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Originally published In Press as doi:10.1074/jbc.M003387200 on August 18, 2000

J. Biol. Chem., Vol. 275, Issue 45, 35448-35456, November 10, 2000
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Binding of a Large Chondroitin Sulfate/Dermatan Sulfate Proteoglycan, Versican, to L-selectin, P-selectin, and CD44*

Hiroto KawashimaDagger , Mayumi HiroseDagger , Jun HiroseDagger , Daisuke NagakuboDagger , Anna H. K. Plaas§, and Masayuki MiyasakaDagger

From the Dagger  Department of Bioregulation, Biomedical Research Center, Osaka University Graduate School of Medicine 2-2, Yamada-oka, Suita 565-0871, Japan and the § Cell Biology Laboratory, Shriners' Hospital for Children, Florida 33612

Received for publication, April 20, 2000, and in revised form, August 15, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44. The binding of versican to L- and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A cross-blocking study indicated that L- and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L- and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteoglycans are a heterogeneous family of macromolecules found in all tissues. They provide structural integrity to tissues and mediate cell proliferation, differentiation, and migration. Proteoglycans consist of a core protein to which one or more glycosaminoglycan (GAG)1 side chains are covalently attached. There are several types of GAGs, which include heparin or heparan sulfate (HS), chondroitin sulfate (CS), dermatan sulfate (CS B), and keratan sulfate (KS). Several of the biological activities of proteoglycans are attributable to the GAGs. For example, heparin binds anti-thrombin III (1), and heparin and HS bind fibroblast growth factor (2) and various chemokines (3-5). Furthermore, CS binds a subset of chemokines, such as RANTES (regulated on activation normal T cell expressed and secreted) (6) and PF-4 (platelet factor-4) (7).

Versican, also known as PG-M, is a large CS proteoglycan that is expressed in cultured fibroblasts (8), proliferating keratinocytes (9), and arterial smooth muscle cells (10), and is found in the kidney, skin, brain, and other tissues (11). Versican has a hyaluronic acid (HA)-binding domain at its amino terminus and a set of epidermal growth factor (EGF)-like, lectin-like, and complement regulatory protein-like domains at its carboxyl terminus (8). It also has a CS-bearing domain in its middle portion that contains two alternatively spliced domains called GAG-alpha and GAG-beta (12). As a result of alternative splicing, versican has four distinct isoforms that contain different numbers of CS chains (13). The amino-terminal domain of versican binds HA with high affinity (14). On the other hand, the carboxyl-terminal lectin-like domain binds simple sugars such as fucose and GlcNAc (15) and sulfated glycolipids (16). The lectin-like domain also binds extracellular matrix proteins such as tenascin-R (17) and fibulin-1 (18), apparently through protein-protein interactions. The EGF-like domain of versican enhances cell proliferation, at least in part through binding to the EGF receptor (19). Until now, little has been known about the counter receptors that interact with the CS chain of versican in vivo.

We have previously shown that versican, derived from a renal adenocarcinoma cell line, ACHN, interacts with a leukocyte adhesion molecule, L-selectin, through versican's CS chains (20). L-selectin is a member of the selectin family, which is characterized by an amino-terminal lectin-like domain followed by an EGF-like domain and complement regulatory protein-like domains. Studies of the carbohydrate-based ligands for L-selectin initially identified two sialomucins, GlyCAM-1 (21) and CD34 (22), which are expressed on the high endothelial venules of lymph nodes. Subsequent studies revealed that these molecules interact with L-selectin via a sialyl Lewis X (sLeX)-like carbohydrate structure, 6-sulfo-sLeX (23). Other sialomucins, such as podocalyxin-like protein (24), sulfated glycoprotein (Sgp) 200 (25), and PSGL-1 (26) also bind to L-selectin in a carbohydrate-dependent manner. PSGL-1 also binds to the endothelial P- and E-selectin (26, 27).

