Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity

Dermatan sulfate increases the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 1000-fold by providing a catalytic template to which both the inhibitor and the protease bind. Dermatan sulfate is a linear polymer of D-glucuronic acid (GlcA) or L-iduronic acid (IdoA) alternating with N-acetyl-D-galactosamine (GalNAc) residues. Heterogeneity in dermatan sulfate results from varying degrees of O-sulfation and from the presence of the two types of uronic acid residues. To characterize the HCII-binding site in dermatan sulfate, we isolated the smallest fragment of dermatan sulfate that bound to HCII with high affinity. Dermatan sulfate was partially N-deacetylated by hydrazinolysis, cleaved with nitrous acid at pH 4, and reduced with [3H]NaBH4. The resulting fragments, containing an even number of monosaccharide units with the reducing terminal GalNAc converted to [3H]2,5-anhydro-D-talitol (ATalR), were size-fractionated and then chromatographed on an HCII-Sepharose column. The smallest HCII-binding fragments were hexasaccharides, of which approximately 6% bound. Based on ion-exchange chromatography, the bound material appeared to comprise a heterogeneous mixture of molecules possessing four, five, or six sulfate groups per hexasaccharide. Subsequently, hexasaccharides with the highest affinity for HCII were isolated by overloading the HCII-Sepharose column. The high-affinity hexasaccharides were fractionated by strong anion-exchange chromatography, and one major peak representing approximately 2% of the starting hexasaccharides was isolated. The high-affinity hexasaccharide was cleaved to disaccharides that were analyzed by anion-exchange chromatography, paper electrophoresis, and paper chromatography. A single disulfated disaccharide, IdoA(2-SO4)----ATalR(4-SO4) was observed, indicating that the hexasaccharide has the following structure: IdoA(2-SO4)----GalNAc(4-SO4)----IdoA(2-SO4)---- GalNAc(4-SO4)----IdoA(2-SO4)----ATalR(4-SO4). Since IdoA(2-SO4)----GalNAc(4-SO4) comprises only approximately 5% of the disaccharides present in intact dermatan sulfate, clustering of these disaccharides must occur during biosynthesis to form the high-affinity binding site for HCII.     

Molecular size of dermatan sulfate oligosaccharides required to bind and activate heparin cofactor II

Heparin cofactor II (HCII) inhibits thrombin rapidly in human plasma in the presence of heparin or dermatan sulfate. To determine the minimum structure of dermatan sulfate required to activate HCII, the glycosaminoglycan was partially degraded by sequential treatment with periodate, [3H]borohydride, and sulfuric acid. Labeled oligosaccharide fragments were separated by gel filtration chromatography. Purified fragments were then applied to a column of HCII bound to concanavalin A-Sepharose, and bound oligosaccharides were eluted with a gradient of sodium chloride. Di-, tetra-, and hexasaccharide fragments did not bind to HCII, while 15% of the octasaccharides and up to 45% of larger fragments bound. Octasaccharides that bound to the HCII column had a greater negative charge than the run-through material based on anion-exchange chromatography, suggesting that they contained a greater number of sulfate groups per molecule. Fragments of dermatan sulfate containing a minimum of 12-14 sugar residues accelerated inhibition of thrombin by HCII. Fragments of this length that bound to the column of immobilized HCII had molar specific activities greater than those of the fragments that did not bind. These studies suggest that HCII is activated by dermatan sulfate fragments greater than or equal to 12 residues in length that contain a specific octasaccharide sequence required for binding to the inhibitor.     

Binding of heparin or dermatan sulfate to thrombin is essential for the sulfated polysaccharide-accelerated inhibition of thrombin by heparin cofactor II

Heparin cofactor II (HC II) and thrombin were chemically modified with pyridoxal 5'-phosphate, and their effects on the inhibition of thrombin by HC II in the presence of heparin or dermatan sulfate were studied. The inhibition of thrombin by HC II was enhanced about 7000-fold in the presence of heparin or dermatan sulfate. However, this enhancement by heparin dwindled to 110- and 9.6-fold when the modified HC II and the modified thrombin, respectively, were substituted for native proteins. Essentially identical results were obtained from the experiments using dermatan sulfate. These results indicate that the binding of heparin or dermatan sulfate to both thrombin and HC II is required for the sulfated polysaccharide-dependent acceleration of the thrombin inhibition by HC II, and the binding to thrombin is more essential for the reaction.     

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.     

