Structural Recognition by Recombinant Human Heparanase That Plays Critical Roles in Tumor Metastasis
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m206510200
ISSN1083-351X
AutoresYukihiko Okada, Shuhei Yamada, Minako Toyoshima, Jian Dong, Motowo Nakajima, Kazuyuki Sugahara,
Tópico(s)Carbohydrate Chemistry and Synthesis
ResumoHuman heparanase is an endo-β-d-glucuronidase that degrades heparan sulfate/heparin and has been implicated in a variety of biological processes, such as inflammation, tumor angiogenesis, and metastasis. Although the cloned enzyme has been demonstrated to have a critical role in tumor metastasis, the substrate specificity has been poorly understood. In the present study, the specificity of the purified recombinant human heparanase was investigated for the first time using a series of structurally defined oligosaccharides isolated from heparin/heparan sulfate. The best substrates were ΔHexUA(±2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-GlcUA-GlcN(NS,6S) and ΔHexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S) (where ΔHexUA, GlcN, GlcUA, NS, 2S, and 6S represent unsaturated hexuronic acid,d-glucosamine, d-glucuronic acid, 2-N-sulfate, 2-O-sulfate, and 6-O-disulfate, respectively). Based on the percentage conversion of the substrates to products under identical assay conditions, several aspects of the recognition structures were revealed. 1) The minimum recognition backbone is the trisaccharide GlcN-GlcUA-GlcN. 2) The target GlcUA residues are in the sulfated region. 3) The -GlcN(6S)-GlcUA-GlcN(NS)- sequence is essential but not sufficient as the cleavage site. 4) The IdoUA(2S) residue, located two saccharides away from the target GlcUA residue, claimed previously to be essential, is not indispensable. 5) The 3-O-sulfate group on the GlcN is dispensable and even has an inhibitory effect when located in a highly sulfated region. 6) Based on these and previous results, HexUA(2S)-GlcN(NS,6S)-IdoUA-GlcNAc(6S)-GlcUA-GlcN(NS,±6S)-IdoUA(2S)-GlcN(NS,6S) (where HexUA represents hexuronic acid) has been proposed as a probable physiological target octasaccharide sequence. These findings will aid establishing a quantitative assay method using the above tetrasaccharide and designing heparan sulfate-based specific inhibitors of the heparanase for new therapeutic strategies. Human heparanase is an endo-β-d-glucuronidase that degrades heparan sulfate/heparin and has been implicated in a variety of biological processes, such as inflammation, tumor angiogenesis, and metastasis. Although the cloned enzyme has been demonstrated to have a critical role in tumor metastasis, the substrate specificity has been poorly understood. In the present study, the specificity of the purified recombinant human heparanase was investigated for the first time using a series of structurally defined oligosaccharides isolated from heparin/heparan sulfate. The best substrates were ΔHexUA(±2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-GlcUA-GlcN(NS,6S) and ΔHexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S) (where ΔHexUA, GlcN, GlcUA, NS, 2S, and 6S represent unsaturated hexuronic acid,d-glucosamine, d-glucuronic acid, 2-N-sulfate, 2-O-sulfate, and 6-O-disulfate, respectively). Based on the percentage conversion of the substrates to products under identical assay conditions, several aspects of the recognition structures were revealed. 1) The minimum recognition backbone is the trisaccharide GlcN-GlcUA-GlcN. 2) The target GlcUA residues are in the sulfated region. 3) The -GlcN(6S)-GlcUA-GlcN(NS)- sequence is essential but not sufficient as the cleavage site. 4) The IdoUA(2S) residue, located two saccharides away from the target GlcUA residue, claimed previously to be essential, is not indispensable. 