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• W1484166075 abstract "The lymphocyte adhesion molecule CD44 recognizes a non-hyaluronate proteoglycan, gp600, secreted by mouse T cell line CTLL2. We now demonstrate that gp600 is identical to serglycin, a member of the small proteoglycan family stored in intracellular secretory granules of lymphoid, myeloid, and some tumor cells. Purified gp600 has the ability to bind specifically to CD44, and the binding is dependent on activation of CD44. The CD44-binding elements on gp600 or serglycin are glycosaminoglycans consisting of chondroitin 4-sulfate. Serglycin is readily exocytosed, and its interaction with active form CD44 augments the CD3-dependent degranulation of CD44 positive CTL clones. We conclude that the serglycin secreted from secretory granules of hematopoietic cells is a novel ligand for CD44, and could regulate lymphoid cell adherence and activation. The lymphocyte adhesion molecule CD44 recognizes a non-hyaluronate proteoglycan, gp600, secreted by mouse T cell line CTLL2. We now demonstrate that gp600 is identical to serglycin, a member of the small proteoglycan family stored in intracellular secretory granules of lymphoid, myeloid, and some tumor cells. Purified gp600 has the ability to bind specifically to CD44, and the binding is dependent on activation of CD44. The CD44-binding elements on gp600 or serglycin are glycosaminoglycans consisting of chondroitin 4-sulfate. Serglycin is readily exocytosed, and its interaction with active form CD44 augments the CD3-dependent degranulation of CD44 positive CTL clones. We conclude that the serglycin secreted from secretory granules of hematopoietic cells is a novel ligand for CD44, and could regulate lymphoid cell adherence and activation. INTRODUCTIONCD44, which exhibits significant sequence homology to the phylogenically conserved amino-terminal domain of cartilage link proteins, is an important cell surface adhesion molecule expressed on lymphoid cells, myeloid cells, fibroblasts, epithelial cells, and endothelial cells (Jalkanen et al., 1986; Stamenkovic et al., 1989; Goldstein et al., 1989; for reviews, see Haynes et al.(1989) and Lesley et al. (1993a)). Recent studies reveal that this molecule has many isoforms with various inserts in the membrane proximal portion (for review, see Herrlich et al.(1993)). Ligands for CD44 have been shown to be extracellular matrix components such as hyaluronic acid (Aruffo et al., 1990), fibronectin (Jalkanen and Jalkanen, 1992), and collagen types I and VI (Wayner et al., 1987; Carter and Wayner, 1988). In addition, the chondroitin sulfate-modified invariant chain has recently been suggested to be a ligand for CD44 (Naujokas et al., 1993). The CD44 molecule is thought to participate in various adhesive events including lymphocyte recirculation (Jalkanen et al., 1987), lymphohemopoiesis (Miyake et al., 1990), and tumor cell invasiveness (Gunthert et al., 1991). Monoclonal antibodies directed against the CD44 molecule enhance the proliferation of T cells (Shimizu et al., 1989; Denning et al., 1990). In addition, some anti-CD44 monoclonal antibodies can trigger effector functions of murine and human T cell clones (Seth et al., 1991; Galandrini et al., 1993). Therefore, one of the important functions of CD44 in the immune system seems to be the activation of lymphocytes, although the natural ligand interacting with CD44 remains to be determined.An anti-CD44 monoclonal antibody, Hermes-3, that does not interfere with hyaluronate binding (Culty et al., 1990), inhibits the binding of human lymphocytes to high endothelial venules on frozen lymph node sections, indicating the involvement of CD44 in lymphocyte homing (Jalkanen et al., 1987). It was also reported that the binding of murine lymphocytes to high endothelial venules is resistant to hyaluronidase treatment (Culty et al., 1990), indicating that hyaluronate is not a ligand for CD44 in lymphocyte-high endothelial venule interaction. In the search for a novel ligand for CD44, we identified a sulfated macromolecule, gp600, in the culture supernatant of a murine T cell line, and reported that gp600 is a proteoglycan consisting of a small core protein (18-22 kDa) and chondroitin sulfate-like glycosaminoglycans (Toyama-Sorimachi and Miyasaka, 1994).Proteoglycans found ubiquitously in tissues are composed of a protein core and glycosaminoglycan side chains (for reviews, see Ruoslahti (1989) and Kolset and Gallagher(1990)). In hematopoietic cells, a member of the proteoglycan family termed serglycin is found in the secretory granules (for review, see Stevens et al.