Human Ryudocan from Endothelium-like Cells Binds Basic Fibroblast Growth Factor, Midkine, and Tissue Factor Pathway Inhibitor (∗)

  • Tetsuhito Kojima
    Correspondence
    To whom correspondence and reprint requests should be addressed: First Dept. of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan . Tel.: 81-52-741-2111 (ext. 2203); Fax: 81-52-741-1612
    Affiliations
    First Department of Internal Medicine, Nagoya University Hospital, Nagoya 466
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  • Akira Katsumi
    Affiliations
    First Department of Internal Medicine, Nagoya University Hospital, Nagoya 466
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  • Tomio Yamazaki
    Affiliations
    First Department of Internal Medicine, Nagoya University Hospital, Nagoya 466
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  • Takashi Muramatsu
    Affiliations
    Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466,
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  • Tetsuro Nagasaka
    Affiliations
    Division of Pathology, Clinical Laboratory, Nagoya University Hospital, Nagoya 466
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  • Kazuoki Ohsumi
    Affiliations
    Mitsubishi Yuka Bio-Clinical Laboratories, Inc., Tokyo 174
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  • Hidehiko Saito
    Affiliations
    First Department of Internal Medicine, Nagoya University Hospital, Nagoya 466

    Aichi Juridical Foundation for Blood Disease Research, Nagoya 463, Japan
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  • Author Footnotes
    ∗ This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture, the Ministry of Health and Welfare, and Funds for Comprehensive Research on Aging and Health, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Ryudocan, a heparan sulfate proteoglycan, was isolated from human endothelium-like EAhy926 cells by a combination of ion-exchange and immunoaffinity chromatography. Purified human ryudocan has biochemical properties similar to those of rat ryudocan isolated from microvascular endothelial cells. Human ryudocan contains only heparan sulfate (HS) glycosaminoglycan chains along with a core protein with an apparent molecular mass of 30 kDa. We evaluated the interactions between purified human ryudocan and several extracellular ligands by using a solid-phase binding assay. We found that basic fibroblast growth factor (bFGF), midkine (MK), and tissue factor pathway inhibitor (TFPI) exhibit significant ryudocan binding. Heparitinase (but not chondroitin ABC lyase) treatment destroyed the ability of ryudocan binding to bFGF, MK, and TFPI. Heparin and HS, but not chondroitin sulfate, inhibited such ryudocan binding. Thus, the HS chains of ryudocan appear to be responsible for its binding to bFGF, MK, and TFPI. The apparent dissociation constants for purified ryudocan were as follows: bFGF, 0.50 nM; MK, 0.30 nM; and TFPI, 0.74 nM. Immunohistochemical analysis revealed that ryudocan was expressed in fibrous connective tissues, peripheral nerve tissues, and placental trophoblasts. These findings suggest that ryudocan may possess multiple biological functions, such as bFGF modulation, neurite growth promotion, and anticoagulation, via HS-binding effectors in the cellular microenvironment.