5) The 3-O-sulfate group on the GlcN is dispensable and even has an inhibitory effect when located in a highly sulfated region. 6) Based on these and previous results, HexUA(2S)-GlcN(NS,6S)-IdoUA-GlcNAc(6S)-GlcUA-GlcN(NS,±6S)-IdoUA(2S)-GlcN(NS,6S) (where HexUA represents hexuronic acid) has been proposed as a probable physiological target octasaccharide sequence. These findings will aid establishing a quantitative assay method using the above tetrasaccharide and designing heparan sulfate-based specific inhibitors of the heparanase for new therapeutic strategies. heparan sulfate proteoglycan 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid fluorescein isothiocyanate high performance liquid chromatography glycosaminoglycan heparin 4-deoxy-α-l-threo-hex-4-enepyranosyluronic acid 2-N-sulfate 2-O-sulfate 3-O-sulfate 6-O-sulfate extracellular matrix Interactions between adherent cells and the extracellular environment influence maintenance of cellular functions such as proliferation, differentiation, and migration. Heparan sulfate proteoglycans (HS-PGs)1 are covalently linked protein-HS glycosaminoglycan (GAG) conjugates found in extracellular matrix (ECM) and on the cell surface of most cells and have been demonstrated to be key components of the cell-cell and cell-ECM interactions (for reviews, see Refs. 1David G. Biochem. Soc. Trans. 1991; 19: 816-820Crossref PubMed Scopus (14) Google Scholar, 2Iozzo R.V. Annu. Rev. Biochem. 1998; 67: 609-652Crossref PubMed Scopus (1359) Google Scholar, 3Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Annu. Rev. Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2361) Google Scholar, 4Tumova S. Woods A. Couchman J.R. Int. J. Biochem. Cell Biol. 2000; 32: 269-288Crossref PubMed Scopus (315) Google Scholar). Most of the biological properties of HS-PGs are conferred by the HS moiety, which is a sulfated polydisperse copolymer of alternating GlcN and HexUA (GlcUA or IdoUA) residues (for reviews, see Refs. 5Salmivirta M. Lidholt K. Lindahl U. FASEB J. 1996; 10: 1270-1279Crossref PubMed Scopus (398) Google Scholar and 6Lindahl U. Kusche-Gullberg M. Kjellén L. J. Biol. Chem. 1998; 273: 24979-24982Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). HS-GAG binds to and co-localizes with structural proteins, such as fibronectin and collagen in the ECM, providing a framework for matrix organization (for reviews, see Refs. 7Oldberg Å. Antonsson P. Hedbom E. Heinegård D. Biochem. Soc. Trans. 1990; 18: 789-792Crossref PubMed Scopus (47) Google Scholar, 8Iozzo R.V. Cohen I.R. Grassel S. Murdoch A.D. Biochem. J. 1994; 302: 625-639Crossref PubMed Scopus (344) Google Scholar, 9Erickson A.C. Couchman J.R. J. Histochem. Cytochem. 2000; 48: 1291-1306Crossref PubMed Scopus (245) Google Scholar). Both cell surface and ECM HS-PGs also tether various growth/differentiation factors and cytokines as storage depots of bioactive signaling molecules. There is clear evidence that an association of a ligand with HS-GAGs can activate or stabilize the ligand and also facilitate signal transduction via its high affinity receptor (for reviews, see Refs. 10Stringer S.E. Gallagher J.T. Int. J. Biochem. 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Paraskeva C. Turnbull J.E. Deakin J.A. Gallagher J.T. J. Biol. Chem. 1998; 273: 51-57Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 16Nurcombe V. Ford M.D. Wildschut J.A. Bartlett P.F. Science. 1993; 260: 103-106Crossref PubMed Scopus (378) Google Scholar, 17Brickman Y.G. Nurcombe V. Ford M.D. Gallagher J.T. Bartlett P.F. Turnbull J.E. Glycobiology. 