(1988)). Serglycins can be classified into 2 groups on the basis of the nature of the glycosaminoglycans, heparan sulfate serglycins and chondroitin sulfate serglycins. Various types of serglycin have been characterized, e.g. a heparin sulfate type in serosal mast cells, a chondroitin 4,6-sulfate type in mouse bone marrow-derived mast cells, a chondroitin 4-sulfate type in natural killer cells, eosinophils, and HL-60 leukemic promyelocytes, and a chondroitin 6-sulfate type in megakaryocytes and platelets (Stevens et al., 1988a; Kolset and Gallagher, 1990). Serglycin is distinct from all other cell surface- and matrix-localized proteoglycans both in its high degree of sulfation and its resistance to proteolysis. Although the molecular masses of these proteoglycans are heterogenous (60-750 kDa) due to differences in their glycosaminoglycan side chains, the gene responsible for the peptide core, which is composed primarily of tandem serine-glycine repeats, is a single gene (Tantravahi et al., 1986). The serglycin peptide core is estimated to be Mr 16,000-18,000 (Bourdon et al., 1985; Stevens et al., 1988b; Avraham et al., 1989), similar to that of gp600 (Toyama-Sorimachi and Miyasaka, 1994). The expression of serglycin seems to be restricted to the yolk sac, hematopoietic cells, and some tumor cells. Serglycin has been suggested to participate in the packaging of basically charged serine proteases in secretory granules and the regulation of their enzymatic activity (Stevens et al., 1988a). It has also been postulated that serglycin plays a role in cell-mediated cytotoxicity, since these proteoglycans are exocytosed when an effector cell kills tumor target cells (MacDermott et al., 1985). However, the functions of serglycin have not been fully defined.In the present study, since the various characteristics of gp600 so far identified remarkably resemble those of chondroitin sulfate serglycin, we isolated and biochemically characterized gp600 in detail to determine whether gp600 is indeed serglycin. We show that the amino acid sequence of the core protein of purified gp600 is identical to that of serglycin and that chondroitin 4-sulfate, a major glycosaminoglycan of gp600, is essential for CD44 binding. Furthermore, we indicate that CD44-serglycin interaction is involved in lymphoid cells adhesiveness and activation. This study provides further understanding not only of the physiological functions of CD44 but also those of serglycin.EXPERIMENTAL PROCEDURESAntibodies and CellsKM201 is directed against mouse CD44 and inhibits CD44 binding to hyaluronate (Miyake et al., 1990). Anti-CD44 monoclonal antibody IRAWB14 (Lesley et al. 1992), which induces hyaluronate binding to CD44, was kindly provided by Dr. J. Lesley (Department of Cancer Biology, The Salk Institute). Human IgG was purchased from Cappel. F(ab′)2 fraction of biotin-conjugated goat anti-human IgG was purchased from Zymed Laboratories Inc.CTLL2, CTLL2 transfectants (Toyama-Sorimachi and Miyasaka, 1994) of mouse CD44, and mouse thymoma cell line BW5147 were grown in RPMI 1640 supplemented with 10% fetal calf serum (Iansa), 10 mM Hepes, 2 mML-glutamine, 1 mM sodium pyruvate, 10−4M 2-mercaptoethanol, 1% (v/v) 100 × nonessential amino acids (Flow Laboratories), 100 units/ml penicillin, and 100 μg/ml streptomycin (complete medium). For the culture of CTLL2 and its transfectants, 1 nM recombinant mouse interleukin-2 was added to the complete medium (Karasuyama et al., 1989). In the case of large scale culture of CTLL2, serum-free medium EX-cell 300™ (JRH Bioscience) was used. The culture of bone marrow-derived mast cells was performed as described