CD44, a distinct class of carbohydrate-binding molecules, is expressed on a variety of cell types, including leukocytes, fibroblasts, endothelial cells, and epithelial cells. Its best characterized ligand is probably HA (28), whereas other types of ligands have also been described. Previous work from our laboratory shows that a CS proteoglycan, serglycin, derived from a T cell line, binds CD44 to stimulate granzyme release from a CD44-positive cytotoxic cell line (29). The CS form of the invariant chain has also been reported to bind CD44 (30). These carbohydrate-based ligands interact with the link module present on the amino terminus of CD44 (31). The link module appears to have a close structural similarity to the C-type lectin domain (32), suggesting an evolutionary relationship and a possible functional link between CD44 and the selectins.

The present study was performed to determine whether versican binds to other selectin family members, besides L-selectin, and to CD44. Our data show that versican that is derived from ACHN renal adenocarcinoma cells binds L-selectin, P-selectin, and CD44 through its CS chains and that, among the various GAG chains, CS B, CS E, and HS bind L- and P-selectin, whereas HA, CS A, CS B, CS C, CS D, CS E, and chondroitin (CH) bind CD44.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents

Human L-selectin-Ig and human E-selectin-Ig were provided by Dr. S. R. Watson (Genentech, Inc., South San Francisco, CA). Human P-selectin-Ig was provided by Dr. M. Omata (Suntory Co., Osaka, Japan). Each selectin-Ig chimera consists of the amino-terminal lectin-like domain, the EGF-like domain, the first two complement regulatory protein-like domains, and the Fc domain of human IgG1. Human CD44-Ig was produced as described previously (29). Neuraminidase (Arthrobacter ureafaciens) was purchased from Roche Molecular Biochemicals. CH, CS A (whale cartilage), CS B (pig skin), CS C (shark cartilage), CS D (shark cartilage), CS E (squid cartilage), KS (bovine cornea), HS (bovine kidney), biotinylated sLeX polymeric-probe (sLeX BP-probe), chondroitinase ABC (Proteus vulgaris), chondroitinase AC II (Arthrobacter aurescens), chondroitinase B (Flavobacterium heparitinum), hyaluronidase SD (Streptococcus dysgalactiae), hyaluronidase (Streptomyces hyalurolyticus), anti-sLeX mAb KM-93, anti-versican mAb 2B1, anti-GAG mAbs (CS-56, 2-B-6, 3-B-3, 1-B-6), and biotinylated HABP (hyaluronic acid binding protein) were purchased from Seikagaku Kogyo Co. (Tokyo, Japan). SLeX-BSA was purchased from Oxford GlycoSciences (Bedford, MA). SLeX-sphingolipid was purchased from Nisshin Oil Mills Ltd. (Tokyo, Japan). Anti-sLeX mAb CSLEX-1 was purchased from Becton Dickinson (San Jose, CA). Anti-sLeX mAb 2H5 was provided by Dr. R. Kannagi (Aichi Cancer Center, Nagoya, Japan). HA (human umbilical cord) and human IgG1 were purchased from Sigma (St. Louis, MO). Anti-human L-selectin mAbs were obtained as follows: MHL-1, MHL-2, and MHL-3 from Seikagaku Kogyo Co., TQ1 from Coulter-Immunotech (Miami, FL), DREG-56 from PharMingen (San Diego, CA), 4G8 from R&D Systems (Minneapolis, MN). Anti-human P-selectin mAbs were obtained as follows: G1 from Bender MedSystems (Vienna, Austria), CLB-Thromb/6 and 1.2B6 from Coulter-Immunotech, AK4 from PharMingen, and ARP 2-4 from Dr. S. Tojo of Sumitomo Pharmaceutical Co. (Osaka, Japan). Anti-human CD44 mAbs were obtained as follows: F10-44-2 from Southern Biotechnology Associates Inc. (Birmingham, AL), BU-75 from Ancell Immunology Research Products (Bayport, MN), BRIC235 from the International Blood Reference Laboratory (Bristol, UK), and RAMBM 5.5.8 from Dr. J. Lesley (Department of Cancer Biology, The Salk Institute, San Diego, CA). Anti-mouse CD44 mAb KM201 was provided by Dr. K. Miyake (Saga University Medical School, Saga, Japan).

