed to chronic UV irradiation.

The hormonally active form of vitamin D (1,25(OH)2D3) is known to be a physiological regulator of the proliferation and differentiation of skin cells including keratinocytes, fibroblasts, and adipocytes. In the present study, the efficacy of 1,25(OH)2D3 on the conversion of adipose tissue to fibrous tissue in photodamaged skin was investigated in a murine animal model. Groups of hairless mice were exposed to solar-simulating UV irradiation (lambdamax, 352 nm; UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) for 20 weeks at a dose of 10.8 J/cm2 five times weekly on weekdays. At the end of 20 weeks irradiation, wrinkling in the dorsal skin was induced. The histological and biochemical studies indicated that UV irradiation caused a disappearance of adipocytes and concomitant accumulation of the extracellular matrix components (fibrosis), including collagen, hyaluronan, and chondroitin/dermatan, which are synthesized by fibroblasts. Application of 1,25(OH)2D3 on the dorsal skin prior to UV irradiation dramatically prevented both the disappearance of adipocytes and the accumulation of extracellular matrix components in the lower dermis, resulting in antiwrinkling. These findings indicate that 1,25(OH)2D3 prevents the UV-induced abnormal differentiation and proliferation of adipocytes and fibroblasts, which arise from a common progenitor, mesenchymal cells.

Probing the interaction of dengue virus envelope protein with heparin: assessment of glycosaminoglycan-derived inhibitors.

A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.

Analytical method of heparan sulfates using high-performance liquid chromatography turbo-ionspray ionization tandem mass spectrometry.

We established a highly sensitive quantitative analytical method of heparan sulfates (HS) by LC-MS-MS. It became possible to determine the unsaturated disaccharides produced by the enzyme digestion of HS, and to perform the whole analyses on one sample within 3 min by use of a short column of CAPCELL PAK NH2 UG80 (35 mm x 2 mm I.D.). The assay method was validated and showed the satisfactory sensitivity, precision and accuracy, which enabled the quantitation up to picomol level. By employing this method, we performed the analyses of HS in mouse brain and liver, and tumor tissues of tumor-bearing mouse transplanted subcutaneously with Meth A fibrosarcoma cells. The compositions of the unsaturated disaccharide units derived from HS were found to be somewhat different among those tissues. It is assumed that the site of sulfation in HS may be controlled by certain regulatory mechanisms. The quantitative method developed in this study is believed to be a very useful method for the determination of compositional profiles of constitutive disaccharide units of tissue HS.

Analytical method for keratan sulfates by high-performance liquid chromatography/turbo-ionspray tandem mass spectrometry.

We established a highly sensitive LC/MS/MS method for the analysis of the disaccharides produced from keratan sulfates (KS). It was revealed that the disaccharides produced by keratanase II enzymatic digestion of KS could be determined with high sensitivity by the negative-ion mode of multiple reaction monitoring. Furthermore, monosulfated and disulfated disaccharides can be separated using a short column of Capcell Pak NH2 UG80 (35 mm x 2 mm i.d.). The complete analysis of one sample can be performed within 5 min. The assay method was validated and showed satisfactory sensitivity, precision, and accuracy, which enabled quantitation at subpicomole levels. From the results of analyses of KS obtained from cornea, nasal cartilage, and brain, it was found that the degree of sulfation at the C-6 position of the galactose residues differed among those samples in the following order: nasal cartilage > cornea > brain. Our analytical method is very useful for the analyses of KS in various biological materials and for comparison of the degree of sulfation of KS from various biological samples.

Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides.

Hyaluronan was partially depolymerized on a large-scale quantity using bacterial hyaluronidase (E.C. 4.2.2.1) for preparation of chemically fully O-sulfated oligosaccharides. The hyaluro-oligosaccharide (HAoligo) mixture obtained by partial digestion was repeatedly applied to low pressure gel permeation chromatographic separation to purify the size-unified oligosaccharide ranged from 4- to 20-mer. The purity and size of each HAoligo was confirmed by using proton nuclear magnetic resonance ((1)H NMR) spectroscopy, capillary electrophoresis (CE) on normal polarity mode, and a newly established separation method by normal phase chromatography with Amide-80 column. The purified HAoligos ranged 4- to 20-mer were applied to chemically fully O-sulfation. Characterization of chemically fully O-sulfated HAoligos was performed by both chemical compositional analyses after hydrolysis and (1)H NMR spectroscopy. While the anti-factor IIa activity of 4- to 20-mer O-sulfated HAoligos was less than 3.1 units/mg, the inhibitory action for hyaluronidase (bovine testicular hyaluronidase (E.C.3.2.1.35)) of the oligosaccharides ranged 16- to 20-mer were corresponding to 79% of that shown by fully O-sulfated hyaluronan (MW 100 kDa) through both competitive and noncompetitive effects.