Molecular basis for the susceptibility of fibrin-bound thrombin to inactivation by heparin cofactor ii in the presence of dermatan sulfate but not heparin

Although fibrin-bound thrombin is resistant to inactivation by heparin.antithrombin and heparin.heparin cofactor II complexes, indirect studies in plasma systems suggest that the dermatan sulfate.heparin cofactor II complex can inhibit fibrin-bound thrombin. Herein we demonstrate that fibrin monomer produces a 240-fold decrease in the heparin-catalyzed rate of thrombin inhibition by heparin cofactor II but reduces the dermatan sulfate-catalyzed rate only 3-fold. The protection of fibrin-bound thrombin from inhibition by heparin.heparin cofactor II reflects heparin-mediated bridging of thrombin to fibrin that results in the formation of a ternary heparin.thrombin.fibrin complex. This complex, formed as a result of three binary interactions (thrombin.fibrin, thrombin.heparin, and heparin.fibrin), limits accessibility of heparin-catalyzed inhibitors to thrombin and induces conformational changes at the active site of the enzyme. In contrast, dermatan sulfate binds to thrombin but does not bind to fibrin. Although a ternary dermatan sulfate. thrombin.fibrin complex forms, without dermatan sulfate-mediated bridging of thrombin to fibrin, only two binary interactions exist (thrombin.fibrin and thrombin. dermatan sulfate). Consequently, thrombin remains susceptible to inactivation by heparin cofactor II. This study explains why fibrin-bound thrombin is susceptible to inactivation by heparin cofactor II in the presence of dermatan sulfate but not heparin.     

Osmotic stress regulates the anticoagulant efficiency of dermatan sulfate.

Glycosaminoglycans (GAGs) in pericellular and interstitial spaces help to maintain local water homeostasis and blood coagulation balance. This study explored whether dehydrating microenvironment conditions influence dermatan sulfate's (DS) anticoagulant activity. Water transfer during antithrombin activation by dermatan sulfate was measured using osmotic stress techniques. Anticoagulant activity was determined from the change in the rate of coagulation factor Xa (fXa) inhibition. Osmotic stress accelerated reaction rates, indicating water transfer from reactants to bulk. The net volume transferred, measured using osmotic probes similar in size to the reacting proteins, was approximately 2500 mol of water per mole of fXa inhibited. The reaction efficiency, V(sat)/K 1/2 (rate at saturation/concentration resulting in half-maximal rates), determined in titrations with monosulfated dermatan sulfate and disulfated dermatan sulfate (DDS), were 4x10(4) and 2x10(5) M-1 s-1 under osmotic stress and in the presence of calcium, corresponding to 34- and 81-fold increases over efficiency measured under standard conditions. These results indicate that dermatan sulfate can contribute significantly to antithrombin activation, and that in dehydrating environments and depending of ionic conditions, its anticoagulant efficiency can exceed that of heparan sulfate (HS).     

N-Acetylgalactosamine 4,6-O-sulfate residues mediate binding and activation of heparin cofactor II by porcine mucosal dermatan sulfate

Dermatan sulfate (DS) accelerates the inhibition of thrombin by heparin cofactor II (HCII). A hexasaccharide consisting of three l-iduronic acid 2-O-sulfate (IdoA2SO3)-->N-acetyl-D-galactosamine 4-O-sulfate (GalNAc4SO3) subunits was previously isolated from porcine skin DS and shown to bind HCII with high affinity. DS from porcine intestinal mucosa has a much lower content of this disaccharide but activates HCII with potency similar to that of porcine skin DS. Therefore, we sought to characterize oligosaccharides from porcine mucosal DS that interact with HCII. DS was partially depolymerized with chondroitinase ABC, and oligosaccharides containing 2-12 monosaccharide units were isolated. The oligosaccharides were then fractionated by anion-exchange and affinity chromatography on HCII-Sepharose, and the disaccharide compositions of selected fractions were determined. We found that the smallest oligosaccharides able to bind HCII were hexasaccharides. Oligosaccharides 6-12 units long that lacked uronic acid (UA)2SO3 but contained one or two GalNAc4,6SO3 residues bound, and binding was proportional to both oligosaccharide size and number of GalNAc4,6SO3 residues. Intact DS and bound dodecasaccharides contained predominantly IdoA but little D-glucuronic acid. Decasaccharides and dodecasaccharides containing one or two GalNAc4,6SO3 residues stimulated thrombin inhibition by HCII and prolonged the clotting time of normal but not HCII-depleted human plasma. These data support the hypothesis that modification of IdoA-->GalNAc4SO3 subunits in the DS polymer by either 2-O-sulfation of IdoA or 6-O-sulfation of GalNAc can generate molecules with HCII-binding sites and anticoagulant activity.     

Formation of dermatan sulfate by cultured human skin fibroblasts. Effects of sulfate concentration on proportions of dermatan/chondroitin

[3H,35S]Dermatan/chondroitin sulfate glycosaminoglycans produced during culture of fibroblasts in medium containing varying concentrations of sulfate were tested for their susceptibility to chondroitin ABC lyase and chondroitin AC lyase. Chondroitin ABC lyase completely degraded [3H]hexosamine-labeled and [35S] sulfate-labeled dermatan/chondroitin sulfate to disaccharides. Chondroitin AC lyase treatment of the labeled glycosaminoglycans produced different results. With this enzyme, dermatan/chondroitin sulfate formed at high concentrations of sulfate yielded small glycosaminoglycans and larger oligosaccharides but almost no disaccharide. This indicated that the dermatan/chondroitin sulfate co-polymer contained mostly iduronic acid with only an occasional glucuronic acid. As the medium sulfate concentration was progressively lowered, there was a concomitant increase in the susceptibility to degradation by chondroitin AC lyase. Thus, the labeled glycosaminoglycans formed at the lowest concentration of sulfate yielded small oligosaccharides including substantial amounts of disaccharide. The smaller chondroitin AC lyase-resistant [3H,35S]dermatan/chondroitin sulfate oligosaccharides were analyzed by gel filtration. Results indicated that, in general, the iduronic acid-containing disaccharide residues present in the undersulfated [3H,35S]glycosaminoglycan were sulfated, whereas the glucuronic acid-containing disaccharide residues were non-sulfated. This work confirms earlier reports that there is a relationship between epimerization and sulfation. Moreover, it demonstrates that medium sulfate concentration is critical in determining the proportions of dermatan to chondroitin (iduronic/glucuronic acid) produced by cultured cells.     

