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N-Acetylglucosamine-6-sulfate sulfatase activity was assayed by incubation of the radiolabeled monosaccharide N-acetylglucosamine [1-14C]6-sulfate (GlcNAc6S) with homogenates of leukocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with N-acetylglucosamine-6-sulfate sulfatase deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls and other mucopolysaccharidosis types. The level of enzymatic activity toward GlcNAc6S was compared with that toward a sulfated disaccharide and a sulfated trisaccharide prepared from heparin. The disaccharide was desulfated at the same rate as the monosaccharide and the trisaccharide at 30 times that of the monosaccharide. Sulfatase activity toward glucose 6-sulfate and N-acetylmannosamine 6-sulfate was not detected. Sulfatase activity in fibroblast homogenates with GlcNAc6S exhibited a pH optimum at pH 6.5, an apparent Km of 330 mumol/liter, and inhibition by both sulfate and phosphate ions. The use of radiolabeled GlcNAc6S substrate for the assay of N-acetylglucosamine-6-sulfate sulfatase in leukocytes and skin fibroblasts for the routine enzymatic detection of the Sanfilippo D syndrome is recommended.
A simple and efficient approach has been set up for fabricating highly active sulfated titania-silica (SO(4)(2-)/TiO(2)-SiO(2)): Ti(SO(4))(2) was hydrolyzed in the presence of silica, making it possible to sulfate titania and form titania-silica mixed oxide in one step. This study was focused on investigating the roles of sulfate species and silica in improving the physicochemical properties and photoactivity of SO(4)(2-)/TiO(2)-SiO(2) through comparison with sulfated titania (SO(4)(2-)/TiO(2)) and sulfate-free catalysts (TiO(2) and TiO(2)-SiO(2)). Various characterization methods, including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and surface photovoltage spectroscopy (SPS), were employed to test these materials. The results revealed that for SO(4)(2-)/TiO(2) and TiO(2)-SiO(2) the sole presence of either sulfate species or silica imposes negative effects on the photocatalysis behavior of titania, leading them to have negligible photoactivities. On the contrary, in the case of SO(4)(2-)/TiO(2)-SiO(2), sulfate species and silica were proved to act in a cooperative manner; therefore, the following enhanced structure and surface properties of SO(4)(2-)/TiO(2)-SiO(2) were obtained: (i) relatively well-crystallized and smaller-size (15.4 nm) anatase-phase titania was formed upon 500 degrees C calcination without forming rutile phase and (ii) the formation of active surface sulfate species promotes the separation of photoinduced electron-hole pairs and therefore accelerates the photocatalysis reaction. Therefore, its photoactivity is enhanced as a result of the favorable synergic effects between sulfate species and silica due to their simultaneous presence.
The C-terminal region of sulfate transporters from plants and animals belonging to the SLC26 family members shares a weak but significant similarity with the Bacillus sp. anti-anti-sigma protein SpoIIAA, thus defining the STAS domain (sulfate transporter and anti-sigma antagonist). The present study is a structure/function analysis of the STAS domain of SULTR1.2, an Arabidopsis thaliana sulfate transporter. A three-dimensional model of the SULTR1.2 STAS domain was built which indicated that it shares the SpoIIAA folds. Moreover, the phosphorylation site, which is necessary for SpoIIAA activity, is conserved in the SULTR1.2 STAS domain. The model was used to direct mutagenesis studies using a yeast mutant defective for sulfate transport. Truncation of the whole SULTR1.2 STAS domain resulted in the loss of sulfate transport function. Analyses of small deletions and mutations showed that the C-terminal tail of the SULTR1.2 STAS domain and particularly two cysteine residues plays an important role in sulfate transport by SULTR1.2. All the substitutions made at the putative phosphorylation site Thr-587 led to a complete loss of the sulfate transport function of SULTR1.2. The reduction or suppression of sulfate transport of the SULTR1.2 mutants in yeast was not due to an incorrect targeting to the plasma membrane. Both our three-dimensional modeling and mutational analyses strengthen the hypothesis that the SULTR1.2 STAS domain is involved in protein-protein interactions that could control sulfate transport.
We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.
