Separation and properties of five glycosaminoglycan sulfatases from rat skin.

Chondroitin sulfates, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and oligosaccharides derived from these sulfated glycosaminoglycans have been used for the measurement of sulfatase activity of rat skin extracts. Chromatographic fractionation of the extracts followed by specificity studies demonstrated the existence of five different sulfatases, specific for 1) the nonreducing N-acetylglucosamine 6-sulfate end groups of heparin sulfate and keratan sulfate, 2) the nonreducing N-acetylgalactosamine (or galactose) 6-sulfate end groups of chondroitin sulfate (or keratan sulfate), 3) the nonreducing N-acetylgalactosamine 4-sulfate end groups of chondroitin sulfate and dermatan sulfate, 4) certain suitably located glucosamine N-sulfate groups of heparin and heparan sulfate, or 5) certain suitably located iduronate sulfate groups of heparan sulfate and dermatan sulfate. Two arylsulfatases, one of which was identical in its chromatographic behaviors with the third enzyme described above, were also demonstrated in the extracts. These results taken together with those previously obtained from studies on human fibroblast cultures suggest that normal skin fibroblasts contain at least five specific sulfatases and diminished activity of any one may result in a specific storage disease.     

Direct observation of fibrinogen-heparinoid complexes formation using surface plasmon resonance.

We analyzed the binding of heparinoid or heparin with fibrinogen by real-time measurement using surface plasmon resonance technology. Poly(glucosyloxyethyl methacrylate) sulfate [poly(GEMA) sulfate] and dextran sulfate were used as heparinoids. The binding ability of each sulfated polymer was estimated by having each polymer-containing buffer interact with the sensor chip surfaces that had immobilized fibrinogen. Dextran sulfate and poly(GEMA) sulfate showed high affinity to the fibrinogen in this experiment, while the heparin did not. All of the dextran sulfates were desorbed from its surface, while about 30% of the poly(GEMA) sulfate remained on the immobilized fibrinogen upon the addition of NaCl to the buffer which was done in order to analyze the desorption of poly(GEMA) sulfate or dextran sulfate from the surface of the fibrinogen. These data show that the type of binding between fibrinogen-poly(GEMA) sulfate was different from that of dextran sulfate, indicating that the interaction between fibrinogen and poly(GEMA) sulfate was caused not only by an electrostatic but also by a hydrophobic force. These results suggest that the interaction mechanism of heparinoids with fibrinogen was different from that of heparin.     

Sulfate transport in human lung fibroblasts (IMR-90)

Sulfate transport in a fibroblast cell line derived from human lung (IMR-90) occurred mainly via high- and low-affinity, SITS-sensitive pathways and to a lesser extent by an SITS-insensitive mechanism. In low-ionic-strength media (sucrose substituted for salts) the apparent Km of the carrier-mediated sulfate influx was 1 mM. At 0.3 mM, the sulfate concentration normally found in human serum, the contribution of the SITS-insensitive pathway was negligible. In physiological salts solution, an SITS-sensitive, high-affinity (Km 34 +/- 14 microM) sulfate influx system was observed at extracellular sulfate concentrations less than 100 microM. Between 100 and 500 microM sulfate, the range normally found in human serum, sulfate influx occurred via an SITS-sensitive, low-affinity pathway and to a small extent by an SITS-insensitive mechanism. Extracellular chloride inhibited the influx and stimulated the efflux of sulfate. Bicarbonate and thiosulfate inhibited sulfate influx but had no effect on sulfate efflux. Phosphate, arsenate, or Na+ did not affect sulfate uptake. These results indicate that in human lung fibroblast IMR-90 cells sulfate is transported mainly via an SO4(2-)/Cl- exchange system independent of the phosphate or Na+ transport. Since sulfate concentration as high as 50 mM only slightly increased sulfate efflux, SO4(2-)/SO4(2-) exchange is probably a minor component of sulfate uptake.     