      INTRODUCTION

      Ryudocan is an integral membrane heparan sulfate proteoglycan (HSPG) (
      The abbreviations used are: HSPG
      heparan sulfate proteoglycan
      HS
      heparan sulfate
      bFGF
      basic fibroblast growth factor
      GAG
      glycosaminoglycan
      MK
      midkine
      TFPI
      tissue factor pathway inhibitor
      CS
      chondroitin sulfate
      ATIII
      antithrombin III
      CSF
      colony-stimulating factor
      PAGE
      polyacrylamide gel electrophoresis
      CHAPS
      3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
      BSA
      bovine serum albumin
      PBS
      phosphate-buffered saline.
      ) that was originally isolated from endothelial cells as an anticoagulant molecule(
      • Kojima T.
      • Leone C.W.
      • Marchildon G.A.
      • Marcum J.A.
      • Rosenberg R.D.
      ,
      • Kojima T.
      • Shworak N.S.
      • Rosenberg R.D.
      ). This compound is a member of the syndecan family composed of four cell membrane intercalated HSPGs: syndecan (syndecan-1), fibroglycan (syndecan-2), N-syndecan (syndecan-3), and ryudocan (syndecan-4), which have homologous transmembrane and intracellular domains, but very distinct extracellular regions(
      • Bernfield M.
      • Kokenyesi R.
      • Kato M.
      • Hinkes M.
      • Spring J.
      • Gallo R.L.
      • Lose E.J.
      ). The best characterized molecule of this family is syndecan, the prototypical member that was isolated from mouse mammary epithelial cells(
      • Saunders S.
      • Jalkanen M.
      • O'Farrell S.
      • Bernfield M.
      ). Syndecan selectively binds via its heparan sulfate (HS) chains to a variety of matrix components, including fibrillar collagens(
      • Koda J.E.
      • Rapraeger A.
      • Bernfield M.
      ), fibronectin(
      • Saunders S.
      • Bernfield M.
      ), thrombospondin(
      • Sun X.
      • Mosher D.F.
      • Rapraeger A.
      ), tenasin(
      • Salmivirta M.
      • Elenius K.
      • Vainio S.
      • Hofer U.
      • Chiquet-Ehrismann R.
      • Thesleff I.
      • Jalkanen M.
      ), and amphoterin(
      • Salmivirta M.
      • Rauvala H.
      • Elenius K.
      • Jalkanen M.
      ), which suggests that syndecan may be an extracellular matrix receptor.
      Syndecan also binds to the heparin-binding growth factors, such as basic fibroblast growth factor (bFGF)(
      • Kiefer M.C.
      • Stephans J.C.
      • Crawford K.
      • Okino K.
      • Barr P.J.
      ,
      • Bernfield M.
      • Hooper K.C.
      ,
      • Ishihara M.
      • Tyrrell D.J.
      • Kiefer M.C.
      • Barr P.J.
      • Swiedler S.J.
      ,
      • Elenius K.
      • Määttä A.
      • Salmivirta M.
      • Jalkanen M.
      ,
      • Salmivirta M.
      • Heino J.
      • Jalkanen M.
      ). The potential importance of this interaction is underscored by recent reports demonstrating the involvement of cell-surface HSPGs in bFGF signaling mechanisms(
      • Yayon A.
      • Klagsbrun M.
      • Esco J.D.
      • Leder P.
      • Ornitz D.M.
      ). N-Syndecan is another member of the syndecan family. This molecule was recently identified and cloned in rat Schwann cells and chicken embryos(
      • Carey D.J.
      • Evans D.M.
      • Stahl R.C.
      • Asundi V.K.
      • Conner K.J.
      • Garbes P.
      • Cizmeci-Smith G.
      ,
      • Gould S.E.
      • Uphold W.B.
      • Kosher R.A.
      ). N-Syndecan possesses a high degree of specificity for bFGF through its glycosaminoglycan (GAG) chains(
      • Chernousov M.A.
      • Carey D.J.
      ). N-Syndecan was also isolated as a heparin binding growth-associated molecule (pleiotrophin) receptor and was anticipated to mediate the neurite outgrowth-promoting signal from a heparin binding growth-associated molecule to the cytoskeleton of growing neurites(
      • Raulo E.
      • Chernousov M.A.
      • Carey D.J.
      • Nolo R.
      • Rauvala H.
      ).
      Several syndecan-4 cDNAs have been characterized for the rat (ryudocan(
      • Kojima T.
      • Shworak N.S.
      • Rosenberg R.D.
      )), human (ryudocan (
      • Kojima T.
      • Inazawa J.
      • Takamatsu T.
      • Rosenberg R.D.
      • Saito H.
      ) and amphiglycan(
      • David G.
      • van der Schueren B.
      • Marynen P.
      • Cassiman J.-J.
      • van den Berghe H.
      )), and chicken (
      • Baciu P.C.
      • Acaster C.
      • Goetinck P.E.
      ) forms. Little is known, however, about the biological function of this molecule. Thus, we purified human ryudocan from endothelium-like cells (EAhy926 cells) and examined its interactions with various biological ligands by using a solid-phase binding assay. The ligands tested for ryudocan binding were bFGF, midkine (MK), and tissue factor pathway inhibitor (TFPI). Among them, MK is a heparin-binding growth factor unrelated to fibroblast growth factor(
      • Kadomatsu K.
      • Tomomura M.
      • Muramatsu T.
      ,
      • Tomomura M.
      • Kadomatsu K.
      • Tomomura M.
      • Matsubara S.
      • Muramatsu T.
      ), promotes neurite outgrowth (
      • Muramatsu H.
      • Shirahama H.
      • Yonezawa S.
      • Maruta H.
      • Muramatsu T.
      ) and neuronal cell survival(
      • Michikawa M.
      • Kikuchi S.
      • Muramatsu H.
      • Muramatsu T.
      • Kim S.U.
      ), and enhances plasminogen activator activity in aortic endothelial cells(
      • Kojima S.
      • Muramatsu H.
      • Amanuma H.
      • Muramatsu T.
      ). TFPI is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa- and factor VIIa-tissue factor activity(
      • Rapaport S.I.
      ,
      • Broze G.J.
      ). We also investigated the expression of human ryudocan by using an immunohistochemical method utilizing a specific anti-human ryudocan antibody.