previously (Razin et al., 1984).Purification of Gp600Gp600 was monitored by the presence of uronic acid, and also by its ability to bind to a soluble fusion protein of CD44 and IgG (CD44-IgG) (Aruffo et al., 1990). Conditioned medium from CTLL2 cells was centrifuged for 20 min at 5000 × g, and supernatant was recovered. The sample was concentrated in a Millipore concentrator (Minitan™ system, Mr 30,000 cut-off). The initial volume of 16 liters was concentrated to 450 ml, diluted with a buffer containing 4 M urea, 20 mM Tris (pH 8.0), and 0.2 M NaCl (TSKG-DEAE equilibration buffer), and concentrated again to 450 ml. The concentrated sample was loaded onto a TSKG-DEAE (Toso) at a flow rate of 1 ml/min, and eluted with a 60-ml linear NaCl gradient (0.2-1 M) in the same buffer. Gp600 was observed at approximately 0.5 M NaCl. Fractions containing gp600 were pooled, concentrated using a Centriprep-30 concentrator (Amicon), and dialyzed against distilled H2O. The dialyzed sample was adjusted to 6 M guanidine, 20 mM Tris (pH 8.0), and 0.1% CHAPS, 1The abbreviations used are: CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidELISAenzyme-linked immunosorbent assayBSAbovine serum albuminPBSphosphate-buffered salineHPLChigh performance liquid chromatographyCTLcytotoxic T cellBCECF2′,7′-bis(2-carboxyethyl)-5(and −6)-carboxyfluorescein. and loaded at a flow rate of 0.5 ml/min onto a TSKG-3000 column (Toso) previously equilibrated with 6 M guanidine, 20 mM Tris (pH 8.0), and 0.1% CHAPS. In this step, gp600 was recovered in the void fraction. The fractions containing gp600 were pooled, concentrated, and dialyzed against distilled H2O. The dialyzed sample was treated with DNase I to remove contaminating nucleic acid. The resulting sample was diluted with the same volume of buffer containing 6 M guanidine, 20 mM Tris (pH 8.0), and 0.1% CHAPS, and applied onto TSKG-3000 at a flow rate of 0.5 ml/min. Gp600 was recovered in the void fraction. Fractions containing gp600 were concentrated, dialyzed against 10 mM sodium phosphate buffer, and subjected to hydroxylapatite chromatography (HA-1000). By this hydroxylapatite column chromatography, the gp600 activities were clearly resolved into two fractions, the flow-through fraction and a fraction eluting at approximately 150 mM phosphate buffer. Since the first fraction was purer than the second, we focused on the flow-through fraction. The fraction was dialyzed against distilled H2O, adjusted to 4 M urea, 20 mM Tris (pH 8.0), and 0.2 M NaCl, and loaded onto a TSKG-DEAE column. Gp600 was eluted with a linear gradient of NaCl (0.2-1 M) in the same buffer. The final preparation was dialyzed against distilled H2O and lyophilized. The purity of the preparation was assessed by electrophoresis after radiolabeling the sample with Na125I. Uronic acid was determined according to the method of Bitter and Muir(1962).Preparation of CD44-IgG Fusion ProteinThe CD44-IgG expression plasmid (Aruffo et al., 1990) was kindly provided by Dr. B. Seed (Department of Molecular Biology, Massachusetts General Hospital). CD44-IgG fusion protein was generated as described previously (Aruffo et al., 1990). Purification was performed using Protein G-Sepharose column chromatography.ELISAPurified gp600 and defined glycosaminoglycans were coated onto 96-well microtiter plates overnight at 4°C. Nonspecific sites were blocked with 1% BSA in PBS at room temperature for 2 h. CD44-IgG fusion protein or control human IgG (20 μg/ml final concentration) was added and the plates were incubated for 1 h at 4°C. The plates were washed and incubated with biotinylated goat anti-human IgG for 30 min at room temperature, followed by incubation with avidin-peroxidase for 30 min. The plates were developed after the addition of the o-phenylendiamine substrate and read with a microplate reader. A background value defied as the signal in the absence of CD44-IgG was subtracted from the experimental values to yield the specific signal.Cell Adhesion AssayThe cell adhesion assay was performed using BCECF-labeled cells as described previously (Toyama-Sorimachi et al., 1993). Gp600 (100 μg/ml) was