Biosynthesis of heparin/heparan sulfate: kinetic studies of the glucuronyl C5-epimerase with N-sulfated derivatives of the Escherichia coli K5 capsular polysaccharide as substrates

Åsa Hagner-McWhirter, Helgi H.Hannesson, Patrick Campbell2, John Westley3, Lennart Rodén2, Ulf Lindahl and Jin-Ping Li1

Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 582, S-751 23, Uppsala, Sweden, 2Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA, and 3Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA

Received on May 10, 1999; revised on July 5, 1999; accepted on July 22, 1999.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-3H]glucose. Following complete or partial (~50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine·SO3 complex, which yielded products that were recognized as substrates by the epimerase and released tritium from C5 of the D-glucuronyl residues upon incubation with the enzyme. Comparison of the kinetic properties of the two substrates showed that the fully N-sulfated derivative was the best substrate in terms of its Km value, which was significantly lower than that of its partially N-acetylated counterpart. The Vmax values for the E.coli polysaccharide derivatives were essentially the same but were both lower than that of the O-desulfated [3H]heparin used in our previous studies. Surprisingly, the apparent Km values for all three substrates increased with increasing enzyme concentration. The reason for this phenomenon is not entirely clear at present. Partially purified C5-epimerase preparations from the Furth mastocytoma and bovine liver, respectively, behaved similarly in terms of their reactivity towards the various substrates, but the variation in apparent Km values with enzyme concentration precluded a detailed comparison of their kinetic properties.

Key words: Escherichia coli K5/heparan sulfate/heparin/glucuronyl C5-epimerase/mastocytoma


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The assembly of the polysaccharide chains of heparin and heparan sulfate proteoglycans is a multi-step process effected by at least 11 different enzymes (Lindahl, 1989Go; Salmivirta et al., 1996Go). After initial formation of a specific carbohydrate-protein linkage region and a polysaccharide backbone composed of alternating D-glucuronic acid and N-acetylglucosamine units, several modifications of the latter region take place, which include deacetylation of the N-acetylglucosamine residues and subsequent N-sulfation, C5-epimerization of D-glucuronic (GlcA) to L-iduronic acid (IdoA) residues, and O-sulfation in four positions. The C5-epimerization is unique among the metabolic sugar interconversions inasmuch as it does not occur at the level of small metabolites but only after the GlcA residues have already been incorporated into the growing polysaccharide chains. A GlcA residue is not recognized as a substrate for the epimerase until at least one of the adjacent N-acetylglucosamine residues, specifically the residue linked to C4 of the target uronic acid, has been deacetylated and N-sulfated (Jacobsson et al., 1984Go). Whereas N-sulfate groups are thus an essential part of the substrate structure, O-sulfate groups are not, and, indeed, O-sulfation at C6 of the glucosamine residues on either side of the uronic acid, or at C2 of the uronic acid unit itself, renders the latter inaccessible to the epimerase.

During the epimerization, the C5 hydrogen of the hexuronic acid is abstracted and replaced by hydrogen from water (Jacobsson et al., 1979aGo; Prihar et al., 1980Go). This feature of the reaction is the basis for the assay of epimerase activity currently used (Campbell et al., 1983Go; Jensen et al., 1983Go). The substrate for the enzyme is prepared by N-deacetylation and complete desulfation of heparin, followed by N-sulfation, and incubation with the epimerase in the presence of 3H2O then yields a product in which tritium has been incorporated at C5 of both GlcA and IdoA residues. Upon incubation of the labeled polysaccharide with epimerase preparations to be analyzed, the tritium is released as 3H2O, and the rate of this reaction provides a measure of the activity of the enzyme.

The glucuronyl C5-epimerase catalyzing formation of IdoA residues in the course of heparan sulfate biosynthesis has previously been purified to homogeneity from bovine liver (Campbell et al., 1994Go), and its primary structure has been determined by cDNA cloning (Li et al., 1997Go). Some of its basic molecular and catalytic properties, however, have not yet been elucidated; e.g., we have only scant knowledge of the kinetics of the epimerase reaction.

In the present study, we have developed a new procedure for substrate preparation based on metabolic labeling of the capsular polysaccharide of Escherichia coli K5, which has the same structure, [GlcAß1,4-GlcNAc{alpha}1,4]n, as the nonsulfated precursor polysaccharide in heparin/heparan sulfate biosynthesis (Vann et al., 1981Go). The bacterial polysaccharide is labeled at C5 of the GlcA (and N-acetylglucosamine) residues by growing E.coli K5 bacteria in the presence of D-[5-3H]glucose, and the isolated polysaccharide is then converted into an epimerase substrate by chemical N-deacetylation and N-sulfation. Since glucuronic acid is its sole uronic acid constituent, the derivative of the bacterial polysaccharide is more akin to the physiological substrates of the epimerase than the O-desulfated [5-3H]uronyl-heparin used previously (Jensen et al., 1983Go), which contains a large proportion of iduronic acid residues. Using the metabolically labeled K5 polysaccharides as substrate, we have examined the kinetics of the epimerase reaction in some detail and have also, with the use of partially N-deacetylated/N-sulfated derivatives, determined the effect of N-acetylated glucosamine residues located in the vicinity of the glucuronic acid residues targeted by the enzyme. Furthermore, we have compared the kinetic properties of epimerase preparations from mouse mastocytoma and bovine liver with a view to exploring further the problem why connective-tissue type mast cells make heparin (Enerbäck, 1989Go; Nader and Dietrich, 1989Go) while all other cell types, with an apparently similar synthetic apparatus, produce the closely related but less extensively modified heparan sulfates (Gallagher and Walker, 1985Go; Gallagher et al., 1986Go; Kjellén and Lindahl, 1991Go). An additional incentive for undertaking this comparison was the finding that multiple forms of the N-deacetylase/N-sulfotransferase occur in various tissues (Hashimoto et al., 1992Go; Eriksson et al., 1994Go; Orellana et al., 1994Go), pointing to the possibility that similar polymorphism may apply to the epimerase so as to modulate the biosynthetic process toward the formation of either heparan sulfate or heparin.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The present investigation addresses the following specific questions concerning the kinetic properties of the GlcA C5-epimerase involved in the biosynthesis of heparin and heparan sulfate: What are the basic kinetic parameters of the fully N-deacetylated/N-sulfated E.coli K5 polysaccharide? What effect, if any, do N-acetylated (rather than N-sulfated) glucosamine residues in the immediate vicinity of a glucuronic acid residue have on its reactivity in the epimerase reaction? Does the size of the polysaccharide substrate influence the kinetics of the reaction? Is the bacterial polysaccharide derivative a better substrate than the O-desulfated heparin used previously? Is there any difference in kinetic behavior between liver and mastocytoma epimerase? By and large, these questions have been answered by the experiments described in the following account. Of particular interest is the finding that the kinetic behavior of the epimerase reaction does not conform to that expected for the simplest steady-state model.