Competitive Mechanisms for Inhibition of Sulfate Reduction and Methane Production in the Zone of Ferric Iron Reduction in Sediments

Mechanisms for inhibition of sulfate reduction and methane production in the zone of Fe(III) reduction in sediments were investigated. Addition of amorphic iron(III) oxyhydroxide to sediments in which sulfate reduction was the predominant terminal electron-accepting process inhibited sulfate reduction 86 to 100%. The decrease in electron flow to sulfate reduction was accompanied by a corresponding increase in electron flow to Fe(III) reduction. In a similar manner, Fe(III) additions also inhibited methane production in sulfate-depleted sediments. The inhibition of sulfate reduction and methane production was the result of substrate limitation, because the sediments retained the potential for sulfate reduction and methane production in the presence of excess hydrogen and acetate. Sediments in which Fe(III) reduction was the predominant terminal electron-accepting process had much lower concentrations of hydrogen and acetate than sediments in which sulfate reduction or methane production was the predominant terminal process. The low concentrations of hydrogen and acetate in the Fe(III)-reducing sediments were the result of metabolism by Fe(III)-reducing organisms of hydrogen and acetate at concentrations lower than sulfate reducers or methanogens could metabolize them. The results indicate that when Fe(III) is in a form that Fe(III)-reducing organisms can readily reduce, Fe(III)-reducing organisms can inhibit sulfate reduction and methane production by outcompeting sulfate reducers and methanogens for electron donors.     

Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells

Bovine corneal endothelial (BCE) cells seeded and grown on plastic dishes were labeled with 35S-sulfate or 3H-glucosamine for 48 h at various phases of growth of the cultures. Newly synthesized proteoglycans were isolated from the culture medium and from the extracellular matrix (ECM) produced by the BCE cells, and the glycosaminoglycan (GAG) component of the proteoglycans was analyzed. Cells actively proliferating on plastic surfaces secreted an ECM that contained heparan sulfate as the major 35S-labeled GAG (86%) and dermatan sulfate as a minor component (13%). Upon reaching confluence, the BCE cells incorporated 35S-labeled chondroitin sulfate (20%), as well as heparan sulfate (66%) and dermatan sulfate (14%), into the EC. Seven-day postconfluent cells incorporated newly synthesized heparan sulfate and dermatan sulfate into the matrix in approximately equal proportions. Dermatan sulfate was the main 35S-labeled GAG (60-65%) in the medium of both confluent and postconfluent cultures. 35S-Labeled chondroitin sulfate (20-25%) and heparan sulfate (15%) were also secreted into the culture medium. The type of GAG incorporated into newly synthesized ECM was affected when BCE cells were seeded onto ECM-coated dishes instead of plastic. BCE cells actively proliferating on ECM-coated dishes incorporated newly synthesized heparan sulfate and dermatan sulfate into the ECM in a ratio that was very similar to the ratio of these GAGs in the underlying ECM. Addition of mitogens such as fibroblast growth factor (FGF) to the culture medium altered the type of GAG synthesized and incorporated into the ECM by BCE cells seeded onto ECM-coated dishes if the cells were actively growing, but had no effect on postconfluent cultures.     

Anticoagulant and antithrombin activities of oversulfated fucans.

Three species of oversulfated fucans having different sulfate contents (the ratio of sulfate/total sugar residues, 1.38-1.98) were prepared by chemical sulfation of a fucan sulfate (sulfate/sugar ratio, 1.28) isolated from the brown seaweed Ecklonia kurome. The anticoagulant activities of the oversulfated fucans were compared with that of a parent fucan with respect to activated partial thromboplastin time (APTT) and thrombin time (TT) in plasma. The respective activities (for APTT and TT) of the oversulfated fucans increased to 110-119% and 108-140% of the original values with increase in their sulfate content. The anticoagulant activity with respect to APTT (173 units/mg) of an oversulfated fucan (sulfate/sugar ratio, 1.98) was higher than that (167 units/mg) of heparin used as a standard. The heparin cofactor II-mediated antithrombin activity of the oversulfated fucans also increased significantly with increase in sulfate content. The maximum activity was higher than those of the parent fucan and heparin. However, the increment of the anticoagulant and the antithrombin effects gradually decreased with increase in the sulfate content of the fucans. These results indicate that the effects of the fucan sulfate are dependent on its sulfate content until a plateau is reached.     

Proteoheparan sulfate from human skin fibroblasts. Evidence for self-interaction via the heparan sulfate side chains

We have studied the affinity between fibroblast proteoheparan sulfate (medium- and cell surface-derived species) and heparan sulfate-agaroses by affinity chromatography. The evidence for an interaction between the heparan sulfate side chains of the proteoglycans and the immobilized heparan sulfate are as follows: (a) the individual side chains released from the proteoglycan by papain bind to the affinity matrix, (b) the bound proteoglycans are desorbed by a solution of cognate heparan sulfate chains, and (c) the core protein obtained by heparan sulfate-lyase digestion of the proteoglycan does not bind to the affinity matrix. The proteoglycans interact only with one subtype of heparan sulfate. The binding of free heparan sulfate chains to the affinity matrix is completely abolished by heparan sulfate oligosaccharides provided they are composed of both iduronate- and glucuronate-containing disaccharide sequences.     

