THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255, No. 11, Issue of June 10, pp. 5094-5100,1980 Printed an U.S.A.

Biosynthesis of Heparin CONCERTED ACTION OF LATE F O L Y M ~ R - M O ~ I F ~ ~ A T I O N REACTIONS*

(Received for publication, December 5,1979)

Ingvar Jacobsson and Ulf Lindahl From the DeDartment of Medical Chemistry, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, S-

”

751 23 Vppsala, Sweden ”

The substrate specificity of 0-sulfotr~sferases in- volved in the biosynthesis of heparin was studied by incubating exogenous polysaccharide acceptors with mouse mastocytoma microsomal fraction in the pres- ence of phosphoadenyIyl[a5S]sulfate. Characterization of the labeled products showed that 0-sulfation occurs preferentially in the vicinity of N-sulfate groups; that 2-0-sulfation of L-iduronic acid residues occurs pref- erentially or exclusively in the absence of a 6-0-sulfate group on adjacent D-glUCOSamine units; and that 6- 0-sulfation of D-glucosamine residues occurs readily in the presence of 2-0-sulfate groups on adjacent L- iduronic acid units. Furthermore, structural analysis of microsomal heparin-precursor polysaccharides showed a distinct intermediate species that contained 2-0-sulfated L-iduronic acid units but essentially no 6- 0-sulfate groups on the (N-sulfated) D-glucosamine res- idues. The results suggest that 2-0-sulfation of L-idu- ronic acid units is tightly coupled to the formation of these units (by 5-epimerization of D-glucuronic acid residues) and, furthermore, that both processes are completed before 6-0-sulfation of the polysaccharide molecule i s initiated. ~ - ~ ~ u c u r o n o s y ~ 5-epimerization not accompanied by 2-0-sulfation occurs at a still ear- lier stage of polymer modification; the resulting tidu- ronic acid units appear to remain nonsulfated through- out the subsequent ~ ~ ~ c a t j o ~ reactions.

The biosynthesis of heparin involves a series of polymer- modification reactions that lead, in a cell-free biosynthetic system, to the formation of a number of microsomal polysac- charide intermediates. The reactions take place in a stepwise manner, such that the resulting intermediates are distinctly different structurally and may be separated by ion exchange chromatopaphy (3, 4; see also Fig. 4 of the present report). The nature of each reaction was deduced initially from the structures of these intermediates (see Fig. 1 in Ref. 5) but has also been defined by use of specific assay procedures (5-8). The sequence of polymer modifcations is as follows. Polym- erization of D-glUCUrO& acid and N-acetyl-D-glucosamine residues in alternating sequence yields the intermediate, PS- MAC,’ which subsequentiy undergoes partial N-deacetylation

* This work was supported by grants from the Swedish Medica1 Research Council (2309), the Swedish University of AgricuIturai Sciences and AB Kabi, Stockholm. This is Paper X in a series in which the preceding reports are Refs. 1 and 2. The costs of Publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

See Fig. I in Ref. 5 for abbreviations of polysaccharides that axe intermediates in heparin biosynthesis. Other abbreviations used are: N-sulfate, sulfamino group; @sulfate, ester sulfate group; PAPS, 3”

and is converted into a polymer, PS-NH,+, that contains D- glucosamine residues with unsubstituted amino groups. In the next step, PS-NHs+ is transformed into PS-NSOa-, which contains N-sulfated as well as N-acetylated D-glucosamine residues, D-glucuronic acid and some L-iduronic acid units, but no 0-sulfate groups. Finally, a major portion of the D- glucuronic acid units undergo 5-epimerization, to L-iduronic acid residues, along with the incorporation of 0-sulfate groups at C-2 of L-iduronic acid and at C-6 of D-glucosamine units. The product of these final reactions, PS-N/O-SO,, is a mix- ture of polysaccharides that includes the end product of the overall biosynthetic process, i.e. heparin. The present report describes the inter-relation between the final reactions, giu- curonosyl5-epimerization and 0-sulfation. A tentative scheme on the sequential order of the various polymer-modification reactions will be presented.

EXPERIMENTAL PROCEDURES

Materials-The preparations of chemically modified (desulfated, N-deacetylated, and N-sulfated) heparin and of heparan sulfate (iso- lated from human aorta) were as described (5). The heparan sulfate was treated with acetic anhydride (9) in order to acetylate any free amino groups present. Analytical data are given in Table I of Ref. 5 and in Table I of the present report.

UDP-~-[’~C3glucuronic acid (321 ~ ~ i / ~ r n o l ) , Nar3H]BHs (247 &i/ gmol), and Nag(35S)S0, (carrier-free) were obtained from the Radi- ochemical Centre, Buckinghamshire, United Kingdom. UDP-N-ace- tyl-n-[6-3H]glucosamine (approximately 7 mCi/pmol) and unlabeled UDP-N-acetyl-D-glucosamine were purchased from New England Nuclear Co., Dreieichenhain, West Germany, and from Sigma Chem- ical Go., St. Louis, Mo., respectively. Unlabeled PAPS was prepared as described earlier (5) . ”S-labeled PAPS was synthesized in a similar manner, except that a 100,OOO X g supernatant of the rat liver homogenate was used as enzyme source (instead of proteins precipi- tated with ammonium sulfate), and carrier-free NadYSS)S04 was added (1 mCi/BO-pl incubation mixture) instead of u ~ a b e ~ e d sulfate.