Characterization of tritium-labeled E.coli K5 polysaccharide and its N-deacetylated/ N-sulfated derivatives
The polysaccharide isolated from a 45-ml culture incubated with 3 mCi of D-[5-3H]glucose (see Materials and methods) contained a total of 1.5 mg of HexA and had a specific radio­activity of 70 x 106 cpm/mg HexA. Upon analytical anion-exchange chromatography on DEAE-Sephacel of a sample of the labeled polysaccharide in mixture with the isomeric glycosaminoglycan, hyaluronan, both polysaccharides were eluted in essentially the same position (Figure 1A). As expected, the ~50% and ~100% N-deacetylated/N-sulfated derivatives were eluted in more retarded positions. Gel chromatography of the native K5 polysaccharide and the two derivatives on Superose 6 showed that the hydrazinolysis procedure had resulted in substantial depolymerization (Figure 1B). The peak elution positions of the rather broad peaks corresponded to species of ~57 kDa for the native K5 polysaccharide (~150 disaccharide units), ~30 kDa for the ~50% N-sulfated derivative (~75 disaccharide units), and ~16 kDa for the fully N-sulfated derivative (~40 disaccharide units). This effect of hydrazinolysis is in accord with previous observations (Shaklee and Conrad, 1984Go). Deaminative cleavage of the ~100% N-sulfated derivative at pH 1.5 yielded predominantly nonsulfated disaccharides, as indicated by the gel chromatography profile shown in Figure 2A. Small amounts of tetrasaccharides and larger oligosaccharides were also formed, suggesting that the N-deacetylation had not been complete. However, part of the tetra- and larger oligosaccharides were due to "anomalous" ring contraction during the deaminative cleavage, as indicated by repeated gel chromatography following mild acid treatment (data not shown) (Shively and Conrad, 1976bGo). When this was taken into account, the proportion of N-sulfated residues in the modified K5 polysaccharide was calculated to be ~92%. The ~50% N-sulfated species also yielded disaccharides but, in addition, appreciable amounts of tetrasaccharides and larger oligosaccharides (Figure 2B). Calculations based on the proportions of disaccharides and larger oligosaccharides indicated that approximately 50% of the GlcN units were N-sulfated. However, in view of the incomplete separation of the larger oligosaccharides, this ratio could not be established with certainty. Elution patterns similar to those in Figure 2 were observed when the partially and essentially completely N-deacetylated (1.5 and 5 h hydrazinolysis, respectively) but not yet N-sulfated K5 samples were reacted with HNO2 at pH 3.9 (data not shown) (see Shively and Conrad, 1976aGo).



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Fig. 1. Characterization of [5-3H]GlcA-labeled E.coli K5 polysaccharide and its derivatives by anion-exchange chromatography (A) and gel chromatography (B). (A) Samples (20,000 cpm) of native (solid circles), ~50% N-deacetylated/N-sulfated (open diamonds), and ~100% N-deacetylated/N-sulfated (open circles) polysaccharide were analyzed by chromatography on DEAE-Sephacel. Effluent fractions were analyzed for radioactivity. The elution positions of hyaluronan (HA) and heparin, determined by carbazole analysis after chromatography of unlabeled polysaccharide standards, are indicated by arrows. (B) Samples (20,000 cpm) of native and modified polysaccharides (for symbols, see A) were analyzed on a Superose 6 column. The elution positions of molecular-weight standards derived from heparin and hyaluronan are indicated by arrows. The effluent fractions containing the ~100% N-sulfated K5 polysaccharide were pooled as indicated to yield material with ~100 disaccharides (pool 1) and 8–18 disaccharides (pool 2). For additional information, see Materials and methods.

 


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Fig. 2. N-Substituent analysis of modified, [5-3H]GlcA-labeled K5 polysaccharides. Samples of metabolically labeled K5 polysaccharide, ~100% (A) or ~50% N-deacetylated/N-sulfated (B), were deaminated by treatment with HNO2 at pH 1.5, and the products were reduced with NaBH4. The resultant material was subjected to gel chromatography on a Superdex 30 column. Effluent fractions were analyzed for radioactivity. The approximate elution positions of disaccharides (2), tetrasaccharides (4), hexasaccharides (6), octasaccharides (8), and larger oligosaccharides (>8) are indicated. For additional information see Materials and methods.

 
To determine the distribution of the 3H-label between the GlcN and GlcA units of the K5 polysaccharide, a sample of the disaccharide GlcA-aManR obtained by deaminative cleavage (Figure 2A) was digested with ß-D-glucuronidase, and the products were subjected to high-voltage paper electrophoresis at pH 5.3 (Figure 3A). Analysis of the electropherogram showed that ~60% of the 3H comigrated with GlcA mono­saccharide, while ~40% remained at the origin and presumably consisted of anhydromannitol derived from GlcN units in the intact polysaccharide.