coated onto 96-well microtiter plates (Sumitomo H-plate). After incubation overnight at 4°C, the wells were filled with PBS containing 1% BSA and incubated for 2 h at room temperature to block nonspecific protein absorption. Cell lines were washed and resuspended in serum-free RPMI containing 5 μM BCECF-AM. After incubation for 45 min at 37°C, the cells were washed with RPMI containing 10% fetal calf serum, and then resuspended in Ca2+ and Mg2+-free PBS. BCECF-labeled cells (2 × 105 cells/well) were then added and the plates were incubated in the presence or absence of anti-CD44 antibody or control antibodies. Each experiment was run in duplicate or triplicate. Nonadherent cells were removed by inverting the plate. Adherent lymphocytes were solubilized with 0.1% Nonidet P-40 in PBS, and the fluorescence intensity of each well was measured with a Fluoroscan II (Flow Laboratories). The background level, defined as the binding to BSA, was subtracted from all values to yield the specific signal.Enzyme TreatmentEnzymes used for proteoglycan digestion were as follows; chondroitinase ABC (from Proteus vulgaris), chondroitinase ACII (from Arthrobacter aurescens), hyaluronidase (from Streptomyces hyalurolytics), heparinase (from Flavobacterium heparinum), heparitinase (from Flavobacterium heparinum), and α-L-fucosidase (from Charonia lampus). All these enzymes were purchased from Seikagaku Kogyo Co. Treatment of immobilized gp600 with chondroitinase ABC or hyaluronidase was carried out as described previously (ToyamaSorimachi and Miyasaka, 1994). Chondroitinase ABC and ACII were used at 20 μg/ml and 100 milliunits/ml, respectively, and hyaluronidase was used at 20 μg/ml (40 turbidity reducing units/ml). Heparinase, heparitinase, and fucosidase were used at 10 milliunits/ml.Amino Acid Sequence AnalysisAmino acid sequence analysis of the purified gp600 core protein was performed with an Applied Biosystems model 475A gas-phase sequencer. The analysis was kindly performed by Dr. S. Tsubuki, Department of Molecular Biology, the Tokyo Metropolitan Institute of Medical Science.Flow Cytometry AnalysisFlow cytometry analysis was performed as described previously (Toyama-Sorimachi and Miyasaka, 1994). In a competition assay (Table 1), cells were preincubated with various concentrations of proteoglycans, and then incubated with 5 μg/ml fluorescein isothiocyanate-conjugated hyaluronic acid (Seikagaku Kogyo). Chondroitin sulfates E and K were kindly provided by Dr. N. Seno, Department of Molecular Biology, the Tokyo Metropolitan Institute of Medical Science. At least 10,000 cells/sample were analyzed on an EPICS-CS flow cytofluorometer (Coulter).Tabled 1 Open table in a new tab Disaccharide AnalysisAfter treatment of the prepared gp600 with chondroitinase ABC, unsaturated disaccharide analysis was carried out by high-performance liquid chromatography (HPLC) as reported previously (Sugawara et al., 1989, 1992). HPLC analysis was performed with a Waters model 600E (Millipore Corp.) on an amino silica gel column (NH2-125-N, Senshu Scientific, Tokyo; 4.6-mm inner diameter × 150 mm), with a programmed gradient elution from 16 to 500 mM NaH2PO4.Immunofluorescence Staining of Secretory GranulesCells were mounted onto slide glass by cytospin and air dried. After fixation with 3% formalin, cells were incubated with 10 μg/ml purified CD44-IgG at room temperature for 60 min. After washing with PBS containing 0.1% of BSA, the samples were incubated with affinity purified biotin-conjugated anti-human IgG at 1:200 dilution. After washing, the samples were incubated with fluorescein isothiocyanate-labeled avidin at 1:1000 dilution. Samples were examined by fluorescence microscopy.Granzyme Release AssayMouse CTL clones 557 and 560 were kindly provided by Dr. S. Aizawa (Department of Physiology and Pathology, National Institute of Radiological Science). Release of granzyme A from CTL cytoplasmic granules was induced by plastic immobilized anti-CD3. ELISA plates were first coated overnight at 37°C with 50 μl of various concentrations of anti-CD3 (2C11) in PBS. Wells were washed with PBS, and triplicate aliquots