1998; 8: 463-471Crossref PubMed Scopus (37) Google Scholar, 18Feyzi E. Saldeen T. Larsson E. Lindahl U. Salmivirta M. J. Biol. Chem. 1998; 273: 13395-13398Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Another way to alter the functional state of HS-PGs is to degrade and release HS-GAGs from the core proteins, which is achieved by the specific action of an endoglycosidase, heparanase (for reviews, see Refs. 19Bame K.J. Glycobiology. 2001; 11: 91-98Crossref PubMed Scopus (144) Google Scholar and 20Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: 99-108PubMed Google Scholar). Heparanase is the name of mammalian endoglucuronidase capable of specifically cleaving HS-GAGs and differs from bacterial eliminases such as heparinase and heparitinase (21Nakajima M. Irimura T., Di Ferrante N. Nicolson G. J. Biol. Chem. 1984; 259: 2283-2290Abstract Full Text PDF PubMed Google Scholar). The extracellular heparanase has been implicated in basement membrane remodeling after injury or at inflammation sites by destroying HS-GAG chains and in the regulation of cell growth and differentiation by releasing growth factors that are bound to extracellular HS-PGs. There is also evidence that the heparanase activity correlates with the metastatic potential of tumor cells in animal models and increases in the sera of human patients with metastatic cancers (22Nakajima M. Irimura T., Di Ferrante D., Di Ferrante N. Nicolson G.L. Science. 1983; 220: 611-613Crossref PubMed Scopus (312) Google Scholar, 23Nakajima M. Irimura T. Nicolson G.L. J. Cell. 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A direct role of the heparanase in tumor cell invasion was confirmed by the transfection of the sense and antisense heparanase cDNA into cells, which acquired highly and poorly metastatic phenotypes, respectively (26Vlodavsky I. Friedmann Y. Elkin M. Aingorn H. Atzmon R. Ishai-Michaeli R. Bitan M. Pappo O. Peretz T. Michal I. Spector L. Pecker I. Nat. Med. 1999; 5: 793-802Crossref PubMed Scopus (735) Google Scholar, 30Uno F. Fujiwara T. Takata Y. Ohtani S. Katsuda K. Takaoaka M. Ohkawa T. Naomoto Y. Nakajima M. Tanaka N. Cancer Res. 2001; 61: 7855-7860PubMed Google Scholar). High expression of the heparanase mRNA was also observed in advanced stage tumors and metastatic cell lines derived from various tissues (27Hulett M.D. Freeman C. Hamdorf B.J. Baker R.T. Harris M.J. Parish C.R. Nat. Med. 1999; 5: 803-809Crossref PubMed Scopus (492) Google Scholar, 28Kussie P.H. Hulmes J.D. Ludwig D.L. Patel S. Navarro E.C. Seddon A.P. Giorgio N.A. Bohlen P. Biochem. Biophys. Res. Commun. 1999; 261: 183-187Crossref PubMed Scopus (184) Google Scholar, 31Gohji K. Okamoto M. Kitazawa S. Toyoshima M. Dong J. Katsuoka Y. Nakajima M. J. Urol. 2001; 166: 1286-1290Crossref PubMed Scopus (102) Google Scholar,32Mikami S. Ohashi K. Usui Y. Nemoto T.G. Katsube K. Yanagishita M. Nakajima M. Nakamura K. Koike M. Jpn. J. Cancer Res. 2001; 92: 1062-1073Crossref PubMed Scopus (105) Google Scholar). The aim of the present study was to explore the substrate recognition property of the human recombinant heparanase. Previously, several attempts were made to define the substrate specificity of the heparanase partially purified from different animal sources (for reviews, see Refs. 19Bame K.J. Glycobiology. 2001; 11: 91-98Crossref PubMed Scopus (144) Google Scholar and 20Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: 99-108PubMed Google Scholar). Most approaches have involved the structural analysis of the fragments generated by enzymatic cleavage of polymer HS from various tissues and heparin (Hep) polysaccharides derived from the lung and intestine. Specificity studies on human heparanase using structurally defined oligosaccharide substrates have been limited to those performed using partially purified enzyme preparations (33Thunberg L. Bäckström G. Wasteson Å. Robinson H.C. Ögren S. Lindahl