Endocan Is a Novel Chondroitin Sulfate/Dermatan Sulfate Proteoglycan That Promotes Hepatocyte Growth Factor/Scatter Factor Mitogenic Activity
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m108395200
ISSN1083-351X
AutoresDavid Béchard, Thibaut Gentina, Maryse Delehedde, Arnaud Scherpereel, Malcolm Lyon, Marc Aumercier, Rosemay Vazeux, Colette Richet, Pierre Degand, Brigitte Jude, Anne Janin, David G. Fernig, André‐Bernard Tonnel, Philippe Lassalle,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoProteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458–20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression. Proteoglycans that modulate the activities of growth factors, chemokines, and coagulation factors regulate in turn the vascular endothelium with respect to processes such as inflammation, hemostasis, and angiogenesis. Endothelial cell-specific molecule-1 is mainly expressed by endothelial cells and regulated by pro-inflammatory cytokines (Lassalle, P., Molet, S., Janin, A., Heyden, J. V., Tavernier, J., Fiers, W., Devos, R., and Tonnel, A. B. (1996) J. Biol. Chem. 271, 20458–20464). We demonstrate that this molecule is secreted as a soluble dermatan sulfate (DS) proteoglycan. This proteoglycan represents the major form either secreted by cell lines or circulating in the human bloodstream. Because this proteoglycan is specifically secreted by endothelial cells, we propose to name it endocan. The glycosaminoglycan component of endocan consists of a single DS chain covalently attached to serine 137. Endocan dose-dependently increased the hepatocyte growth factor/scatter factor (HGF/SF)-mediated proliferation of human embryonic kidney cells, whereas the nonglycanated form of endocan did not. Moreover, DS chains purified from endocan mimicked the endocan-mediated increase of cell proliferation in the presence of HGF/SF. Overall, our results demonstrate that endocan is a novel soluble dermatan sulfate proteoglycan produced by endothelial cells. Endocan regulates HGF/SF-mediated mitogenic activity and may support the function of HGF/SF not only in embryogenesis and tissue repair after injury but also in tumor progression. dermatan sulfate proteoglycan chondroitin sulfate dermatan sulfate heparan sulfate proteoglycan chondroitin sulfate/dermatan sulfate proteoglycan glycosaminoglycan endocan wild-type hepatocyte growth factor/scatter factor platelet-rich plasma platelet-poor plasma small leucine-rich repeat Δ4,5-unsaturated hexuronate Δ4,5-unsaturated hexuronate 2-sulfate β-d-N-acetylgalactosamine β-d-N-acetylgalactosamine 4-sulfate β-d-N-acetylgalactosamine 6-sulfate 6-OSO3), β-d-N-acetylgalactosamine 4,6-disulfate β-d-glucuronate α-l-iduronate fibroblast growth factor Dulbecco's modified Eagle's medium Chinese hamster ovary monoclonal antibody polyacrylamide gel electrophoresis human umbilical vein endothelial cell enzyme-linked immunosorbent assay phosphate-buffered saline activated partial thromboplastin time thrombin clotting time In the last few years, the vascular endothelium has been shown to play a crucial role in inflammation, coagulation, angiogenesis, and tumor invasion, primarily through the fine regulation of receptor-ligand interactions and secretion of different mediators. Endothelial cells also express several proteoglycans such as decorin, biglycan, PG-100, glypican, and members of the syndecan family that regulate intercellular interactions and activation processes. Proteoglycans are complex macromolecules that consist of a polypeptide with one or more glycosaminoglycan chains covalently bound to a serine (or rarely a threonine) residue. Different families of proteoglycans have been described (1Ruoslahti E. J. Biol. Chem. 1989; 264: 13369-13372Abstract Full Text PDF PubMed Google Scholar, 2Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Crossref PubMed Scopus (1676) Google Scholar, 3Hardingham T.E. Fosang A.J. FASEB J. 1992; 6: 861-870Crossref PubMed Scopus (1012) Google Scholar) as follows: heparan sulfate, chondroitin sulfate, dermatan sulfate, and keratan sulfate proteoglycans. 