of 2 × 105 cytolytic clonal T cells were dispensed into the wells in 100 μl of RPMI 1640 containing 10 μg/ml IRAWB14 and 1% BSA in the presence or absence of 50 μg/ml purified gp600. After centrifugation at 1000 × g for 1 min, the plates were incubated for 3 h at 37°C. Control cells were incubated in the absence of anti-CD3. Granzyme A activity was tested by adding 180 μl of the substrate (0.2 M Tris-HCl, pH 8.0, 2 × 10−4M Nα-CBZ-L-lysine thiobenzyl ester (BLT), 2.2 × 10−4M dithiobis(nitrobenzoic acid)) to 20 μl of cell supernatant or to cell lysates solubilized with 0.1% Nonidet P-40 (Pasternack and Eisen, 1985). The absorbance at 405 nm was determined using an ELISA reader after a 1-h incubation at room temperature. The amount of granzyme secreted over the spontaneous release was plotted as the percent of the total enzyme content of the effector cells.RESULTSPurification of Gp600As a ligand for CD44, we recently identified a chondroitinase-sensitive proteoglycan, gp600, found in the culture supernatant of the mouse CTL line, CTLL2 (Toyama-Sorimachi and Miyasaka, 1994). To further characterize gp600, we purified it from the conditioned medium of CTLL2. In the final purification step, gp600 eluted as a single peak from a TSKG-DEAE column (Fig. 1). Nucleic acid represented less than 1% of the material in this peak. Overall, 1.21 mg of the proteoglycan was obtained from 16 liters of CTLL2 conditioned medium with 3.4% recovery. The activity to bind to CD44 was concentrated by 9,500-fold (Table 1).Figure 1DEAE-ion exchange chromatography profile of gp600 obtained by hydroxylapatite chromatography. The gp600 fraction from hydroxylapatite chromatography was applied to a TSKG-DEAE column. After washing, the column was eluted at 20°C with a linear gradient of NaCl as indicated. Fractions were analyzed for uronic acid; A530 (•), A280 (○), and A260 (□).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Specific Binding of CD44 to Gp600We first investigated the binding ability of purified gp600 to CD44 by ELISA. This was performed by examining the binding of soluble CD44-IgG to gp600 immobilized on a plastic plate. CD44-IgG bound to gp600 in a dose-dependent manner, while no significant binding was observed with control human-IgG (Fig. 2).Figure 2Binding of soluble CD44 to purified gp600. Increasing amounts of purified gp600 were immobilized on an ELISA plate, and the binding of CD44-IgG (•) and control human IgG (□) to gp600 was assessed as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then examined CD44 binding to gp600 by a cell binding assay. CD44 positive or negative cell lines were labeled with fluorescent dye, and the adherence of cells to immobilized gp600 was assessed by measuring fluorescence intensity. CD44 positive BW5147 cells adhered to both gp600 and hyaluronate in the absence of Ca2+ and Mg2+ cations (Fig. 3). In contrast, CD44 negative CTLL2 cells failed to adhere to either gp600 or hyaluronate, but transfection of CTLL2 cells with CD44 cDNA (Toyama-Sorimachi and Miyasaka, 1994) resulted in a marked increase in adhesion. The binding of CD44 positive cells to both gp600 and hyaluronate was completely inhibited by an anti-CD44 monoclonal antibody KM201 (Fig. 3) but not by control rat IgG or isotype-matched monoclonal antibodies (data not shown), indicating that cell adhesion to gp600 is mediated by CD44. Hyaluronidase had no effect on the binding of CD44 positive cell lines to gp600, although the binding to hyaluronate was completely eliminated by enzyme treatment (Fig. 3, right column), clearly indicating that hyaluronate is not involved in the interaction between CD44 and gp600.Figure 3CD44-dependent cell adhesion to immobilized gp600. CD44 positive (BW5147 and CTLL2MCD44-1) and negative (CTLL2) cell lines were labeled with fluorescent dye, and their binding to immobilized gp600 (left column) or hyaluronate (right column) was examined. Lane 1, control binding to immobilized materials in the absence of anti-CD44; lane 2, lane 1 plus 50 μg/ml anti-CD44 (KM201); lane 3, hyaluronidase treatment of immobilized materials.