U. J. Biol. Chem. 1982; 257: 10278-10282Abstract Full Text PDF PubMed Google Scholar, 34Pikas D.S., Li, J. Vlodavsky I. Lindahl U. J. Biol. Chem. 1998; 273: 18770-18777Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). In the present study, we systematically investigated the specificity of the purified recombinant human heparanase using a number of structurally defined oligosaccharides as substrates and revealed the hitherto unreported specificity of the enzyme, which will aid establishing the quantitative assay methods and designing inhibitors for new therapeutic strategies in highly metastatic cancer and other heparanase-related diseases, notably inflammatory or cardiovascular diseases. HS (sodium salt) from bovine kidney and concanavalin A-agarose was purchased from Seikagaku Corp. (Tokyo, Japan). Stage 14 Hep (sodium salt) from porcine intestinal mucosa was obtained from American Diagnostic Inc. (New York). TSK-GEL G3000SWXL was from TOSOH Corp. (Tokyo, Japan). Sephacryl S-300HS and Hep-Sepharose CL-6B columns as well as a prepacked disposable PD-10 column containing Sephadex G-25 medium were purchased from Amersham Biosciences. Fluorescein isothiocyanate (FITC) was from Sigma-Aldrich. Structurally defined oligosaccharides were isolated from porcine intestinal Hep or bovine kidney HS, and their homogeneity was judged by capillary electrophoresis as described previously (35Yamada S. Yoshida K. Sugiura M. Sugahara K. Khoo K.H. Morris H.R. Dell A. J. Biol. Chem. 1993; 268: 4780-4787Abstract Full Text PDF PubMed Google Scholar, 36Yamada S. Sakamoto K. Tsuda H. Yoshida K. Sugahara K. Khoo K.H. Morris H.R. Dell A. Glycobiology. 1994; 4: 69-78Crossref PubMed Scopus (43) Google Scholar, 37Sugahara K. Tohno-oka R. Yamada S. Khoo K.H. Morris H.R. Dell A. Glycobiology. 1994; 4: 535-544Crossref PubMed Scopus (64) Google Scholar, 38Yamada S. Murakami T. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1995; 270: 8696-8705Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 39Tsuda H. Yamada S. Yamane Y. Yoshida K. Hopwood J.J. Sugahara K. J. Biol. Chem. 1996; 271: 10495-10502Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 41Yamada S. Sakamoto K. Tsuda H. Yoshida K. Sugiura M. Sugahara K. Biochemistry. 1999; 38: 838-847Crossref PubMed Scopus (33) Google Scholar). HS (sodium salt) from bovine kidney and Hep (sodium salt) from porcine intestinal mucosa were labeled with FITC as described previously (25Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Human melanoma A375M cells were cultured as monolayers in RPM-I1640 (Nissui, Tokyo, Japan) supplemented with heat-inactivated 10% fetal bovine serum, 2 mml-glutamine, penicillin (100 units/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 mg/ml) in humidified 95% air, 5% CO2 at 37 °C. The 1632-bp-long cDNA coding for a human heparanase was inserted into an expression vector, pcDNA3.1/Hygro (Invitrogen), and the vector was transfected into human melanoma A375M cells using the LipofectAMINETMreagent (Invitrogen). The transfectants were selected by hygromycin resistance and a stable transfectant cell line expressing a high level of recombinant human heparanase was established. After the cells had grown to confluence, they were harvested and homogenized in a lysis buffer (50 mm Tris-HCl, pH 7.5, containing 150 mm NaCl, 0.5% Triton X-100, 0.2 mm4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 1 μg/ml aprotinin). The cell lysate was then loaded onto a Hep-Sepharose column (5.0 × 10 cm) preequilibrated with 50 mm Tris-HCl, pH 7.5, containing 150 mmNaCl at a flow rate of 3.0 ml/min, and heparanase was eluted with the same buffer, containing 1.0 m NaCl as described previously (25Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The 1.0 m NaCl-eluted