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Thus HS (35Deakin J.A. Lyon M. J. Cell Sci. 1999; 112: 1999-2009Crossref PubMed Google Scholar, 36van der Voort R. Taher T.E. Wielenga V.J. Spaargaren M. Prevo R. Smit L. David G. Hartmann G. Gherardi E. Pals S.T. J. Biol. Chem. 1999; 274: 6499-6506Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 37Sergeant N. Lyon M. Rudland P.S. Fernig D.G. Delehedde M. J. Biol. Chem. 2000; 275: 17094-17099Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 38Sakata H. Stahl S.J. Taylor W.G. Rosenberg J.M. Sakaguchi K. Wingfield P.T. Rubin J.S. J. Biol. Chem. 1997; 272: 9457-9463Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and DS (35Deakin J.A. Lyon M. J. Cell Sci. 1999; 112: 1999-2009Crossref PubMed Google Scholar) can potentiate HGF/SF signaling. We previously described an endothelial cell-specific molecule 1 (ESM-1) that is interestingly restricted to endothelial cells and to the lung and kidney (39Lassalle P. Molet S. Janin A. Heyden J.V. Tavernier J. Fiers W. Devos R. Tonnel A.B. J. Biol. Chem. 1996; 271: 20458-20464Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). The synthesis and secretion of ESM-1 are up-regulated by tumor necrosis factor, interleukin-1, and lipopolysaccharide and down-regulated by interferon-γ (40Bechard D. Meignin V. Scherpereel A. Oudin S. Kervoaze G. Bertheau P. Janin A. Tonnel A. Lassalle P. J. Vasc. Res. 2000; 37: 417-425Crossref PubMed Scopus (161) Google Scholar). ESM-1 can regulate CD11a/CD18 integrin (LFA-1)-mediated activation of leukocytes through binding to the LFA-1 and its ability to inhibit ICAM-1 binding (41Bechard D. Scherpereel A. Hammad H. Gentina T. Tsicopoulos A. Aumercier M. Pestel J. Dessaint J.P. Tonnel A.B. Lassalle P. J. Immunol. 2001; 167: 3099-3106Crossref PubMed Scopus (206) Google Scholar). We now demonstrate that the major form of ESM-1 consists of a heavily glycosylated 165-amino acid mature polypeptides. The carbohydrate moiety of ESM-1 is a single chain of the glycosaminoglycan DS covalently linked to serine 137. ESM-1 promotes the HGF/SF-mediated proliferation of human embryonic kidney cells, in a similar way to heparin. This activity is strictly dependent on the presence of the DS chain. These findings indicate that ESM-1 may be directly and specifically involved in the regulation of HGF/SF activities. Because ESM-1 is specifically secreted by endothelial cells as a proteoglycan we propose to rename ESM-1 as "endocan." CHO cells were cultured in α-minimum Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum. SV40-transfected human endothelial cells (SV1 cells) (42Lassalle P. LaGrou C. Delneste Y. Sanceau J. Coll J. Torpier G. Wietzerbin J. Stehelin D. Tonnel A.B. Capron A. Eur. J. Immunol. 1992; 22: 425-431Crossref PubMed Scopus (39) Google Scholar) were cultured in RPMI 1640, containing 2 mml-glutamine and 10% fetal calf serum. Human embryonic kidney cells (293 cell line) were cultured in DMEM with 10% fetal calf serum. 