Prevention of the photodamage in the hairless mouse dorsal skin by kojic acid as an iron chelator.

Kojic acid, a fungal metabolic product, has been used as a skin-depigmenting agent in skin care products marketed in Japan. Iron in the skin is known to be involved in wrinkling as a result of chronic photodamage. Kojic acid was expected to have anti-wrinkling activity, since it possesses iron-chelating activity. We now evaluated the anti-wrinkling activity of kojic acid by using hairless mice exposed to chronic solar-simulating ultraviolet (UV) irradiation as model animal. At the end of a 20-week irradiation period, topical application of kojic acid before UV irradiation was observed to dramatically prevent: (1) the wrinkling, (2) hyperplasia of the epidermis, (3) fibrosis of the lower dermis, and (4) the increase of extracellular matrix components in the upper dermis. These findings indicate that kojic acid is a typical agent preventing wrinkling of the skin due to chronic photodamage.

Comparison of oligosaccharides derived from salivary mucin of Japanese secretor and non-secretor individuals of blood group type-A.

Comparison of oligosaccharide components derived from salivary mucin was performed between secretor and non-secretor individuals. Salivary mucin was collected from four secretors and three non-secretors having blood group type-A. Compositional analysis showed that the contents of galactose and N-acetylglucosamine in the non-secretor were higher than those in the secretor. The O-linked oligosaccharides obtained by treatment with alkaline borohydride were separated by gel filtration using Sephadex G-50. The results indicated that the size of the type-A active oligosaccharides from the secretor was similar to or smaller than that of the non-secretor. Ion-exchange chromatography showed that the secretors had strong type-A activities in both the neutral and acidic fractions but the non-secretors showed type-A activity mainly in the neutral fraction. These results suggest that compositional differences in blood group substances exist between secretors and non-secretors.

Determination of N-acetyl- and N-glycolylneuraminic acids in gangliosides by combination of neuraminidase hydrolysis and fluorometric high-performance liquid chromatography using a GM3 derivative as an internal standard.

A highly sensitive method for quantification of sialic acids in gangliosides was developed. The sialic acids, released by hydrolysis of gangliosides, were converted to fluorescent derivatives with 1,2-diamino-4,5-(methylenedioxy)benzene (DMB) and separated on a reversed-phase C18 column with an isocratic elution. As little as 0.1-1.0 nmol of sialic acid in ganglioside was quantified. The use of acetate buffer instead of water in the mobile phase could prevent damage on the column and reduce background peaks derived from the reagents. When gangliosides were subjected to acid hydrolysis, the velocity of hydrolysis varied depending on their structures and a part of the sialic acid liberated decomposed with prolonged heating time. Therefore gangliosides were hydrolyzed by Arthrobacter ureafaciens neuraminidase in the presence of sodium cholate after addition of an internal standard. For the internal standard, GM3 with N-propionylneuraminic acid (GM3(NeuPr)) was synthesized from GM3(NeuAc) by N-deacylation followed by N-propionylation. Folch partition was used to decrease lipophilic materials included in the sample, and the sialic acids released were recovered from the upper phase. The present method has a satisfactory sensitivity in the simultaneous quantification of NeuAc and NeuGc in purified gangliosides as well as in crude lipid fractions containing a variety of gangliosides.

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

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

A new sulfated beta-galactan from clams with anti-HIV activity.

A new polysaccharide composed of galactan sulfate with a beta-(1-->3)-glycosidic linkage has been isolated from the marine clam species Meretrix petechialis. The polysaccharide was homogeneous in its composition containing D-galactose. The glycosidic linkage was examined by 2D DQF-COSY and 2D NOESY spectroscopy. The coupling constant of anomeric proton was 7.8 Hz, suggesting a beta-galacto configuration. The downfield shift of H-2 of galactose residue demonstrated the presence of 2-O-sulfonate group. TQF-COSY confirmed that the C-6 position was substituted with a sulfonate group. The anti-HIV activity of the polysaccharides has been evaluated by the inhibition of syncytia formation. The fusion index and percentage fusion inhibition of sulfated galactan were 0.34 and 56% at 200 micrograms/mL.

Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans.

