Proc. Nati. Acad. Sci. USA Vol. 84, pp. 3565-3569, June 1987 Biochemistry Heparin sequences in the chains of an endothelial cell (heparitinase I/heparitinase iH/heparinase/copolymer/disaccharide sequence) H. B. NADER*, C. P. DIETRICH*, V. BUONASSISI, AND P. COLBURN W. Alton Jones Cell Science Center, Lake Placid, NY 12946 Communicated by Gordon H. Sato, January 13, 1987

ABSTRACT The structure ofthe glycosaminoglycan chain MATERIALS AND METHODS of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using Substrates, Enzymes, and Materials. Heparin from bovine various degradative enzymes (heparitinase I, heparitinase II, intestinal mucosa and heparan sulfate from bovine pancreas heparinase, glycuronidase, sulfatases) from Flavobacterium were gifts from P. Bianchini (Opocrin Research Laborato- heparinum. This proteoglycan inhibits the thromboplastin- ries, Modena, Italy). Chondroitin 4- and 6-sulfates were activated pathway of coagulation; as a consequence, the cata- purchased from Miles. Heparinase, heparitinases, disaccha- lytic conversion of prothrombin to thrombin is arrested. ride sulfoesterase, and glycuronidase were prepared from Heparitinase I (EC 4.2.2.8), an enzyme with specificity re- induced F. heparinum cells, and mono-, di-, and tetrasac- stricted to the heparan sulfate portion of the polysaccharide, charides were prepared from heparin and heparan sulfates as releases fragments with the electrophoretic mobility and the described (11-13). Ethylenediamine (1,2-diaminoethane) was structure of heparin. Conversely, an assessment of the size and purchased from Aldrich. L-[ring-2,3,4,5,6-3H]phenylalanine distribution ofthe heparan sulfate regions has been provided by (106.3 Ci/mmol; 1 Ci = 37 GBq), D-[1,6-3H(N)]glucosamine the use of heparinase (EC 4.2.2.7), which, by degrading the hydrochloride (42.5 Ci/mmol), and carrier-free [35S]sulfuric heparin sections of the chain, releases two segments that exhibit acid were purchased from New England Nuclear. the structure of heparan sulfate. One of these segments is Preparation of the Heparan Sulfate Proteoglycan (HSPG) attached to the core. On the basis of these findings, the Synthesized by Endothelial Cell Cultures. An established heparan sulfate chain can be defined as a copolymer containing endothelial cell line derived from rabbit aorta (14) was used heparin regions in its structure. The combined use of these for these studies. To obtain labeled in their enzymes has made it possible to establish the disaccharide moieties, postconfluent cell cultures were in- sequence of parts of the glycosaminoglycan moiety of this cubated for 24-48 hr in F-12 tissue culture medium supple- proteoglycan. mented with 5% fetal bovine serum and either 150 uCi of carrier free [35S]sulfuric acid or 10 ,uCi of [35S]sulfuric acid and 10 ,Ci of [3H]glucosamine per ml. Proteoglycans labeled Heparan sulfate proteoglycans are complex macromolecules in their protein core were obtained by supplementing the that consist of a protein backbone to which heparan sulfate culture medium with 10 ,Ci of [3H]phenylalanine per ml. The chains are covalently linked (1). They are ubiquitous com- HSPG was isolated from the conditioned medium by Seph- pounds found in a wide variety ofvertebrate and invertebrate arose CL-6B gel filtration followed by ion-exchange chro- tissues (2) and are actively synthesized by cells in culture (3). matography on DEAE-cellulose as described (15). When These proteoglycans have been found to be present on the indicated, protein-free heparan sulfate