fractions were diluted with an equal volume of the dilution buffer (50 mm sodium acetate buffer, pH 6.0, containing 0.4% CHAPS) and applied to a concanavalin A-agarose column (2.5 × 10 cm) at a flow rate of 0.8 ml/min. Heparanase was eluted with 50 mm sodium acetate buffer, pH 6.0, containing 0.5 m NaCl, 0.2% CHAPS, and 0.7m α-methylmannose as described previously (25Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The concanavalin A-agarose-eluted fractions were diluted 3.3-fold with 50 mm sodium acetate buffer, pH 6.0, containing 0.2% CHAPS, and loaded onto an immunoaffinity column (1.0 × 5 cm) containing the anti-heparanase polyclonal antibody (42Gohji K. Hirano H. Okamoto M. Kitazawa S. Toyoshima M. Dong J. Tatsuoka Y. Nakajima M. Int. J. Cancer. 2001; 95: 295-301Crossref PubMed Scopus (111) Google Scholar) at a flow rate of 0.18 ml/min. Heparanase was eluted with 0.1 m glycine-HCl, pH 2.7, containing 0.05% CHAPS at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected, and aliquots were used for heparanase assay and SDS-PAGE, which was performed under reducing conditions employing 4–20% gradient polyacrylamide gels (84 × 91 mm) in the Tris-glycine buffer system (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (215642) Google Scholar). After electrophoresis, gels were subjected to silver staining. The human heparanase protein was quantified by the BCA protein assay (Pierce). Human heparanase activity was determined from the gel permeation HPLC chromatogram of the enzyme digest of FITC-HS by measuring a forward half area of the peak of the intact FITC-HS as described previously (25Toyoshima M. Nakajima M. J. Biol. Chem. 1999; 274: 24153-24160Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The decrease in this peak area observed after heparanase treatment was measured using an integrator, and the amount of the degraded FITC-HS was calculated from the decrease in fluorescence intensity. One unit was defined as the quantity of the enzyme that degraded 10 ng of FITC-HS/min. Enzyme reactions were carried out in 100 μl of 0.1m sodium acetate buffer, pH 4.2, containing 1 μg of FITC-Hep, at 37 °C for 3 h and terminated by the addition of 100 μg of nonlabeled porcine intestinal Hep and subsequent heating at 100 °C for 5 min. The digests were centrifuged at 15,000 rpm for 5 min to precipitate the insoluble materials. The supernatant fluids were analyzed by gel filtration HPLC on a TSK-GEL G3000SWXLcolumn (0.78 × 30 cm) preequilibrated with 50 mmTris-HCl, pH 7.5, containing 150 mm NaCl and 0.05% (v/w) NaN3 at a flow rate of 1 ml/min. Structurally defined tetra- or hexasaccharide (0.3 nmol each) isolated from porcine intestinal Hep or bovine kidney HS was digested with 0.88 or 0.44 units of the purified human heparanase in a total volume of 100 μl of 0.1 m sodium acetate buffer, pH 4.2, at 37 °C for 21 h or the indicated periods. The enzymatic reactions were terminated as described above. As control experiments, each oligosaccharide was incubated with the heat-inactivated heparanase under the same conditions. Each enzyme digest was analyzed by HPLC on an amine-bound silica column as reported previously (44Sugahara K. Okumura Y. Yamashina I. Biochem. Biophys. Res. Commun. 