Domain structure of heparan sulfates from bovine organs.

Samples of heparan sulfate, isolated from bovine aorta, lung, intestine, and kidney, were degraded by digestion with a mixture of heparitinases or by treatment with nitrous acid, with or without previous N-deacetylation. Analysis of the resulting oligosaccharides showed that the various heparan sulfate samples all contained regions of up to 8 or 9 consecutive N-acetylated glucosamine residues, as well as contiguous N-sulfated sequences. L-Iduronic acid accounted for a remarkably constant proportion, 50-60%, of the total hexuronic acid units within the latter structures. Of the total iduronic acid units, 36-55% were located outside the contiguous N-sulfated regions, presumably in sequences composed of alternating N-acetylated and N-sulfated disaccharide residues. While most of the iduronic acid units within the N-sulfated blocks were 2-O-sulfated, those located outside were almost exclusively nonsulfated. The heparan sulfate preparations differed markedly with regard to the content of 6-O-sulfated glucosamine units, more than half of which were located outside the N-sulfated block regions. These findings suggest that the formation of iduronic acid residues and their subsequent 2-O-sulfation are coupled within but not outside the contiguous N-sulfated regions of the heparan sulfate chains and, furthermore, that the 2-O- and 6-O-sulfotransferase reactions are differentially regulated during heparan sulfate biosynthesis.

The sulfated glycosaminoglycans generally occur in the tissues as proteoglycans, in which the polysaccharide chains are covalently bound to a core protein (reviewed in Ref. 1). Heparan sulfate (HS) 1 proteoglycans are abundant on cell surfaces and in the extracellular matrices of most tissues and have been attributed a multitude of biological activities. These effects are mostly believed to reflect binding of HS chains to specific proteins (2)(3)(4). While the physiological or pathophysiological significance of such interactions is subject to increasing attention, their structural basis is generally unclear. Thus, with the exception of the antithrombin-binding pentasaccharide sequence in heparin/HS (reviewed in Ref. 5) and more recently described, still incompletely defined, binding regions for some growth factors (6 -10), little is known regarding the saccharide sequences involved in protein binding. In fact, since most HSbinding proteins bind to heparin, i.e. the most densely sulfatesubstituted member of the glycosaminoglycan family, the interactions are often tacitly assumed to be nonspecific in character. On the other hand, indirect observations imply an appreciable degree of specificity in interactions between HS and various proteins such as cytokines (11) or selectins (12), and it has been proposed that specific protein-binding regions that occur in "hidden" form in heparin are selectively expressed by different HSs (3,4,8).
The sequence characteristics of HSs produced by different cells, or occurring in different tissues, are poorly defined. No methodology is yet available for routine sequence analysis of HS chains. Furthermore, the problem is complicated by the inherent heterogeneity of even highly purified HS preparations. The biosynthetic reactions required to generate different HS sequences have been defined and include the formation of a (GlcA␤1,4-GlcNAc ␣1,4-) n polysaccharide, that is subsequently modified through N-deacetylation/N-sulfation of GlcNAc units, C-5 epimerization of GlcA to IdoA units, and O-sulfation at different positions. The O-sulfate substitution may occur at C-2 of IdoA and some GlcA units, and at C-6 (to some extent C-3) of GlcN units (3). Due to the sequential order of these reactions, and the substrate specificities of the enzymes involved, the initial distribution of N-sulfate groups will heavily influence the location of IdoA units and O-sulfate groups as well. N-Sulfate and residual N-acetyl groups tend to form block structures (13,14). Generally, the degree of N-sulfation correlates with the IdoA and O-sulfate contents of a heparin/HS type polysaccharide (15). However, few attempts have been made to assess, in a more comprehensive fashion, the overall domain structure of HS, with regard to N-substituents as well as the products of "downstream" polymer-modification reactions. The present study was initiated to obtain such information, by structural analysis of HSs from different bovine organs.