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Fig. 3. Location of 3H radioactivity in metabolically labeled K5 polysaccharide. (A) Distribution of radiolabel between the two monosaccharide moieties. The GlcA-aManR disaccharide pool from the experiment shown in Figure 2A was desalted by lyophilization, and the labeled disaccharide was digested with ß-D-glucuronidase from bovine liver as described previously (Jacobsson et al., 1979bGo). The digest was treated with 0.25 M NH4OH to prevent lactonization of the free GlcA and subjected to high-voltage paper electrophoresis on Whatman No. 3 MM paper in 0.83 M pyridine, 0.5 M acetic acid, pH 5.3, at 80 V/cm for 40 min. Paper strips were cut into 1 cm segments that were eluted with ~1 ml of water and analyzed for radioactivity. The migration positions of standards are indicated by arrows: 1, aManR monosaccharide; 2, intact GlcA-aManR disaccharide; 3, GlcA monosaccharide. (B) Heparitinase digestion of ~50 % N-deacetylated/N-sulfated K5 [3H]polysaccharide. A sample (80,000 cpm) of the 3H-labeled polymer was mixed with 10,000 cpm of 14C-labeled, unmodified K5 polysaccharide and incubated at 43°C for 23 h with 5 mU of heparitinase in 150 µl of 50 mM Hepes, pH 7.0, containing 1 mM CaCl2. The digest was chromatographed on a column (1 x 190 cm) of Sephadex G-25 in 0.2 M NH4HCO3, and the effluent was analyzed for 3H (open circles) and 14C (solid circles).

 
The location of the radioactivity in the GlcA units was examined by digestion of the 50% N-sulfated derivative with bacterial heparitinase I. This enzyme cleaves linkages between N-acetylated or N-sulfated glucosamine residues and GlcA, yielding disaccharides with a {Delta}4,5-unsaturated uronic acid at the nonreducing terminus (Linhardt et al., 1990Go). In the course of the reaction, the C5 hydrogen of the GlcA is released and converted to water. Upon gel chromatography of the heparitinase digest, two major radioactive peaks were observed, one in the position of disaccharides and the other in the same position as 3H2O (Figure 3B). The relative amounts of radioactivity in the two peaks were ~40 and ~60% of the total radioactivity, respectively. This result indicated that at least 60% of the tritium was located at C5 of the GlcA units and was in agreement with the 60/40 distribution of the radioactivity between GlcA and GlcN indicated above. As expected, a metabolically 14C-labeled K5 polysaccharide, included in the heparitinase incubation mixture as an internal control, yielded labeled disaccharides but no further retarded, labeled products (Figure 3B).

Product formation as a function of time
Three enzyme preparations were used in the experiments reported here, i.e. pure bovine liver epimerase and partially purified preparations from bovine liver and the Furth mouse mastocytoma. With the fully N-deacetylated/N-sulfated as well as the 50% N-deacetylated/N-sulfated E.coli K5 poly­saccharide as substrates, the reaction with all three enzyme preparations proceeded linearly with time for at least 30 min at all substrate concentrations used in the kinetic experiments described below (data not shown).

Km and Vmax values
Determination of the Km and Vmax values for the fully and partially N-sulfated K5 polysaccharide at three different concentrations of the pure liver epimerase gave the results summarised in Table I. Surprisingly, the values increased substantially with increasing enzyme concentration, ranging from 4 to 19 µM HexA for the fully N-sulfated K5 derivative and from 11 to 33 µM HexA for the partially N-sulfated K5 derivative (Table I; see also Figure 5). This increase was in general agreement with the results of a number of single measurements, carried out earlier at different times and at different enzyme concentrations, which had given values ranging from 3.4 to 78 µM HexA for the fully N-sulfated K5 derivative, an almost 25-fold variation. In contrast to the Km measurements, the calculations of Vmax values gave the expected results and showed good proportionality to the enzyme concentration (Table I).


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Table I. Apparent Km and Vmax values for the purified bovine liver epimerase using different substrates
 


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Fig. 5. Effect of substrate size on apparent Km and Vmax values. The fully N-sulfated K5 derivative was size fractionated on a Superose 6 column, yielding fractions containing ~100 (pool 1) and 8–18 (pool 2) disaccharides (see Figure 1B). Different amounts of pure epimerase were incubated under standard assay conditions with various amounts of pool 1 (open triangles), pool 2 (open circles), and unfractionated polysaccharide (solid circles), and released 3H2O was quantified. (A) values; (B) Vmax values.

 
Dependence of reaction rate on enzyme concentration
Since Km measurements are valid only if product formation is directly proportional to enzyme concentration at all substrate concentrations used for the calculations, we examined the data from experiments shown in Table I more closely to determine whether this was indeed the case. It then became apparent that the expected linear increase occurred only at the highest substrate concentrations tested, while an increasing deviation from linearity was observed as the substrate concentrations decreased (Figure 4). Even though this phenomenon could be ascribed, in part, to consumption of substrate during the reaction—at the highest enzyme concentration and the lowest substrate concentration 22% of the substrate had reacted at the end of the incubation—the magnitude of the deviation was such that it could not be explained simply by substrate depletion. Consideration of the Michaelis-Menten equation shows that a downward deviation such as that observed in our experiments will result in a progressive increase in the calculated values with increasing enzyme concentration. Possible explanations to this phenomenon will be discussed below.



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Fig. 4. Tritium release as a function of enzyme concentration at different substrate concentrations. For each concentration of ~100% N-sulfated K5 [3H]polysaccharide used in the experiments shown in Table I, the released 3H was plotted against the enzyme concentration. The substrate concentrations, in µM HexA, are indicated in the graph.