glycosaminoglycan plasma membrane and in the extracellular matrix (4, 5) and chains were prepared from the proteoglycan by incubation exhibit a peculiar structural variability according to the tissue with 0.1 mg of Superase (protease from Pfizer) per ml for 4 and species of origin (2, 6). Despite their wide occurrence, hr at 60°C. After incubation the mixture was heated for 7 min little is known of their biological function. They have been at 100°C, and the radiolabeled glycosaminoglycan was pre- implicated in several biological processes such as cell-cell cipitated with 2 volumes of methanol at -20°C in the recognition (7), tissue differentiation (8), organization of presence of carrier heparan sulfate. extracellular matrices (9), and cell-matrix and cell-substrate Enzymatic Degradation of the Glycosaminoglycan Chains of adhesion (10). the HSPG. A typical incubation mixture contained 0.1 unit of The availability of two heparitinases (11, 12) and a hepar- enzymes, 20-50 x 103 cpm of HSPG with 50 ,ug each of inase (EC 4.2.2.7) from Flavobacterium heparinum (12, 13), heparan sulfate and heparin, and other additions as indicated which can be used in conjunction to elucidate the distribution in 0.05 M ethylenediamine acetate buffer (pH 7.0) in a final and grouping in the polymeric chain of disaccharides with volume of 30 ul. The incubation mixtures were spotted in various degrees of sulfation and with different hexuronic acid Whatman no. 1 paper and subjected to chromatography in moieties, has enabled us to undertake the structural study of isobutyric acid/1 M NH3, 5:3 (vol/vol), or isobutyric acid/ a proteoglycan isolated from the conditioned medium of 1.25 M NH3, 5:3.6, for 48 hr. Electrophoresis of the degra- endothelial cell cultures that appears to be highly character- dation products was performed in Whatman 3MM paper in istic of this cell type. Using these enzymes, we have deter- 0.25 M (NH4)HCO3 buffer (pH 8.5). The unsaturated pro- mined that the glycosaminoglycan chain of this proteoglycan contains heparin segments and have developed a strategy for Abbreviations: IdoA, iduronic acid; AIdoA, 0-(4-deoxy-hex-4-eno- the elucidation of the sequence of the disaccharide repeating pyranosyliduronic acid; AIdoA-2S, AIdoA 2-sulfate; GlcA, glucu- units that may be applicable to the study of other structurally ronic acid; AGlcA, 0-(4-deoxy-hex-4-enopyranosylglycuronic acid; related compounds. GlcNS, 2-sulfamino-D-glucose; GlcNS-6S, GlnNS 6-sulfate; Gln- NAc, 2-acetamido-D-glucose; GlcNAc-6S, GlcNAc 6-sulfate; (1-4), glycosidic linkage (1--4); HSPG, endothelial cell heparan sulfate The publication costs of this article were defrayed in part by page charge proteoglycan. payment. This article must therefore be hereby marked "advertisement" *On leave from Departamento Bioquimica, Escola Paulista de in accordance with 18 U.S.C. §1734 solely to indicate this fact. Medicina, C.P. 20372 Sao Paulo, S.P. Brazil. Downloaded by guest on September 25, 2021 3565 3566 Biochemistry: Nader et A Proc. Natl. Acad. Sci. USA 84 (1987)

* _ HEPARIN - iG~cA(1-4)GlcNAc-6S _ CHONDROrTIN SULFATE .* HEPARAN SULFATE K AGIcA(1-4)GIcNS

GIcNS-6S from e AIdoA01-4)GlcNS-6S