TJA ”-L aMan disaccharides, unlabeled or with 3H-labeled anhydro- mannitol residues, were isolated after d e ~ i n a t i v e cleavage of heparin or heparan sulfate with nitrous acid (10).

ous gift from Dr. A. Wasteson, University of Uppsala, Sweden. The a-L-Iduronate sulfatase, isolated from human serum, was a gener-

preparation of microsomal fraction from mouse mastocytoma t i m e has been described (5). Solubilization of the microsomal preparation was carried out by treatment with detergent and salt, according t.o the procedure of Schwartz and Roden (11). Microsomal fraction, sedimented by centrifugation at 100,OOO X g for 60 min, was suspended in 0.05 M Tris/acetate buffer, pH 5.5, containing 0.05 M KCi, 0.001 M EDTA, and 0.5% Nonidet P-40, to yield a final protein concentration of 40 mg/ml. After 30 min at 4”C, the material was centrifuged at

phosphoadeny~y~ulfate; Hepes, 4-(2-hy~oxyethy~)-l-piper~~neeth- anesulfonic acid; UA, unspecified uronic acid; GlcUA, D-glucuronic acid; IdUA, L-iduronic acid GlcNHs’, 2-deoxy-2-amino-~-glucose; GlcNAc, 2-deoxy-2-acetamido-~-glucose; GlcNS03-, 2-deoxy-2-sulf- amino-D-glucose; aMan, 2,5-anhydro-~-mannitol. The location of 0- sulfate goups is indicated in parentheses; glycosidic linkages are indicated by m o w s (4).

______ ” - ” __I____ ”

5094

Biosynthesis of Heparin

100,ooO x g for 60 min; the resulting pellet was resuspended in the buffer described above, by sonication, and potassium chloride was added to a final concentration of 0.5 M. After 15 min at 4"C, the mixture was centrifuged as before, and the supernatant (10 mg of protein/ml) was dialyzed against 0.05 M Hepes, pH 7.4, containing 0.25 M sucrose and 0.5% Nonidet P-40.

Analyt~cal Methods-Methods for the dete~ination of protein and radioactivity have been described (4). Uronic acid was determined by the method of Bitter and Muir (121, with D-glucuronolactone as standard. Ratios of ~-['~C]glucuronic acid to ~-['~C]iduronic acid were estimated as described ( 5 ) . The disaccharide composition of N-sul- fated polysaccharides was determined by analysis of deamination products (10, 13).

Paper electrophoresis and paper c ~ m a ~ ~ a p h y were c d e d out as described previously (10).

Incubation of Mastocytoma Microsomal Fraction-Sulfated mi- crosomal heparin-precursor polysaccharides were prepared by incu- bating, per ml of 0.05 M Hepes, pH 7.4: IO pCi of UDP-D-[14C]- glucuronic acid, 100 pCi of UDP-N-acetyl-~-[6-~H]g~ucosamine, 35 nmol of unlabeled UDP-N-acetyl-D-glucosamine, 10 mg of microsomal protein, 10 pmol of MnCh; 10 pmol of MgC12, and 5 pmol of CaC12. Incubation was carried out a t 37OC for 3 h, and 1 pmol of unlabeled PAPS was added/& of incubation mixture at the start and after 1 h of incubation.

[?!$]Sulfate groups were incorporated into exogenous polysaccha- ride acceptors by i n c u b a ~ ~ g , per ml of 0.05 M Hepes, pH 7.4: 0.8 mg of polysaccharide substrate (heparan sulfate or modified heparin); 0.2 pmol of ["SJPAPS (300 pCi/pmol); 2 mg of particulate or solubilized microsomal protein; 10 pmol of MnCh, 10 pmol of MgCh; and 5 pmol of CaC12. The mixture was incubated at 37°C for 2 h.

Incubations were terminated by heating at 100°C for 2 min. Labeled polysaccharides were isolated after digestion with papain, as described (5).

RESULTS

The inter-relationship of the final polymer-modification reactions in heparin biosynthesis was studied in two types of experiments. First, the substrate specificities of the two 0- sulfotransferases were investigated with regard to the effects of pre-existing sulfate groups in the polysaccharide acceptor molecule, the rationale being that the results could give a clue to the sequential order of the corresponding reactions in the biosynthetic process. Secondly, the expression of these reac- tions in a cell-free biosynthetic system was studied by struc- tural analysis of microsomal polysaccharide intermediates.

