Biosynthesis of heparan sulfate in rat liver. Characterization of polysaccharides obtained with intact cells and with a cell-free system.

A rat liver microsomal fraction was incubated with UDP-[’4C]glucuronic  acid and UDP-N-acetylglucosamine. Chromatography of the resulting labeled polysaccharide on DEAE-cellulose yielded two distinct components, a fully N-acetylated and a partially N-deacetylated polysaccharide, respectively. Addition of 3’phosphoadenylylsulfate to a microsomal fraction containing such preformed, nonsulfated polysaccharide resulted in the conversion of the partially N-deacetylated component into sulfated products. These products were partially separated, by ion exchange chromatography, into a less retarded, essentially N-sulfated fraction, and a more retarded, Nand 0-sulfated fraction. In the two fractions, [‘4C]iduronic acid comprised 35 and 65%, respectively, of the total labeled hexuronic acid. In either fraction about 25% of the glucosamine residues were Nacetylated. The isolated polysaccharides were highly similar to compounds previously implicated as intermediates in the biosynthesis of heparin (see Jacobsson, I., and Lindahl, U. (1980) J. BioZ. Chem 255,5094-5100). Incubation of isolated rat liver cells with [‘4C]glucosamine yielded labeled heparan sulfate (see Oldberg, A., Hook, M., Obrink, B., Pertoft, H., and Rubin, K. (1977) Biochem J. 164, 75-81). This material displayed less extensive polymer modification (40 to 50% of the glucosamine residues remained N-acetylated) than did the product of cell-free biosynthesis. A microsomal fraction prepared from the same batch of liver cells produced polysaccharides that were indistinguishable from those obtained with the microsomes from whole liver. These results suggest that the mechanism of hepa r m sulfate biosynthesis is similar to that of heparin biosynthesis. However, in the cell-free system, the regulation of the polymer-modification process is defective, resulting in the formation of an extensively modified, heparin-like polysaccharide. The regulatory mechanism apparently depends on the integrity of the intact cell.

A rat liver microsomal fraction was incubated with UDP-['4C]glucuronic acid and UDP-N-acetylglucosamine. Chromatography of the resulting labeled polysaccharide on DEAE-cellulose yielded two distinct components, a fully N-acetylated and a partially N-deacetylated polysaccharide, respectively. Addition of 3'phosphoadenylylsulfate to a microsomal fraction containing such preformed, nonsulfated polysaccharide resulted in the conversion of the partially N-deacetylated component into sulfated products. These products were partially separated, by ion exchange chromatography, into a less retarded, essentially N-sulfated fraction, and a more retarded, Nand 0-sulfated fraction. In the two fractions, ['4C]iduronic acid comprised 35 and 65%, respectively, of the total labeled hexuronic acid. In either fraction about 25% of the glucosamine residues were Nacetylated. The isolated polysaccharides were highly similar to compounds previously implicated as intermediates in the biosynthesis of heparin (see Jacobsson, I.,  J. BioZ. Chem 255,5094-5100).

Incubation of isolated rat liver cells with ['4C
]glucosamine yielded labeled heparan sulfate (see Oldberg, A., Hook, M., Obrink, B., Pertoft, H., and Rubin, K. (1977) Biochem J. 164, 75-81). This material displayed less extensive polymer modification (40 to 50% of the glucosamine residues remained N-acetylated) than did the product of cell-free biosynthesis. A microsomal fraction prepared from the same batch of liver cells produced polysaccharides that were indistinguishable from those obtained with the microsomes from whole liver. These results suggest that the mechanism of hepa r m sulfate biosynthesis is similar to that of heparin biosynthesis. However, in the cell-free system, the regulation of the polymer-modification process is defective, resulting in the formation of an extensively modified, heparin-like polysaccharide. The regulatory mechanism apparently depends on the integrity of the intact cell.
Heparan sulface proteoglycans are produced by most cells grown in vitro. The molecule appears to be preferentially located in the micro-environment of cells, either directly as-sociated with the cell surface (1,2) or incorporated into basement membranes (3) or related structures (4,5 ) with which the cells interact. Due to the pericellular location of heparan sulfate, this polysaccharide has been hypothetically implicated in t k e regulation of cell proliferation and of cell adhesion.
The polysaccharide portion of the heparan sulfate proteoglycan is composed of alternating D-glucosamine and hexuronic (D-glucuronic or L-iduronic) acid units. Sulfate substituents occur either as N-sulfate' groups at C-2 of the glucosamine residues, or as 0-sulfate groups at C-6 of the glucosamine or at C-2 of the iduronic acid units (for reviews, see Refs, 6 and 7). Heparan sulfate is structurally related to heparin, which is produced by mast cells. Contrary to heparin, heparan sulfate has little or no blood anticoagulant activity. While the two polysaccharides generally contain the same structural elements, heparan sulfate is recognized by its relatively higher glucuronic acid and N-acetyl contents and lower iduronic acid and (Nand 0-) sulfate contents. However, the composition of the two polysaccharides varies considerably with different species and tissues of origin, and the classification of polysaccharides as heparin or heparan sulfate is sometimes difficult.
Our knowledge regarding the biosynthesis of this group of polysaccharides is essentially restricted to the formation of heparin. A biosynthetic scheme has been established in cellfree experiments using microsomal fractions from transplantable mouse mastocytomas (see Refs. 7-10 and references therein). The polysaccharide chains are initially formed by polymerization of D-glucuronic acid and N-acetylglucosamine residues in dternating sequence. The resulting nonsulfated polymer subsequently undergoes a series of modification reactions, including N-deacetylation and N-sulfation of glucosamine residues, C5-epimerization of D-glUCurOniC acid to Liduronic acid units, 2-O-sulfation of the latter units, and, finally, 6-0-sulfation of glucosamine residues.* These reactions take place in a stepwise manner, leading to the formation of a number of distinct polymeric intermediates that may be separated by ion exchange chromatography. Structural modifications introduced early in the process are prerequisite to substrate recognition by enzymes catalyzing subsequent reactions. On the other hand, d disaccharide units are not