ORIGIN _SUPERASE AIdoA-2S(1-4)GlcNS + HTASE I HEPASE NONE ENYME * a.GIcA(1 -4)GlcNS-6S FIG. 1. Electrophoretic behavior ofHSPG and its heparinase and heparitinase I degradation products. About 20,000 cpm of[31S]HSPG * -.1IdoA-2S(1-4)GlcNS-6S was incubated with Superase (lanes +), a proteolytic enzyme, or with V¢ buffered solution containing protease inhibitors (lanes -) in the presence of 50 ,ug of carrier heparan sulfate for 2 hr at 60°C in a final 9 - Unknown volume of 20 A.l. The incubation mixtures were then heated at 100°C for 7 min. To the mixtures, 0.1 unit ofheparinase (HEPASE), 0.1 unit _ K_ AIdoA-2S(1-4)GlcNS-6S- of heparitinase I (HTASE I), or buffer (NONE) were added, and the jA *g L GlcA(1-4)GlcNS-6S mixtures were incubated further for 3 hr in 0.05 M ethylenediamine acetate buffer (pH 7.0) at 30°C in a final volume of 30 ,ul. Aliquots (5 ,ul) were applied to the agarose gel and subjected to electrophoresis in 0.05 M sodium phosphate buffer (pH 8.5) for 30 min at 120 V. After Origin fixing and staining, a radioautogram was prepared by exposing the 1 2 3 4 dried gel to x-ray film. Lane St shows a standard mixture of heparan sulfate, , and heparin. FIG. 2. Degradation products formed from [35S]HSPG by action of heparinase, heparitinase II, and heparitinase I. About 20,000 cpm ducts formed were detected by short-wave UV lamp. The of [35S]HSPG in the presence of 100 ,ug of carrier heparan sulfate radioactive 35S-labeled products were located by exposure of were incubated with 0.1 unit of heparitinase I (lane 1) or the chromatograms to Kodak x-ray film (SB-5) for 3-15 days. heparinase/glycuronidase (lanes 2 and 3) or in the absence of They were quantitated by assaying the paper containing the enzymes (lane 4) in 0.05 M ethylenediamine acetate buffer (pH 7.0) radioactive compounds in 0.5% 2,5-diphenyloxazole in tol- in a final volume of 30 1.l for 4 hr at 300C. After incubation, the mixtures were heated at 100'C for 1 min, and 0.1 unit of heparitinase uene in a liquid scintillation spectrometer. One of the prod- II was added to lanes 1 and 3. The mixtures were incubated an ucts, 0-(4-deoxy-hex-4-enopyranosylglycuronic acid)-(1-. additional 4 hr at 30°C. The incubation mixtures were applied to 4)-2-acetamido-D-glucose [AGlcA(1-4)GlcNAc] contains on- Whatman no. 1 filter paper and chromatographed for 48 hr with ly [3H]glucosamine and was located with the help ofthe same isobutyric acid/1.25 M NH3, 5:3.6 (vol/vol), as the descending disaccharide formed from the carrier heparan sulfate by solvent. A radioautogram was then prepared from the chromatogram action of heparitinase I (see below). with x-ray film. Fractionation of HSPG Degradation Products Prepared by the Action of the Enzymes. About 50,000 cpm of [3H]gluco- ages where the glucosamine is sulfated at the 2 position. The samine and [35S]HSPG with carriers heparin and heparan heparitinase I is specific for N-acetyl or N-sulfate glucosa- sulfate (100 ,g) were incubated with 0.1 unit of heparinase in minido-glucuronic acid linkages. This enzyme only acts on the presence of 0.02 M MnCl2 or heparitinase I in a final heparan sulfate regions where the N-acetyl or N-sulfate volume of 50 Al as described above. After incubation 500 jig glucosamine is not sulfated at the 6 position. The heparitinase of heparan sulfate was added to the mixture and applied to a II is a relatively nonspecific enzyme acting preferentially Sephadex G-50 superfine column (1 x 120 cm) previously upon glucosaminido-glucuronic acid linkages where the N- equilibrated with 1 M acetic acid. The products were eluted acetyl or N-sulfate glucosamine is sulfated at the 6 position. from the column in 1-ml fractions with 1 M acetic acid. None of the enzymes act on N-desulfated glucosaminido- Aliquots of the different fractions (see below) were used to uronic acid linkages. Also, the disaccharide sulfoesterase and determine the amount of radioactivity