      EXPERIMENTAL PROCEDURES

       Materials

      Heparin, HS from bovine kidney, type A chondroitin sulfate (CS) from whale cartilage, chondroitin ABC lyase, fibronectin, type I collagen, bFGF, epidermal growth factor, insulin-like growth factor, and rabbit anti-human insulin-like growth factor IgG were all purchased from Seikagaku Co. (Tokyo). Human antithrombin III (ATIII) and rabbit anti-human ATIII antibody were provided by Hoechst Japan Ltd. (Tokyo). Rabbit IgGs of anti-human collagen (type I) and anti-human epidermal growth factor were from Cosmo Bio (Tokyo). Chemically synthesized human MK, which has neurite promoting activity (
      • Muramatsu H.
      • Inui T.
      • Kimura T.
      • Sakakibara S.
      • Song X.
      • Maruta H.
      • Muramatsu T.
      ) and enhances plasminogen activator activity in aortic endothelial cells(
      • Kojima S.
      • Inui T.
      • Kimura T.
      • Sakakibara S.
      • Muramatsu H.
      • Amamura H.
      • Maruta H.
      • Muramatsu T.
      ), was from Peptide Institute (Suita, Japan). Anti-human MK serum was obtained from New Zealand White rabbits immunized with synthesized human MK. Human recombinant TFPI (
      • Enjoji K.
      • Miyata T.
      • Kamikubo Y.
      • Kato H.
      ) was obtained from Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan), and rabbit anti-human TFPI IgG was a generous gift from Dr. Hisao Kato (National Cardiovascular Center Research Institute, Suita, Japan). Granulocyte colony-stimulating factor (CSF), granulomonocyte CSF, and interleukin-3 were supplied by Kirin Brewery Co. (Tokyo). Monocyte CSF and rabbit anti-human monocyte CSF IgG were obtained from Morinaga Co. (Tokyo). Rabbit IgGs of anti-human bFGF, anti-human granulocyte CSF, anti-human granulomonocyte CSF, and anti-human interleukin-3 were provided by Mitsubishi Yuka BCL, Inc. (Tokyo). SDS-polyacrylamide gel electrophoresis (PAGE) reagents were from Nacalai Tesque, Inc. (Kyoto, Japan). Prestained protein markers and culture media were obtained from Life Technologies, Inc. DEAE-Sephacel, CNBr-activated Sepharose, and PD-10 columns were provided by Pharmacia Biotech Co. (Tokyo). Phenylmethylsulfonyl fluoride, CHAPS, and bovine serum albumin (BSA; fraction V) were from Sigma. Carrier-free 125I was provided by Amersham Co. (Tokyo). Coomassie Brilliant Blue protein assay reagents were purchased from Pierce.

       Preparation of Poly- and Monoclonal Anti-human Ryudocan Antibodies

      We purchased the synthetic peptide of the human ryudocan ectodomain, human [Tyr25,Cys26]ryudocan (residues 27-39; NH2-YCDEDVVGPGQESDD-COOH (Ryu2)(
      • Kojima T.
      • Inazawa J.
      • Takamatsu T.
      • Rosenberg R.D.
      • Saito H.
      )), from Perkin Elmer Japan Co., Applied Biosystems Division (Urayasu, Japan). This polypeptide was then conjugated to ovalbumin with meta-maleimidobenzoyl-N-hydroxysuccinimide ester (
      • Harlow E.
      • Lane D.
      ) for immunizing rabbits or mice. Rabbit anti-ryudocan serum was obtained from Japanese rabbits immunized every 2 weeks with ovalbumin-Ryu2 peptide. Hybridoma cells producing mouse anti-human ryudocan monoclonal antibody were obtained by fusion of spleen cells from immunized BALB/c mice with ovalbumin-Ryu2 peptide and P3U1 parent cells. Mouse spleen cells (4 × 108) were mixed with P3U1 cells (1 × 108) and fused in the presence of 50% polyethylene 4000. Hybridomas were grown in hypoxanthine/aminopterin/thymidine medium and selected by radioimmunoassay.

       EAhy926 Cell Culture

      The EAhy926 cell line(
      • Edgell C.-J. S.
      • McDonald C.C.
      • Graham J.B.
      ), a hybrid of human umbilical vein endothelial cells and human lung carcinoma cells, was a generous gift from Dr. Cora-Jean S. Edgell (University of North Carolina, Chapel Hill, NC). EAhy926 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate. The flasks were gassed with 5% CO2 at 37°C, and the medium was exchanged every 2 days. Conditioned media were harvested when the cultures attained a stationary density for 7 days.

       Isolation of Human Ryudocan

      Conditioned media were adjusted to contain 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride and filtered through a DEAE-Sephacel column (2.5 × 10 cm) at a flow rate of 20 ml/h. After washing the column with 5 column volumes of 0.4 M NaCl, 1 mM EDTA, and 50 mM sodium acetate, pH 6.0, followed by 5 column volumes of 0.3 M NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.4, bound proteoglycans were eluted with 1 M NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.4.
      After dialysis against 0.15 M NaCl, 1 mM EDTA, and 50 mM Tris-HCl, pH 7.4, CHAPS was added to a concentration of 0.6%, and the proteoglycans eluted from the DEAE-Sephacel column were charged to a monoclonal antibody-Sepharose affinity column (CNBr-activated Sepharose conjugated with anti-human ryudocan monoclonal antibody). The column was washed with 5 column volumes of 0.15 M NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 7.4, and 0.6% CHAPS, and the proteoglycans were eluted with 3 column volumes of 0.15 M NaCl and 50 mM glycine, pH 2.5, followed by immediate neutralization with 0.2 volume of 1 M Tris-HCl, pH 7.8. The eluant from the monoclonal antibody affinity column was then loaded onto a second DEAE-Sephacel column (0.5 ml). This column was washed with 5 column volumes of 0.4 M NaCl and 50 mM sodium acetate, pH 5.0, followed by 5 column volumes of 0.3 M NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Proteoglycans were eluted with 1 M NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Purified human ryudocan was divided into aliquots and stored at −70°C for further use.