Purification of Versican

Versican was purified from the conditioned medium of a human renal adenocarcinoma cell line, ACHN, as described previously (20).

Biotinylation

Purified versican (50 µg/ml) or HA (1 mg/ml) was dialyzed against 0.1 M NaHCO3, pH 8.0, containing 0.1 M NaCl, and coupled overnight with 100 µg/ml NHS-LC-biotin (Pierce Chemical Co., Rockford, IL) at room temperature. The reaction was quenched by the addition of a quarter volume of 1 M Tris-HCl, pH 7.6. The biotinylated material was dialyzed against phosphate-buffered saline (PBS) and stored at -80 °C until use.

Western Blotting Analysis

Biotinylated versican was subjected to SDS-PAGE or SDS-agarose-PAGE (20) and transferred onto an IPVH filter (Millipore Co., Bedford, MA). After blocking with PBS containing 3% BSA and 0.1% NaN3, the blot was probed with either (i) ABC reagent (Vector Laboratories, Inc., Burlingame, CA), or (ii) anti-versican mAb 2B1 (0.5 µg/ml) and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (American Qualex Co., 1:2000), as described previously (20). After washing with PBS containing 0.1% BSA and 0.05% Tween 20, the blot was developed with ECL Western blotting detection reagents (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) according to the instructions provided by the manufacturer.

Preparation of Lipid-derivatized GAGs

Lipid-derivatized GAGs were prepared according to the method of Stoll et al. (33) with some modifications. Briefly, 1 mg of each GAG was dissolved in 100 µl of distilled water. To this, 1.9 ml of dipalmitoyl phosphatidylethanolamine (5 mg/ml) in chloroform/methanol (1:1, v/v) was added, and the reaction mixture was sonicated for 10 min, then incubated at 60 °C for 2 h. After the incubation, 200 µl of NaBH3CN (10 mg/ml) in methanol was added, and the reaction mixture was incubated at 60 °C for 16 h. The reaction mixture was then lyophilized and dissolved in 2 M NaCl in 15% ethanol, and the insoluble material was removed by centrifugation. The sample was precipitated with five equivalent volumes of ethanol, and the precipitate was dissolved in 0.2 M NaCl and applied to a TSKgel Phenyl Toyopearl 650M column (TOSOH, Tokyo, Japan) equilibrated with 0.2 M NaCl. The lipid-derivatized GAG was eluted with 30% methanol. The eluate was lyophilized and redissolved in distilled water before use.

Enzyme Treatment

Chondroitinase ABC or chondroitinase B treatment was performed at 37 °C for 2 h in 50 mM Tris-HCl, 15 mM sodium acetate, pH 8.0. Neuraminidase, hyaluronidase SD (S. dysgalactiae), hyaluronidase (S. hyalurolyticus), or chondroitinase ACII treatment was performed at 37 °C, for 2 h in 50 mM sodium acetate (pH 6.0).

Enzyme-linked Immunosorbent Assay

Method 1-- Recombinant Ig-chimeras, the control human IgG1, or the mAbs against sLeX or GAGs in PBS (25 µl/well) were added to 96-well flat-bottomed microtiter plates (Costar EIA/RIA plate 3690, Corning Inc., Corning, NY) and kept overnight at 4 °C. The wells were washed with buffer A (0.05% Tween 20, 20 mM HEPES-NaOH, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 6.8) and blocked with Block Ace (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) for 2 h. Biotinylated versican, biotinylated HA, or sLeX BP-probe in buffer A was added to the wells in the presence or absence of mAbs, GAGs, or the soluble Ig-chimeras and incubated for 2 h. After washing the wells with buffer A, alkaline phosphatase-conjugated streptavidin (Promega, Madison, WI), diluted 1:500, was added and incubated for 1 h. To quantify the reaction, Blue Phos substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and the optical density was read at 650 nm in a microtiter plate reader (InterMed Co., Tokyo).