 
Effect of substrate size on kinetic parameters
The following experiment was carried out to determine whether the size of the polymeric substrates influenced their reactivity in the epimerase reaction. 5–3H-Labeled ~100% N-sulfated K5 polysaccharide was chromatographed on Superose 6, and selected fractions of the eluate were pooled as shown in Figure 1, yielding materials with 8–18 and ~100 disaccharide units, respectively. From substrate concentration curves at different enzyme concentrations, Km and Vmax values were calculated for the two fractionated materials as well as the unfractionated polysaccharide. As seen from Figure 5B, the Vmax values at each enzyme concentration were essentially the same for all three preparations (except for one aberrant value) and fell on straight lines which, when extrapolated, passed through the origin. In contrast, the values for the larger fragments (~100 disaccharides) and the unfractionated polysaccharide were substantially lower than those determined for the smaller fragments, indicating that the larger fragments were better substrates (Figure 5A, Table I). For example, at an enzyme concentration of 4 ng per 50 µl incubation mixture, the values were 7 and 36 µM for the larger and smaller fragments, respectively. It is further apparent from Figure 5A that the values increased with increasing enzyme concentration, in accord with the results shown in Table I. The values for the smaller fragments increased more than 3-fold, from 11 to 36  µM, while a smaller increase, from 3.6 to 7.2 µM, was observed for the larger fragments. No attempt was made in these experiments to determine the minimum size of an oligosaccharide recognized as a substrate by the epimerase, but it should be mentioned that, in an earlier investigation, Jacobsson et al. (1984)Go found that a heparin-derived octasaccharide was ~10% as reactive as the macromolecular substrate.

Effect of N-acetyl groups on kinetic parameters
An absolute requirement for substrate recognition by the epimerase is the presence of an N-sulfate group on the glucosamine residue linked to C4 of the glucuronic acid unit targeted by the enzyme (Jacobsson et al., 1984Go). In contrast, the glucosamine residue on the other side of the uronic acid may be either N-sulfated or N-acetylated. It has not been previously established, however, whether there is a quantitative difference in reactivity between uronic acid residues flanked by two N-sulfated glucosamines and those with an N-acetylglucosamine residue on the reducing terminal side. In the present study, this problem was approached by experiments of two types, i.e., competition studies with nonradioactive E.coli K5 polysaccharide derivatives of appropriate structure and direct determination of Km and Vmax values with [5-3H]GlcA-labeled K5 polysaccharide derivatives as substrates.

In the first type of experiments, reaction mixtures contained partially purified liver epimerase and O-desulfated [5-3H]HexA-labeled heparin as the substrate, and two nonradioactive K5 polysaccharide derivatives (one essentially completely N-deacetylated and N-sulfated, and the other containing 25% N-acetylated and 75% N-sulfated GlcN residues) were tested for their ability to compete with the labeled substrate. As shown in Figure 6, the presence of these polysaccharides in the reaction mixtures led to decreased formation of 3H2O from the 3H-labeled substrate. The fully N-sulfated derivative caused 50% inhibition at a concentration of ~0.1 µg per assay mixture (Figure 6A), while the partially N-acetylated polysaccharide was ~10-fold less efficient and yielded the same degree of inhibition only at a concentration of ~1.3 µg per assay mixture (Figure 6B). Thus, the results obtained are those expected if the presence of N-acetyl groups in the substrate polysaccharide weakens its interaction with the enzyme.



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Fig. 6. Effect of unlabeled K5 polysaccharide derivatives on epimerase-catalyzed release of 3H from [3H]heparin. Liver epimerase was incubated under standard assay conditions with 10,000 cpm of the [5-3H]HexA-labeled O-desulfated heparin substrate along with the indicated amounts of unlabeled ~100% (A) or ~75% N-deacetylated/N-sulfated K5 polysaccharide (B). The results are expressed as percent inhibition of the 3H release occurring in the absence of competitive substrate. The approximate amounts of unlabeled polysaccharide yielding 50% inhibition are indicated.

 
The results of the second type of experiment, which have already been reported in Table I, showed that the Vmax values for the fully and partially N-sulfated K5 polysaccharides were essentially the same at each enzyme concentration used and that they increased linearly with increasing enzyme con­centration. The values for the 50% N-acetylated/N-sulfated derivative, however, were two to three times as high as those for the fully N-sulfated derivative, indicating that the latter was the better substrate. Like the values for the fully N-sulfated polysaccharide, the values for the 50% N-acetylated/N-sulfated substrate increased substantially with increasing enzyme concentration (Table I).

Comparison of O-desulfated [3H]heparin and metabolically labeled E.coli K5 polysaccharide derivatives as epimerase substrates
Determination of the kinetic parameters for the heparin-derived epimerase substrate showed that comparable values obtained at different enzyme concentrations were substantially higher than those of the K5 polysaccharide derivatives (Table I). This was true for all three enzyme concentrations used in these experiments, and, again, a significant increase in values was observed with increasing enzyme concentration. Somewhat surprisingly, the Vmax values for the heparin-derived substrate were 2- to 4-fold higher (see Tables I, II) than those determined for the fully N-sulfated K5 polysaccharide derivative, although the latter may be considered to be more akin to the physiological epimerase substrates.


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Table II. Apparent Km and Vmax values of epimerase preparations from mouse mastocytoma and bovine liver using different substrates
 
In view of these results, it was of interest to compare the structure of the heparin-derived substrate with that of the K5 polysaccharide derivatives and, in particular, to determine its uronic acid composition and the distribution of label between IdoA and GlcA. Compositional analysis was carried out as described in Materials and methods. The unlabeled O-desulfated heparin gave the results shown in Figure 7A, from which a uronic acid composition of 83% IdoA and 17% GlcA was calculated. After incubation of the O-desulfated heparin with liver epimerase and 3H2O for 1 week, the GlcA content had increased to 56%, indicating that back-epimerization of IdoA residues had occurred (Figure 7B). (The radioactivity incorporated at C5 of the HexA residues during the incubation did not interfere with the compositional analysis, since it amounted to less than 0.1% of the total radioactivity incorporated from the [3H]borohydride). Determination of the distribution of radioactivity between the two uronic acids in the enzymatically [5-3H]HexA-labeled polysaccharide gave similar results, as shown by analysis of the products of nitrous acid treatment at pH 1.5 and reduction with nonradioactive borohydride. Thus, 59% of the label was found in GlcA and 41% in IdoA (Figure 7C).