1989; 162: 189-197Crossref PubMed Scopus (80) Google Scholar). Eluates were monitored at 232 nm. The sensitivity of each oligosaccharide to the enzyme was judged by the peak shift to earlier elution positions. The area of the individual peaks was compared before and after heparanase treatment, and the amount of the degraded oligosaccharide was calculated from the decrease in the intact peak area using an integrator. The pcDNA3/Hygro vector containing the full-length human heparanase cDNA clone was expressed in human melanoma A375M cells. The cell lysate prepared from the stable transfectant cells was subjected to affinity chromatographies using the matrices Hep-Sepharose, concanavalin A-agarose, and anti-heparanase antibody-immobilized Affi-Gel 10 to purify the recombinant enzyme. Aliquots of fractions 1–17 eluted from the anti-heparanase antibody column were subjected to the heparanase assay and SDS-PAGE followed by silver staining (Fig. 1). The strong catalytic activity toward FITC-HS was detected in fractions 6–8. Heparanase is initially synthesized as an inactive 65-kDa glycoprotein that is cleaved at the N terminus to generate an active 50-kDa enzyme (for reviews, see Refs. 19Bame K.J. Glycobiology. 2001; 11: 91-98Crossref PubMed Scopus (144) Google Scholar and 20Parish C.R. Freeman C. Hulett M.D. Biochim. Biophys. Acta. 2001; 1471: 99-108PubMed Google Scholar). Being consistent with this, a 50-kDa band was detected evidently on SDS-PAGE in the fractions containing HS-degrading activity. Silver staining showed a 30-kDa protein band in fractions 6–8 and the faint 65-kDa protein band, which corresponded to the IgG light chain, probably leaking from the antibody-immobilized column and the enzyme precursor, respectively. Fractions 6–8 were combined and used to investigate the substrate specificity of the heparanase. The protein concentration and the heparanase activity of the pooled fractions were measured. The specific activity was 1.6 units/μg of protein. The FITC-labeled Hep was incubated with the purified recombinant heparanase to confirm the enzyme activity and also the presence of the heparanase recognition sequences in bovine kidney HS and porcine intestinal Hep chains. The digest was analyzed by gel filtration HPLC on a column of TSK-GEL G3000SWXL as shown in Fig. 2. Although both FITC-HS and -Hep were degraded by the enzyme, the latter appeared to be more susceptible to the heparanase-catalyzed cleavage than the former. Since porcine intestinal Hep contains the heparanase cleavable sites, experiments were undertaken to elucidate the substrate recognition property of the heparanase using a series of structurally defined sulfated oligosaccharides, which were isolated from the repeating disaccharide region of porcine intestinal Hep and bovine kidney HS (41Yamada S. Sakamoto K. Tsuda H. Yoshida K. Sugiura M. Sugahara K. Biochemistry. 1999; 38: 838-847Crossref PubMed Scopus (33) Google Scholar, 45Yamada S. Sugahara K. Trends Glycosci. Glycotech. 1998; 10: 95-123Crossref Scopus (33) Google Scholar) and included 1 tri-, 28 tetra-, 1 penta-, 9 hexa-, and 3 octasaccharides. The substrate specificity of the human heparanase was investigated using 8 hexa- and 12 tetrasaccharides derived from Hep/HS (Tables I andII). Their fine structures have been established by 500-MHz NMR analysis (35Yamada S. Yoshida K. Sugiura M. Sugahara K. Khoo K.H. Morris H.R. Dell A. J. Biol. Chem. 1993; 268: 4780-4787Abstract Full Text PDF PubMed Google Scholar, 36Yamada S. Sakamoto K. Tsuda H. Yoshida K. Sugahara K. Khoo K.H. Morris H.R. Dell A. Glycobiology. 1994; 4: 69-78Crossref PubMed Scopus (43) Google Scholar, 37Sugahara K. Tohno-oka R. Yamada S. Khoo K.H. Morris H.R. Dell A. Glycobiology. 1994; 4: 535-544Crossref PubMed Scopus (64) Google Scholar, 38Yamada S. Murakami T. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1995; 270: 8696-8705Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 39Tsuda H. Yamada S. Yamane Y. Yoshida K. Hopwood J.J. Sugahara K. J. Biol. Chem. 1996; 271: 10495-10502Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 41Yamada S. Sakamoto K. Tsuda H. Yoshida K. Sugiura M. Sugahara K. Biochemistry. 