       Assay and Radioiodination of Proteins

      Protein concentrations were determined by the Bradford (
      • Bradford M.M.
      ) method by using the Coomassie Brilliant Blue protein assay kit. The A595 measurements for 50 μl of sample were carried out in a DU7500 spectrophotometer (Beckman Instruments) with BSA as a standard. Purified ryudocan was radiolabeled with Na125I by the chloramine-T method as described previously (
      • Kojima T.
      • Leone C.W.
      • Marchildon G.A.
      • Marcum J.A.
      • Rosenberg R.D.
      ). The radiolabeled components were separated from free Na125I by gel filtration chromatography through a PD-10 desalting column and on a DEAE-Sephacel column (0.3 ml) as described above. The radiolabeled species were stored at −70°C until use.

       SDS-PAGE

      SDS-PAGE was carried out through a linear gradient (4-15%) by using a minigel apparatus (Nippon Eido, Tokyo) and the buffer system of Laemmli(
      • Laemmli U.K.
      ). Prestained molecular weight markers were employed as molecular size markers. Before electrophoresis, samples were adjusted to 1% SDS and boiled for 10 min. Samples run under reducing conditions were supplemented with 1%β-mercaptoethanol. After electrophoresis, the gels were fixed with 30% methanol, dried, and then analyzed by autoradiography with Hyperfilm-MP (Amersham Co.).

       Identification of GAG Chains on Purified Human Ryudocan

      125I-Radiolabeled ryudocan (~4 × 106 cpm/μg) was characterized by employing polysaccharidases of known substrate specificity. Samples (3 × 104 cpm) were incubated either for 5 h at 37°C with 1 unit of purified Flavobacterium heparitinase or for 5 h at 37°C with 0.1 unit of chondroitin ABC lyase as described previously (
      • Kojima T.
      • Leone C.W.
      • Marchildon G.A.
      • Marcum J.A.
      • Rosenberg R.D.
      ). After enzyme treatment, samples were subjected to SDS-PAGE and analyzed by autoradiography as described above.

       Solid-phase Binding Assay

      A solid-phase ligand binding assay was performed as described by Chernousov and Carey(
      • Chernousov M.A.
      • Carey D.J.
      ), with a minor modification. Briefly, 0.25-5 μg/well concentrations of various proteins (in 100 μl of 50 mM NaHCO3, pH 9.2) were coated onto microtiter plate wells (Falcon 3911) by overnight incubation at 4°C. After three washes with 100 μl of phosphate-buffered saline (PBS; 0.137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) containing 0.1% BSA (0.1% BSA/PBS), the wells were incubated with 1% BSA in PBS for 4 h at room temperature to block nonspecific binding sites. The wells were then rinsed three times with 0.1% BSA/PBS and incubated with 125I-radiolabeled ryudocan (3000-7000 cpm/well) in 100 μl of 0.1% BSA/PBS at 4°C overnight. After three rinses with 0.1% BSA/PBS to remove any unbound material, the wells were dried and individually separated. The amount of bound radiolabeled ryudocan in each well was quantified with an Aloka γ-counter (Aloka Co. Ltd., Tokyo).
      To assure that the applied proteins stuck to the wells, we performed an immunodetection assay as follows. After coating with the proteins (0.5 μg/well) and blocking nonspecific binding sites as described above, the wells were sequentially incubated with a specific rabbit antibody against each ligand and a peroxidase goat anti-rabbit IgG antibody (Dako Japan Co. Ltd., Kyoto, Japan) in 0.1% BSA/PBS containing 0.05% Tween 20. After washing five times with distilled water, the wells were incubated with 6.2 mMo-phenylenediamine dihydrate and 0.01% (v/v) H2O2 in 75 μl of 0.1 M citrate/phosphate buffer, pH 5.0. The reaction was stopped by adding 75 μl of 3 M H2SO4, and the absorbance at A492 of the sample in each well was measured. In addition, we measured the amount of protein remaining in the well after removal of ligand solution using a 125I-labeled tracer for each protein. We labeled each ligand protein with 125I as described above and verified the purity of each 125I-labeled protein by SDS-PAGE and autoradiography. The wells, which were coated with each ligand containing 125I-labeled tracer as described above, were washed, and the amount of bound radioactivity was quantified.
      To verify that unlabeled ryudocan exhibits similar binding properties, we also tested the competitive ability of unlabeled ryudocan against 125I-ryudocan binding. Thus, a solid-phase binding assay was performed in the presence of serial amounts of unlabeled ryudocan. After coating with 0.5 μg/well bFGF, MK, or TFPI, the microtiter plate wells were incubated with 0.1 ng (1 × 103 cpm)/well 125I-ryudocan containing unlabeled ryudocan (0.1, 1, or 10 ng/well). The wells were washed, and the amount of bound radiolabeled ryudocan in each well was quantified as described above.