Glycosaminoglycans including dermatan sulphate, hyaluronan, heparan sulphate and heparin were chemically modified by O-sulphonation. By altering the reaction conditions, products having a different degree of O-sulphonation could be obtained. Glycosaminoglycan derivatives were prepared having no free hydroxyl groups, with sulphoester group/disaccharide unit ratios of 4.0 for dermatan sulphate and hyaluronan, and sulphoester and sulphamide group/disaccharide unit ratios of 4.22 and 4.88 for heparan sulphate and heparin, respectively. 1H NMR spectroscopy showed that the fully O-sulphonated hyaluronan derivative had a glucuronate residue with an altered conformation. Since glycosaminiglycans and their derivatives are often used as anticoagulant/antithrombotic agents, their anti-amidolytic activities were determined. The anti-factor IIa activity of fully O-sulphonated dermatan sulphate, hyaluronan and heparan sulphate ranged from 40 to 80 units/mg, while no anti-factor Xa activity of the fully O-sulphonated glycosaminoglycans was detected. These values are lower than those reported for low-molecular-weight heparins and are consistent with the requirement of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. Interestingly, the anti-factor Xa of heparin is lost by chemical O-sulphonation.

Effect of fully sulfated glycosaminoglycans on pulmonary artery smooth muscle cell proliferation.

Fully sulfated heparin and other glycosaminoglycans, namely heparan, chondroitin, and dermatan sulfates, and hyaluronan have been prepared by using sulfur trioxide under mild chemical conditions. All these derivatives were assayed for antiproliferative activity on cultured bovine pulmonary artery smooth muscle cells (BPASMCs). No appreciable difference was found between heparin and fully sulfated heparin. Chondroitin and dermatan sulfates actually stimulated BPASMCs growth but full sulfonation made them strongly antiproliferative. Native hyaluronan was not antiproliferative but became strongly so after sulfonation. Neither acharan sulfate nor N-sulfoacharan sulfate had any antiproliferative activity. This suggests that O-sulfonation of the polysaccharide is critical for antiproliferative activity, whereas N-sulfonation of glucosamine residues is not.

Structural characteristics of oversulfated chondroitin/dermatan sulfates in the fibrous lesions of the liver with cirrhosis.

The structural characteristics of oversulfated chondroitin/dermatan sulfates (C/DSs) in the fibrous lesions of the rat liver with cirrhosis were examined. Long-Evans Cinnamon rats were subjected to the present study as the model animals with cirrhosis. The serial polyester wax sections of liver with cirrhosis were processed into the fibrous lesions and the nonfibrous lesions. The oversulfated C/DSs in the tissue sections on a glass slide were degraded to unsaturated disaccharides by chondroitinase ABC and ACII digestion in the presence of bacterial collagenase. Subsequently, the resulting unsaturated disaccharides were determined by the reversed-phase ion-pair high-performance liquid chromatography with fluorometric postcolumn derivatization using 2-cyanoacetamide as a reagent. Through these in situ investigations, we found some facts as follows: (i) in the fibrous lesion, the remarkable increase of the oversulfated C/DSs content and the decrease of the oversulfation degree of the C/DSs were observed compared with those in the nonfibrous lesion, (ii) the proportion of the iduronic acid content in the C/DSs in the fibrous lesion was significantly low compared with that in the nonfibrous lesion, and (iii) in the nonfibrous lesion close to the fibrous lesion, both quantitative and qualitative alterations of C/DSs were not observed at all. These findings indicate that the oversulfated C/DSs with low iduronic acid content are possible marker for the fibrogenesis of liver with cirrhosis.

Inhibition of hyaluronidase by fully O-sulfonated glycosaminoglycans.

We report a new flow injection assay (FIA) method for determining hyaluronidase activity and the inhibitory effects of chemical fully O-sulfonated glycosaminoglycans on this enzyme. The products of enzymatic action on hyaluronidase can be detected by FIA using fluorometric detection with the fluorogenic reagent 2-cyanoacetamide. The major products derived from hyaluronan by the action of mammalian testicular hyaluronidase (a hydrolyase) were confirmed by (1)H NMR spectroscopy and capillary electrophoresis. The FIA method was next applied to the assay of hyman urinary hyaluronidase activity and the screening of hyaluronidase inhibitors. The human urinary hyaluronidase activity measured ranged from 46 to 59 turbidity reducing units/mg protein. Among the glycosaminoglycans only heparin showed hyaluronidase inhibition. Chemically O-sulfonated glycosaminoglycans showed IC(50) values of hyaluronidase inhibition that correlated with the degree of O-sulfonation. Heparin was found to inhibit hyaluronidase activity noncompetitively, while chemically O-sulfonated HA strongly inhibited hyaluronidase through both competitive and noncompetitive effects.