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Fig. 7. Paper chromatography of disaccharides from unlabeled and [5-3H]HexA-labeled O-desulfated heparin. (A) Unlabeled O-desulfated heparin, used as starting material for the preparation of [5-3H]HexA-labeled substrate, was treated with HNO2 at pH 1.5, and the cleavage products were reduced with NaB3H4. After isolation by chromatography on Sephadex G-15 and lyophilization, the disaccharides were subjected to paper chromatography. (B) [5-3H]HexA-labeled epimerase substrate, generated by incubation of O-desulfated heparin with epimerase in the presence of 3H2O, was analyzed as in (A). (C) The same heparin-derived [5-3H]HexA-labeled substrate was analyzed as in (A), except that the reduction was carried out with unlabeled borohydride. The migration positions of D-glucuronyl-2,5-anhydro-D-[1-3H]mannitol (GM) and L-iduronyl-2,5-anhydro-D-[1-3H]mannitol (IM) are indicated.

 
Despite the extensive back-epimerization during the labeling procedure, the heparin-derived substrate apparently still contained a large proportion of IdoA residues and differed structurally from the bacterial polysaccharide derivatives mainly in this respect. Since the epimerase reaction is readily reversible and the D-gluco configuration is favored over the L-ido configuration under the experimental conditions chosen, it may then be suggested that the C5 hydrogen is released more readily from IdoA residues than from GlcA residues and that this may be the reason for the higher Vmax values exhibited by the O-desulfated [3H]heparin. Presently, however, we have no knowledge of the rates of the intermediate steps in the epimerase reaction.

Kinetic studies on partially purified Furth mastocytoma and liver epimerase
One of the objectives of the present investigation was to compare the kinetic properties of epimerase preparations from the Furth mouse mastocytoma, which produces heparin, and bovine liver, which synthesizes heparan sulfate but not heparin. It was not practically feasible to prepare sufficient quantities of pure epimerase from the mastocytoma, and the comparison therefore had to be limited to partially purified materials. The results are shown in Figure 8 and Table II, from which it is apparent that the kinetic behavior of the masto­cytoma enzyme vis-à-vis the three substrates used was similar to that of the liver enzyme. Thus, the fully N-sulfated K5 polysaccharide derivative exhibited the lowest value, while that of the 50% N-acetylated/N-sulfated derivative was more than twice as high and the heparin-derived substrate gave an intermediate value. Essentially similar Vmax values were observed for the two K5 polysaccharide substrates, while that of the O-desulfated [3H]heparin was about 2 to 3 times as high. The increase in values with increasing enzyme concentration reported for the pure liver enzyme was observed also for the mastocytoma enzyme (data not shown) and the partially purified liver enzyme (see Table II). In view of this variation, it was not possible to make a valid comparison between the partially purified enzymes with respect to their true Km values, but it may be noted that the values for the mastocytoma enzyme fell within the same range as that observed for the liver enzyme (see Tables I and II).



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Fig. 8. Substrate concentration curves and Lineweaver-Burk plots for partially purified mastocytoma and liver epimerase. Mastocytoma (A, C, and E; solid circles) and liver (B, D, and F; open circles) epimerase were incubated under standard assay conditions with various amounts of O-desulfated [3H]heparin (A, B), ~100% (C, D), or ~50% N-deacetylated/N-sulfated [5-3H]GlcA-labeled K5 polysaccharide (E, F), and released 3H2O was quantified. The velocity (V) is expressed as cpm 3H released per 30 min and the substrate concentration ([S]) as µM HexA (equal to the concentration of disaccharide units), determined by the carbazole reaction. Duplicate or triplicate incubations were performed for all three substrates. The Km and Vmax values are presented in Table II.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Studies of the GlcA C5-epimerase involved in the biosynthesis of heparin and heparan sulfate generally depend on a radiochemical assay procedure which is based on the abstraction of the hydrogen atom at C5 (i.e., the chiral center) that occurs during the epimerase reaction (Lindahl et al., 1976Go; Jacobsson et al., 1979aGo; Prihar et al., 1980Go). When tritium is present at C5, enzyme action will result in the release of label as 3H2O, which is subsequently quantified, e.g., by distillation (Jacobsson et al., 1979aGo), biphasic liquid scintillation counting (Campbell et al., 1983Go), or by the anion-exchange column procedure used in the present study. The labeled substrate can be generated by a procedure previously designed by Jensen et al. (Jensen et al., 1983Go), who incubated chemically O-desulfated heparin with a preparation of liver epimerase in the presence of 3H2O, thus generating a polysaccharide in which both GlcA and IdoA residues were labeled with tritium at C5. The new method for substrate preparation developed in the present study has several advantages, practical as well as theoretical, over the procedures described above. The basic requirements are simple, and the labor involved is not excessive. Apart from a facility for growing bacteria on a small scale, only readily available laboratory equipment and chemicals are needed, and the isolation and chemical modifications (N-deacetylation and N-sulfation) of the polysaccharide may be completed in a few days. It should also be noted that the amount of radioactive product generated from ~1 mCi of D-[5-3H]glucose was of the same order of magnitude as that obtained from 1 Ci of 3H2O by the method of Campbell et al. (1994)Go, and the new procedure therefore eliminates the need for handling of large amounts of radioactive materials. Furthermore, since the E.coli polysaccharide contains only GlcA and no IdoA, its N-deacetylated/N-sulfated derivative is more akin to the physiological epimerase substrate than is the O-desulfated [3H]heparin substrate (although it should be remembered that the modifications of the polymer, including epimerization, probably begin before polymerization has been completed; see Lidholt and Lindahl, 1992Go; Salmivirta et al., 1996Go).

A major but seemingly simple task in the present investigation was to determine the Km values for the N-deacetylated/N-sulfated derivatives of the E.coli K5 polysaccharide in the GlcA C5-epimerase reaction. This proved more difficult than we had anticipated, mainly because the experimentally determined values varied with the concentration of epimerase in the reaction mixtures. Since, however, the increase in apparent Km values with increasing enzyme concentration shown in Table I was close to linear (and perfectly linear in another experiment with a 1 h rather than a 30 min incubation time), it seemed reasonable to assume that a true Km value could be determined by extrapolation to zero enzyme concentration (see also legend to Figure 9). From the data in Table I, a Km of 2–3 µM was thus obtained for the fully N-sulfated derivative. If, instead of the uronic acid concentration, the concentration of polysaccharide molecules was used as the basis for the calculation of the Km values, the numbers were obviously substantially lower. For example, if the extrapolated value for the polysaccharide fraction with ~100 disaccharides per chain is taken to be ~2.5 mM HexA (see Figure 5A), then the value based on polysaccharide concentration is ~25 nM. This is an unusually low Km value and suggests (but does not prove) a high affinity of the enzyme for its substrate.