       Immunohistochemistry

      Tissues obtained from four patient autopsies (two adults and two fetuses), placenta, and coronary atheromatous plaques were used in this study. Tissue sections were prepared from Formalin-fixed paraffin-embedded blocks and treated as described previously with minor modifications(
      • Nagasaka T.
      • Sobue M.
      • Niwa M.
      • Yasui C.
      • Nara Y.
      • Fukatsu T.
      • Nakashima N.
      • Takeuchi J.
      ). Briefly, the deparaffinized tissue sections were soaked in methanol containing 0.3% (v/v) H2O2 to block the pseudo-peroxidase reaction and washed with Tris-buffered saline. Sections were incubated with normal rabbit serum (1:100 dilution) for 20 min, followed by treatment with rabbit anti-human ryudocan serum (IgG fraction, 1:1000 dilution) for 30 min at room temperature. Following another washing step, the bound IgGs were labeled with biotinylated anti-rabbit immunoglobulin and subsequently incubated with peroxidase-conjugated streptavidin (Histofine SAB-PO kits, Nichirei Co., Tokyo) according to the manufacturer's protocols. After washing with Tris-buffered saline, the tissue sections were incubated in 0.05 M sodium acetate/acetic acid buffer, pH 5.0, containing 0.02% (w/v) 3amino-9-ethylcarbazole and 0.014% (w/v) H2O2 and allowed to react for 5-10 min. Sections were finally counterstained with hematoxylin and mounted in glycerin/gelatin solution (glycerin, 20% gelatin solution of 1:1). As a negative control, PBS was used instead of rabbit anti-human ryudocan antibody. To assure the specificity of the signals, we also used an excess amount of Ryu2 peptide to compete with the native ryudocan molecule in the specific binding of anti-human ryudocan IgG. Thus, we incubated rabbit anti-human ryudocan IgG with an excess amount of Ryu2 peptide (molar ratio of 3000:1) for 20 h at 4°C prior to treatment of samples following the immunostaining procedures as described above.

      RESULTS

       Purification and Characterization of Human Ryudocan from EAhy926 Cells

      We purified human ryudocan from EAhy926 cells by using a combination of anion-exchange and immunoaffinity chromatography (see “Experimental Procedures”). The overall yields of human ryudocan from conditioned media of EAhy926 cells for the entire purification procedure averaged 2.5 ng of protein/106 cells. These results were similar to previously reported purification yields of rat ryudocan from rat fat pad microvascular endothelial cells(
      • Kojima T.
      • Leone C.W.
      • Marchildon G.A.
      • Marcum J.A.
      • Rosenberg R.D.
      ).
      To determine the purity of the proteoglycan preparation and to identify the attached glycosaminoglycan chains, this material was subjected to enzymatic degradation. After radiolabeling with 125I, the human ryudocan preparation was treated with purified Flavobacterium heparitinase or chondroitin ABC lyase and analyzed by SDS-PAGE through a 4-15% gradient (Fig. 1). Autoradiography of the resulting material showed a diffuse band with an apparent molecular mass of 160 kDa. Treatment of the sample with purified Flavobacterium heparitinase resulted in a single distinct band with an apparent molecular mass of 30 kDa. This corresponded to the previously described core protein size of rat ryudocan from microvascular endothelial cells(
      • Kojima T.
      • Leone C.W.
      • Marchildon G.A.
      • Marcum J.A.
      • Rosenberg R.D.
      ). In contrast, chondroitin ABC lyase treatment did not affect the migration of the iodinated 160-kDa molecule. These data indicate that the purification procedure yielded pure human ryudocan, which was predominantly composed of heparan sulfate GAG chains.
      Figure thumbnail gr1
      Figure 1:Characterization of human ryudocan from EAhy926 cells. After radiolabeling with 125I, purified human ryudocan was subjected to heparitinase or chondroitin ABC lyase treatment as indicated, analyzed by electrophoresis through a 4-15% SDS-polyacrylamide gradient gel, and autoradiographed.