Substrate Specificity of 0-Sulfotransferases Role of N-Sulfate Groups-A low sulfated heparan sulfate

was used as substrate for mastocytoma microsomal sulfotrans- ferases. The D-glucosamine residues of this sulfate acceptor were largely N-acetylated, only about 30% containing sulfated amino groups (5). The low degree of N-sulfation is reflected in the extended elution pattern of the deaminated polysaccha- ride2 on gel chromatography (Fig. 1). About 10% of the deam- ination products were disaccharides, with IdUA(2-OS03-) + aMan as the dominating component (Table I), corresponding to (-+GlcNS03-) -+ IdUA(2-OS03-) -+ GlcNS03- 3 se- quences in the intact polysaccharide (where the glucosamine residue in parentheses would be present in the intact polysac- charide but would not be represented in the disaccharide recovered after deamination*). After incubation with micro- somal fraction in the presence of [35S]PAPS, the reisolated

a Polysaccharides were deaminated by treatment with nitrous acid, under conditions leading to specific attack of N-sulfated glucosamine residues (IO). These residues are converted to 2,5-anhydromannose units, with loss of the N-sulfate group as inorganic sulfate, and cleavage of the corresponding glucosaminidic linkages; N-acetylated glucosamine residues are unaffected. A uronic acid unit located be- tween two N-sulfated glucosamine residues will thus be recovered in a UA + aMan disaccharide; an isolated N-acetylated glucosamine moiety will give rise to a UA + GlcNAc + UA -+ aMan tetrasaccha- ride, etc.

0.3 -

5 0 m In 0.2 - I- U W

5095

1 5

'0 m '0

E

c x

P 0

5 ; ; ; Y) m

60 80 100 120 EFFLUENT VOLUME (ml)

tvo FIG. 1. Gel chromatography on Sephadex G-25 of heparan

sulfate, after incubation with mastocytoma microsomal frac- tion in the presence of [35S]PAPS, and deamination with ni- trous acid. Incubation with ["SIPAPS was performed as described under "Experimental Procedures"; the reisolated polysaccharide was treated with nitrous acid at pH 1.5 and the products were reduced (10). The deaminated and reduced material was applied to a column (1 X 185 cm) of Sephadex (2-25, that was eluted with 0.2 M NH4HCOs at a rate of 4 ml/h. Effluent fractions of 2 ml were collected and analyzed for uronic acid by the carbazole reaction (0) or for '"S (0). The uronic acid-containing material with peak elution position at 103 ml is disaccharides. The most retarded % peak corresponds to inorganic ["'S]sulfate. The 35S-labeled disaccharides were pooled as indicated by the vertical lines, lyophilized, and further analyzed as described in the text.

polysaccharide contained about 2400 cpm of 35S/gg of uronic acid. Treatment with nitrous acid released a major portion of this label as inorganic [35S]sulfate (Fig. l), representing N- [35SJsulfate groups in the intact polysaccharide?. O-[35S]Sul- fate groups were mainly associated with the disaccharide and, to a lesser extent, with the tetrasaccharide fractions, but decreased in amount in larger oligosaccharides containing consecutive N-acetylated disaccharide units (Fig. 1). 0- Sulfate groups had thus been incorporated preferentially into N-sulfated, rather than into N-acetylated, regions of the ac- ceptor polysaccharide molecule. The high yield of labeled disaccharide suggests that (-+GlcNS03-) -+ UA "F GlcNS03- "+ sequences are particularly efficient acceptors of 0-sulfate groups. However, the isolation of labeled components larger than disaccharide indicates that 0-sulfation may also involve N-acetylated structures of the type (+GlcNS03-) -+ [UA -+

-+ ClcNAc]= + UA + GlcNS03- +. The data available do not show whether both the N-acetylated and the N-sulfated disaccharide units of such sequences can become 0-sulfated. Since the 0-sulfate acceptor ability apparently decreases with increasing values for n, it appears likely that 0-sulfation of N- acetylated disaccharide units will only occur (if at all) in juxtaposition to N-sulfated disaccharide units.

,' The incorporation of N-["S]suifate groups into polysaccharides that contain acetylated but no unsubstituted amino groups requires concerted N-deacetylation and N-sulfation reactions (J. Riesenfeld, M. Hook, and U. Lindahl, unpublished observation). Such N-35S sulfation was often observed but was not a constant finding in exper- iments of the type described (see Ref. 6); the reason for this irregu- larity is unknown,

5096 Biosynthesis of Heparin

Role of 0-Sulfate Groups-The relation of incorporated 0- ["S]sulfate groups to pre-existing, unlabeled 0-sulfate groups was studied by further analysis of the labeled disaccharides isolated in the experiment described in the preceding section. Separation of these components by paper electrophoresis at pH 1.7 showed 87% di-0-sulfated and 13% mono-0-sulfated disaccharides. After digestion of the di-0-sulfated disaccha- ride with a-L-iduronate sulfatase, labeled mono-0-sulfated disaccharide and undigested di-0-sulfated disaccharide were detected, but no inorganic ["5S]sulfate (Fig. 2). The di-0- sulfated, labeled disaccharide should therefore have consisted exclusively of IdUA(2-OS03J + aMan(6-035S03-). The cor- responding labeled disaccharide units in the intact polysac- charide would thus have been formed by 6-0-[35S]sulfation of preformed "+ IdUA(2-OS03-) + GlcNS03- + structures, and not by 2-0-[35S]sulfation of preformed -+ IdUA -+ GlcNSOs-(6-OS03-) + structures. These findings raised the question as to the substrate specificity of the iduronosyl2-0- sulfotransferase; can L-iduronic acid residues become sulfated if the adjacent D-glucosamine unit (bound to C-1 of the L- iduronic acid residue) already carries a 6-O-sulfate group? Owing to the low proportion of + IdUA -+ GlcNS03-(6- OSOS-) + disaccharide units, as compared to -+ IdUA(2-