Biosynthesis of Heparan
Sulfate 7051 involved in each reaction. This incomplete polymer modification is characteristically reflected in the structural microheterogeneity of the final product. While the biosynthesis of heparan sulfate has been demonstrated in various cultured cells, little is known of its organization at the subcellular level. Levy et al. (12) observed the formation of an Nand 0-sulfated polysaccharide on incubating a microsomal fraction from pig aorta with UDP-glucuronic acid, UDP-N-acetylglucosamine and PAPS. They also noted that N-sulfation appeared to precede 0-sulfation. In the present investigation, the biosynthesis of heparan sulfate was studied in more detail, using a cell-free rat liver system. The results indicate striking similarities with heparin biosynthesis and point to novel aspects regarding the regulation of polymer modification.
Liver cells were isolated from male Sprague-Dawley rats by perfusion of the livers in situ with a collagenase solution (15). The resulting cell suspension was washed in a Ca2+-and Mg*+-containing buffer as described (16). More than 90% of the cells obtained by this procedure attach to a Petri dish coated with fibronectin (17) where they acquire a morphology characteristic of hepatocytes (18). Furthermore, at least 90% of the cells attach to a culture dish coated with asialo~eruloplasmin,~ indicating the presence of the hepatocytespecific receptor involved in the clearance of asialoglycoproteins from the plasma (19). Microscopic inspection of the cells after staining with toluidine blue (20) failed to show any metachromatically staining mast cells (5,000 cells counted).
A microsomal fraction was prepared from whole rat liver as follows. Two livers were finely divided with a pair of scissors in 100 rnl of 0.25 M sucrose, containing 2 mM Tris-HC1, pH 7.4, and were then homogenized with a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 1,000 X g for 20 min, and the supernatant obtained was then recentrifuged for another 20-min period, at 20,000 X g. A microsomal fraction was collected by centrifugation of the 20,000 X g supernatant at 100,000 X g for 2 h. The pelleted material was washed once by suspension in 50 ml of the buffered sucrose, followed by recentrifugation at 100,000 X g for 2 h. The resulting pellet was suspended in buffered sucrose. For the preparation of microsomal fraction from isolated hepatocytes, cells from four livers were suspended in 350 rnl of 1 m~ NaHC03, containing 0.5 mM CaC12, and left for 90 min in an ice bath. The lysed cells were then collected in a small volume by centrifugation at 2,000 X g for 3 min, and were homogenized in a 50-ml Dounce homogenizer by 25 strokes with a B pestle. The homogenate was mixed with the remainder of the supernatant from the centrifugation, and a microsomal fraction (sedimenting between 20,000 X g and 100,000 X g) was collected by centrifugation as described above.
Analytical Methods-The methods used to determine uronic acid, protein, radioactivity, and ratios of ~-['~C]glucuronic acid to L-['~C] iduronic acid were as indicated in a previous report (13). High voltage paper electrophoresis was performed on Whatman 3 " paper in 1.6 M formic acid, pH 1.7, at 40 volts/cm. Ion exchange chromatography of glycosaminoglycans on DEAE-cellulose (Whatman DE-52) was carried out as described earlier (13; see also the legend to Fig. 1).
The N-substituent pattern of heparin-related polysaccharides was analyzed by selective cleavage with nitrous acid followed by gel chromatography of the products. Polysaccharides were treated with nitrous acid at pH 1.5 or at pH 3.9, for deamination of N-sulfated or N-unsubstituted glucosamine residues, respectively (21). N-Acet,ylated glucosamine residues are resistant to deamination. Susceptible M. Hook, K. Ruhin, and B. Obrink, unpublished observation. glucosamine residues are converted into 2,5-anhydromannose units, with cleavage of the corresponding glucosaminidic linkages. The resulting oligosaccharides were analyzed by gel chromatography on a column (0.8 X 200 cm) of Sephadex G-25, eluted with 1 M NaCl at a rate of 4 ml/h. In order to convert polysaccharides into oligosaccharides with exclusively N-acetylated glucosamine residues, a procedure involving deamination at pH 1.5 followed by a second reaction period at pH 3.9 was employed, as described in detail elsewhere (22). A tetrasaccharide (hexuronosyl+ N-acetylglucosaminyl+ hexuronosyl + 2,5-anhydromannose) resulting from such treatment will represent an isolated N-acetylated glucosamine unit in the intact polysaccharide chain, whereas hexa-and octasaccharides correspond to sequences with two and three consecutive N-acetylated disaccharide units, respectively. The oligosaccharide composition of deamination products, as determined by gel chromatography, will thus define the N-substituent pattern of the polysaccharide molecule (see also Ref. 13).
Incubation Procedures-Isolated rat liver cells were incubated with radioactively labeled D-glucosamine according to Oldberg et al. (23), with the exception that I4C-labeled glucosamine (5 pCi/ml of incubation medium) was substituted for [3H]glucosamine. Labeled polysaccharide was isolated by gel chromatography following papain digestion, as described. The product was digested with chondroitinase ABC (24); the enzyme-resistant polysaccharide was reisolated by ion exchange chromatography on DEAE-cellulose.
Incubations of microsomal fraction contained, per ml of 0.05 M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4 25 pCi of UDP-['4C]glucuronic acid; 2.5 pmol of UDP-N-acetylglucosamine; 10 mg of microsomal protein; 10 pmol of MnClt; 10 pmol of MgClz; and 5 pmol of CaC12. After incubation at 37 "C for 60 min, a portion of the incubation mixture was withdrawn for isolation of nonsulfated polysaccharide. To the remainder, 5 pmol of unlabeled UDP-glucuronic acid was added per ml of incubation mixture to interrupt further incorporation of radioactivity, followed by 0.1 pmol of PAPS; the addition of PAPS was repeated every 3 min for a total period of 60 min.' Incubations were terminated by heating at 100 "C for 5 min. Labeled polysaccharides were isolated by gel chromatography following digestion with papain, as described (13). The products were digested with chondroitinase ABC (24) and were then separated by chromatography on DEAE-cellulose.