Altered dermatan sulfate structure and reduced heparin cofactor II-stimulating activity of biglycan and decorin from human atherosclerotic plaque

Biglycan and decorin are small dermatan sulfate-containing proteoglycans in the extracellular matrix of the artery wall. The dermatan sulfate chains are known to stimulate thrombin inhibition by heparin cofactor II (HCII), a plasma proteinase inhibitor that has been detected within the artery wall. The purpose of this study was to analyze the HCII-stimulatory activity of biglycan and decorin isolated from normal human aorta and atherosclerotic lesions type II through VI and to correlate activity with dermatan sulfate chain composition and structure. Biglycan and decorin from plaque exhibited a 24-75% and 38-79% loss of activity, respectively, in thrombin-HCII inhibition assays relative to proteoglycan from normal aorta. A significant negative linear relationship was observed between lesion severity and HCII stimulatory activity (r = 0.79, biglycan; r = 0.63, decorin; p < 0.05). Biglycan, but not decorin, from atherosclerotic plaque contained significantly reduced amounts of iduronic acid and disulfated disaccharides DeltaDi-2,4S and DeltaDi-4,6S relative to proteoglycan from normal artery. Affinity coelectrophoresis analysis of a subset of samples demonstrated that increased interaction of proteoglycan with HCII in agarose gels paralleled increased activity in thrombin-HCII inhibition assays. In conclusion, both biglycan and decorin from atherosclerotic plaque possessed reduced activity with HCII, but only biglycan demonstrated a correlation between activity and specific glycosaminoglycan structural features. Loss of the ability of biglycan and decorin in atherosclerotic lesions to regulate thrombin activity through HCII may be critical in the progression of the disease.     

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.     

Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan

We purified dermatan sulfate proteoglycan (PG) from the capsule of human ovarian fibroma for use as an immunogen. A monoclonal antibody, designated 6B6, was produced which reacts to the intact molecule of dermatan sulfate PG and the chondroitinase AC-treated core molecule on Western-blotted nitrocellulose membrane. Localization of materials showing crossreactivity to this antibody was studied in human tissues by indirect immunohistochemistry. The interstitial elements of almost all tissues examined were positive for the antibody: dermis, submucosal layer of digestive tract, perichondral layer, perivascular connective tissue, perineurium, adventitia of aorta, vessel wall of vein, pleura, and fibrous capsule of kidney and liver. Positive staining was also observed in fibrous elements at post-necrotic foci of cardiac muscle and pancreas, and at atherosclerotic lesions of aorta. The distribution of the antigen, core protein of the dermatan sulfate PG, revealed with 6B6 was compared to that of the dermatan sulfate side chain, which was demonstrated with antibody 9A-2 (Couchman et al.: Nature 307:650, 1984) after treatment with chondroitin sulfate B-lyase. The distribution of both antigens, core protein, and dermatan sulfate side chains showed the same pattern, with minor exceptions. The antibody 6B6 will be a useful tool to study the immunohistochemical localization of dermatan sulfate PG.     

Binding of chondroitin sulfate, dermatan sulfate and fat-storing cell-derived proteoglycans to rat hepatocytes

1. The interaction of isolated rat hepatocytes with exogenous 3H-labeled chondroitin-4-sulfate and dermatan sulfate and with biosynthetically 35S-labeled proteoglycans secreted by cultured rat liver fat-storing cells has been studied. 2. All ligands are bound by hepatocytes in a concentration-dependent manner. Scatchard-plot analysis of the data revealed the existence of high- and low-affinity binding modes. 3. The cell-bound exogenous [3H]glycosaminoglycans could be displaced by each unlabeled ligand and by heparin, whereas displacement of the endogenous material was less effective. 4. Binding of all ligands to hepatocytes increased with time. For the exogenous glycosaminoglycans the two- to threefold amount was retained at 37 degrees C as compared to 4 degrees C; it was markedly reduced by pretreatment of the cells with trypsin. 5. Degradation of the exogenous ligands could be detected neither for the cell-bound fraction nor for the free glycosaminoglycans in the culture medium. 6. The binding of the ligands to hepatocytes is viewed as a cell-matrix interaction. Its possible pathobiochemical relevance in liver fibrosis or neoplasia is discussed.     

Regioselectivity in the sulfation of dermatan sulfate and methyl 4,6-O-benzylidene-alpha-D-idopyranoside.

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.