TABLE I Analysis of total disaccharides formed on deamination of heparin- related polysaccharides, and of 35S-labeled disaccharides obtained on deamination of the same polysaccharides after incubation with mastocytoma microsomal fraction, in the presence of [3'SJPAPS

Polysaccharide

Heparan sulfate Modified heparin Disaccharide

Total di- b:E2& Total di- b:Efa&i. saccha- rides" saccha- saccha-

ridesh rides" rides*

GlcUA + aMan 12 25 IdUA + aMan 3 38 GlcUA + aMan(G-OSOs-) 2 3 10 IdUA + aMan(6-OS03-) 3

IdUA(2-OS03-) -+ 12 87 3 26 IdUA(2-OS08-) + aMan 68 1 1 3 2: 44 21

aMan(6-OSO:lJ Polysaccharides were deaminated with nitrous acid and the prod-

ucts were reduced with Na[3H]BH4 (10). Disaccharides, isolated by gel chromatography on Sephadex G-25, comprised 10% and 74% of the heparan sulfate and the modified (partially desulfated, N-deace- tylated, and N-sulfated) heparin starting materials, respectively. The disaccharides were separated into the components indicated by a combination of paper electrophoresis and paper chromatography, as described (lo), and were quantified by the 3H-label in the anhydro- mannitol units. The results are expressed as per cent of total disac- charides in the deamination products.

* Polysaccharides were incubated with mastocytoma microsomal fraction in the presence of [3'S]PAPS, as described under "Experi- mental Procedures." The reisolated heparan sulfate and modified heparin both contained 2400 cpm of %3/pg of uronic acid. Deamina- tion of the heparan sulfate released 58% of the label as inorganic [3sS]sulfate (corresponding to N-["'S]sulfate groups in the intact polysaccharide), as shown by gel chromatography on Sephadex G-25 (see Fig. 1 and the text for further information). Of the remaining (0)-[JsS]sulfate groups, 61% were recovered in a disaccharide fraction. Deamination of the modified heparin yielded about 10% of the label as inorganic [3sS]sulfate; 85% of the 0-[:'"S]sulfate groups were re- covered in disaccharides. The 35S-labeled disaccharides were sepa- rated into mono-0- and di-0-sulfated components by paper electro- phoresis at pH 1.7. The mono-0-sulfated disaccharides obtained from the modified heparin were separated further by paper chromatogra- phy (10). The results are expressed as per cent of total "S-labeled disaccharides in the deamination products, assuming only one 0- [35SJsulfate group per labeled di-0-sulfated disaccharide molecule (see "Results").

8 -

6 -

0 0 I I , m I 0 0 E ,

0 10 20 30 MIGRATION DISTANCE (cm )

FIG. 2. Paper electrophoresis at pH 1.7 of 35S-labeled di-0- sulfated disaccharide, after digestion with a-L-iduronate sul- fatase. Disaccharides were isolated from deaminated, 35S-labeled heparan sulfate as described in the legend to Fig. 1. The di-0-sulfated component (87% of the total labeled disaccharide) was isolated by paper electrophoresis, and a sample (240 X 10' cpm) was incubated with 0.3 mg of a-L-iduronate sulfatase in 50 pl of 0.05 M acetate buffer, pH 4.5, at 37°C for 72 h. The digest was heated at 100°C for 2 min and was then directly applied to the Whatman No. 3MM electropho- resis paper. The standards shown below the tracing are: I , UA + aMan; II, UA + aMan(6-OSOsJ; IIZ, I~UA(~-OSOS-) + aMan(6- OSOS-); IV, inorganic ["'S]sulfate.

OSOe-) + GlcNS03- "-$ units, in the heparan sulfate starting material (Table I), the results shown in Fig. 2, although suggestive, were not considered conclusive. Therefore, the experiment was repeated with a different sulfate-acceptor polysaccharide, a chemically modified heparin preparation, that contained larger amounts of nonsulfated L-iduronic acid residues, in non-0-sulfated as well as in 6-0-sulfated disac- charide units, and relatively smaller amounts of -+ IdUA(2- OSOJ -+ GlcNS03- + units (Table I). The total amount of [3sS]sulfate incorporated (2400 cpm of ?3/pg of uronic acid) was similar to that obtained with heparan sulfate as acceptor, but a larger proportion of the label went into mono-0-sulfated disaccharide units (Table I). The mono-0-sulfated species formed was largely "+ IdUA(2-035S03-) -+ GlcNS03- -+ units, thus demonstrating that the microsomal preparation con- tained active a-L-iduronosyl 2-0-sulfotransferase. However, digestion of the labeled di-0-sulfated disaccharide with a - ~ - iduronate sulfatase, followed by paper electrophoresis, again indicated IdUA(2-OS03-) -+ aMan(6-03'S03-) as the only component present. These results suggest that sulfation of L- iduronic acid units occurs preferentially, or exclusively, in the absence of 6-0-sulfate groups on the adjacent (at C-1) D- glucosamine residue^.^

The effect of a 6-0-sulfate group on the D-glUCOSamine residue bound to C-4 of the potential target L-iduronic acid unit cannot be evaluted by the present results. If, as suggested later in this paper, 2- 0-sulfation of L-iduronic acid units occurs only in conjunction with the formation of such units (by 5-epimerization of D-glUCUrOniC acid residues), then the inhibitory effect of 6-0-sulfate groups on the 2-0- sulfation reaction may be secondary to their inhibitory effect on the 5-epimerization reaction.5 ' I. Jacobsson, U. Lindahl, J. Jensen, L. Roden, H. Prihar, and D.