       Ryudocan Binding to Potential Ligands

      The binding of 125I-labeled human ryudocan to potential ligands was examined by using the solid-phase assay as described under “Experimental Procedures.” Microtiter wells (precoated with a 1 μg/well concentration of the proteins) were incubated with 125I-labeled human ryudocan. After washing the wells, the amount of bound radioactive material was determined. The results indicate that bFGF, MK, and TFPI exhibited significant 125I-ryudocan binding (Fig. 2). In contrast, fibronectin, type I collagen, epidermal growth factor, insulin-like growth factor, granulocyte CSF, monocyte CSF, granulomonocyte CSF, interleukin-3, and ATIII showed no significant affinity for ryudocan, even if the concentrations of these proteins were increased to 5 μg/well (data not shown).
      Figure thumbnail gr2
      Figure 2:Binding of 125I-labeled human ryudocan to potential ligands. Microtiter wells were coated with 1 μg/well fibronectin (FN), type I collagen (Col I), bFGF, epidermal growth factor (EGF), insulin-like growth factor (IGF), MK, granulocyte CSF (G-CSF), monocyte CSF (M-CSF), granulomonocyte CSF (GM-CSF), interleukin-3 (IL-3), ATIII, TFPI, or BSA and incubated with 6.9 × 103 cpm/well 125I-ryudocan. After washing, the amount of bound radioactivity was counted. Data from triplicate samples (mean ± S.D.) are presented, and similar data were obtained in two additional experiments.
      To assure that the applied proteins stuck to the wells, we performed an immunodetection assay as described under “Experimental Procedures.” The significant signals of absorbance at A492 were obtained from all ligands tested, compared with those from the BSA control (data not shown). In addition, we measured the amount of protein remaining in the wells after removal of ligand solution by using a 125I-labeled tracer for each protein. The amounts of the remaining ligands in the wells ranged from 6.0 to 20.7%. These data suggest that a lack of ryudocan binding could not result from a failure of the applied proteins to stick to the wells.
      We also performed a competition assay to verify that unlabeled ryudocan exhibits similar binding properties as described under “Experimental Procedures.” Unlabeled ryudocan inhibited dose-dependently the binding of 125I-ryudocan to bFGF, MK, and TFPI (Fig. 3). Presumably, due to excess of coated ligands, a relatively large amount of unlabeled ryudocan would be needed to block the binding of 125I-ryudocan. Thus, the observed 125I-ryudocan binding might not be a consequence of the iodination reaction.
      Figure thumbnail gr3
      Figure 3:Inhibition of 125I-ryudocan binding by unlabeled ryudocan. Microtiter wells were coated with bFGF, MK, or TFPI (0.5 μg/well) and then incubated with 0.1 ng (1.1 × 103 cpm)/well 125I-ryudocan containing unlabeled ryudocan (0.1 (open columns), 1 (hatched columns), or 10 (closed columns) ng/well) as indicated. After washing, the amount of bound radioactivity was counted. Data from duplicate samples (mean ± S.D.) are presented, and similar data were obtained in an additional experiment. The absolute counts corresponding to 100% binding were 696 cpm for bFGF, 692 cpm for MK, and 216 cpm for TFPI.
      To investigate the involvement of ryudocan GAG chains in ligand binding, we digested the GAG chains of 125I-ryudocan with purified Flavobacterium heparitinase or chondroitin ABC lyase prior to the solid-phase binding assay (Fig. 4). Heparitinase treatment destroyed the ability of 125I-ryudocan binding to bFGF, MK, and TFPI. In contrast, chondroitin ABC lyase treatment did not affect 125I-ryudocan binding to these potential ligands. These data indicate that the HS chains, not the core protein of ryudocan, are responsible for binding bFGF, MK, and TFPI.
      Figure thumbnail gr4
      Figure 4:Glycosaminoglycan chains govern ryudocan binding to bFGF, MK, and TFPI. Microtiter wells were coated with bFGF (0.5 μg/well), MK (0.5 μg/well), or TFPI (0.5 μg/well) and subsequently incubated with 3.4 × 103 cpm/well intact 125I-ryudocan preparation (open columns) or that which was pretreated with purified Flavobacterium heparitinase (closed columns) or chondroitin ABC lyase (hatched columns) as indicated. After washing, the amount of bound radioactivity was counted. Data from duplicate samples (mean ± S.D.) are presented, and similar data were obtained in two additional experiments. The absolute counts corresponding to 100% binding were 1690 cpm for bFGF, 1241 cpm for MK, and 162 cpm for TFPI.
      We also tested the inhibition of ryudocan binding by heparin, HS, and CS (Fig. 5). We was found that heparin distinctly inhibited the binding of 125I-ryudocan to bFGF, MK, and TFPI. HS also moderately inhibited 125I-ryudocan binding to these molecules, but at high concentrations. In contrast, CS had no inhibitory effect on 125I-ryudocan binding to these ligands.
      Figure thumbnail gr5
      Figure 5:Inhibition of ryudocan binding by glycosaminoglycans. Microtiter wells were coated with bFGF (0.25 μg/well), MK (0.25 μg/well), or TFPI (2.5 μg/well) and then incubated with 6.6 × 103 cpm/well 125I-ryudocan in the presence of heparin (Hp), HS, or CS at the indicated concentration. After washing, the amount of bound radioactivity was counted. Data from duplicate samples (mean ± S.D.) are presented, and similar data were obtained in two additional experiments. The absolute counts corresponding to 100% binding were 1615 cpm for bFGF, 1040 cpm for MK, and 1006 cpm for TFPI.
      We examined the concentration-dependent binding of ryudocan to bFGF, MK, and TFPI by a solid-phase assay as described under “Experimental Procedures.” We found that the binding of ryudocan to bFGF, MK, and TFPI was saturable (Fig. 6). Scatchard analyses showed that the apparent Kd values were 0.50 nM for bFGF, 0.30 nM for MK, and 0.74 nM for TFPI (Fig. 6, inset).
      Figure thumbnail gr6
      Figure 6:Concentration-dependent binding of ryudocan to bFGF, MK, and TFPI. Microtiter wells were coated with bFGF (open circles; 0.25 μg/well), MK (closed circles; 0.25 μg/well), or TFPI (open squares; 2.5 μg/well) and then incubated with various concentrations of 125I-ryudocan (specific activity of 4.2 × 105 cpm/μg). After washing, the amount of bound radioactivity was counted. The insets show Scatchard analyses of 125I-ryudocan binding to immobilized bFGF (open circles), MK (closed circles), and TFPI (open squares).