EXPERIMENTAL PROCEDURES
Materials-Preparations of HS were obtained in principle as described (16). Briefly, the various samples were isolated from bovine aorta, lung, intestine, and kidney by a procedure involving protease digestion of boiled and minced tissue, alkali treatment, removal of nucleic acids by low pH precipitation and nuclease digestion, anionexchange chromatography, and removal of galactosaminoglycans by digestion with chondroitinase ABC. The ion-exchange chromatography used to recover HSs from aorta, lung, and intestine was done using a column of Dowex 1-X2, that was first washed to eliminate material dissociated from the resin in 0.75 M NaCl and then eluted with 1.0 M (aorta) or 1.25 M (lung, intestine) NaCl. The kidney HS was separated into two fractions, preparation A that was eluted between 0.7 M and 1.1 M NaCl, and preparation B that was eluted between 1.1 M and 1.25 M NaCl. The M r ranges for the various preparations were estimated to ϳ12-44 ϫ 10 3 (aorta), ϳ9 -30 ϫ 10 3 (lung), ϳ10 -30 ϫ 10 3 (intestine), ϳ9 -21 ϫ 10 3 (kidney A) and ϳ10 -32 ϫ 10 3 (kidney B), by gel permeation HPLC using chondroitin sulfates as standards (data not shown).
Methods-Compositional analysis of HS was performed using Flavobacterium heparinum HS lyases. About 100 g of HS was digested with a mixture of heparitinases I and II (EC 4.2.2.7 and EC 4.2.2.8; 20 milliunits each; Seikagaku) in 40 l of 20 mM acetate buffer, pH 7.0, 2 mM calcium acetate, at 37°C for 2 h. The reaction was terminated by boiling for 30 s. The yield of resultant unsaturated disaccharides was estimated by gel permeation HPLC using connected columns of TSK-gel 4000, 3000, 2500 PWXL, equipped with a refractive index monitor. The disaccharides were separated further on a CarboPac PA-1 HPLC column (Dionex), eluted with a LiCl gradient as described in the legend to Fig. 2, and monitored by absorbance at 230 nm, essentially as described (17).
Deaminative cleavage of HS with nitrous acid was done according to two alternative procedures. In one approach, 0.5 mg of polysaccharide was treated with 500 l of HNO 2 reagent at pH 1.5, followed by reduction of the products with 0.5 mCi of NaB 3 H 4 , essentially as described (18). Under these conditions, N-sulfated GlcN units are selectively attacked, with cleavage of the corresponding glucosaminidic link (19). N-Acetylated GlcN residues are not affected; thus, a single Nacetylated disaccharide unit surrounded by two N-sulfated units will be recovered as a tetrasaccharide, whereas consecutive N-acetylated units will give rise to larger oligosaccharides. All oligosaccharides formed will contain a [1-3 H]aMan R unit at their reducing end. Unincorporated 3 H was removed by passage through a column of Sephadex G-15 (1 ϫ 190 cm, eluted in 0.2 M NH 4 HCO 3 ). Prior to further analysis, the oligosaccharides were pooled and subjected to mild acid treatment (25 mM H 2 SO 4 , 80°C, 30 min) to eliminate sequences containing products of "anomalous" deaminative ring contraction (20). The 3 H-labeled saccharides were analyzed further as described in the text or in the legends to figures.
Alternatively, the HS preparations were N-deacetylated by hydrazinolysis before deamination (21)(22)(23). Samples of 1 mg were dissolved in 1 ml of hydrazine hydrate and 1% (w/v) hydrazine sulfate, and were then heated in sealed glass tubes at 96°C for 4 h. After repeated evaporation to dryness, the N-deacetylated polysaccharides were recovered by passage through columns (1 ϫ 90 cm) of Sephadex G-15, equilibrated in 10% ethanol. The excluded material was pooled and lyophilized, and samples corresponding to 100 g of polysaccharide (as determined by the carbazole reaction (24), assuming a hexuronic acid content of 33%) were deaminated with HNO 2 , first at pH 1.5 (cleavage at N-sulfated GlcN units) and then at pH 3.9 (cleavage at N-unsubstituted GlcN units; Ref. 25), followed by reduction of the products with 100 Ci of NaB 3 H 4 . The resultant labeled disaccharides thus would represent the entire initial, N-acetylated as well as N-sulfated, saccharide sequences.
The HexA-[1-3 H]aMan R disaccharide fractions were analyzed by anion-exchange HPLC (26), as described in more detail in the legend to Fig. 1. The nonsulfated disaccharides, GlcA-aMan R and IdoA-aMan R , were not resolved in this procedure and were instead separated by descending paper chromatography (ethyl acetate/acetic acid/H 2 O, 3:1: 1), following isolation by preparative paper electrophoresis (80 V/cm) on Whatman 3MM paper, in 0.083 M pyridine, 0.05 M acetic acid, pH 5.3. Tetrasaccharides obtained after deamination at pH 1.5/NaB 3 H 4 treatment were separated further by high voltage paper electrophoresis (40 V/cm) in 1.6 M formic acid (pH 1.7). Paper strips were cut into 1-cm segments that were eluted with water, and the aqueous extract was analyzed by scintillation counting.