S. Feingold, manuscript in preparation.

Biosynthesis of Heparin

An interesting difference was noted between the sulfation patterns obtained with intact and with solubilized micro- somes, respectively. With solubilized microsomal enzymes 2- 0-sulfation of L-iduronosyl moieties was drastically reduced in relation to 6-0-sulfation of D-glucosaminyl moieties. This is illustrated in Fig. 3, which shows a paper chromatographic separation of the m~no-O-[~~S]sulfated disaccharides isolated after incubation of modified heparin and [35S]PAPS with intact (Fig. 3A) or solubilized (Fig. 3B) microsomal prepara- tion. The significance of this finding is discussed below.

Sequential Order of Glucuronosyl5-Epimerization and Sulfate Transfer Reactions

Sulfated heparin-precursor polysaccharides were prepared by incubating mastocytoma microsomal fraction with UDP- ~-[‘~C]glucuronic acid and UDP-N-acetyl-~-[6-~H]glucosa- mine. in the presence of unlabeled PAPS. Fractionation of the

/ ’ I -7, I 10 20 30 40

MIGRATION DISTANCE ( c m ) FIG. 3. Paper chromatography of m~no-O-[~~S]sulfated di-

saccharides isolated after deamination of modified heparin, incubated with [36S]PAPS and ( A ) particulate; (B) solubilized microsomal enzymes. Solubilization of microsomal fraction and incubations of polysaccharide were carried out as described under “Experimental Procedures.” The reisolated polysaccharides con- tained 2400 ( A ) and 430 ( B ) cpm of %/pg of uronic acid, respectively. Degradation of the labeled polysaccharides and isolation of mono-0- [”S]sulfated disaccharides were carried out as described in the legend to Table I. The disaccharides were separated into two fractions, containing 6-0-sulfated D-glucosamine and 2-0-sulfated L-iduronic acid residues, respectively, by paper chromatography (Solvent B in Ref. 10). The standards shown below the chromatograms are: Z, GlcUA + aMan(G-OSO$-) and IdUA + aMan(G-OSOs-); ZZ, IdUA(2- OSOS-) + aMan.

PS-NH; PS-NS0; PS-Nn-SOq- A c

PS-NAc A

5097

1.5

- I

1.0 I- U E I- z IAI u z 0 u

0.5 u d

-I

t t t HA c s HeP

FRACTION NO. FIG. 4. Chromatography on DEAE-cellulose (5) of sulfated

polysaccharides (0) obtained by incubating mastocytoma mi- crosomal fraction with radioactively labeled nucleotide sugars and unlabeled PAPS (see “Experimental Procedures”). For comparison, the elution pattern of the nonsulfated components (0) obtained in the absence of PAPS is also indicated. Effluent fractions were analyzed for 14C and were then pooled as indicated at the top of the figure. The fractions obtained were desalted by passage through a column of Sephadex G-25, eluted with 10% aqueous ethanol. The arrows indicate the peak elution positions of hyaluronic acid (HA), chondroitin 4-sulfate (CS), and heparin (Hep), respectively. - - -, LiCl concentration.

products by ion exchange chromatography yielded three peaks of labeled material (Fig. 4). The least retarded of these com- ponents, PS-NS03-, was previously shown to contain N-sul- fated (and a minor proportion of N-acetylated) D-glUCOSamine residues, D-glucuronic acid residues, and a small amount of L-iduronic acid units, but no 0-sulfate groups (3,5). The more retarded components both contained N-sulfate as well as 0- sulfate groups and were therefore, in keeping with previous terminology, denoted PS-N/O-S03--a and PS-N/O-S03--b, respectively. The composition of the polysaccharides was determined by analysis of UA + aMan disaccharides formed by treatment with nitrous acid (10); the various disac- charides were quantified by the 3H-label introduced into the polysaccharides with the glucosamine residues and recovered in the anhydromannitol units of the disaccharides. Each of the three polysaccharide fractions showed about 80% conver- sion to disaccharides, indicating that most of the D-glUCOSa- mine residues were N-sulfated? The predominant disaccha- ride unit in PS-NS03- was + GlcUA + GlcNS03- +, with a small but significant contribution of -+ IdUA + GlcNS03- + units (Table 11). In PS-N/O-S03--a, the -+ GlcUA + GlcNS03- + units were decreased by more than half, as compared to PS-NS03-, and were replaced by + IdUA(2- OSOJ + GlcNS03- + units. PS-N/O-S03--b, finally, was largely composed of three disaccharide units, + GlcUA -+

GlcNS03-(6-OS03-) +, + IdUA + GlcNS03-(6-OS03-)

5098 Biosynthesis of Heparin

TABLE I1 Analysis of deamination products from microsomal heparin-

precursor p o l y s a c c h a ~ ~ ~ s

Analysis" Polysaccharide

".

