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Cell Proteoglycan (Heparitinase I/Heparitinase Ih/Heparinase/Copolymer/Disaccharide Sequence) H

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Cell Proteoglycan (Heparitinase I/Heparitinase Ih/Heparinase/Copolymer/Disaccharide Sequence) H Proc. Nati. Acad. Sci. USA Vol. 84, pp. 3565-3569, June 1987 Biochemistry Heparin sequences in the heparan sulfate chains of an endothelial cell proteoglycan (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 protein 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 proteoglycans labeled in their enzymes has made it possible to establish the disaccharide carbohydrate 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, chondroitin 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.
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