Seven mutants of Chlorella pyrenoidosa (Emerson strain 3) impaired for sulfate utilization have been isolated after treatment of the wild-type organism with nitrosoguanidine by replica plating on media containing thiosulfate and l-methionine. These mutants fall into three classes based on their ability to grow on sulfate, accumulate compounds labeled from sulfate-(35)S, and reduce adenosine 3'-phosphate 5'-phosphosulfate-(35)S (PAPS-(35)S) to thiosulfate-(35)S. Mutant Sat(2) (-) cannot grow on sulfate, but it accumulates thiosulfate-(35)S and homocysteic acid-(35)S from sulfate-(35)S in vivo. In addition, extracts of mutant Sat(2) (-) reduce PAPS-(35)S to thiosulfate-(35)S, indicating the possession of enzyme fractions S and A, both of which are required for thiosulfate formation. Mutants Sat(1) (-), Sat(3) (-), Sat(4) (-), Sat(5) (-), and Sat(6) (-) cannot grow on sulfate, and their extracts lack the ability to reduce PAPS-(35)S to thiosulfate-(35)S. Mutant Sat(7) (-)R(1), a probable revertant, can grow on sulfate but still lacks the ability to reduce PAPS-(35)S to thiosulfate-(35)S in vitro. Complementation experiments in vitro show that the block in formation of acid-volatile radioactivity in every case is due to the absence of activity associated with fraction S. All mutants can grow on thiosulfate and all possess the activating enzymes which convert sulfate to PAPS. Through a comparison of nutritional and enzymatic characteristics, the first outlines of a branched and complicated pathway for sulfate reduction in Chlorella are beginning to emerge.
Sulfate reducers and related organisms which had previously been found to reduce Fe(III) with H(2) or organic electron donors oxidized S to sulfate when Mn(IV) was provided as an electron acceptor. Organisms catalyzing this reaction in washed cell suspensions included Desulfovibrio desulfuricans, Desulfomicrobium baculatum, Desulfobacterium autotrophicum, Desulfuromonas acetoxidans, and Geobacter metallireducens. These organisms produced little or no sulfate from S with Fe(III) as a potential electron acceptor or in the absence of an electron acceptor. In detailed studies with Desulfovibrio desulfuricans, the stoichiometry of sulfate and Mn(II) production was consistent with the reaction S + 3 MnO(2) + 4H-->SO(4) + 3Mn(II) + 2H(2)O. None of the organisms evaluated could be grown with S as the sole electron donor and Mn(IV) as the electron acceptor. In contrast to the other sulfate reducers evaluated, Desulfobulbus propionicus produced sulfate from S in the absence of an electron acceptor and Fe(III) oxide stimulated sulfate production. Sulfide also accumulated in the absence of Mn(IV) or Fe(III). The stoichiometry of sulfate and sulfide production indicated that Desulfobulbus propionicus disproportionates S as follows: 4S + 4H(2)O-->SO(4) + 3HS + 5 H. Growth of Desulfobulbus propionicus with S as the electron donor and Fe(III) as a sulfide sink and/or electron acceptor was very slow. The S oxidation coupled to Mn(IV) reduction described here provides a potential explanation for the Mn(IV)-dependent sulfate production that previous studies have observed in anoxic marine sediments. Desulfobulbus propionicus is the first example of a pure culture known to disproportionate S.
The absorption, excretion, and metabolism of [1-14C]lauryl sulfate as either the sodium or propionyl erythromycin salt has been studied in the rat and man. In the rat 88% of the radiolabel from propionyl erythromycin [1-14C]lauryl sulfate was excreted in the urine in the 24-hr period following a single oral dose. More than 95% of the radiolabel was excreted as the metabolite butyric acid 4-sulfate. This metabolite was shown to be derived from carbons 1-4 of the lauryl sulfate moiety. There was no evidence of sulfate cleavage in the rat. In man 51-59% of the administered ratioactivity was recovered in the urine. The major excretion product was butyric acid 4-sulfate accounting for approximately 95% of urinary radioactivity. The remainder was unchanged lauryl sulfate. A significant portion of the radiolabeled propionyl erythromycin lauryl sulfate was converted to 14CO2 indicating that the sulfate linkage was cleaved. A minimum value of 9-16% of the dose was metabolized in this manner.