1999; 38: 838-847Crossref PubMed Scopus (33) Google Scholar). Eighteen contained GlcUA residue(s) at the internal position of each oligosaccharide sequence, whereas the other two contained only ΔHexUA and IdoUA but not GlcUA residues as uronic acid components. These 20 oligosaccharides (0.3 nmol each) were individually incubated with the purified recombinant heparanase under identical conditions (using 0.88 units of the enzyme, at 37 °C for 21 h), and the reaction products from each digestion were analyzed by HPLC on an amine-bound silica column for identification and quantification to compare the relative susceptibilities of the oligosaccharide substrates with the enzyme. Since the elution profile was monitored by absorbance at 232 nm of the ΔHexUA residue at the nonreducing terminus, only the intact substrate and the product derived from the nonreducing side of each substrate were detected, but the product from the reducing side was not due to the lack of ΔHexUA.Table IThe structure of human heparanase-sensitive oligosaccharidesFraction No.aThe fraction numbers refer to those cited in Ref. 45.StructurePercentage cleavagebCalculated based on the proportion of the starting oligosaccharide remaining in the digest.ReferencecThe fraction numbers refer to those designated in the original studies.%↓dThe arrows indicate the presumable cleavage sites of human heparanase in the oligosaccharide substrates.Hexa-1ΔHexUA(2S)-GlcN(NS,6S)-IdoUA- GlcNAc(6S)-GlcUA- GlcN(NS,3S,6S)26Fractions 6–34 in Ref. 40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar↓Hexa-4ΔHexUA(2S)-GlcN(NS,6S)-IdoUA- GlcNAc(6S)-GlcUA- GlcN(NS,6S)85Fractions 6–27 in Ref. 40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar↓ (↓)Hexa-7ΔHexUA(2S)-GlcN(NS,6S)-GlcUA- GlcN(NS,6S)-GlcUA- GlcN(NS,6S)>95Fractions 6–31 in Ref.40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar↓ (↓)Hexa-7SeThis hexasulfated hexasaccharide was prepared by the digestion of ΔHexUA(2S)-GlcN(NS,6S)-GlcUA-GlcN(NS,6S)-GlcUA-GlcN(NS,6S) with 2-sulfatase.ΔHexUA-GlcN(NS,6S)-GlcUA- GlcN(NS,6S)-GlcUA- GlcN(NS,6S)>95Fraction b-19S in Ref.39Tsuda H. Yamada S. Yamane Y. Yoshida K. Hopwood J.J. Sugahara K. J. Biol. Chem. 1996; 271: 10495-10502Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar↓Hexa-8ΔHexUA(2S)-GlcN(NS,6S)-IdoUA- GlcNAc(6S)-GlcUA- GlcN(NS,3S)60Fractions 6–32 in Ref. 40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar↓Hexa-15ΔHexUA(2S)-GlcN(NS,6S)-IdoUA- GlcNAc(6S)-GlcUA- GlcN(NS)59Fractions 6–26 in Ref.40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar↓Hexa-16ΔHexUA(2S)-GlcN(NS,6S)-IdoUA(2S)- GlcNAc-GlcUA- GlcN(NS,6S)30Fractions 6–30 in Ref. 40Yamada S. Yamane Y. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1998; 273: 1863-1871Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar↓Tetra-1ΔHexUA(2S)-GlcN(NS,6S)-GlcUA- GlcN(NS,6S)>95Fraction VIII in Ref. 38Yamada S. Murakami T. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1995; 270: 8696-8705Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar↓Tetra-6ΔHexUA(2S)-GlcN(NS)-GlcUA- GlcN(NS,6S)44Fraction IV in Ref. 38Yamada S. Murakami T. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1995; 270: 8696-8705Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar↓Tetra-10ΔHexUA-GlcNAc(6S)-GlcUA- GlcN(NS,3S)21Fraction III-9 in Ref. 35Yamada S. Yoshida K. Sugiura M. Sugahara K. Khoo K.H. Morris H.R. Dell A. J. Biol. Chem. 1993; 268: 4780-4787Abstract Full Text PDF PubMed Google Scholar↓Tetra-28ΔHexUA-GlcN(NS,6S)-GlcUA- GlcN(NS,6S)40Fraction II in Ref. 38Yamada S. Murakami T. Tsuda H. Yoshida K. Sugahara K. J. Biol. Chem. 1995; 270: 8696-8705Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar↓Tetra-29ΔHexUA(2S)-GlcNAc(6S)-GlcUA- GlcN(NS,6S)56Fraction V in Ref.38Yamada S. Murakami T. Tsuda H. Yoshida K. Sugahara
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