Method 2-- Lipid-derivatized GAGs or sLeX-sphingolipid (100 µg/ml, 25 µl/well) were immobilized on 96-well flat-bottomed microtiter plates by drying at 60 °C for 5 h. After blocking with Block Ace, human IgG1 (5 µ g/ml), L-selectin-Ig (5 µg/ml), P-selectin-Ig (3 µg/ml), or CD44-Ig (1 µg/ml) was added to the wells and incubated for 2 h. The binding was detected with alkaline phosphatase-labeled goat anti-human IgG (American Qualex, San Clemente, CA) diluted 1:1000 and Blue Phos substrate as described above.

Method 3-- Versican (2 or 4 µg/ml, 25 µl/well) or HA (4 µg/ml, 25 µl/well) was added to 96-well flat-bottomed microtiter plates and kept overnight at 4 °C. After blocking with Block Ace, chondroitinase ABC, chondroitinase ACII, chondroitinase B, or hyaluronidase (S. hyalurolyticus) was added to the wells and incubated at 37 °C for 2 h. After washing the wells with PBS containing 0.05% Tween 20 and 0.1% BSA, mAb CS-56, 2-B-6, 3-B-3, 1-B-5, or biotinylated HABP (5 µg/ml) was added to the wells and incubated for 1 h. The binding was detected with HRP-labeled goat anti-mouse IgG+M (American Qualex) diluted 1:500 and o-phenylenediamine (0.4 mg/ml) as described previously (20). When biotinylated HABP was used as a primary reagent, the binding was detected with HRP-labeled streptavidin (Zymed Laboratories Inc. Laboratories, San Francisco, CA).

FACE Analysis

FACE (fluorophore-assisted carbohydrate electrophoresis) was performed as described previously (34). In brief, 730 ng of purified versican was digested in 50 µl of 0.1 M ammonium acetate, pH 7.3, containing 0.5 unit/ml each of chondroitinase ABC and ACII, for 18 h at 37 °C. The digestion products were separated from enzymes and the deglycosylated core protein by ethanol precipitation, fluorotagged with 2-aminoacridone (Molecular Probes Inc., Eugene, OR), and then analyzed by FACE. The molar ratio of Delta Di-0S:Delta Di-6S:Delta Di-4S was assessed by the Eagle Eye documentation system (Stratagene, La Jolla, CA) with illumination at 300 nm.

Cell Binding Assay

Mouse T lymphomas BW5147 and EL-4 obtained from ATCC (American Type Culture Collection; Rockville, MD) or EL-4 transfected with human L-selectin cDNA (20) was labeled with 5 µM 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Molecular Probes Inc., Eugene, OR) for 30 min at 37 °C. After washing, the cells were suspended in buffer B (20 mM HEPES-NaOH, 0.14 M NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 6.8) with or without 20 µg/ml anti-mouse CD44 mAb KM201 (rat IgG1), or 30 µg/ml anti-human L-selectin mAbs TQ-1 or DREG-56 (mouse IgG1). After incubation at 4 °C for 10 min, the cells were directly applied to the wells of a 96-well microtiter plate (5 × 104 cells/well; Costar EIA/RIA plate 3690) that had been coated with 4 µg/ml versican or HA and subsequently blocked with Block Ace. In some experiments, versican- or HA-coated wells were pretreated with 5 milliunits/ml or 100 milliunits/ml of chondroitinase ABC, or 40 TRU/ml of hyaluronidase (from S. hyalurolyticus) at 37 °C for 2 h before applying the cells. The cells were allowed to bind for 40 min at 4 °C. The wells were then filled with buffer B, and the plate was inverted and placed for 30 s. After removal of the unbound cells by gentle aspiration, 1% Nonidet P-40 in PBS was added to each well, and the plate was read at 485 nm of excitation and 538 nm of emission in Labosystems Fluoroskan II (Labosystems Japan, Tokyo).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Versican Binds L-selectin, P-selectin, and CD44-- L-selectin-reactive versican was purified from the conditioned medium of a renal adenocarcinoma cell line, ACHN, as described previously (20), labeled with NHS-LC-biotin, and subjected to Western blotting analysis (Fig. 1A). When analyzed on SDS-PAGE (Fig. 1A, left), the purified versican was retained at the top of the gel and was specifically reactive with the anti-versican mAb 2B1. On SDS-agarose-PAGE (20), the purified versican migrated as a broad band of approximately 1600 kDa that was also reactive with mAb 2B1. The purified versican was used throughout this study.