The hydrolysis of 4-methylumbelliferyl sulfate by liver sulfatases to free fluorescent 4-methylumbelliferone and the subsequent formation of the glucuronide conjugate were studied in the isolated perfused rat liver. In livers from fed, phenobarbital-treated rats, 4-methylumbelliferyl sulfate (0.25-1.5 mM) was hydrolyzed rapidly to free 4-methylumbelliferone at maximal rates of about 5 mumol/g/hr. A major fraction of the free 4-methylumbelliferone formed was converted to the glucuronide at maximal rates around 20 mumol/g/hr. Similar rates of hydrolysis were observed in livers from fasted, phenobarbital-treated or normal rats, although the ratio of glucuronide to free product was decreased markedly by fasting. In liver homogenates, however, rates of organic sulfate hydrolysis exceeded those observed in the perfused liver by at least 2-fold, suggesting that 4-methylumbelliferyl sulfate content is an important determinant of rates of hydrolysis in the perfused liver. There was a good correlation (r = 0.91) between rates of product formation and fluorescence of 4-methylumbelliferone detected from the liver surface with fiber optic light guides. Fluorescence of 4-methylumbelliferone produced from hydrolysis of 4-methylumbelliferyl sulfate was also monitored with micro-light guides placed on periportal and pericentral areas of the liver lobule for the estimation of local rates of product formation. When perfusions were in the anterograde direction, desulfation of 4-methylumbelliferyl sulfate was about 50% higher in pericentral (28.8 +/- 9.3 mumol/g/hr) than in periportal (18.2 +/- 2.7 mumol/g/hr) areas. Furthermore, 4-methylumbelliferyl sulfate content determined in microdissected samples was 1.5- to 2-fold higher in pericentral than in periportal regions of the liver lobule but the activity of 4-methylumbelliferyl sulfate sulfatase was identical in both zones of the liver lobule. We conclude, therefore, that the local substrate content is an important determinant of rates of 4-methylumbelliferyl sulfate hydrolysis in sublobular zones of the liver.
Sulfated glycoconjugates were ultrastructurally localized within embryonic chick marrow by using the high iron diamine-silver proteinate stain. Stain was concentrated in the extravascular, granulopoietic compartment, indicating that granulopoiesis, but not erythropoiesis, proceeded in a highly sulfated environment. It was likely that most of the stainable material represented sulfated proteoglycans since staining was abrogated by predigesting tissue with enzymes and other treatments known to degrade specific glycosaminoglycan chains. Chondroitinase/hyaluronidase digestion resulted in the removal of most of the stainable material associated with the extracellular matrix and a portion of the stainable material associated with fibroblastic cell surfaces. Unaffected material lay in close proximity to fibroblastic cell membranes. Heparitinase/heparinase digestion had essentially the opposite effect. Sulfated material associated with matrix components was largely unaffected, but the fibroblastic plasmalemmal material was now absent. These results suggest that there are at least two categories of sulfated proteoglycans in the granulopoietic compartment, each differentially distributed. The plasmalemmal material likely represented heparan sulfate which in this tissue appeared to be associated in a uniform layer with fibroblastic stromal cell membranes and not with blood or endothelial cell membranes. Material identified as chondroitin sulfates was found within patches of amorphous matrix that was located on fibroblastic stromal cell surfaces and that was interspersed with fibrils in the extracellular matrix. Chondroitin sulfates were sparsely distributed on granulocytic cell surfaces.
Previous investigations have identified proteoglycans in the central nervous system during development and have implicated some proteoglycans as axon guidance molecules that act by inhibiting axon extension. The present study investigated the pattern of immunoreactivity for several glycosaminoglycans common to certain proteoglycans relative to growing retinal axons in the developing chick visual system and in retinal explant cultures. Immunostaining for chondroitin-6-sulfate, chondroitin-4-sulfate, and keratan sulfate was observed to colocalize with retinal axons throughout the retinofugal pathway during the entire period of retinal axon growth. The proteoglycan form of collagen IX, however, was only observed in the retina, primarily peripheral to the areas with actively growing axons. The pattern of immunostaining for chondroitin sulfate in tissue sections suggested that the retinal axons might be a source for some of the chondroitin sulfate immunostaining in the developing visual pathway. This was confirmed in that chondroitin sulfate immunostaining was also observed on neurites emanating from cultured retinal explants. These findings indicate that retinal axons grow in the presence of chondroitin sulfate and keratan sulfate proteoglycans and that these proteoglycans in the developing chick visual pathway have functions other than to inhibit axon growth.
Two cDNAs, LeST1-1 (AF347613) and LeST1-2 (AF347614), encoding sulfate transporters have been cloned from tomato (Lycopersicon esculentum Mill.) by reverse transcription-polymerase chain reaction and their expression characterised. Sharing 76% identity at the amino acid level, the transporters are phylogenetically associated with the Group-1, high-affinity plant sulfate transporters. Both were shown to have high affinity for sulfate by uptake kinetic analysis using a yeast (Saccharomyces cerevisiae) sulfate-transporter mutant. Km values of 11.5 microM and 9.8 microM were calculated for LeST1-1 and LeST1-2, respectively, the same order of magnitude as those previously reported for several other Group-1 high-affinity sulfate transporters. In situ hybridisation to S-deficient tomato roots showed LeST1-1 to be expressed in the epidermis and pericycle, whereas LeST1-2 expression was located to the epidermis only. Northern analysis shows that the mRNA abundances of both LeST1-1 and LeST1-2 are upregulated in the root in response to sulfate deprivation. LeST1-1 is specifically expressed in root tissue, a characteristic of Group-1 sulfate transporters. LeST1-2, however, was also detected in tomato leaves and stems and is upregulated and expressed to a similar extent in these tissues under conditions of sulfate deprivation. Induction of LeST1-2 expression was also observed in the vascular tissues of a resistant line of tomato infected with the vascular wilt pathogen Verticillium dahliae.