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Gp600 Is Identical to SerglycinTo further characterize gp600, we prepared the core protein by treating gp600 with chondroitinase ABC (Fig. 4A; and Toyama-Sorimachi and Miyasaka(1994)), and determined its NH2-terminal amino acid sequence. Ten amino acid residues were identified, identical to those found in the published sequence of mouse serglycin (Fig. 4B) (Avraham et al., 1989). Furthermore, a remarkable sequence similarity to rat (Bourdon et al., 1985) and human (Stevens et al., 1988b) serglycin was observed. No other significant sequence homology was found in the homology search (EMBL protein data base, release 27.0). The determined sequence contained a portion of the tandem serine-glycine repeats highly conserved among human, mouse, and rat serglycin. The fact that the assumed serine residues were not detected by amino acid sequencing may be due to extensive glycosylation of the serine residues and/or a relatively lower recovery of serine derivatives. A minor sequence, YDDYG, found in mouse serglycin, was also identified. Corroborating these findings, the molecular size of the core protein of gp600 is 18-22 kDa, quite similar to that of serglycin (Toyama-Sorimachi and Miyasaka, 1994). Polymerase chain reaction analysis confirmed transcription of the serglycin gene in CTLL2 cells as well as its CD44 transfectants (data not shown). These results indicate that the core protein of gp600 is identical to that of serglycin.Figure 4Identification of gp600 core protein as serglycin. A, preparation of the gp600 core protein by degradation of the glycosaminoglycans with chondroitinase ABC. The core protein of gp600 was radiolabeled with Na125I by the lactoperoxidase method. A small amount of radiolabeled gp600 (104 cpm) was mixed with 1 mg of purified, unlabeled gp600 for detection of the core protein. After overnight treatment with chondroitinase ABC, the core protein was precipitated by adding acetone/ethanol. Gp600 (•) or gp600 treated with chondroitinase ABC (○) was applied to a gel filtration column. Fractions (No. 39-46) from gel filtration chromatography were pooled for NH2-terminal amino acid sequence analysis. B, comparison of the amino acid sequences (single letter code) of the gp600 core protein with serglycin peptide cores expressed in mouse bone marrow-derived mast cells (BMMC), rat basophilic leukemia-1 (RBL) cells, and human HL-60 cells. Numbers indicate the residue numbers in the respective sequences.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Serglycin is known to localize in the secretory granules of granular leukocytes. Therefore, we examined immunohistologically whether soluble CD44 binds to intracellular secretory granules in CTLL2. It was revealed that CD44-IgG stained the cytoplasmic granules of CTLL2 distinctly while no significant fluorescence was observed with control human IgG (Fig. 5). A similar observation was obtained with interleukin-3-dependent mouse bone marrow-derived mast cells (Fig. 5), which produce chondroitin sulfate-type serglycin in their secretory granules (Razin et al., 1982; Stevens et al., 1985). These results support the notion that CD44 binds to serglycin.Figure 10Immunofluorescence staining of intracellular granules of CTLL2 and mouse bone marrowderived mast cells with CD44-IgG. Cytospin samples of cells fixed with 3% formalin were incubated with 10 μg/ml CD44-IgG or control human IgG.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Chondroitin Sulfate Glycosaminoglycans on Gp600 Are Necessary for CD44 BindingPrevious studies suggest that CD44 recognizes chondroitin sulfate (Aruffo et al., 1990; Sy et al., 1991; Naujokas et al., 1993). To determine whether the chondroitinase-sensitive sugar chain on gp600 is necessary for CD44 binding, binding of CD44-IgG to gp600 was assayed by ELISA before and after the treatment of gp600 with various mucopolysaccharide degrading enzymes. The CD44-IgG reactivity was lost following digestion of gp600 with chondroitinase ABC or ACII but not with heparinase, heparitinase, fucosidase, or hyaluronidase (Fig. 6A). The effect of chondroitinase ABC was abolished in the presence of