Changes in heparan sulfate structure during transition from the proliferating to the non-dividing state of cultured arterial smooth muscle cells

Cultured arterial smooth muscle cells synthesize a cell-associated heparan sulfate proteoglycan which consists of a 92 kDa core protein with 3 to 4 heparan sulfate side chains covalently attached. Biosynthesis of the cell-associated heparan sulfate proteoglycan was compared in proliferating and in non-dividing vascular smooth muscle cells which are preincubated in the presence of [35]sulfate or a combination of [35S]methionine and [3H]glucosamine. The Mr of the core protein was identical in either growth state, but changes in the structure of the heparan sulfate side chains were observed. Non-dividing (postconfluent) arterial smooth muscle cells form longer heparan sulfate chains with a higher proportion of hydrophobic (N-acetyl) groups than proliferating (preconfluent) cells as judged from gel filtration experiments, hydrophobic interaction chromatography and heparitinase degradation. An enzyme preparation from proliferating cells catalyzes deacetylation and N-sulfation of heparan sulfate at a 5-fold higher activity than from non-dividing cells. Cell density-dependent structural differences of heparan sulfate are related to the finding that heparan sulfate isolated from non-dividing cells has a 10-fold higher antiproliferative potency than heparan sulfate from proliferating (preconfluent) cells.     

Sulfate Uptake in Saccharomyces cerevisiae: Biochemical and Genetic Study

Sulfate uptake is the first step of the sulfate assimilation pathway, which has been shown in our laboratory to be part of the methionine biosynthetic pathway. Kinetic study of sulfate uptake has shown a biphasic curve in a Lineweaver-Burk plot. The analysis of this plot indicates that two enzymes participate in sulfate uptake. One (permease I) has a high affinity for the substrate (K(m) = 0.005 mM); the other (permease II) shows a much lower affinity for sulfate (K(m) = 0.35 mM). Regulation of the synthesis of both permeases is under the control of exogenous methionine or S-adenosylmethionine. It was shown, moreover, that synthesis of sulfate permeases is coordinated with the synthesis of the other methionine biosynthetic enzymes thus far studied in our laboratory. An additional specific regulation of sulfate permeases by inhibition of their activity by endogenous sulfate and adenosyl phosphosulfate (an intermediate metabolite in sulfate assimilation) has been shown. A mutant unable to concentrate sulfate has been selected. This strain carried mutations in two independent genes. These two mutations, separated in two different strains, lead to modified kinetics of sulfate uptake. The study of these strains leads us to postulate that there is an interaction in situ between the products of these two genes.     

Transforming growth factor (type beta) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia

Cultured monolayers of NMuMG mouse mammary epithelial cells have augmented amounts of cell surface chondroitin sulfate glycosaminoglycan (GAG) when cultured in transforming growth factor-beta (TGF-beta), presumably because of increased synthesis on their cell surface proteoglycan (named syndecan), previously shown to contain chondroitin sulfate and heparan sulfate GAG. This increase occurs throughout the monolayer as shown using soluble thrombospondin as a binding probe. However, comparison of staining intensity of the GAG chains and syndecan core protein suggests variability among cells in the attachment of GAG chains to the core protein. Characterization of purified syndecan confirms the enhanced addition of chondroitin sulfate in TGF-beta: (a) radiosulfate incorporation into chondroitin sulfate is increased 6.2-fold in this proteoglycan fraction and heparan sulfate is increased 1.8-fold, despite no apparent increase in amount of core protein per cell, and (b) the size and density of the proteoglycan are increased, but reduced by removal of chondroitin sulfate. This is shown in part by treatment of the cells with 0.5 mM xyloside that blocks the chondroitin sulfate addition without affecting heparan sulfate. Higher xyloside concentrations block heparan sulfate as well and syndecan appears at the cell surface as core protein without GAG chains. The enhanced amount of GAG on syndecan is partly attributed to an increase in chain length. Whereas this accounts for the additional heparan sulfate synthesis, it is insufficient to explain the total increase in chondroitin sulfate; an approximately threefold increase in chondroitin sulfate chain addition occurs as well, confirmed by assessing chondroitin sulfate ABC lyase (ABCase)-generated chondroitin sulfate linkage stubs on the core protein. One of the effects of TGF-beta during embryonic tissue interactions is likely to be the enhanced synthesis of chondroitin sulfate chains on this cell surface proteoglycan.     