present, and the glycuronidase were used to distinguish two types of disul- different peaks obtained were combined, lyophilized, and fated disaccharides formed by action of heparinase upon subjected to enzymatic analyses. HSPG-O-(4-deoxy-hex-4-enopyranosyliduronic acid 2-sul- Other Methods. N-desulfation of [35S]HSPG and [3H]glu- fate)-(1--4)-2-sulfamino-D-glucose [AIdoA-2S(1-4)GlcNS] cosamine-labeled HSPG was performed in 0.04 M HCl at and 0-(4-deoxy-hex-4-enopyranosyliduronic acid)-(1-4)- 100°C for 2 hr in the presence of carrier heparan sulfate. GlcNS 6-sulfate [AIdoA(1-4)GlcNS-6S]. Only AIdoA-2S(1-4) Agarose gel electrophoresis in phosphate buffer was per- GlcNS is a substrate for the sulfatase producing AIdoA(1-4) formed as described (16). GlcNS. The AIdoA(1-4)GlcNS-6S is a substrate for the glycuronidase producing GlcNS-6S. For further details see refs. 11-13 and 17. RESULTS Fragmentation of HSPG by Heparinase, Heparitinase I, and Most ofthe structural studies reported in this paper are based Heparitinase II. The electrophoretic migration of the purified on the specificities of the enzymes prepared from Flavo- HSPG (before and after proteolysis) as well as the oligosac- bacterium heparinum. Their action has been studied in detail charide products formed from this compound by heparinase by different laboratories (for a review, see ref. 17). Briefly, and heparitinase I are shown in Fig. 1. The heparitinase I the heparinase acts upon glucosaminido-iduronic acid link- oligosaccharides have the same electrophoretic migration as Downloaded by guest on September 25, 2021 Biochemistry: Nader et al. Proc. Natl. Acad. Sci. USA 84 (1987) 3567

Table 1. Disaccharides formed by action of heparitinases upon heparinase degradation products of HSPG Mole ratio of disaccharide (disacch.)/fragmentt AIdoA-2S- AIdoA-2S- AIdoA- AGlcA- AGlcA- AGlcA- AGlcA- Mol of fragment/ (1-4)- (1-4)- (1-4)- (1-4)- (1-4)- (1-4)- (1-4)- Fragment mol of heparan sulfate* GlcNS-6S GlcNS GlcNS-6S GlcNS-6S GlcNAc-6S GlcNS GlcNAc Oligo 1 4.0 1.02 0.95 5.95 6.30 Oligo 2 5.0 0.88 1.04 1.82 3.00 - Tetra 1 5.0 1.04 0.96 Tetra 2 5.0 - 1.02 1.06 Disacch. 1 5.0 1.00 - - Disacch. 2 4.7 0.94 Disacch. 3 3.7 0.74 *Moles of fragment per mole of heparan sulfate. tNo value indicates <0.3 mol/mol of fragment. .3 heparin in this system, whereas the oligosaccharides pro- 31 A duced by heparinase have about the same migration of the intact HSPG. No significant changes in migration were 2' 2 observed by proteolytic treatment of the compounds. These initial experiments suggested that HSPG is a copolymer containing a heparin-like oligosaccharide in a heparan sulfate 1' -1 chain. ,ft I Further analysis ofthe heparin portion ofthe glycosamino- nWIE. -- _ Ln chain was performed by using the combined action of 20 K30 40 50 60 70 80 heparinase and glycuronidase. The small molecular weight products formed by action of the enzymes upon [35S]HSPG are shown in Fig. 2. Tetrasaccharides, tri-, and disulfated B OLK;O TETRA disaccharides and GlcNS-6S [resulting from AIdoA(1-4) 1 2 1 2 1.0 GlcNS-6S by action of the glycuronidase] are formed from A; ;; 1 2 3 HSPG by action of a of these two c4 *0.8 mixture enzymes. These 80 cv, results imply that the heparan sulfate chain of the proteogly- V- 0..6 *o can contains iduronic acid in different regions of its structure 1- as in heparin. The trisulfated disaccharide [AIdoA-2S(1-4) a.E 0..4 x GlcNS-6S], characteristic of heparin, accounts for >10o of U -0..2 E. the disaccharides that compose this proteoglycan (Table 1). w U z By combining these enzymes with heparitinases I and II, a 01 I - . . . , 20 30 _R~~~V.40 50 60 70 80 w decrease of the tetrasaccharides and an increase of tri- and n disulfated disaccharides are observed besides the formation (04 0 U., of other products (Fig. 2). 