293 cells used for the test of proliferation were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10 μg/ml insulin and 10 μg/ml transferrin. Proteinase K and chondroitinase ABC were purchased from Roche Molecular Biochemicals; chondroitinases B, ACI, and C were from Sigma; and heparinase III was from Grampian Enzymes (Orkney, UK). Human HGF/SF was from R & D Systems (Abingdon, UK), and heparin was from Sigma. Anti-endocan monoclonal antibodies were produced and purified as described previously (40Bechard D. Meignin V. Scherpereel A. Oudin S. Kervoaze G. Bertheau P. Janin A. Tonnel A. Lassalle P. J. Vasc. Res. 2000; 37: 417-425Crossref PubMed Scopus (161) Google Scholar). The full-length endocan cDNA was excised from the pCDM8 vector, purified, and inserted intoXho I-Hin dIII-digested pcDNA3 expression vector (Invitrogen, Groningen, The Netherlands). The construct was transfected into CHO and 293 cells using LipofectAMINE (Life Technologies, Inc.), followed by selection with G418 (1000 or 300 μg/ml, respectively). Stably transfected cell lines were obtained by limit dilution, and the resulting cell lines were named CHO-Endocan and 293-Endocan. Two potential O- glycosylation sites in the endocan polypeptide, serine 137 and threonine 120, were predicted by the NetOglyc 2.0 Prediction Server. The codons for serine 137 and threonine 120 in the endocan cDNA were each mutated to an alanine codon by PCR with the QuickChange site-directed mutagenesis kit, according to the manufacturer's recommendations (Stratagene, Cambridge, UK). The resulting alanine mutations in the cDNA were confirmed by sequencing on an ABI Prism 377 automated DNA sequencer (PE Biosystems, Courtaboeuf, France). The mutated cDNAs were then transfected into 293 cells to obtain transient and stable transfectants (293-S137A/endocan and 293-T120A/endocan cell lines). The cell lines were cultured in suspension in medium without fetal calf serum (CHO-SFM II and 293-SFM, Life Technologies, Inc.). After 3–4 days in culture, the medium was collected and stored at −20 °C until use. The pH of the medium was adjusted to pH 8, before application to a column (2.5 × 10 cm) of DEAE-Sepharose (Amersham Pharmacia Biotech). The column was washed with 0.2 m NaCl, 50 mm Tris, pH 8, and then eluted with a gradient of 0.8–1m NaCl in the same buffer. Collected fractions were adjusted to 0.2 m NaCl, 50 mm Tris, pH 8.0, and applied to an immunoaffinity column made by immobilizing an anti-endocan mAb (MEC4) on Affi-Gel Hz Hydrazide gel, following the recommendations of the manufacturer (Bio-Rad). After washing with 0.2m NaCl, 50 mm Tris, pH 8.0, endocan was eluted with 3 m MgCl2, concentrated, and dialyzed against the same solution using an Ultrafree 10-kDa molecular mass cut-off membrane (Millipore, Bedford, MA). The final material was then quantified by immunoassay for endocan, and its purity was verified by SDS-PAGE followed by Coomassie Blue or Alcian Blue staining. The nonglycosylated form of endocan (S137A/endocan) was purified in only one step by immunoaffinity chromatography. The degree of purity of WT/endocan and S137A/endocan was checked by gel filtration chromatography on Superdex 75/200 HR 10/30 columns (0.13 × 30 cm;Amersham Pharmacia Biotech) and yielded a single peak. The preparations were shown to be endotoxin-free by the limulus amebocyte lysate test (BioWhittaker, Verviers, Belgium). Eight hundred ml of spent plasma, kindly provided by the Etablissement de Transfusion Sanguine (Lille, France), was first precipitated with 60% ammonium sulfate. The precipitate was dissolved and dialyzed against 0.2m NaCl, 50 mm Tris, pH 8.0. The dialysate was applied to a 50-ml Affi-Gel pre-column (Bio-Rad) prior to immunoaffinity chromatography, which was then performed as described above. Cells in 80-cm2 flasks were lysed by incubation in PBS containing 0.5% Nonidet P-40 and an anti-protease mixture (Roche Molecular Biochemicals) for 30 min at 4 °C with agitation. Cell lysates were cleared by centrifugation at 10,000 × g for 15 min. One μg of anti-endocan mAb (MEP19) or ICAM-1 