Isolation and characterization of proteoglycans secreted by normal and malignant human mammary epithelial cells

The proteoglycans secreted by a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal human breast cell line (HBL-100). The physicochemical characteristics of these proteoglycans were established by hexosamine analysis, chemical and enzymatic degradations, and dissociative cesium chloride density gradient centrifugation, and by gel filtration before and after alkaline beta-elimination. Both cell lines secreted approximately 70% of the synthesized proteoglycans, which were composed of 20% heparan sulfate and 80% chondroitin sulfate proteoglycans. The MDA cell line secreted large hydrodynamic size (major) and small hydrodynamic size heparan sulfate proteoglycan. In contrast HBL cells secreted only one species having a hydrodynamic size intermediate to the above two. The chondroitin sulfate proteoglycans from MDA medium were slightly larger than the corresponding polymers from HBL medium. All proteoglycans except the small hydrodynamic size heparan sulfate proteoglycan from MDA medium were of high buoyant density. The proteoglycans of both cell lines contained significant proportions of disulfide-linked lower molecular weight components which were more pronounced in the proteoheparan sulfate polymers, particularly those from MDA medium, than in chondroitin sulfate proteoglycans. The glycosaminoglycans of heparan sulfate proteoglycans from MDA medium were more heterogeneous than those from HBL medium. The glycosaminoglycan chains of large hydrodynamic size heparan sulfate proteoglycans from MDA medium were larger in size than those from HBL medium while small hydrodynamic size heparan sulfate proteoglycans contained shorter glycosaminoglycan chains. In contrast to the glycosaminoglycans derived from chondroitin sulfate proteoglycans of both MDA and HBL medium were comparable in size. The heparan sulfate as well as chondroitin sulfate proteoglycans of both cell lines contained both neutral (di- and tetrasaccharides) and sialylated (tri- to hexasaccharides) O-linked oligosaccharides.     

Inhibition studies of soybean and human 15-lipoxygenases with long-chain alkenyl sulfate substrates

Lipoxygenases are currently potential targets for therapies against asthma, artherosceloris, and cancer. Recently, inhibition studies on both soybean (SLO) and human lipoxygenase (15-HLO) revealed the presence of an allosteric site that binds both substrate, linoleic acid, and inhibitors; oleic acid (OA) and oleyl sulfate (OS). OS (K(D) approximately 0.6 microM) is a approximately 30-fold more potent inhibitor than OA (K(D) approximately 20 microM) due to the increased ionic strength of the sulfate moiety. To further investigate the role of the sulfate moiety on lipoxygenase function, SLO and 15-HLO were assayed against several fatty sulfate substrates (linoleyl sulfate (LS), cis-11,14-eicosadienoyl sulfate, and arachidonyl sulfate). The results demonstrate that SLO catalyzes all three fatty sulfate substrates and is not inhibited, indicating a binding selectivity of LS for the catalytic site and OS for the allosteric site. The 15-HLO, however, manifests parabolic inhibition kinetics with increasing substrate concentration, and it is irreversibly inhibited by these fatty sulfate substrates at high concentrations. The inhibition can be stopped, however, by the addition of detergent to the fatty sulfate mixture prior to the addition of 15-HLO. These results, combined with the modeling of the kinetic data, indicate that the inhibition of 15-HLO is due to a substrate aggregate. These substrate aggregates, however, do not inhibit SLO and could present a novel mode of inhibition for 15-HLO.     