4 - C -2 - To gather information on the length and possible distribu- CD tion of these disaccharide units in HSPG, this material was 3 ~~~HEXA labeled with [3H]glucosamine and [35S]sulfuric acid, subject- cv, cn ed to the action ofheparinase, and fractionated by molecular- &-l sieving chromatography. Several peaks were obtained after fractionation on Sephadex G-50 (Fig. 3B): two peaks with elution volumes corresponding to two oligosaccharides ofMr 1- =6500 and Mr =3000 and five major peaks with the same 0 elution profiles of tetra- and disaccharides. The fractions 0 L - containing these different compounds were pooled and sub- 20 30 40 50 60 70 80 jected to degradation with heparitinases I and II. The types and molar ratios of the disaccharides obtained from each one 3- D DISACCH. -1.2 of the peaks are shown in Table 1. Sequence of the Oligo- and Tetrasaccharides Obtained from HSPG by Heparinase Degradation. Table 1 shows that oligo- 1~ ~~ saccharide 1 contains -1 mol of IdoA-2S(1-4)GlcNS-6S, 1 mol of GlcA(1-4)GlcNS-6S, and 6 mol each of GlcA(1-4) ~~~ ~ ~ ~ ~ ~ ~ ~ ~ . GlcNAc and GlcA(1-4)GlcNS. The sum of these disaccha- 1 - 0.4 rides would give a Mr of6000, which is close to the molecular 0. weight value obtained for this oligosaccharide by gel filtration 20 30 40 50 60 70 80 chromatography with Sephadex G-50. Likewise, the oligo- FRACTION NUMBER saccharide 2 contains approximately 1 mol each of IdoA- 2S(1-4)GlcNS and GlcA(1-4)GlcNS-6S, 2 mol of GlcA(1-+4)- FIG. 3. Fractionation by Sephadex G-50 chromatography of GlcNAc 6-sulfate [GlcA(1-4)GlcNAc-6S], and 3 mol of [3H]glucosamine/[35S]-labeled HSPG degradation products formed The sum by the action of the different enzymes. The experiments were GlcA(1-4)GlcNS. of these disaccharides gives a Mr performed as described. (A) Control, HSPG. (B) HSPG with of -3500. This again is in close agreement with the elution heparinase. (C) Oligosaccharide 1 (fractions 32-41 from B) with profile obtained for this oligosaccharide by molecular-sieving heparitinase I. (D) HSPG with heparitinase I. U, uncharacterized chromatography. peak; disacch., disaccharide. Downloaded by guest on September 25, 2021 3568 Biochemistry: Nader et al. Proc. Natl. Acad. Sci. USA 84 (1987)

IdoA-2S-GlcNS-6S OLIGO 1 Regarding oligosaccharide 2, the order of GlcA(1-4)GlcNAc- 6S and GlcA(1-4)GlcNS-6S could not be established. IdoA-2S-GlcNS 5 6 Oligosaccharides Obtained from HSPG by Heparitinase I OLIGO 2 ^ o-cj IdoA-GlcNS-6S Degradation. The results of the chromatographic analysis of the products formed from HSPG by heparitinase I are shown * TETRA 1 GIcA-GlcNS-6S in Fig. 3D. Two main peaks were eluted at the volumes corresponding to Mr 3500 and 3000 plus another main peak in - GIcA-GlcNAc-6S TETRA 2 0 GIcA-GIcNS the disaccharide region. As expected, analyses of the disac- 0-Y DISACCH 1 X charide peak by paper chromatography revealed a mixture of *-0 GlcA-GlcNAc DISAACH ?;G AGlcA(1-4)GlcNS and AGlcA(1-4)GlcNAc. The fractions containing the two main oligosaccharides DISACCH 3 O-f0 were combined and degraded by heparinase followed by heparitinase II. The type and amount of products formed FIG. 4. Structure of HSPG degradation products formed by from these compounds by the combined action of the en- action of heparinase. Designations