       Immunohistochemical Analysis of Ryudocan Expression

      To investigate the localization of ryudocan expression, we analyzed several tissues by immunohistochemical staining as described under “Experimental Procedures.” We found that cytotrophoblasts of placental villi as well as Schwann cells of peripheral nerve tissues were distinctly stained (Fig. 7, A and B). Interestingly, the fetal pulmonary endothelium and endocardium, but not the adult counterparts, were stained clearly (Fig. 7, C and D). We also found that human ryudocan was significantly expressed in some pathological tissues, including fibrous regions of renal Bowman's capsules and coronary atheromatous plaques (Fig. 7, E and F).
      Figure thumbnail gr7
      Figure 7:Immunostaining of human ryudocan in various human tissues. The tissue sections were immunostained with anti-human ryudocan as described under “Experimental Procedures.” Cytotrophoblasts (A; magnification × 400), peripheral nerve tissues (B; × 200), fetal lung capillaries (C; × 200), fetal endocardium (D; × 200), fibrous regions of Bowman's capsules (E; × 200), and fibrous regions of coronary atheromatous plaques (F; × 200) were distinctly stained. To assure the specificity of the signals, the tissue sections were also immunostained with anti-human ryudocan antibody pretreated (+) or nontreated(-) with an excess amount of Ryu2 peptide as described under “Experimental Procedures.” Cytotrophoblasts (G; × 100) and fetal lung capillaries (H; × 100) were distinctly stained with nontreated antibody(-), whereas the specific signals faded away when anti-human ryudocan IgG was preincubated with an excess amount of Ryu2 peptide (+).
      To demonstrate the specificity of the signals, we used Ryu2 peptide as a competitor with the native ryudocan molecule as described under “Experimental Procedures.” The signals of cytotrophoblasts as well as the fetal pulmonary endothelium faded away when rabbit anti-human ryudocan IgG was incubated with an excess amount of Ryu2 peptide prior to immunostaining (Fig. 7, G and H). These results thus suggest that the stained signals would be specific for the human ryudocan molecule.