Cadmium-induced sulfate uptake in maize roots

The effect of cadmium (Cd) on high-affinity sulfate transport of maize (Zea mays) roots was studied and related to the changes in the levels of sulfate and nonprotein thiols during Cd-induced phytochelatin (PC) biosynthesis. Ten micromolar CdCl(2) in the nutrient solution induced a 100% increase in sulfate uptake by roots. This was not observed either for potassium or phosphate uptake, suggesting a specific effect of Cd(2+) on sulfate transport. The higher sulfate uptake was not dependent on a change in the proton motive force that energizes it. In fact, in Cd-treated plants, the transmembrane electric potential difference of root cortical cells was only slightly more negative than in the controls, the external pH did not change, and the activity of the plasma membrane H(+)-ATPase did not increase. Kinetics analysis showed that in the range of the high-affinity sulfate transport systems, 10 to 250 microM, Cd exposure did not influence the K(m) value (about 20 microM), whereas it doubled the V(max) value with respect to the control. Northern-blot analysis showed that Cd-induced sulfate uptake was related to a higher level of mRNA encoding for a putative high-affinity sulfate transporter in roots. Cd-induced sulfate uptake was associated to both a decrease in the contents of sulfate and glutathione and synthesis of a large amount of PCs. These results suggest that Cd-induced sulfate uptake depends on a pretranslational regulation of the high-affinity sulfate transporter gene and that this response is necessary for sustaining the higher sulfur demand during PC biosynthesis.     

Mass transfer limitation of sulfate in methanogenic aggregates

The role of mass transfer limitation of sulfate as a factor governing the competition between sulfate reducing and methane producing bacteria in methanogenic aggregates was theoretically evaluated by the calculation of steady-state sulfate microprofiles using a reference set of parameters obtained from the literature. The shooting method was used as a numerical technique for solving the mathematical model. The effect of the parameters on mass transport limitation was tested by varying each reference value of the parameters with a factor of 3. Sulfate limitation within granules prevailed at moderate (0.1 kg m(-3)) and low sulfate concentrations in the bulk liquid, at high maximum sulfate utilization rates (3.73 x 10(-5) kg SO(4) (2-) kg(-1) VSS S(-1) or biomass concentrations (40 KG VSS m(-3)), and in large aggregates (radius of 7.5 10(-4) m). The effective diffusion coefficient of sulfate and the affinity constant were less determinative for the penetration depth of sulfate within a granule. (c) 1994 John Wiley & Sons, Inc.     

The susceptibility of the giant freshwater prawn Macrobrachium rosenbergii to Lactococcus garvieae and its resistance under copper sulfate stress.

Addition of copper sulfate (0.1 to 0.4 mg l(-1)) to tryptic soy broth (TSB) had no effect on growth rate of the bacterial pathogen Lactococcus garvieae. Giant freshwater prawns Macrobrachium rosenbergii were injected with L. garvieae (4 x 10(6) colony-forming units [cfu] prawn(-1)) grown in TSB or TSB containing copper sulfate at 0.1, 0.2, 0.3 or 0.4 mg l(-1). After 48 h, the cumulative mortality was significantly (p < 0.05) higher for prawns exposed to L. garvieae grown in 0.4 mg l(-1) copper sulfate than at the lower concentrations examined. In other experiments, prawns were injected with TSB-grown L. garvieae (4 x 10(6) and 2 x 10(5) cfu prawn(-1)), then held in water containing copper sulfate. After 8 h the mortality of L. garvieae-exposed prawns held in water containing 0.4 mg l(-1) copper sulfate was significantly higher than prawns held in water containing 0.2 and 0.3 mg l(-1) copper sulfate. At the lower L. garvieae density, cumulative mortality of prawns increased directly with ambient copper sulfate concentrations in the range of 0.2 to 0.4 mg l(-1). All prawns survived a 168 h exposure to 0.1 mg l(-1) copper sulfate. Prawns exposed to different concentrations of copper sulfate were examined for hemocyte density, phenoloxidase activity and respiratory burst. No significant differences in hemocyte density were observed among treatments. In prawns following a 48 h exposure to 0.1 mg l(-1) copper sulfate, phenoloxidase activity was decreased, but respiratory burst was increased. In conclusion, copper sulfate increased the virulence of L. garvieae to M. rosenbergii and modulated its immune system. Copper sulfate at 0.1 mg l(-1) decreased susceptibility of M. rosenbergii to L garvieae infection, whereas at 0.2 mg l(-1) the susceptibility was increased. The generation of superoxide anion by M. rosenbergii exposed to copper sulfate at a concentration higher than 0.2 mg l(-1) was considered to be cytoxic.     