of the glycosidic linkage have zymes is shown in Table 2. been omitted. The uronic acid of the disaccharide located at the Heparin-Like Regions ofHSPG. The results shown in Fig. 3D nonreducing end is unsaturated. suggests that the oligosaccharides with Mr 3500 and 3000 contain the heparin-like regions of HSPG. In an attempt to The results shown in Table 1 and Fig. 3 together with the obtain information on the possible sequence of these two knowledge of the specificity of the heparinase and segments, the oligo-, tetra-, and disaccharides obtained by heparitinases enable one to order the different disaccharides heparinase degradation of HSPG (Fig. 4) were assembled in in a specific sequence as shown in Fig. 4. Since the such a way as to include the two main oligosaccharide products heparinase acts upon glucosaminido-iduronic acid linkages, formed by the action of heparitinase I from HSPG (Fig. 3D). the unsaturated iduronic acid-containing disaccharides will The combination that best approaches the disaccharide com- be located at the nonreducing end of all the fragments. In the position ofthe oligosaccharides (Table 2) together with the sites case of the tetrasaccharides, the subsequent disaccharide of action ofheparinase and heparitinase I is shown in Fig. 5. In units are at the reducing end as shown in Fig. 4. this model two oligosaccharides with Mr 4900 and 4500 could To assign the next disaccharide unit from the nonreducing be produced by the action of the heparitinase I. The arrange- end of oligosaccharide 1, the compound was degraded with ment of the different tetra- and disaccharides used in the heparitinase I [which degrades the regions containing GlcA(1- assembly of Fig. 5 is aleatoty, and their proper order could not 4)GlcNS and GlcA(1-4)GlcNAc] and subjected to molecular- be established by the present methodology. sieving chromatography (Fig. 3C). This oligosaccharide was Distance of Heparin Sequence from the Protein Core of completely degraded to small fragments and disaccharides. The HSPG. To answer this question, HSPG labeled in the protein fractions containing the hexasaccharide were combined, further core with [3H]phenylalanine was degraded with heparitinase degraded by heparitinase II, and subjected to chromatography I or heparinase and subjected to analysis by agarose gel on paper. Three disaccharides were identified-namely, electrophoresis. As shown earlier (Fig. 1) in this system, the AIdoA-2S(1-4)GlcNS-6S, AGlcA(1-4)GlcNS-6S, and AGlcA(1- intact proteoglycan migrates with an electrophoretic mobility 4)GlcNS in about the same molar ratios. This indicates that similar to that of the heparan sulfate chains. If the heparin GlcA(1-4)GlcNS-6S is the vicinal disaccharide of AIdoA-2S(1- segment is separated from the protein core by a heparan 4)GlcNS-6S and is followed by GlcA(1-4)GlcNS, which con- sulfate type of structure of a sizable length, treatment with tains the linkage susceptible to heparitinase I (Fig. 4). The heparinase should affect the mobility of the protein core only disaccharide peak proved to be, by paper chromatography, a to a limited extent. Conversely, treatment with heparitinase mixture of AGlcA(1-4)GlcNAc and AGlcA(1-4)GlcNS. A third I should effectively decrease the amount oftritium label in the smaller peak was eluted between the hexa- and disaccharides heparan sulfate area of the agarose gel if the segment of (Fig. 3C), and it was not degraded by any of the enzymes, glycosaminoglycan close to the glycopeptide linkage is rep- remaining unidentified. resented