      DISCUSSION

      Ryudocan is one of the syndecan family members that are type I integral membrane HSPGs. HS binds a variety of proteins, including peptide growth factors, extracellular matrix components, cell adhesion molecules, lipolytic enzymes, protease inhibitors, and circulating lipoproteins. Syndecan, the best characterized molecule in its family, has been shown to selectively bind a variety of extracellular matrix molecules, suggesting that syndecan may be an extracellular matrix receptor(
      • Koda J.E.
      • Rapraeger A.
      • Bernfield M.
      ,
      • Saunders S.
      • Bernfield M.
      ,
      • Sun X.
      • Mosher D.F.
      • Rapraeger A.
      ,
      • Salmivirta M.
      • Elenius K.
      • Vainio S.
      • Hofer U.
      • Chiquet-Ehrismann R.
      • Thesleff I.
      • Jalkanen M.
      ,
      • Salmivirta M.
      • Rauvala H.
      • Elenius K.
      • Jalkanen M.
      ).
      Recently, it was shown that cell-surface HS appears to be required for the binding of bFGF to its high affinity receptor(
      • Yayon A.
      • Klagsbrun M.
      • Esco J.D.
      • Leder P.
      • Ornitz D.M.
      ). Syndecan from mammary epithelial cells bound bFGF via its HS chains and was considered a good candidate for a cell-surface HSPG low affinity receptor(
      • Bernfield M.
      • Hooper K.C.
      ). However, overexpression of syndecan at the surface of NIH 3T3 cells has been shown to increase fibronectin binding and yet inhibit bFGF-induced cell proliferation(
      • Mali M.
      • Andtfolk H.
      • Miettinen H.M.
      • Jalkanen M.
      ). It was also reported that syndecan as well as glypican and fibroglycan block heparin-dependent bFGF receptor binding due to competitive inhibition(
      • Aviezer D.
      • Levy E.
      • Safran M.
      • Svahn C.
      • Buddecke E.
      • Schmidt A.
      • David G.
      • Vlodavsky I.
      • Yayon A.
      ). In addition, perlecan, a large basal lamina proteoglycan, has been identified as a major candidate for a bFGF low affinity accessory receptor as well as an angiogenic modulator by virtue of its differential HS structure(
      • Aviezer D.
      • Hecht D.
      • Safran M.
      • Eisinger M.
      • David G.
      • Yayon A.
      ). Nevertheless, it is still possible that bFGF high affinity interactions in vivo require cooperative effects between these cellular HSPGs. In this study, we showed that ryudocan has a specific binding affinity for bFGF via its HS chains, with an apparent Kd of 0.50 nM. We also demonstrated that ryudocan is distinctly expressed in several fibrous tissues as well as in fetal lung capillaries and endocardium. These data suggest that ryudocan may participate in fibroblast growth and fetal angiogenesis through the bFGF interaction either as a low affinity receptor or as an inhibitor of pathogenesis or cellular development.
      MK showed the highest affinity (apparent Kd = 0.30 nM) for ryudocan among the three ligands tested. MK promotes both neurite outgrowth and the survival of various embryonic neurons, and the neurite promoting activity is in the COOH-terminal half of the MK molecule, which has heparin binding activity(
      • Muramatsu H.
      • Inui T.
      • Kimura T.
      • Sakakibara S.
      • Song X.
      • Maruta H.
      • Muramatsu T.
      ). MK has ~50% sequence identity to pleiotrophin(
      • Li Y.-S.
      • Milner P.J.
      • Ghauhan A.K.
      • Watson M.A.
      • Hoffman R.M.
      • Kodner C.M.
      • Milbrandt J.
      • Deuel T.F.
      ), also called heparin binding growth-associated molecule(
      • Merenmies J.
      • Rauvala H.
      ). MK and pleiotrophin have similar functions and constitute a new family of heparin-binding proteins involved in regulation of cellular growth and differentiation (
      • Muramatsu T.
      ). The neurite promoting activity was strongly inhibited by heparin and only weakly by HS. (
      N. Kaneda, unpublished data.
      ) This mode of inhibition is quite similar to that of ryudocan binding to MK described in this paper. We also found that ryudocan is expressed abundantly in peripheral nerve bundles. These findings imply that the interaction of HS chains on ryudocan with MK may participate in the formation of a neural network in peripheral nerve tissues. In this context, it is noteworthy that N-syndecan was reported to be the receptor of pleiotrophin in the promotion of neurite outgrowth of embryonic neurons(
      • Raulo E.
      • Chernousov M.A.
      • Carey D.J.
      • Nolo R.
      • Rauvala H.
      ). N-Syndecan is also considered to be a potential co-receptor for bFGF during nerve tissue development(
      • Chernousov M.A.
      • Carey D.J.
      ).
      Several investigators have shown that HSPG is present on the endothelial cell surface and functions as an anticoagulant(
      • Marcum J.A.
      • Atha D.H.
      • Fritze L.M.S.
      • Nawroth P.
      • Stern D.
      • Rosenberg R.D.
      ). Interestingly, ryudocan was originally isolated from rat microvascular endothelial cells as an anticoagulant HSPG(
      • Kojima T.
      • Leone C.W.
      • Marchildon G.A.
      • Marcum J.A.
      • Rosenberg R.D.
      ,
      • Kojima T.
      • Shworak N.S.
      • Rosenberg R.D.
      ). Immunohistochemical analysis revealed that ryudocan is distinctly expressed in placental cytotrophoblasts as well as in fetal lung capillaries. Placental tissues are known to be resources for tissue factor involved in blood coagulation, and placental dysfunction may cause disseminated intravascular coagulopathy. It has been reported that TFPI is detected specifically in macrophages in the villi of term placenta (
      • Werling R.W.
      • Zacharski L.R.
      • Kisiel W.
      • Bajaj S.P.
      • Memoli V.A.
      • Rousseau S.M.
      ) and that heparin enhances the rate of factor Xa inhibition by recombinant TFPI in the presence of Ca2⁺(
      • Wesselschmidt R.
      • Likert K.
      • Huang Z.
      • MacPhail L.
      • Broze Jr., G.J.
      ). In this study, we demonstrated that purified ryudocan from EAhy926 cells possesses significant affinity for TFPI, but not for ATIII. These data suggest that human ryudocan may have an anticoagulant activity through its TFPI interaction. This may be one physiological function of ryudocan in the placental villus cytotrophoblasts.
      The variation in structure and binding affinity of cell-surface HS on syndecan is a differentiated characteristic of each cell type(
      • Kato M.
      • Wang H.
      • Bernfield M.
      • Gallagher J.T.
      • Turnbull J.E.
      ). This enables cells to respond to the specific HS-binding effectors in the cellular microenvironment. The attachment of different numbers of HS and CS chains to syndecan family members may alter interactions with specific proteins and hence modify the biological function of these components. It has been reported that ryudocan possesses three functional GAG attachment sites, that the sites are always occupied with GAG chains, and that each site is capable of bearing either HS or CS(
      • Shworak N.W.
      • Shirakawa M.
      • Mulligan R.C.
      • Rosenberg R.D.
      ). In addition, it was noted that the distribution of ryudocan isoforms and the length of ryudocan HS chains are altered when cells shift from the exponentially growing to the post-confluent state(
      • Shworak N.W.
      • Shirakawa M.
      • Mulligan R.C.
      • Rosenberg R.D.
      ,
      • Colliec-Jouault S.
      • Shworak N.W.
      • Liu J.
      • de Agostini A.L.
      • Rosenberg R.D.
      ). These variations in structure of GAG chains may serve to expand the functional versatility of ryudocan and to allow it to participate in many different biological processes. We demonstrated in this report that purified human ryudocan, obtained from endothelium-like cells (EAhy926 cells), has specific affinities for such ligands as bFGF, MK, and TFPI. Ryudocan is distinctly expressed in peripheral nerve tissues, fibrous tissues, and placental trophoblasts. These results thus suggest that ryudocan may function as a bFGF modulator, as a neurite growth promoter, and as an anticoagulant via the HS-binding effectors present in the cellular microenvironment.

      Acknowledgments

      We thank Drs. Robert D. Rosenberg and Cora-Jean S. Edgell for generously providing the EAhy926 cells, Dr. Hisao Kato for supplying human recombinant TFPI and anti-human TFPI rabbit IgG, Drs. Haruo Hirayama and Makoto Hirai for providing dissection samples of coronary atheromatous plaques, and Drs. Norio Kaneda and Motohiro Hamaguchi for helpful discussions. We also thank Satoshi Suzuki and Chika Wakamatsu for excellent technical help.

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