mAb (clone 164B) was added to the cleared lysates and cell supernatants and incubated overnight at 4 °C with agitation. Fifty microliters of anti-mouse immunoglobulin conjugated to agarose beads (Sigma) was added to the samples at 4 °C for 90 min, and then the agarose beads were collected by centrifugation, washed twice with lysis buffer, and then twice with PBS. The beads were resuspended in 20–40 μl of SDS-PAGE sample buffer, boiled for 2 min, centrifuged, and the polypeptides separated by SDS-PAGE. Following SDS-PAGE, polypeptides were transferred to nitrocellulose membranes according to standard procedures. After a blocking step, the membranes were incubated for 1 h with endocan mAb (MEP14) at 1 μg/ml, followed by a 1-h incubation with an anti-mouse Fc horseradish peroxidase-conjugated secondary antibody (Sigma). Immunoreactivity was detected by ECL (Amersham Pharmacia Biotech). For amino acid sequence analysis, purified endocan was transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) after SDS-PAGE and stained with 0.1% Coomassie Blue. The protein band of 50 kDa was excised from the membrane, and the N-terminal sequence was determined by automated Edman degradation on an ABI 473A protein sequencer. To determine the size of the glycosaminoglycan chains, purified endocan was digested with proteinase K with an enzyme:endocan ratio of 1:50 (w/w) in 0.2 m NaCl, 10 mm Tris, pH 8.0, at 56 °C for 3 h. The sample was then incubated overnight with 0.5 ml of DEAE-Sepharose at 4 °C. The DEAE-Sepharose was poured into a Bio-Spin chromatography column (0.5 × 3 cm; Bio-Rad) and washed with 10 column volumes of buffer. GAG chains were eluted with 1 ml of 1.2 m NaCl, 50 mm Tris, pH 8, and then aliquoted and stored at −70 °C. Losses of GAG chains during purification were estimated by applying untreated endocan to the same volume of DEAE-Sepharose; from an input of 120 μg, 100 μg of endocan was recovered in 1 ml of elution buffer. Thus, total endocan input to the digest was adjusted to give a final estimated GAG concentration of 200 μg/ml. Samples were analyzed on 12% SDS-PAGE, followed by Coomassie Blue or Alcian Blue staining. To determine the nature of the glycosaminoglycan substitution, purified endocan was digested with several specific enzymes, namely chondroitinase ABC (0.5 unit/mg in 40 mm Tris-HCl, 40 mm sodium acetate, pH 8.0, at 37 °C for 3 h), chondroitinase B (200 unit/mg in 20 mm Tris-HCl, 50 mm NaCl, 4 mm CaCl2, 0.01% (w/v) bovine serum albumin, pH 7.5, at 25 °C for 2 h), chondroitinase ACI (1 unit/ml in 250 mm Tris-HCl, 75 mm sodium acetate, pH 7.3, at 37 °C for 2 h), chondroitinase C (80–120 units/ml in 50 mm Tris-HCl, pH 8.0, at 25 °C for 3 h), and heparinase III (0.1 mm calcium acetate, 1 mm sodium acetate, 100 μg/ml bovine serum albumin, pH 7, overnight at room temperature). Samples were analyzed by SDS-PAGE and Western blotting. 293-Endocan cells were cultured in suspension in 250 ml of 293 SFM medium until the end of the log phase of cell division. The cells were then centrifuged and resuspended in 250 ml of fresh medium containing 5 μCi/ml d-[6-3H]glucosamine hydrochloride (Amersham Pharmacia Biotech). After 48 h of incubation at 37 °C, the cell supernatants were processed for the purification of endocan as described above, in two steps including DEAE-Sepharose ion-exchange chromatography followed by immunoaffinity chromatography. This yielded a specific activity of 24,000 cpm/μg of endocan. The 3H-labeled GAG chains were released from endocan by β-elimination with 50 mm NaOH, 1 mNaBH4 at 45 °C for 24 h. After neutralization with 1 m acetic acid, the GAG was precipitated by addition of 3 volumes of ethanol containing 1.3% (w/v) potassium acetate