GlcUA "+ aMan 77 35 10 IdUA + aMan 16 la <5 GlcUA "+ aMan(6-OSOJ <5 126 31 IdUA + aMan(6-OSO3.-} <5 6 18 IdUA(2-OS03-) "+ aMan <5 33 7 IdUA(2-OS03J -.) aMan- (6- <5 <5 32

OSOS')

Total iduronic acid' 20 (18) 53 (45) 62 (56) Iduronic acid 2-sulfate" <5 35 39 Anhydromannitol &sulfated <5 2Ob 81

e The labeled ([14.Gonic acids, ['H]glucosamine) polysaccharides were treated with nitrous acid, and the deamination products were reduced. Disaccharides, about 80% of the total deamination products, were isolated by gel chromatography and fractionated by a combl- nation of paper electrophoresis and paper chromatography (10). The separated products were quantified by the 3H-label in the anhydro- mannitol units, the results are expressed as per cent of total disaccha- rides.

The disaccharide, GlcUA- aMan(G-OSOs-), from PS.N/O-S03-- a appeared as a double peak on paper chromatography, owing to the presence of an unknown 3H-Iabeled component. The values given are therefore probably too high. It is not known whether the presence of small amounts of 6-0-sulfated D-glucosamine residues in fraction PS- N/0-SO3--a reflects an actual structural feature of this in~rmediate or a contamination with PS-N/O-S03--b molecules.

Proportion of total disaccharides that contain iduronic acid (ob- tained by summation of values for IdUA -+ aMan, IdUA -+ aMan(6- OS03-), IdUA(2-OS03-) -+ aMan, and IdUA(2-OS03-) --* aMan(6- OS03-)). The values in parentheses give the ratio, in per cent, of ["CIIdUA to (['4CJIdUA + ['4C]GlcUA), as determined after degra- dation of the polysaccharides to monosaccharides and separation of the ['4CJuronic acids by paper chromatography.

Proportion of total disaccharides that contain iduronic acid 2-0- sulfate (sum of IdUA(2-OS03-) aMan + IdUA(2-OS03C) -+ aMan(G-OSOs-)) and anhydromannitoi &sulfate (sum of GlcUA -+ aMan(G-OSOs-) + IdUA + aMan(G-OSOa-) + ldUA12-OS03-) -r, aMan(G-OSOa-)), respectively.

-

*, and 3 IdUA(2-OS03-) -+ GlcNS03-(6-0SOa-) +, all containing 6-0-sulfated D-glucosamine residues. Table I1 also shows the amounts in each poiysaccharide fraction of total L-iduronic acid, 2-0-sulfated L-iduronic acid, and 6-0-sulfated N-sulfo-D-glucosamine units. It is seen that PS-N/O-S03--a and PS-N/O-S03--b contain about the same amounts of L- iduronic acid and the same proportion of 2-O-sulfated L-idu- ronic acid units, but differ considerably with regard to 6-0- sulfated D-glucosamine residues. If it is assumed that PS-N/ O-SOs--a is an intermediate in the conversion of PS-NS03- into PS-N/O-SOS--b, then it must also be concluded that the formation and 2-0-sulfation of L-iduronic acid residues pre- cede the 6-0-sulfation of D-glucosamine units. Furthermore, during the conversion of PS-NS03- (which contains nonsd- fated L-iduronic acid residues) into PS-N/O-SOS--a, the intro- duction of 2-0-sulfate groups is accompanied by a correspond- ing increase in L-iduronic acid content,. While D-glucuronosyl 5-epimerization is thus not necessarily followed by 2-0-sulfa- tion of the resulting L-iduronic acid residues (see also Ref. 5), the latter reaction would seem to be tightly coupled to the e p ~ e r ~ a t i o n process.

DISCUSSION

Previous studies on the biosynthesis of heparin suggested that the stepwise nature of the polymer-mod~lcationreactions

applies also to the final stages of this process. An N-sulfated microsomal polysaccharide, PS-NS03-, devoid of 0-sulfate groups, was thus identified as an in t e~ed ia t e in the formation of the N- and 0-sulfated product, PS-N/O-SO,- (3). The conversion of PS-NS03- into PS-N/O-S03- involves not only 0-sulfation in two different positions but also extensive 5- epimerization of D-glucuronic acid residues into L-iduronic acid units. Studies on the substrate specificities of the corre- sponding enzymes have yielded information bearing on the functional interrelationship of these reactions (Table 111). The D-giucuronosyl 5-epimerase wiu only attack residues that are adjacent to N-sulfated D-glucosamine units (5): Since the resulting L-iduronic acid residues may become 2-0-sulfated whereas D-ghCurOniC acid residues may not (8), it is obvious that the 2-0-sulfotransferase reaction will be similarly con- fined to the N-sulfated regions of the polymer. Finally, the results of the present study indicate that also the D-glucosa- minyl6-0-sulfotransferase operates preferentially in the vicin- ity of N-sulfate groups. Taken together, these findings suggest that in the biosynthesis of heparin-like polysaccharides, the overall extent of polymer modification will be determined primarily by the initial modifkation reactions, the N-dea- cetylation and N-sulfation of D-glucosamine residues; only N- sulfated regions of the resulting biosynthetic intermediate, PS-NSOa-, have the appropriate structure required for further modification in direction of the final product. This conclusion is in excellent agreement with the structural properties estab- lished for heparin-like polysaccharides, in which L-iduronic acid units and 0-sulfate groups are both accumulated in N - sulfated regions of the molecule (8).