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  		Fig. 1.   Purified versican. A, versican was isolated as described previously (20), labeled with NHS-LC-biotin, subjected to SDS-PAGE (left) or SDS-agarose-PAGE (right), and transferred to a polyvinylidene difluoride membrane. The sample was probed using ABC reagent or anti-versican mAb 2B1 as a primary antibody and HRP-goat anti-mouse IgG as a secondary antibody. Mouse IgG1 (mIgG1) was used as a control. B and C, binding of biotinylated versican (B) or sLeX BP-probe (C) (0-3 µg/ml) to the wells coated with 5 µg/ml anti-sLeX mAb 2H5 (open circle ), anti-sLeX mAb KM-93 (), anti-sLeX mAb CSLEX-1 (), anti-chondroitin sulfate mAb CS-56 (triangle ), or control mouse IgM (black-square) was determined by ELISA (Method 1) as described under "Experimental Procedures."

Because the glycoprotein ligands for L-selectin so far identified bear sLeX-like carbohydrates, we examined whether versican also possesses these carbohydrate epitopes. As shown in Fig. 1 (B and C), versican bound minimally, if at all, to immobilized anti-sLeX mAbs (2H5, KM-93, CSLEX-1), whereas the positive control, the sLeX BP-probe, bound well. On the other hand, versican bound to immobilized anti-CS mAb CS-56, whereas the sLeX BP-probe did not. These results indicate that, unlike the other known glycoprotein ligands for L-selectin, versican has CS chains but little or none of the sLeX determinants.

We then explored the possibility that versican binds not only L-selectin but also other selectin family members and CD44 (Fig. 2). As shown in Fig. 2A, biotinylated versican reacted with not only L-selectin-Ig but also P-selectin-Ig and CD44-Ig in a dose-dependent manner, but with neither E-selectin-Ig nor control human IgG. Biotinylated versican did not bind irrelevant Ig chimeras such as CD2-Ig or poliovirus receptor-Ig (data not shown). As expected, sLeX BP-probe bound to the immobilized L-, P-, and E-selectin-Igs (Fig. 2B), consistent with previous reports that sLeX is a common ligand for the selectins (35). Biotinylated HA reacted with CD44-Ig but not with the selectin-Igs (Fig. 2C).


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  		Fig. 2.   Binding of biotinylated versican to L-selectin-, P-selectin-, and CD44-Igs. Binding of biotinylated versican (A), sLeX-BP probe (B), or biotinylated HA (C) (0-3 µg/ml) to the immobilized human IgG1 (), L-selectin-Ig (black-triangle), E-selectin-Ig (), P-selectin-Ig (black-square), or CD44-Ig (open circle ) (2 µg/ml) was determined by ELISA (Method 1) as described under "Experimental Procedures."