Additional analytical procedures are described in the legends to figures.

RESULTS
Overall Composition of Heparan Sulfate Chains-Samples of HS isolated from bovine aorta, lung, intestine, and kidney (two preparations, designated A and B, separated by anion-exchange chromatography; see "Materials") were N-deacetylated by hydrazinolysis and deaminated, under conditions leading to cleavage at N-unsubstituted as well as at N-sulfated GlcN units. A completely N-deacetylated HS chain thus would be quantitatively degraded to disaccharides. Reduction of the products with NaB 3 H 4 , followed by gel chromatography on Sephadex G-15 revealed labeled tetrasaccharides as a minor component (7-11% of the total labeled products) in addition to disaccharides. Some of these tetrasaccharides were due to "anomalous" deaminative ring contraction (20), since mild acid hydrolysis treatment reduced the proportion of labeled tetrasaccharides (data not shown). The HexA-aMan R disaccharides thus consistently accounted for Ն90% of the total radioactivity, and were considered to appropriately reflect the overall composition of the HS samples (see Ref. 23 regarding nonselectivity of the "anomalous" ring contraction). Separation of the disaccharides by anion-exchange HPLC ( Fig. 1) and paper chromatography (data not shown) gave the results summarized in Table I. All HS samples yielded predominantly non-O-sulfated GlcA-aMan R disaccharide, which represents a -GlcA-GlcNR-structure in the intact polysaccharide, where R stands for either a N-acetyl or a N-sulfate group. Interestingly, the . The peak marked * represents 3 H-labeled impurities originating from the NaB 3 H 4 reduction. Peaks marked ** represent tetrasaccharide contaminants. The GlcA(2-OSO 3 )-aMan R peaks (arrow 3) were occasionally partly obscured by unidentified components of low abundance. To ascertain the identity of this monosulfated disaccharide, samples of labeled disaccharides were first separated by paper electrophoresis at pH 5.3 (see "Methods") and the monosulfated fraction was eluted from the paper and subjected to HPLC analysis. The resultant chromatograms (data not shown) confirmed the occurrence of component 3, in about the same proportions relative to components 4 -6 as estimated before electrophoresis, and without any interference of unknown components (presumably nonsulfated tetrasaccharides). The structure of component 8, IdoA second most abundant disaccharide deamination product varied between the different HS samples, and was IdoA-aMan R from aorta HS, GlcA-aMan R (6-OSO 3 ) from the lung and the two kidney HSs and IdoA(2-OSO 3 )-aMan R from the intestinal HS. Moreover, the remaining disaccharides were obtained in markedly different proportions. Thus, the order of abundance between the various disaccharides differed from one HS sample to another, and was not the same for any two samples.
The HS preparations were also digested with a mixture of heparitinases, again resulting in essentially complete degradation of the HS chains to disaccharides. The yield of disaccharides ranged between 96% and 99% on a weight basis, except for the intestinal HS (90%). Separation of these 4,5-unsaturated species by anion-exchange HPLC (Fig. 2) gave the results shown in Table II. Compared to the deaminative cleavage, lyase digestion entails some loss of information, since the asymmetric configuration at C-5 of the HexA units is eliminated; hence, GlcA and IdoA units yield the same 4,5-unsaturated structure. On the other hand, the N-substituents of the GlcN units are retained, resulting in the generation of Nsulfated and N-acetylated disaccharides. To compare the results of the two procedures, the appropriate disaccharides obtained by deamination were combined as indicated in the legend to Table II. The two sets of data are in good agreement, thus confirming the validity of the methods applied for compositional analysis of HS preparations.
The quantitative analysis of disaccharides enabled an assessment of the overall contents of IdoA and O-sulfate groups in the various HS samples (Fig. 3A). The total IdoA contents (including nonsulfated as well as 2-O-sulfated units) varied between 21% and 35%, and the 2-O-sulfated IdoA between 8.3% and 21%, of the total HexA units. The extent of GlcN 6-O-sulfation ranged from 9.6% to 38% of the total GlcN units.