by an N-acetylated region typical ofheparan sulfate. To establish the order of the two remaining disaccharide The results of these experiments have shown that, only after groups, GlcA(1-4)GlcNAc and GlcA(1-4)GlcNS of oligosac- heparitinase I degradation, there is a marked reduction of the charide 1, the HSPG was N-desulfated by mild acid hydrol- amount of label present in the heparan sulfate region, ysis and subjected to degradation with heparitinase I. The suggesting that the heparin oligosaccharides are separated amounts ofAGlcA(1-4)GlcNAc formed from this HSPG were from the core protein by a heparan sulfate region. comparable to the amounts of this disaccharide formed from a nonhydrolyzed HSPG. This indicates that these disaccha- DISCUSSION rides are clustered in the molecule as shown in Fig. 4. If this were not the case, either no degradation should occur, or Iduronic acid-containing tri- and disulfated disaccharides and N-desulfated tetra- and oligosaccharides should be produced. tetra- and pentasulfated tetrasaccharides typical of heparin These results also lead to the conclusion that the remaining are also conspicuously present in the HSPG of rabbit disaccharides [GlcA(1-4)GlcNS] also have to be vicinal to endothelial cells in culture. As judged by the amounts of each other as shown in Fig. 4. unsaturated products formed by the action of heparinase

Table 2. Disaccharide products formed from heparitinase I-digested oligosaccharides (oligo.) by action of heparinase and heparitinase II Mole ratio of disaccharide/fragment AIdoA-2S- AIdoA-2S- AIdo- AGlcA- AGlcA- AGlcA- (1-4)- (1-4)- (1-4)- (1-4)- (1-4)- (1-4)- Fragment GlcNS-6S GlcNS GlcNS-6S GlcNS-6S GlcNAc-6S GlcNS Oligo. 1 1.10 0.89 1.02 1.85 1.82 2.01 Oligo. 2 1.95 0.93 1.15 1.78 0.25 1.91 Downloaded by guest on September 25, 2021 Biochemistry: Nader et A Proc. Natl. Acad. Sci. USA 84 (1987) 3569

HEPARMNASE I HEPARITINASE I HEPARITINASE I 4,870 4,460

T I T r tA tA t HEPARINASE HEPARINASE FIG. 5. Proposed structure of HSPG and sites of action of heparinase and heparitinase I. R, protein core. Symbols are the same as in Fig. 4, and the molecular weights are indicated.

upon HSPG, -20% of the molecule contains iduronic acid This work was aided by grants from Fundacao de Amparo a residues. Among >20 heparan sulfates from different mam- Pesquisa do Estado de Sao Paulo, Conselho Nacional de Desenvolv- malian and invertebrate tissues and species analyzed by the imento Cientifico e Tecnol6gico, Brazil, and by grants from the same methodology (2, 6), this is the only heparan sulfate Council for Tobacco Research (no. 1414) and R. J. Reynolds, Inc. extensively susceptible to heparinase. The iduronic acid- 1. Roden, L. (1980) in The Biochemistry of and containing disaccharides are clustered in two sulfate-rich Proteoglycans, ed. Lennarz, W. J. (Plenum, New York), pp. oligosaccharide areas separated from the protein core by a 267-371. N-sulfated and N-acetylated region. 2. Nader, H. B., Ferreira, T. M. P. C., Paiva, J. F., Medeiros, The specificity of action of heparinase and heparitinase I M. G. L., Jer6nimo, S. M. B., Paiva, V. M. P. & Dietrich, has made it possible to order most of the disaccharides that C. P. (1984) J. Biol. Chem. 259, 1431-1435. compose the HSPG in a specific sequence as shown in Fig. 3. Dietrich, C. P. & Montes de Oca, M. (1970) Proc. Soc. Exp. Biol. Med.. 134, 955-962. 5. The total number of disaccharides found in these analyses 4. Kraemer, P. M. (1971) Biochemistry 10, 1437-1445. suggests that the heparan sulfate has a Mr of -15,000. 