Isolation and characterization of an asparagine-linked keratan sulfate from the skin of a marine teleost, Scomber japobicus

Keratan sulfate was isolated from the skin of Pacific mackerel (Scomber japonicus) after exhaustive digestion with pronase followed by ethanol precipitation and fractionation on a cellulose column with 0.3% recovery of dried material. The keratan sulfate preparation was separated into four major fractions by Dowex-1 column chromatrography. The chemical and infrared spectrum analyses of the four fractions showed a high degree of heterogeneity in sulfation. Since the carbohydrate-peptide linkage in the teleost skin keratan sulfate was found to be stable in alkali, and asparagine was the predominant amino acid, the asparagine residue in the peptide backbone was most likely to be involved in the N-glycosyl linkage with the carbohydrate moiety. Besides the type of carbohydrate-peptide linkage, the teleost skin keratan sulfate is very similar to corneal keratan sulfate, (keretan sulfate I) in two respects: (1) The teleost skin and bovine corneal keratan sulfates were hydrolyzed much faster by endo-β-galactosidase that the whale nasal cartilage keratan sulfate (keratan sulfate II). (2) Although the teleost skin keratan sulfate showed considerable polydispersity, the molecular weight was in the same range as the corneal keratan sulfate, and it was relatively higher than that of the cartilage keratan sulfate.     

Periplasmic cytochrome c3 of Desulfovibrio vulgaris is directly involved in H2-mediated metal but not sulfate reduction

Kinetic parameters and the role of cytochrome c(3) in sulfate, Fe(III), and U(VI) reduction were investigated in Desulfovibrio vulgaris Hildenborough. While sulfate reduction followed Michaelis-Menten kinetics (K(m) = 220 micro M), loss of Fe(III) and U(VI) was first-order at all concentrations tested. Initial reduction rates of all electron acceptors were similar for cells grown with H(2) and sulfate, while cultures grown using lactate and sulfate had similar rates of metal loss but lower sulfate reduction activities. The similarities in metal, but not sulfate, reduction with H(2) and lactate suggest divergent pathways. Respiration assays and reduced minus oxidized spectra were carried out to determine c-type cytochrome involvement in electron acceptor reduction. c-type cytochrome oxidation was immediate with Fe(III) and U(VI) in the presence of H(2), lactate, or pyruvate. Sulfidogenesis occurred with all three electron donors and effectively oxidized the c-type cytochrome in lactate- or pyruvate-reduced, but not H(2)-reduced cells. Correspondingly, electron acceptor competition assays with lactate or pyruvate as electron donors showed that Fe(III) inhibited U(VI) reduction, and U(VI) inhibited sulfate loss. However, sulfate reduction was slowed but not halted when H(2) was the electron donor in the presence of Fe(III) or U(VI). U(VI) loss was still impeded by Fe(III) when H(2) was used. Hence, we propose a modified pathway for the reduction of sulfate, Fe(III), and U(VI) which helps explain why these bacteria cannot grow using these metals. We further propose that cytochrome c(3) is an electron carrier involved in lactate and pyruvate oxidation and is the reductase for alternate electron acceptors with higher redox potentials than sulfate.     

Structural characterization of human liver heparan sulfate

The isolation, purification and structural characterization of human liver heparan sulfate are described. 1H-NMR spectroscopy demonstrates the purity of this glycosaminoglycan (GAG) and two-dimensional 1H-NMR confirmed that it was heparan sulfate. Enzymatic depolymerization of the isolated heparan sulfate, followed by gradient polyacrylamide gel, confirmed its heparin lyase sensitivity. The concentration of resulting unsaturated disaccharides was determined using reverse phase ion-pairing (RPIP) HPLC with post column derivatization and fluorescence detection. The results of this analysis clearly demonstrate that the isolated GAG was heparan sulfate, not heparin. Human liver heparan sulfate was similar to heparin in that it has a reduced content of unsulfated disaccharide and an elevated average sulfation level. The antithrombin-mediated anti-factor Xa activity of human liver heparan sulfate, however, was much lower than porcine intestinal (pharmaceutical) heparin but was comparable to standard porcine intestinal heparan sulfate. Moreover, human liver heparan sulfate shows higher degree of sulfation than heparan sulfate isolated from porcine liver or from the human hepatoma Hep 2G cell line.     

Heparan sulfate regulates VEGF165- and VEGF121-mediated vascular hyperpermeability

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF(165) and VEGF(121). Because VEGF(121) does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF(165) and VEGF(121). Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF(165) and VEGF(121), suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.     

The disaccharide repeating-units of heparan sulfate

Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.