dermatan sulfate and Zn2+ which together inhibit specifically the enzymatic activity of chondroitinase ABC. These results indicate that the chondroitin sulfate sugar chains on gp600, which can be digested by chondroitinase ABC or ACII, are essential for recognition by CD44.Figure 6Quantitation of CD44-IgG binding to gp600 by ELISA after treatment with mucopolysaccharide degrading enzymes. A, binding of CD44-IgG to immobilized gp600. Purified gp600 (100 μg/ml) was coated on plastic plates before (lane 1) or after treatment with chondroitinase ABC (lane 2), chondroitinase ACII (lane 3), chondroitinase ABC in the presence of 100 mM Zn2+ and 1 mg/ml dermatan sulfate (lane 4), hyaluronidase (lane 5), heparinase (lane 6), heparitinase (lane 7), or fucosidase (lane 8). In lane 9, BSA was used instead of gp600. CD44-IgG binding was examined as described under “Experimental Procedures.” B, binding of CD44-IgG to various glycosaminoglycans. ELISA plates were coated with 1 mg/ml purified gp600 (lane 1), chondroitin (lane 2), hyaluronate (lane 3), chondroitin 4-sulfate (lane 4), chondroitin 6-sulfate (lane 5), dermatan sulfate (lane 6), heparan sulfate (lane 7), keratan sulfate (lane 8), keratan polysulfate (lane 9), heparin (lane 10), or BSA (lane 11) at 4°C overnight.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate the kind of chondroitin sulfate involved in CD44 recognition, disaccharide analysis was performed. Gp600 was completely digested with chondroitinase ABC and the disaccharides obtained were subjected to HPLC analysis. The digest eluted at the position of 4-sulfated disaccharides, and neither non-sulfated nor disulfated disaccharides were detected (Fig. 7). The mass spectrum of the digest also supported this observation (data not shown). These results indicate that gp600 is a chondroitin 4-sulfate type serglycin, and that chondroitin 4-sulfate chains on gp600 are essential for CD44 binding.Figure 7Disaccharide analysis of gp600 glycosaminoglycans by HPLC. The oligosaccharide fraction prepared by chondroitinase ABC treatment of gp600 was chromatographed on an amino silica gel column. The elution positions of authentic unsaturated chondro-disaccharides are indicated. A, oligosaccharide obtained from gp600; B, A plus authentic ΔDi-4S. Disaccharide standard used are: ΔDi-0S, Δ4,5-GlcA(β1-3)GalNAc; ΔDi-6S, Δ4,5-GlcA(β1-3)GalNAc(6-O-sulfate); ΔDi-4S, Δ4,5-GlcA(β1-3)GalNAc(4-O-sulfate); ΔDi-diSD, Δ4,5GlcA(2-O-sulfate)(β1-3)GalNAc(6-O-sulfate); ΔDi-diSE, Δ4,5-GlcA(β1-3)GalNAc(4,6-O-sulfate); ΔDi-triS, Δ4,4-GlcA(2-O-sulfate)(β1-3) GalNAc(4,6-O-disulfate).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We next tested the reactivity of CD44-IgG with various chondroitin sulfates by ELISA. Although the binding of CD44-IgG to gp600/serglycin and hyaluronic acid was readily detected, no significant binding to either chondroitin 4-sulfate or chondroitin 6-sulfate was observed (Fig. 6B). Similarly, these chondroitin sulfate preparations did not interfere with the binding of fluoresceinated hyaluronate to CD44-positive BW5147 cells, although gp600 strongly inhibited hyaluronate binding to CD44-positive cells as assessed by flow cytometry. Dose-dependent blockage of hyaluronate binding was observed with gp600, and almost complete blockage was obtained at 500 μg/ml (Table 2). These results suggest that the gp600 binding domain on CD44 overlaps with or is close to the hyaluronate binding portion. Chondroitin sulfates A and E were slightly effective at 500 μg/ml, but other chondroitin sulfates were inactive. The finding that none of the defined chondroitin 4-sulfates tested so far was recognized by CD44 suggests that the association of chondroitin sulfates with the core protein is important for CD44 binding.T" @default.
• W1484166075 created "2016-06-24" @default.
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• W1484166075 date "1995-03-01" @default.
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• W1484166075 title "A Novel Ligand for CD44 Is Serglycin, a Hematopoietic Cell Lineage-specific Proteoglycan" @default.
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