at −20 °C. The GAG precipitate was collected by centrifugation, washed with 75% (v/v) ethanol, re-centrifuged, air-dried, and then re-dissolved in water. To remove some residual 3H-labeled small oligosaccharides (probably O- linked glycosylation), the sample was applied to a 0.5-ml column of DEAE-Sepharose and washed with PBS. The GAG chains were step eluted with 1 ml of 1.5m NaCl. The sample was then desalted on a PD-10 column equilibrated in distilled water and concentrated by centrifugal evaporation. [3H]GAG chains (40,000 cpm) were exhaustively digested to disaccharides with two additions of 10 mIU of chondroitinase ABC in 50 mm NaCl, 50 mm Tris-HCl, pH 8.0, at 37 °C for 24 h. Digests were boiled and centrifuged to remove protein and then diluted to 1 ml with distilled water adjusted to pH 3.5 with HCl. Samples were applied to a 5-μm Hypersil (0.46 × 25 cm; ThermoQuest, Runcorn UK) strong anion-exchange high pressure liquid chromatography column equilibrated in water pH 3.5. After a wash with pH 3.5 water, the disaccharides were resolved over a linear gradient of 0–0.4m NaCl, pH 3.5, at a flow rate of 1 ml/min. Fractions of 0.5 ml were collected and counted for radioactivity. The3H-labeled disaccharides were identified by comparison with the elution positions of known CS/DS and heparan disaccharide standards monitored by on-line UV detection at 232 nm. Analyses were performed on duplicate enzyme digests. [3H]GAG chains (50,000 cpm) were exhaustively digested with either chondroitinase ACI (40 mIU in 50 mm NaCl, 50 mm Tris-HCl, pH 7.3) or chondroitinase B (8 mIU in 50 mm NaCl, 50 mmTris-HCl, pH 8.0). Digests were initiated with half the enzyme and incubated at 37 °C for 16 h, followed by the addition of the second enzyme aliquot and incubation for a further 2 h. Digests were then boiled and centrifuged to remove the protein, and the cleared supernatants were applied to a Bio-Gel P10 gel filtration column (1 × 170 cm) equlibrated with 0.1 m NaCl and run at a flow rate of 4 ml/h. Fractions of 1 ml were collected and counted for radioactivity. The V o and V tvalues of the column were determined with undegraded GAG and sodium dichromate, respectively. The percentage of galactosaminyl bonds cleaved by each of the specific enzymes was calculated from the distribution of 3H radiolabel across peaks corresponding to known oligosaccharide size fractions, relative to the total eluted3H radiolabel, by the use of a standard algorithm. Control platelet-poor plasma (PPP) was prepared from blood anticoagulated with sodium citrate (30 healthy donors) by centrifugation at 2500 × g for 15 min. All the reagents were purchased from Stago Diagnostica (France). Three parameters were evaluated, by adding endocan or buffer or heparin to PPP. Activated partial thromboplastin time explores the intrinsic pathway of blood coagulation (FI, FII, FV, FVIII, FIX, FX, FXI, and FXII). Deficit or inhibition of one of these factors enhances coagulation time of the reactive mixture (PPP, cephalin, activator, and CaCl2). Thrombin clotting time is performed on PPP + thrombin. With a standard concentration of thrombin, the clotting time of plasma is constant. Abnormalities of fibrin formation induce an increase of the coagulation time. Anti-Xa activity of heparin, or of other inhibitors acting on FXa, is detected by this competitive assay. The studied sample (PPP + endocan, buffer, or heparin) is mixed with FXa and a FXa-specific chromogenic substrate. The final coloration is inversely proportional to the inhibitor concentration. This global sensitive assay can detect platelet or plasmatic abnormality inducing a lag time or a decrease of thrombin generation. In five healthy donors, platelet-rich