Polysaccharide Produced by Microsomal Fraction from
Whole Liver-Incubation of rat liver microsomal fraction with UDP-['4C]glucuronic acid and UDP-N-acetylglucosamine, under the conditions described under "Experimental Procedures," resulted in the formation of 10 to 20 X lo3 cpm of 14C-labeled polysaccharide/ml of incubation mixture and per h. The amount of labeled product increased with increasing concentration of UDP-N-acetylglucosamine within the range tested (up to 2.5 m). About 40% of the labeled material was degraded to oligosaccharides on digestion with chondroitinase ABC. Ion exchange chromatography on DEAEcellulose separated the chondroitinase-resistant ['4C]polysaccharide into two distinct, major peaks, designated MI and MI1 in Fig. 1A. Fraction MI1 co-eluted with standard hyaluronic acid whereas fraction MI was less retarded. The chromatographic behavior of these components was similar to that of the fully N-acetylated and partially N-deacetylated heparinprecursor polysaccharides, PS-NAc and PS-NHy+, respectively, previously characterized in a mastocytoma microsomal system (9,25).
Continued incubation of the microsomal fraction, containing such nonsulfated polysaccharide, in the presence of PAPS, resulted in the disappearance of component MI and the ap-Under the incubation conditions used, PAPS was hydrolyzed at a high rate. In a separate experiment the liver microsomal fraction was incubated with 1 mM [35S]PAPS, ie. a PAPS concentration ten times higher than that resulting from each one of the separate additions of unlabeled PAPS. Electrophoretic analysis showed that after 20 min of incubation, 99% of the radioactivity occurred as inorganic [35S]sulfate. The repetitive addition of PAPS was, therefore, employed to ensure an adequate concentration of the sulfate donor throughout the sulfation period. pearance of two, more anionic, incompletely separated components, MI11 and MIV (Fig. 1B). Fraction MI11 eluted similar to chondroitin $-sulfate, whereas fraction MIV appeared after the peak elution position of chondroitin 4-sulfate but before that of heparin. Fraction MI11 is thus reminiscent of the Nsulfated heparin-precursor polymer, PS-NS03-, while fraction MIV would correspond to the N-and 0-sulfated component,