5. Gowda, D. C., Bhavanandan, V. P. & Davidson, E. A. (1986) Nevertheless, the average molecular weight of the intact J. Biol. Chem. 261, 4926-4934. heparan sulfate chain (after proteolysis or P-elimination) is in 6. Dietrich, C. P., Nader, H. B. & Straus, A. H. (1983) Biochem. the order of 55,000 (unpublished results). This implies that Biophys. Res. Commun. 111, 865-871. four ofthe units shown in Fig. 5 should constitute the heparan 7. Dietrich, C. P., Sampaio, L. O., Toledo, 0. M. S. & Cassaro, sulfate chain. At present no information is available about C. M. F. (1977) Biochem. Biophys. Res. Commun. 75, how these four units are assembled. Perhaps some clues 329-336. could be obtained when the structure ofthe sulfated unknown 8. Kinoshita, S. & Saiga, H. (1979) Exp. Cell Res. 123, 229-236. 9. A. G. in and Molecular of product formed by the joint action of the enzymes upon Ogston, (1970) Chemistry Biology the Intracellular Matrix, ed. Balazs, E. A. (Academic, New HSPG 2 and is accounts (Figs. 3C) obtained. This compound York), Vol. 3, pp. 1231-1240. for 3% of the total sulfated products formed by the action of 10. Laterra, J., Ansbacher, R. & Culp, L. A. (1980) Proc. Natl. the enzymes (about 1 mol of compound per mol of the Mr Acad. Sci. USA 77, 6662-6666. 15,000 unit), has an elution position in Sephadex of a 11. Silva, M. E., Dietrich, C. P. & Nader, H. B. (1976) Biochim. disaccharide, and is not degraded by the glycuronidase or the Biophys. Acta 437, 129-141. disaccharide sulfoesterase. This could be an indication that 12. Silva, M. E. & Dietrich, C. P. (1975) J. Biol. Chem. 250, this "disaccharide" is situated at the nonreducing end of the 6841-6846. chains, since these enzymes do not act upon saturated 13. Dietrich, C. P., Silva, M. E. & Michelacci, Y. M. (1973) J. proves to correct, we Biol. Chem. 248, 6408-6415. disaccharides. If this alternative be 14. Buonassisi, V. & Venter, J. C. (1976) Proc. Natl. Acad. Sci. have to consider the possibility that each of the four units is USA 73, 1612-1616. linked directly to a core resistant to proteolysis and 15. Buonassisi, V. & Colburn, P. (1982) Ann. N. Y. Acad. Sci. 401, a-elimination. Alternatively, our data do not rule out the 76-84. possibility that the carbohydrate chains are branched. 16. Jaques, L. B., Ballieux, R. E., Dietrich, C. P. & kavanagh, Iduronic acid-containing disaccharides have been reported L. W. (1968) Can. J. Physiol. Pharmacol. 46, 351-360. to be present in heparan sulfate species obtained from 17. Dietrich, C. P., Michelacci, Y. M. & Nader, H. B. (1980) in different tissues (18, 19) and from cultured microvascular Mechanism of Saccharide Polymerization and Depolymeriza- endothelial cells (20). In these microvessel-derived cultures, tion, ed. Marshall, J. J. (Academic, New York), pp. 317-329. 18. Cifonelli, J. A. & Dorfman, A. (1960) J. Biol. Chem. 235, as it had been reported for the established endothelial cell line 3283-3288. (15, 21) used in the present studies, heparan sulfate 19. Linker, A. & Hovingh, P. (1974) Carbohydr. Res. 37, 181-192. proteoglycans exhibited anticoagulant activity. The method- 20. Marcum, J. A. & Rosenberg, R. D. (1985) Biochem. Biophys. ology outlined in this report may prove useful for the Res. Commun. 126, 365-372. characterization of the structural features of the glycos- 21. Colburn, P. & Buonassisi, V. (1982) Biochem. Biophys. Res. aminoglycan chain responsible for biological activity. Commun. 104, 220-227, and erratum (1982) 105, 791. Downloaded by guest on September 25, 2021