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Fig. 9. Reaction diagram illustrating a possible mechanism for the distinctive steady-state kinetics of GlcA C5-epimerase. E, Enzyme; A, substrate; (EA), enzyme-substrate complex; X, product; Q, inhibitor competitive with A; and (EQ), enzyme-inhibitor complex. The rate equation for this system is

where vo is the initial velocity, Ao is the initial substrate concentration, Qo is the total inhibitor concentration, , Vmax = k+2Eo (total enzyme concentration, and K3 is the association equilibrium constant for reaction 3. Since = Km (1+ K3Qo) = Km + KmK3Qo, a plot of against Qo must be a straight line that has a slope of KmK3 and yields Km as its ordinate intercept. Therefore, K3 = slope/intercept. When the inhibitor Q is the enzyme itself and the enzyme-inhibitor complex is thus an enzyme dimer with active sites occluded, will be an increasing linear function of enzyme concentration. Calculation of K3 from the data for pool 1 in Figure 6 (using micromolar concentrations of the enzyme) gave a value of 1200 µM-1. From this value it may be calculated, e.g., that in a reaction mixture with Eo = 10–3 µM (in the range of the concentrations used in this work) and Ao = , 30% of the enzyme is present as free monomer and 20% as dimer. (Fifty percent is present as enzyme–substrate complex, since the substrate concentration equals .)

 
The reason for the unusual kinetic behavior of the epimerase is presently not clear. One contributing factor may be the reversible formation of dimers or higher oligomers of the enzyme in such a fashion that the catalytic site is occluded (Figure 9). In fact, upon gel chromatography, the purified epimerase used in this study was eluted in a position corres­ponding to a molecular mass of 200 kDa or greater (data not shown), while the molecular mass determined by SDS-PAGE is ~50 kDa (Campbell et al., 1994Go). Such oligomerization, which would occur to a greater extent as the enzyme concentration increases, would lead to the observed lack of linearity in the relationship between product formation and enzyme concentration. Enzyme oligomerization would be reversed at high substrate concentration, as suggested by the essentially linear relation between Vmax and enzyme concentration. Another, simpler explanation of the unexpected kinetic behavior of the epimerase is that the enzyme molecules do not act independently in the reaction mixtures but, at low substrate concentrations, compete with each other for the same substrate molecules. This assumption seems reasonable in view of the low ratio of substrate to enzyme molecules that pertains even at substrate concentrations close to the apparent Km values. As an example, consider a reaction mixture of 50 µl, containing 4 ng of purified enzyme and 50 nM K5 polysaccharide of the fraction with ~100 disaccharide units per chain (see Figure 5B). On a molar basis, the enzyme concentration is then 1.6 nM (assuming a molecular mass of 50 kDa), and the molar ratio of substrate to enzyme is only 30:1. As an extension of both of the above hypotheses, it may be suggested that, at a low ratio of substrate to enzyme, two or more enzyme molecules may bind to the same polysaccharide chain. This might result in a steric hindrance that lowers the affinity of the enzyme for the substrate (and increases the value), and it may also provide an opportunity for the enzyme molecules, held in close proximity, to interact with each other to form dimers or higher oligomers. The available data, however, do not permit us to arrive at a definitive conclusion as to the reason for the distinctive kinetics of the epimerase at this time.

The use of the fully and partially N-deacetylated and subsequently N-sulfated derivatives of the E.coli K5 polysaccharide made it possible to examine the effect of N-acetylated glucosamine residues on the epimerase reaction. Experiments in which nonradioactive K5 polysaccharide derivatives were found to inhibit tritium release from O-desulfated [3H]heparin suggested that a fully N-sulfated polysaccharide is a better substrate than one in which GlcNAc residues are present in the vicinity of the target GlcA residues. This finding was confirmed by direct measurements of Km values using the [5-3H]GlcA-labeled substrates. Epimerase from either bovine liver or mouse mastocytoma thus consistently showed values for the fully N-sulfated substrate that were substantially lower than those for the 50% N-acetylated derivative (Tables I, II). It should be noted, however, that the values were expressed as total concentration of GlcA in the substrate polysaccharides and that, in the 50% N-acetylated/N-sulfated derivative, not all GlcA residues serve as potential substrates for the enzyme. Previous studies demonstrated that a GlcA residue is susceptible to attack by the enzyme only if it has an N-sulfated GlcN residue on its nonreducing terminal side (Jacobsson et al., 1984Go). It may therefore be argued that the calculation of Km values for the GlcNAc-containing derivative should be based only on the concentration of those GlcA residues that are actually substrates for the epimerase, which is 50% of the total GlcA concentration. Even when calculated on this basis, however, the values for the 50% N-deacetylated/N-sulfated derivative determined in the experiments with the partially purified liver enzyme were close to three times as high as those observed for the fully N-sulfated derivative (Table II). In the experiments with the mastocytoma enzyme (Table II) and the pure liver enzyme (Table I), the difference in values was less pronounced, but in all cases was the fully N-sulfated K5 polysaccharide a better substrate than the GlcNAc-containing derivative in terms of their values. In all but one case (see Table I) this was also true even when the values were calculated on the basis of the concentration of reactive uronic acid residues.