Biosynthesis of Heparan Sulfate
The assumed analogy between the microsomal liver polysaccharides and the mastocytoma heparin-precursor polysaccharides was confirmed by structural characterization of the former species. Fraction MI1 resisted deaminative cleavage under any conditions tested (Fig. 2B) and was thus fully Nacetylated. Fraction MI was partly depolymerized by nitrous acid at pH 3.9 (Fig. 2 A ) but was unaffected at pH 1.5, indicating N-unsubstituted glucosamine residues. In contrast, fractions MI11 (Fig. 2C) and MIV (Fig. 2 0 ) were susceptible to deamination at pH 1.5 but not at pH 3.9, as expected for Nsulfated polysaccharides. Since fraction MI obviously represents a precursor of fractions MI11 and MIV, it is concluded that the formation of N-sulfated polysaccharides in the liver system may proceed via a partially N-deacetylated interme-PS-N/O-SOJ-(9, 25). diate species. The analogy with heparin biosynthesis (22, 25) is further emphasized by the observation that conversion of MI into MI11 and MIV is accompanied by additional Ndeacetylation, as evident from the shift toward more low molecular deamination products (Fig. 2). Moreover, electrophoretic analysis of deamination products showed considerable amounts of 0-sulfate groups in fraction MIV, about 80% of the material migrating toward the anode at pH 1.7 (Fig.  3B). The major sulfated component appeared to be monosulfated hexuronosyl -+ 2,5-anhydromannose disaccharide; no disulfated disaccharide was seen. The much smaller amounts of 0-sulfated components obtained from fraction MI11 (Fig.  3A) probably derives from contaminating MIV material; note that the separation between MI11 and MIV in Fig. 1B is incomplete. ['4C]Iduronic acid comprised 35 and 65% of the total labeled hexuronic acid in fractions MI11 and MIV, respectively. These results further underline the similarity between fraction MI11 and the heparin-precursor polysaccharide PS-NSOC, which contains N-but no 0-sulfate groups and small amounts of iduronic acid. The transformation of PS-NSO3into PS-N/O-S03involves 0-sulfation and further C5-epimerization of D-glucuronic acid into L-iduronic acid units (9,13,25).
Polysaccharide Produced by Isolated Liver Cells-Heparan sulfate produced by cultured rat liver cells (hepatocytes) was characterized in a previous study (23). It differed from fraction MIV, Le. the end product of the cell-free biosynthetic process, by its lower iduronic acid content (40% of the total uronic acid) and, in particular, by its N-substitution pattern, which indicated a blockwise distribution of N-acetylated glucosamine residues, typical for heparan sulfate. In contrast to the extended sequences of consecutive N-acetylated disaccharide units displayed by this polysaccharide (see Fig. 3 in Ref. 23), the N-acetyl groups in fraction MIV occurred largely as isolated units (represented by the tetrasaccharide in Fig. 2 0 ) , and thus showed a distribution characteristic of heparin (26). In summary, these findings would suggest that the polysaccharide synthesized in the cell-free system had been subjected to more extensive polymer modification than that produced by the intact cells.5 On the other hand, it could be argued that cells other than hepatocytes, e.g. mast cells, might have contributed to the microsomal fraction prepared from whole liver, thus affecting the pattern of polymer modification. Such cells would not necessarily be represented in a population of isolated liver cells. In order to eliminate this objection, a microsomal fraction was isolated from the same preparation of liver cells as was used in whole cell biosynthesis experiments.
Intact Cells-Isolated liver cells were incubated with ["C] glucosamine for 16 h. The resulting labeled polysaccharide was isolated and digested with chondroitinase ABC in order to eliminate any hyaluronate or galactosaminoglycan present. Chromatography of the chondroitinase-resistant material on DEAE-cellulose revealed a component that was eluted largely after chondroitin 4-sulfate but before heparin (Fig. 4), similar to the heparan sulfate studied previously (cf. Fig. 1 in Ref. Fig. 4, and each fraction (CI and CII) was subjected to deaminative cleavage followed by gel chromatography (Fig. 5 ) . The resulting chromatograms showed a relatively large proportion of oligosaccharides in the octa-to decasaccharide range, corresponding to three or more N-acetylated disaccharide units in Comparison of the results shown in Fig. 3B with those in Figs. 3 and 4 of Ref. 23 indicates that this conclusion applies also to the incorporation of 0-sulfate groups. The reason for the lack of di-0sulfated disaccharide units in the product of ceu-free biosynthesis is unknown.  Per cent of total glucosamine units, determined as described under "Experimental Procedures.