The substrate specificity studies also provide information on the sequential order of the p o l ~ e r - m o d ~ c a t i o n reactions. The requ~ement for N-sulfate groups in the D-g~UCWOnOSyl 5-epimerization reaction thus established that N-sulfation of D-glucosamine units must precede the formation of L-iduronic acid units (5). Moreover, the presence of 6-0-sulfate groups on D-glucosamine units was found to inhibit not only 5-epi- merization5 but also, as shown in the present study, 2 - 0 - sulfation of adjacent uronic acid residues4 (Table 111). In contrast, the presence of 2-0-sulfate groups on L-iduronic acid units did not prevent 6-0-sulfation of adjacent D-glucosamine residues. These findings can only be rationalized by postulat- ing that D-glUCurOnOSyl 5-epimerization and L-iduronosyl2-0- sulfation take place before D-glucosaminyl6-0-sulfation. Ev- idence confii ing this conclusion was obtained by analysis of the two subfractions of PS-N/O-S03-. The occurrence of both 2-0- and 6-0-sulfate groups in one fraction, PS-N/O-SOz--b, but essentially 2-0-sulfate groups only in the other, PS-N/O- S03--a, strongly indicates that 0-sulfation takes place in a stepwise manner, 2-0-sulfation of a polysaccharide chain being completed before 6-0-sulfation is initiated. This is illus- trated in Fig. 5, which shows a tentative scheme for the formation of the three most abundant disaccharide units in the final product, PS-N/O-SOs--b.

Structural heterogeneity is a conspicuous feature of the CO-

polymeric glycosaminoglycans (14). One expression of this heterogeneity is the blockwise distribution of sugar residues and sulfate groups, such that, in heparin-like polysaccharides, N-sulfate groups, 0-sulfate groups, and L-iduronic acid resi- dues are accumulated in some regions of the polymer chains whereas N-acetyl groups and D-glucuronic acid units predom- inate in other regions. As pointed out above, the formation of such structures may be explained in terms of the substrate specificities of polymer-modifying enzymes. However, super- imposed on the block structures, additional heterogeneity is introduced as a result of incomplete polymer modification. Each modification reaction (with the possible exception of N-

Enzyme Polysaccharide structure"

Accepted as substrate Rejected as substrate " "

D-Glucuronosy1 5-epimerase

L-Iduronosyl2-0-sulfotransferase

D-Glucosaminyl 6-0-sulfotransferase

-+ GlcN-+ GlcUA-+ GlcN-t 4 GlcNAc + GlcUA -+ GlcNAc I I sea- so3-

I soy- s0:r-

6-0-S0:%'

+ GlcN+ GIcUA- G1cNAc-t + GlcNAc + GlcUA + GlcN -+

I

I 4 GlcN-+ GlcUA-t GlcN-+

I I sos- sea-

6-O-SO:j- I

+ TdUA-+ GlcN+ I so:*-

-+ IdUA -+ GlcN +' I so,-

-+ GlcUA+ GlcN+ I sea-

+ IdUA-+ GlcN-t I soy-

-+ 1dUA-t GlcN+ I I 2-0-soy- sos-

" Only structures known to be formed during biosynthesis of heparin are considered; all glycosidic linkages are thus of the appropriate 1

If 2-0-sulfation occurs only in connection with D-giUCWOnOsyl 5-epimerizaiion (see the text) it would be more appropriate to indicate -+

"+ 4 type (p-D-glucuronidic, a-L-iduronidic, a-D-giucosaminidic). The table includes same of the results described in Ref. 5 and Footnote 5.

GlcUA + GlcNS03- -t disaccharide units as substrate for a D-glucuronosyl5-epimerase~~-iduronosyi 2-0-sulfotransferase complex.

PS-NAc

PS-NH:

PS-NS0;

PS-N/O-SO,-a

PS-N/O-SOi-b

CWa0H

O H Y H

coo- CH*OH

. . O H

I \

Unchanged

1 O S O S l l " S 0 ~

Unchanged

CHl0SOj CH,OSOj

ow H M S O j OH " *SO j

FIG. 5. Tentative scheme for the formation of the three most abundant disaccharide units in the final bios~thetic product, PS-N/O-SOs--b. For additional informaiion, see the text.