plasma (PRP) was prepared from blood anticoagulated with sodium citrate by centrifugation at 150 × g for 10 min. Thrombin generation test was performed for each subject, in samples without endocan, with unfractionated calcium heparinate (0.5 IU anti-Xa/ml) or with 0.2, 0.5, or 1 μg/ml endocan (final concentration). Endocan was added 10 min before the assay. At 37 °C, 1 ml of plasma was mixed with 1 ml of CaCl2. Aliquots of 0.1 ml were removed from the reaction each minute for 15 min. Clots formed in the reactive mixture were regularly removed. Aliquots were mixed with 0.2 ml of fibrinogen (Sigma, 4/1000 in Owren buffer) at 37 °C, and clotting time was measured for each aliquot. Thrombin formed in the reactive mixture acts on fibrinogen, inducing fibrin formation. The clotting activity was maximal between 4 and 8 min and then decreased on account of neutralization of thrombin by anti-thrombin. For native endocan, 50 μg of purified WT/endocan or S137A/endocan in 0.5 m NaCl, 50 mm Tris, pH 8, were chromatographed on Superdex 200 or Superdex 75 HR10/30 columns (0.13 × 30 cm; Amersham Pharmacia Biotech), respectively, using a Bio-Rad Biologic Chromatography System (Bio-Rad) at a flow rate of 1 ml/min in the same buffer. Fractions of 1 ml were collected, and endocan was detected by a specific ELISA. The following proteins in the low and high molecular mass calibration kits (Amersham Pharmacia Biotech) were used as standards: ribonuclease A (bovine pancreas, 13.7 kDa), ovalbumin (hen egg, 43 kDa), albumin (bovine serum, 67 kDa), aldolase (rabbit muscle, 158 kDa), ferritin (horse spleen, 440 kDa), and thyroglobulin (bovine thyroid, 669 kDa). Molecular weight standards were run immediately after WT/endocan and S137A/endocan. The elution times of the standards were used to construct a standard linear curve,K av = f (log MR), to determine the apparent molecular mass of WT/endocan and S137A/endocan. Growth-promoting activity was determined by measuring [methyl-3H]thymidine incorporation into 293 cells. Cells were seeded at a density of 1 × 104/well in Techno Plastic Products (TPP) 96-well microplates and maintained for 24 h in DMEM supplemented with transferrin and insulin. Recombinant human HGF/SF was diluted in PBS containing 0.1% bovine serum albumin and added to triplicate wells to obtain a final concentration of 50 ng/ml. WT/endocan, S137A/endocan, and the purified GAG derived from endocan or heparin were added alone or together with HGF/SF, as indicated in the figure legends. After 96 h 0.5 μCi of [methyl- 3H]thymidine/well was added for 16 h and [methyl- 3H]thymidine incorporation into DNA was determined on a TopCount Microplate Scintillation Counter (Packard Instrument Co., Rungis, France). Assays were performed in triplicate. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction test. Binding reactions were carried out at 20 °C in an IAsys Auto+ dual channel resonant mirror biosensor (Affinity Sensors, Saxon Hill, Cambridge, UK) using streptavidin-derivatized aminosilane surfaces as described (43Rahmoune H. Rudland P.S. Gallagher J.T. Fernig D.G. Biochemistry. 1998; 37: 6003-6008Crossref PubMed Scopus (55) Google Scholar, 44Fernig D.G. Iozzo R.V. Proteoglycan Protocols. 171. Humana Press Inc., Totowa, NJ2001: 505-518Google Scholar) with minor modifications. Endocan was biotinylated on the amino groups of the core protein (43Rahmoune H. Rudland P.S. Gallagher J.T. Fernig D.G. Biochemistry. 1998; 37: 6003-6008Crossref PubMed Scopus (55) Google Scholar, 44Fernig D.G. Iozzo R.V. Proteoglycan Protocols. 171. Humana Press Inc., Totowa, NJ2001: 505-518Google Scholar) and then immobilized on the streptavidin-derivatiz
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