Biosynthesis
Calculated from the gel chromatograms (Figs. 2 and 5) of deamination products, as described under "Experimental Procedures." The oligosaccharide composition of each sample was estimated from the areas under the appropriate peaks (calculated after projecting the vertical lines on the curves to the base-line). Obviously, the experimental error in this method may be considerable, and the results, although satisfactory for the purposes of the present study, should be regarded as approximations.
' Synthesized by microsomal fraction prepared from whole liver.
Synthesized by microsomal fraction prepared from isolated liver cells.
e Synthesized by intact liver cells.

Heparan Sulfate
regards the elution patterns on ion exchange chromatography and the distribution of N-acetyl groups within each of the various polymeric intermediates. This is illustrated by a gel chromatogram of deamination products representing fraction MIV of the sulfated microsomal polysaccharide derived from the isolated liver cells (Fig. 5 B ) . The chromatogram is superimposed on the analogous chromatogram pertaining to fraction CII of the polysaccharide produced by the corresponding intact, cells. The similarity between the MIV chromatogram in Fig. 5 B and that in Fig. 2 0 is striking, as is the difference between the MIV and the CI and CII patterns in Fig. 5 (see also Table I). These results demonstrate that the biosynthetic polymer modification is less extensive for a polysaccharide produced by intact liver cells than for a polysaccharide produced by a microsomal fraction from the same cells.

DISCUSSION
Previous studies have demonstrated that isolated rat hepatocytes produce a glycosaminoglycan that, by all current criteria, is identified as a high sulfated heparan sulfate (23, 27). These results were c o n f i i e d in the present investigation. It seemed reasonable to assume that the microsomal fraction prepared from these cells contained the enzymes utilized, by the intact cells, in producing heparan sulfate. Cell-free incubations of such microsomal fraction yielded polymeric intermediates similar to those formed during heparin biosynthesis, using a mouse mastocytoma microsomal fraction (9,25). The polymer-modification reactions involved in the biosynthesis of heparan sulfate are apparently very similar to those leading to the formation of heparin.6 In fact, the isolated intermediates were more heparin-like than anticipated; the cell-free product (fraction MIV) had been subjected to more extensive polymer modification than had the heparan sulfate manufactured by the intact cells.
These results suggest that the regulation of the polymermodification process depends on the integrity of the intact cell. In the cell-free system, a restrictive control function has been lost, leading to the formation of a more extensively modified product. The mechanism of regulation and its relation to other cellular functions are unknown. However, studies on the substrate specificities of the various polymer-modifying enzymes involved in heparin biosynthesis have suggested that the initial reaction, the deacetylation of N-acetylglucosamine residues, has a key regulatory role, since it determines the maximal possible extent of all subsequent reactions (8-10,13). In accord with this postulate, the polymer obtained from the cell-free system contained not only less N-acetyl (and hence more N-sulfate) groups but also more 0-sulfate groups (see Footnote 5) and iduronic acid units. Recent observations indicated that the level of N-deacetylation in heparin biosynthesis may be modulated by a complex interaction between the N-deacetylase and N-sulfotransferase enzymes (22). The concentrations of cofactors (or inhibitors) to these enzymes, or of PAPS, potentially of importance to the interaction, may be subjected to strict intracellular control. Alternatively, the mechanism of regulation may depend on the distribution and spatial arrangement of the polymer-modifying enzymes in the membranes of the endoplasmic reticulum. It seems likely that the organization of the biosynthetic apparatus may be deranged during the preparation of the microsomal fraction.
The corresponding enzymes, including a N-acetylglucosaminyl Ndeacetylase, a uronosyl C5-epimerase, and N-and O-sulfotransferases have been demonstrated in a rat liver microsomal fraction, using specific assay procedures developed for the determination of mastocytomal enzymes (involved in heparin biosynthesis; I. Jacobsson and J. Riesenfeld, unpublished results).
The blood anticoagulant activity of heparin was recently shown to be critically dependent on the fine structure of the polysaccharide molecule (11, 28-30). It seems probable that similar conditions may apply to the interaction between heparan sulfate proteoglycans and other macromolecules at the cell surface or in the pericellular space. These aspects underline the functional significance of the regulatory mechanisms associated with polymer modification in the biosynthesis of heparin-like polysaccharides.