Comparison of the kinetic properties of the epimerase preparations from bovine liver and mouse mastocytoma tissue turned out to be more complicated than we had anticipated because of the unexpected finding that the values varied with the enzyme concentration. Thus, in seven experiments with each of the two enzyme preparations, with the fully N-sulfated K5 polysaccharide as the substrate, the values ranged from 5.8 to 34.2 µM HexA (mastocytoma enzyme) and from 3.4 to 77.9 µM (partially purified liver enzyme) (data not shown). It is therefore apparent that a valid comparison would have to involve the use of pure enzyme from both sources, examined at the same concentrations, but it was not feasible in terms of cost and labor to produce sufficient quantities of mastocytoma tissue for such experiments. If we assume, however, that the kcat values for the liver and mastocytoma enzymes are the same, we may then compare the values obtained in experiments where the Vmax values were the same for both enzymes. One such experiment, with the 50% N-sulfated K5 polysaccharide as the substrate, is seen in Table II, which shows that the values for the two enzymes were almost identical (30 and 29 µM for the mastocytoma and the liver enzyme, respectively). Therefore, if the basic premise is correct, i.e., that the two enzymes have identical kcat values, we may then conclude that there is no significant difference between them with regard to their Km values.

A long-term goal of our investigations of the biosynthesis of heparin and heparan sulfate is to arrive at an understanding of the physiological biosynthetic process. The present study is a step in this direction, but it is important to remember that the polymer-modifying enzymes in the living cell reside in the Golgi apparatus and are membrane-bound. The kinetic parameters established for the soluble experimental systems used in this study therefore do not necessarily reflect the behavior of the biosynthetic apparatus in vivo. Furthermore, it has been proposed that the various enzymes involved in biosynthesis of heparin/heparan sulfate, including the epimerase, act in a processive fashion along the nascent polysaccharide chain that is still growing by addition of GlcA and GlcNAc units at the nonreducing terminus (Lidholt and Lindahl, 1992Go; Salmivirta et al., 1996Go). Evidence for such processivity in the soluble experimental system is lacking, and we have assumed that the interaction between the enzymes and the polysaccharide substrates occurred in a more random fashion (see also Hannesson et al. (1996)Go regarding epimerase action in dermatan sulfate biosynthesis). If, however, the enzyme does indeed act in a processive mode in free solution, processivity might be hampered by "excess" enzyme molecules in the reaction mixture, thus causing a rise in the . Despite these uncertainties, some valuable information has emerged from the present work. In particular, a marked dependence of the values on the concentration of enzyme protein in the reaction mixtures has been observed, which must be taken into account in future studies of the kinetic properties of the enzyme.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
A transplantable mouse mastocytoma, originally described by Furth et al. (Furth et al., 1957Go), was maintained by intra­muscular passage every 10–12 days in the hind legs of (A/SnxLeaden) F1 mice. Calf liver (frozen) was purchased in a local supermarket. Escherichia coli K5 bacteria (O18:K5, Freiburg collection number 2980) were a gift from K.Jann (Max Planck Institut für Immunbiologie, Freiburg, Germany). Two derivatives of the capsular polysaccharide from E.coli K5, in which ~75% or essentially 100% of the N-acetyl groups had been replaced with N-sulfate by hydrazinolysis and treatment with trimethylamine·SO3 (Casu et al., 1994Go), were given by B.Casu (G. Ronzoni Institute, Milan, Italy). Preparations of hyaluronan (Healon) and heparin for use as reference compounds were obtained from Pharmacia and KabiVitrum, respectively. Polysaccharide standards for estimation of Mr values, derived from heparin (3.3 kDa, 8.6 kDa) and from hyaluronan (18.9 kDa, 30 kDa, 43 kDa), were isolated as described previously (Cleland, 1983Go). Heparitinase I (EC 4.2.2.8) was purchased from Seikagaku. ß-D-Glucuronidase from bovine liver (type B-10) was obtained from Sigma. DEAE-Sephacel and Sephadex gels were purchased from Pharmacia Biotech (Uppsala, Sweden). D-[5-3H]Glucose (preparations ranging in specific activity from 12.8 to 16.6 Ci/mmol), D-[14C]glucose (295 mCi/mmol) and NaB[3H]H4 (28 Ci/mmol) were obtained from Amersham. All other chemicals were reagent grade. O-desulfated porcine mucosal heparin, labeled with tritium at C5 of the GlcA and IdoA residues by incubation with GlcA C5-epimerase in the presence of 3H2O, was prepared as described previously (Jensen et al., 1983Go; Campbell et al., 1994Go). The preparation used as epimerase substrate in the present study had a specific activity of 33 x 106 cpm/mg HexA.

GlcA C5-epimerase was purified from bovine liver as described (Campbell et al., 1994Go) and gave one major band on SDS-PAGE. The specific activity of the enzyme preparation was 4.4 x108 cpm 3H released/mg protein/30 min using [5-3H]HexA-labeled O-desulfated heparin as substrate. The partially purified C5-epimerase from bovine liver and mouse mastocytoma were prepared by affinity chromatography on O-desulfated heparin-Sepharose, Procion Red 120-Sepharose, and phenyl-Sepharose as described previously (Campbell et al., 1994Go). The specific activities of the liver and mastocytoma enzyme preparations were 8 x 107 and 6 x 106 cpm 3H released/mg protein/30 min, respectively, as determined by assay with the heparin-derived substrate. Prior to use, the enzyme preparations were passed through PD-10 columns equilibrated with assay buffer.

Preparation of metabolically radiolabeled E.coli K5 polysaccharide
Escherichia coli K5 bacteria (20 µl of a stock suspension) were precultured at 37°C overnight in 2 ml of LB-medium [10 g/l tryptone (Difco), 5 g/l yeast extract (Difco), 10 g/l NaCl]. For preparative labeling, a 1 ml aliquot of the preculture was added to 45 ml of LB medium lacking glucose. After incubation for 70 min (OD600 nm ~0.25), 2 mCi of D-[5-3H]glucose was added along with unlabeled glucose to a final concentration of 0.01%, followed by an additional 1 mCi D-[5-3H]glucose after 2 h of further incubation. The culture was then maintained for a 24 h period and was then centrifuged at 3000 x g for 30 min. The supernatant was applied to a 2 ml column of DEAE-Sephacel in 50 mM LiCl, 50 mM sodium acetate, pH 4.0. The column was washed with the same buffer, and the polysaccharide was eluted with a 50 ml gradient of 0.05–1.5 M LiCl. Fractions containing labeled polysaccharide (near the middle of the gradient) were pooled and dialyzed against water. The 5–3H-labeled polysaccharide obtained had a specific activity of 70 x 106 cpm/mg HexA. A 14C-labeled analog (specific activity 18.5 x 106 cpm/mg HexA) was prepared in a similar fashion, but with D-[1-14C]glucose substituted for D-[5-3H]glucose.