5100 Biosynthesis of Heparin 5 10 15 20 25 30

8 PS - NAc

-GlcNAc 0 -GlcUA - 6-0-sutfate gmup

n -GlcNHj D - IdUA . - 2 - N - or 2-0-sulfata OmUP

-GlcNSOj

FIG. 6. Schematic representation of polymer-modification reactions leading to the formation of a heparin-like polysac- charide. The scheme summarizes the results obtained in the present study and also includes information described elsewhere (3, 5, 7)6 regarding the mechanisms and sequential order of the various reac- tions. In the intermediate polysaccharides, PS-NH,', PS-NSO:I-, and PS-N/O-SOa -a, only those components are shown that represent a change in structure compared to the preceding intermediate. The structure indicated for PS-N/O-S03--b includes most of the disaccha- ride units identified in the isolated material (Table 11) but does not give a quantitative account of its composition; it represents a hypo- thetical sequence of components known to be present in heparin-like polysaccharides (heparin or heparan sulfate). The sequence should be compatible with all structural information available (conclusive evidence for the occurrence of 6-0-sulfated N-acetylglucosamine res- idues, position 9 in the sequence, is still lacking). The reducing terminal of the sequence is to the right. The symbols used are chosen so as to illustrate some of the substrate specificity properties of the polymer-modifying enzymes. It is thus seen (a) that 5-epimerization of D-ghCurOniC acid residues can take place only if the D-glucosamine unit at C-4 is N-sulfated (the o-glucosamine unit at C-1 may be either N-sulfated or N-acetylated) (5)5; and ( b ) that L-iduronic acid residues are accessible to 2-0-sulfation whereas D-glucuronic acid residues are not. The directing influence of the early polymer-modification reac- tions (N-deacetylation and N-sulfation of D-glucosamine residues) is expressed by the accumulation of 0-sulfate groups and L-iduronic acid units in the N-sulfated regions of the polymer. Note incomplete polymer modification (see the text for explanation), for instance the D-glucuronic acid residues in positions 22 and 24 and the nonsulfated L-iduronic acid units in positions 8 and 20. In accord with the results of the present and previous (5) studies, the formation of nonsulfated L7iduronic acid residues is shown to take place before that of 2-0- sulfated L-iduronic acid units. The N-sulfation process involves the formation of a partially N-deacetylated intermediate species, PS- NH:,+, but may also occur by a more concerted N-deacetylation-N- sulfation reaction, as indicated (3).6

sulfation) thus appears to be incomplete, in the sense that a fraction of the potential substrate units escapes modification. The combined effects of block-structure formation and incom- plete polymer modification are illustrated in Fig. 6, which shows the stepwise conversion of the initial polymerization product, PS-NAc, into a hypothetical polysaccharide of highly complex structure, believed to be analogous to that of PS-N/ O-SOs--b. I t is obvious that the number of polymer sequences having similar size but different structures must be exceed- ingly large and, furthermore, that incomplete polymer modi- fication will contribute to this variability. Interactions of phys- iological importance between glycosaminoglycans and other macromolecules may be critically dependent on the fine struc- ture of the polysaccharide chains (14); for instance, it was recently proposed that nonsulfated L-iduronic acid in a specific location is essential for the interaction between heparin and antithrombin, and hence for the blood anticoagulant activity

J. Riesenfeld, M. Hook, and U. Lindahl, unpublished observation.

of the polysaccharide (13, 15). With the above considerations in mind, the question of

regulation assumes vital importance: what factors determine whether a potential substrate residue will undergo or escape a particular polymer-modification reaction? The mode of se- lection is unknown and a random process cannot be excluded. However, the organization of the biosynthetic apparatus is poorly understood and may well involve hitherto unrecognized regulatory mechanisms. Results obtained in the present study may in fact be interpreted in terms of such a mechanism, based on interaction between polymer-modifying enzymes. As noted under "Results," the amount of D-glucuronic acid resi- dues that undergo 5-epimerization during the conversion of PS-NS03- into PS-N/O-SOs--a, is about equal to the amount of 2-0-sulfated L-iduronic acid units in the latter fraction. On the other hand, the nonsulfated L-iduronic acid units in the final product, PS-N/O-SO,--b, appear to be present already in PS-NSOa- (Table 11). These findings suggest that the sulfation of L-iduronic acid residues is tightly coupled to the epimerization reaction that leads to the formation of these residues. Epimerization not accompanied by 2-0-sulfation may occur by a temporally, or spatially separate process, or both, such that the L-iduronic acid units formed will escape sulfation during subsequent steps of polymer modification (Figs. 5 and 6). A biosynthetic machinery may be visualized in which D-glucuronosyl 5-epimerase molecules are attached to the microsomal membranes, either solitary or in complex with L-iduronosyl 2-0-sulfotransferase molecules; the sulfo- transferase would function only in concerted action with the epimerase. Although speculative, this model has some support in experimental data. It was thus noted that whereas mem- brane-bound microsomal sulfotransferases catalyzed the in- corporation of 2-0-sulfate as well as 6-0-sulfate groups into exogenous polysaccharide acceptor, only 6-0-sulfation was obtained with solubilized enzymes (Fig. 3). Solubilization could conceivably result in dissociation of an epimerase. sul- fotransferase complex, thereby inactivating the latter enzyme.

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