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.
The sulfation of dermatan sulfate by SO3-trimethylamine in N,N-dimethylformamide led to substitution initially at HO-6 of residues of 2-acetamido-2-deoxy-beta-D-galactopyranosyl 4-sulfate (1), to produce the 4,6-disulfate (6). When this step reached a level of greater than 50%, sulfation occurred with equal facility at HO-2 and HO-3 of residues of alpha-L-idopyranosyluronic acid (2), giving rise to a mixture of 2-,3-, and 2,3-disulfates. An analogous substitution pattern was observed for HO-2 and -3 of a simpler idopyranose unit, in the sulfation of methyl 4,6-O-benzylidene-alpha-D-idopyranoside (12). This lack of regioselectivity in the reaction of 2 (and 12) contrasts markedly with the high affinity of the reagent for HO-3 of residues of alpha-L-idopyranosyluronic acid present in a modified form of heparin. It is attributed to a difference between the two polymers in the relative orientation of their neighboring amino sugar residues, whereby there is an unobstructed access of the reagent in one instance, and hindrance of HO-2 selectively in the other. Enzymolysis by chondroitinase ABC was found to yield unsaturated disaccharide containing residues of 4,6-disulfate, as well as larger fragments containing unsaturated glycosyl groups derived from L-idopyranosyluronic acid 2-sulfate, evidence of a relatively broad enzyme specificity. The presence of extra sulfate groups in dermatan sulfate did not enhance its weak antithrombotic activity, as measured by anti Xa assay, in disagreement with earlier reports.
35S-heparan sulfate (HS) metabolism by intestinal epithelial cells isolated from streptozotocin-diabetic and control rats was studied. In diabetic cells, a greater amount of 35S-radioactivity was incorporated into HS, however specific radioactivity of this polysaccharide was decreased. Studies into the distribution of sulfate residues in HS after selective deamination of the glucosamine units within the glycosaminoglycan (GAG)-chain, demonstrated that O-sulfate groups are preferentially located in relatively small deamination products: tetrasaccharides and disaccharides. A lower amount of radioactivity related to N-sulfate groups was found in HS from diabetic cells compared to that of control cells demonstrating that, in diabetes, less glucosamine residues within HS chains are subjected to N-sulfation. An increase in the percentage of 35-sulfate and in the percentage amount of uronic acid in tetrasaccharides of HS of diabetic cells indicated that a greater number of tetrasaccharides were generated by deaminative degradation of this HS. Since a decrease in the specific activity of uronic acid in disaccharides as in tetrasaccharides from HS of diabetic cells was observed, it is clear that the degree of O-sulfation of this HS is reduced. It is suggested, that "in vivo" changes in HS metabolism in diabetic intestinal epithelial cells lie in a disturbance in the degree of N- and O-sulfation of disaccharide units within the HS macromolecule.
Histochemical and chemical analyses reveal important differences between tunic and body of the ascidian Styela plicata. The body contains hydroxyproline, nucleic acid and hexuronic acid. Analyses of the hexuronic acid-containing molecules indicates the presence of dermatan sulfate and heparan sulfate. The tunic, on the other hand, contains no hydroxyproline and small amounts of nucleic acid and hexuronic acid. Large amounts of sulfated polysaccharides, identified by agarose gel electrophoresis, are also present in the tunic. In vitro, incorporation of 35S-sulfate and 14C-glucose and 35S-sulfate pulse-chase experiments show that sulfate and glucose are incorporated into the sulfated polysaccharides of the tunic. The radioactivity is associated mainly with the region of the tunic containing the epidermal cells; however, a small amount of radioactivity is also detected in other regions of the tunic, suggesting that the sulfated polysaccharides are synthesized mainly by the epidermal cells and, to a small extent, by other cell types present in the tunic. Conversion of D-glucose to L-galactose, previously observed in the tunic of Styela plicata (Biochemistry 30, 3458-3464, 1990) is more intense in the region of the tunic containing the epidermal cells.