Chemical modification of tritium-labeled K5 polysaccharide
For N-deacetylation of the 5–3H-labeled K5 polysaccharide, samples of 10–15 x 106 cpm were transferred to screw-cap tubes, dried, and treated with 2 mg of hydrazine sulfate and 200 µl of hydrazine hydrate (~36% water) at 96°C. Based on pilot experiments with N-[3H]acetyl-labeled K5 polysaccharide, reaction times of 1.5 and 5 h were chosen to achieve ~50% and essentially complete N-deacetylation. The samples were then cooled, evaporated to dryness in a rotary evaporator, dissolved in water, desalted on PD-10 columns in 10% aqueous ethanol, and finally lyophilized. After lyophilization, the polysaccharides were treated with 0.25 M HIO3 to eliminate hydrazides (Shaklee and Conrad, 1984Go), again desalted on PD-10 columns, and lyophilized. N-Sulfation was carried out by treatment with 26 mg of trimethylamine·SO3 at pH 9.5 in 500 µl of water containing 22 mg NaHCO3 (Levy and Petracek, 1962Go). The reaction mixtures were kept at 55°C under vigorous shaking for 24 h. The products were desalted by gel chromatography and lyophilized.

Enzyme assay
The assay of epimerase activity is based on the release of 3H (recovered as 3H2O) from a substrate polysaccharide of the appropriate structure, 3H-labeled at C5 of HexA units (Jacobsson et al., 1979aGo). Enzyme and substrate, in the amounts stated in the descriptions of the individual experiments, were incubated for 30 min at 37°C in 50 µl of 0.05 M Hepes, 0.015 M EDTA, 0.1 M KCl, 0.015% Triton X-100, pH 7.4. The reaction was stopped by addition of 50 µl of cold 0.05 M sodium acetate, 0.05 M LiCl, pH 4.0, and the reaction mixtures were transferred, along with a 100 µl rinse, to ~400 µl columns of DEAE-cellulose, equilibrated in the same buffer and placed over 6 ml polypropylene scintillation vials. The 3H2O was recovered with an additional 900 µl buffer rinse and quantified by liquid scintillation counting. The background derived from substrate with this method was <1%. Kinetic coefficients were evaluated from steady-state data by nonlinear least squares iterative fitting to a rectangular hyperbolic function by the Enzfitter program (Biosoft). Conversion of Vmax values from cpm to molar terms was done taking into account that only ~60% of the 3H was located at C5 of GlcA units in the K5 polysaccharide.

Analytical methods
The N-substituent patterns of the K5 polysaccharide derivatives were investigated by gel chromatography of degradation products generated by deaminative cleavage. Samples were treated with nitrous acid at pH 1.5, resulting in cleavage of the polysaccharide chains at N-sulfated GlcN units (GlcNAc units being resistant) and conversion of the susceptible units to 2,5-anhydro-D-mannose residues (Shively and Conrad, 1976aGo). The products were reduced with NaBH4, essentially as previously described (Pejler et al., 1987Go), yielding terminal aManR units. They were then analyzed by gel filtration on a prototype of a Superdex 30 column (1.6 x 60 cm), designed for fast performance liquid chromatography (Pharmacia), eluted with 0.5 M NH4HCO3.

The HexA composition of the O-desulfated heparin was assessed by treatment with HNO2 at pH 1.5, and the resultant hexuronyl-anhydromannose disaccharides (~95% disaccharides, data not shown) were reduced with 0.5 mCi NaB3H4 (Maccarana et al., 1996Go) or with 50 µmol NaBH4 (Pejler et al., 1987Go). The labeled HexA-[1-3H]aManR or HexA-[5-3H]aManR disaccharide products were isolated by gel chromatography on a column (1 x 200 cm) of Sephadex G-15 in 0.2 M NH4HCO3, and desalted by repeated lyophilization. The samples were analyzed by paper chromatography on Whatman no.1 paper in ethyl acetate/acetic acid/water (3:1:1 by volume), along with GlcA-[1-3H]aManR and IdoA-[1-3H]aManR standards (Jacobsson et al., 1979bGo). After the separation, the paper strips were dried and cut into 1 cm pieces that were transferred to 6 ml scintillation vials. The samples were extracted in the vials with 1 ml of water for at least 15 min at room temperature and were then analyzed for radioactivity by scintillation counting following the addition of 4 ml of scintillation fluid. The relative proportions of GlcA and IdoA were calculated from the corresponding disaccharide peak areas.

Polysaccharides were quantified by the carbazole reaction for HexA (Bitter and Muir, 1962Go). Protein was determined by the method of Bradford (Bradford, 1976Go). Radioactivity was measured in a Beckman model LS 6000 liquid scintillation counter using Optiphase HiSafe scintillation fluid.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This work was supported by grants from the Swedish Medical Research Council (2309), Polysackaridforskning AB (Uppsala, Sweden), the European Commission (BIO4-CT95-0026), and the National Institutes of Health, USA (DE 08252 and NS 27353). We are indebted to Drs. Torvard Laurent and Kenneth B.Taylor for helpful discussions. The technical assistance by Lena Nylund is gratefully acknowledged.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
aManR, 2,5-anhydro-D-mannitol formed by reduction of terminal 2,5-anhydromannose residues with NaBH4; GlcA, D-glucuronic acid; GlcNAc, 2-deoxy-2-acetamido-D-glucose (N-acetyl-D-glucosamine); GlcN, 2-deoxy-2-amido-D-glucose (D-glucosamine); HexA, hexuronic acid (GlcA or IdoA); IdoA, L-Iduronic acid; HS, heparan sulfate.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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