Amounts and Domain Distribution of N-Sulfate Groups-Information regarding the amounts and distribution of N-sulfated GlcN units was obtained by deamination of native (i.e. not N-deacetylated) HS at pH 1.5, followed by reduction with NaB 3 H 4 and separation of the resultant labeled oligosaccharides by gel chromatography. Under these conditions the chains are cleaved at the sites of N-sulfated GlcN units, whereas N-acetylated units remain intact. Consecutive N-sulfated GlcN residues (in the following referred to as "contiguous" N-sulfated sequences or "N-sulfated blocks") thus will be recovered, as aMan R units, in disaccharides, whereas alternating N-sulfated and N-acetylated GlcN residues ("alternating" sequences) will give rise to tetrasaccharides. Finally, solitary N-sulfate groups ("spaced" sequences) will yield oligosaccharides of at least hexasaccharide size. Gel chromatography on Bio-Gel P-10 provided adequate separation of the various 3 H-labeled oligosaccharides (Fig. 4). All HS samples yielded disaccharides, followed by tetrasaccharides, as major labeled products and, in addition, smaller amounts of well separated oligosaccharides up to 18 -20 saccharide size. Calculation of peak areas afforded estimates of overall N-sulfate/N-acetyl ratios (27,28) as well as proportions of contiguous, alternating, and spaced sequences. While the proportions of contiguous and spaced sequences varied appreciably among the various HS preparations, the proportion of alternating sequence was remarkably constant (Fig. 6A). All samples fell within the range  a Disaccharides were generated by N-deacetylation followed by deaminative cleavage (pH 1.5 and 3.9, resulting in cleavage of all glucosaminidic linkages) and reduction of the products with NaB 3 H 4 , as described under "Methods." The resultant HexA-[1-3 H]aMan R disaccharides were separated by anion-exchange HPLC (Fig. 1) and by paper chromatography (see "Methods"). Values are given as mol % of total labeled disaccharides and were calculated from peak areas. ND, not detected (Յ0.2%).
b The sum of the nonsulfated disaccharides, GlcA-aMan R and IdoA-aMan R , was calculated from anion-exchange HPLC patterns, where the two components emerged together in the peak indicated by arrow 1 in Fig. 1. To obtain the relative proportions of the epimers, the nonsulfated disaccharide fractions were isolated by preparative paper electrophoresis (pH 5.3) and were then separated further by paper chromatography (see "Methods"; data not shown).
of N-sulfation typical for HS (14) and varied from 37% (aorta) to 57% (intestine) of the total disaccharide units (Fig. 3A). Of the total N-sulfate groups in the HS chains, 42-61% were located in contiguous sequence, 24 -33% in alternating structure, and 14 -29% as solitary (spaced) units (Fig. 6B). The aorta sample, with the lowest overall N-sulfate contents (Fig. 3A), showed the lowest proportion of contiguous and the highest proportion of spaced N-sulfate groups, whereas the most highly N-sulfated, intestinal, HS displayed the inverse relation. The occurrence of extended N-acetylated sequences adjacent to the polysaccha-ride-protein linkage (29) could bias the calculations, insofar as such structures are not likely to become radiolabeled following HNO 2 (pH 1.5)/NaB 3 H 4 treatment.

Domain Distribution of Hexuronic Acid and O-Sulfate
Residues-In separate analyses, we determined the composition of disaccharides released by deamination at pH 1.5 only, without prior N-deacetylation of the HS chains ( Fig. 5; Table III). In a Disaccharides, all containing a 4,5-unsaturated hexuronic acid (⌬HexA) unit, were generated by extensive enzymatic digestion of the HS chains, as described under "Methods," and were separated by anion-exchange HPLC (Fig. 2).
b GlcA-aMan R ϩ Ido-aMan R . c IdoA(2-OSO 3 )-aMan R . d GlcA-aMan R (6-OSO 3 ) ϩ IdoA-aMan R (6-OSO 3 ). e IdoA(2-OSO 3 )-aMan R (6-OSO 3 ). N-sulfated blocks (B). A, residue abundance was calculated from the data in Table I and (N-sulfate groups) Fig. 4. B, residue abundance was calculated from the data in Table III. For additional information, see ''Results.'' FIG. 4. Gel chromatography of oligosaccharides produced by deamination at pH 1.5. HS samples were cleaved by treatment with nitrous acid (pH 1.5), and the resultant oligosaccharides were reduced with NaB 3 H 4 and subjected to mild acid treatment to eliminate "anomalous" ring contraction products (see "Methods"). The labeled oligosaccharides were separated by gel chromatography on a column (1 ϫ 150 cm) of Bio-Gel P-10 (superfine), equilibrated with 0.5 M NH 4 HCO 3 . Effluent fractions of ϳ0.5 ml were collected at a rate of 1 ml/h and were analyzed for radioactivity by scintillation counting. The molecular size of the separated oligosaccharides is indicated by the number of monosaccharide units/molecule, shown for each peak of the aorta sample. Fractions corresponding to disaccharides or tetrasaccharides were pooled as indicated by the bars and lyophilized before further analysis.

FIG. 3. Extent of polymer modifications in the entire HS chains (A) and
contrast to the products obtained on complete deaminative cleavage, which were dominated by nonsulfated GlcA-aMan R disaccharide (Table I), the disaccharides released from the contiguous N-sulfated sequences contained predominantly 2-Osulfated IdoA units that were linked to either nonsulfated or 6-O-sulfated aMan R residues. The N-sulfated blocks in the different HS preparations showed a remarkably constant proportion of IdoA, which occurred in 63-69% of the disaccharides units and IdoA(2-OSO 3 ), which occurred in 54 -58% of the disaccharide units (Fig. 3B). Thus, 81-91% of the IdoA residues were 2-O-sulfated. By contrast, the extent of 6-O-sulfation varied widely between 26% and 58% of the contiguous N-sulfated glucosamine units. A comparison of the data in Fig. 3 (Table I) and after cleavage at pH 1.5 (Table  III). A comparison of the data suggests that GlcA 2-O-sulfate is present also in structures outside the N-sulfated blocks.
In order to quantitate the proportion of the different units in the contiguous N-sulfated structures, it was necessary to account for: (i) the distribution of total disaccharide units between contiguous (N-sulfated), alternating, and spaced structures ( Fig. 6A; this information was obtained from the gel chromatograms shown in Fig. 4), and (ii) the abundance of IdoA, IdoA(2-OSO 3 ), and GlcN(6-OSO 3 ) units in the whole chains ( Fig. 3A; compiled from the data in Table I), and in the N-sulfated blocks (Fig. 3B; compiled from the data in Table III). The contiguous N-sulfated regions thus contained about half of the total IdoA units of the chains (Fig. 6D), but almost all of the IdoA 2-O-sulfate units (Fig. 6E). Presumably, most of the remaining (nonsulfated) IdoA occurs in the alternating sequences. By contrast, consistently less than half of the total 6-O-sulfate groups was found in the contiguous sequences (Fig. 6F).
Since most of the total 6-O-sulfate groups appeared to be located outside the N-sulfated block regions, it was of interest to assess the contributions of alternating and spaced sequences to the total O-sulfate contents of the chains. Alternating (Nacetyl/N-sulfate) type structures were recovered as labeled tetrasaccharides following HNO 2 (pH 1.5)/NaB 3 H 4 treatment (Fig. 4) and were separated further by high voltage paper electrophoresis at pH 1.7 (data not shown). The proportions of nonsulfated, mono-O-sulfated, and di-O-sulfated species (no tri-O-sulfated tetrasaccharides were detected) were assessed by determining the distribution of 3 H on the paper strips by scintillation counting (data not shown). Based on these results, it was calculated that the tetrasaccharides recovered from aorta, lung, intestine, kidney A, and kidney B HS contained, on average, 50, 84, 60, 80, and 104 O-sulfate groups/100 tetrasaccharide molecules. Elaborating these data as outlined above in (i) and (ii), it was concluded that between 21% and 32% of the total O-sulfate groups of the various HS preparations occupy alternating sequences (Fig. 6C). Furthermore, 44 -73% of the O-sulfates were located in the contiguous N-sulfated sequences, whereas only two of the samples, from lung and kidney B, showed significant O-sulfation of spaced structures (see also Ref. 30). These findings are compatible with the conclusion that major portions of the 6-O-sulfate groups are located in the alternating sequences, presumably on both N-acetylated and N-sulfated GlcN units (Fig. 7).

DISCUSSION
The N-and 2,6-di-O-sulfated structure -IdoA(2-OSO 3 )-Glc-NSO 3 (6-OSO 3 )-is generally the predominant disaccharide unit in heparin preparations (26), but a minor component in most HSs. Instead, most of the 2-O-and 6-O-sulfate groups are confined to separate disaccharide units, as shown by analysis of products obtained by extensive deaminative cleavage of HS preparations (Table I). There is limited information regarding the distribution of such sulfate groups in relation to functional domains (protein-binding sequences) of HS chains. It has been reported that the binding region for lipoprotein lipase in endothelial cell HS is composed of five consecutive N-and 2,6-di-Osulfated disaccharide repeats (31). By contrast, the minimal binding region for basic fibroblast growth factor contains at least one essential IdoA 2-O-sulfate group, but no 6-O-sulfate residues (7,8); instead, it has been proposed that 6-O-sulfation mediates binding of an adjacent region to the growth factor receptor (32). The antithrombin-binding region in HS (and in heparin) contains a functionally essential 6-O-sulfated GlcNAc or GlcNSO 3 unit that is located 3 monosaccharide residues apart from the nearest Analysis of HS preparations showed an appreciable proportion of the total 6-O-sulfate groups to be located on GlcNAc residues (39,40). Indeed, while 2-O-sulfation of IdoA units was essentially restricted to the N-sulfated blocks, the 6-O-sulfate groups occurred largely outside these regions (Fig. 6F). Furthermore, whereas the extent of 2-O-sulfation within these blocks was essentially the same for all HS preparations analyzed, the degree of 6-O-sulfation varied greatly between the samples (Fig. 3). Nevertheless, the different HS species displayed a a Disaccharides generated by deamination at pH 1.5, resulting in selective cleavage of glucosaminidic linkages associated with N-sulfated GlcN units and reduction of the products with NaB 3 H 4 , as described under "Methods." The resultant HexA-[1-3 H]aMan R disaccharides were separated by anion-exchange HPLC (Fig. 5) and by paper chromatography (see "Methods"). Values are given as mol % of total labeled disaccharides and were calculated from peak areas. ND, not detected (Յ0.2%).
b See footnote b of Table I. remarkably constant proportion of alternating sequence (-Glc-NSO 3 -(HexA-GlcNAc-GlcA-GlcNSO 3 ) n -) (Fig. 6A), rich in 6-Osulfates but essentially devoid of 2-O-sulfate groups (Fig. 6, C,  E, and F; Fig. 7). Such regions could conceivably be designed for specific interactions with proteins through their N-and 6-Osulfate groups. Combining the different structural variables would enable the generation of HS species with distinct composition, sequence characteristics, and domain organization. Selected HS preparations with distinctive structural features have been obtained from rat liver (41), rat kidney (42), murine Reichert's membrane (an extraembryonic basement membrane produced by parietal endoderm cells; Ref. 43), syndecan-1 from different cultured cells (44), and various human organs (45). The scope of the latter study was similar to that of the present report, but was restricted to analysis of contiguous N-sulfated regions. The results suggested that the structural characteristics of HS preparations, isolated from a particular tissue, are highly reproducible between different individuals of the same species. It seems reasonable to conclude that such characteristics are tailored, during HS biosynthesis, to satisfy specific structure/function requirements of the mature polysaccharide, presumably expressed through binding to proteins.
Our current model of heparin/HS biosynthesis features a series of membrane-bound enzymes that simultaneously, in processive yet consecutive fashion, catalyze elongation and modification of a polysaccharide chain (46). Major questions concerning the mechanism and regulation of this process remain unresolved. For instance, the domain-type distribution of N-substituents implies an on-off mode of action, along the polysaccharide chain, for the N-deacetylase/N-sulfotransferase that catalyzes the initial modification reactions. What is the mechanism behind such modulation, and how is it related to the two genetically distinct forms of this enzyme (47,48)? Moreover, what is the molecular relation between the GlcA C-5 epimerase (purified from bovine liver; Ref. 49) and IdoA 2-Osulfotransferase enzymes, that apparently may operate either in concert or separately, depending on the N-sulfation pattern? Finally, which of the enzymes involved in the overall biosynthetic process catalyze more than one reaction? Previous findings indicate that the N-deacetylation/N-sulfation process, as well as the GlcA-and GlcNAc-transfer reactions required for chain elongation, are catalyzed by enzymes with dual activities (50,51). It has been suggested that the IdoA 2-O-and GlcN 6-O-sulfation reactions in heparin biosynthesis may indeed both be catalyzed by the same ϳ60-kDa enzyme (52). On the other hand, the discrepant distribution of 2-O-and 6-O-sulfate groups in HS strongly suggests that the two major O-sulfotransferase reactions are independently regulated. It is noted that a GlcN 6-O-sulfotransferase, purified from the medium of HS-producing cells, appeared devoid of IdoA 2-O-sulfotransferase activity (53). A more definite answer to these questions will require the molecular cloning of all enzymes and essential auxiliary proteins involved and, ultimately, the reconstitution of a functional biosynthetic apparatus using recombinant enzymes/proteins and artificial membrane systems.