Versican Interacts with the Lectin Domain of L- and P-selectin and the Link Module of CD44-- To determine which domains of L- and P-selectin and CD44 are involved in binding versican, we examined the effects of various mAbs with known specificities on the binding of versican or known ligands to L- and P-selectin- and CD44-Igs (Fig. 3). As shown in Fig. 3A, although all anti-L-selectin mAbs that block binding to sLeX (MHL-1, MHL-3, TQ1, DREG-56) inhibited the binding between versican and L-selectin-Ig, non-blocking anti-L-selectin mAbs (MHL-2, 4G8) failed to inhibit the binding. EDTA also inhibited the binding of L-selectin-Ig to versican and sLeX. These results suggest that versican interacts with the lectin domain of L-selectin. Similarly, anti-P-selectin mAbs (G1, CLB-Thromb/6, AK4) and EDTA that block binding to sLeX inhibited the interaction between versican and P-selectin-Ig (Fig. 3B). However, mAb 1.2B6, which has dual specificity for E- and P-selectin (36), inhibited the interaction of P-selectin-Ig with the sLeX BP-probe but not with versican. These results suggest that versican binds to the lectin domain of P-selectin as does sLeX, although the binding sites are not identical. Anti-CD44 mAbs BRIC235 and RAMBM 5.5.8, but not F10-44-2 or BU75, inhibited the binding of both versican and HA to CD44-Ig (Fig. 3C), suggesting that versican, like HA, is recognized by the link module of CD44. BRIC235 recognizes epitope 2a in the link module of CD44 as defined in the Vth Human Leukocyte Differentiation Antigen Workshop (37, 38), whereas RAMBM 5.5.8 recognizes a different epitope in the link module of CD44 (39). When these mAbs were added together, the binding of both versican and HA to CD44-Ig was completely inhibited, whereas doubling the concentration of either mAb added alone did not further increase the extent of inhibition (Fig. 3C). These results suggest that at least two distinct epitopes are involved in the binding of CD44 to versican and to HA.


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  		Fig. 3.   Effects of various mAbs on the binding of versican to L-selectin-, P-selectin-, and CD44-Igs. Biotinylated versican (2 µg/ml in A and B, 0.5 µg/ml in C), sLeX BP-probe (2 µg/ml), or biotinylated hyaluronic acid (HA) (1 µg/ml) was added to wells coated with L-selectin-Ig (2 µg/ml, solid bars in A), P-selectin-Ig (1 µg/ml, solid bars in B), CD44-Ig (0.5 µg/ml, solid bars in C), or the control human IgG1 (open bars) in the presence or absence of 30 µg/ml (A) or 20 µg/ml (B and C) of various mAbs or 10 mM EDTA, washed, and the binding was determined by ELISA (Method 1) as described under "Experimental Procedures." In C, the numbers in parentheses indicate the concentration of mAbs used (µg/ml). Each column represents the mean ± S.D. of triplicate determinations.

Versican Binds L- and P-selectin and CD44 through Its CS Chains-- We next examined whether versican's CS chains are involved in the binding to L- and P-selectin- and CD44-Igs (Fig. 4). The binding of versican to L- and P-selectin- and CD44-Igs was almost completely inhibited by treatment with chondroitinase ABC (Fig. 4, left), whereas the binding of versican to the anti-versican mAb 2-B-1 was not affected (data not shown). As expected, neuraminidase treatment inhibited the binding of the sLeX BP-probe to L- and P-selectin-Igs, whereas hyaluronidase treatment inhibited the binding of HA to CD44-Ig (Fig. 4, right). In contrast, neither of these treatments affected the binding of versican to the L- and P-selectin- and CD44-Igs. These results indicate that versican binds L- and P-selectin and CD44 through its CS side chains. Although chondroitinase ABC can cleave both CS chains and HA, the processing rate for HA is slower (40); thus, we used a concentration of chondroitinase ABC (5 milliunits/ml) that selectively cleaves CS but not HA in Fig. 4C (also see Fig. 9C).


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  		Fig. 4.   Chondroitinase ABC inhibits the binding of biotinylated versican to L-selectin-, P-selectin-, and CD44-Igs. Biotinylated versican (1 µg/ml), sLeX BP-probe (1 µg/ml), or biotinylated hyaluronic acid (HA) (3 µg/ml) treated with or without chondroitinase ABC (in A and B, 100 milliunits/ml; in C, 5 milliunits/ml), hyaluronidase SD (50 milliunits/ml), or neuraminidase (50 milliunits/ml) for 2 h at 37 °C was added to wells coated with L-selectin-Ig (4 µg/ml, solid bars in A), P-selectin-Ig (2 µg/ml, solid bars in B), CD44-Ig (0.5 µg/ml, solid bars in C), or the control human IgG1 (open bars), washed, and the binding was determined by ELISA (Method 1) as described under "Experimental Procedures." Each column represents the mean ± S.D. of triplicate determinations.

Certain GAGs Inhibit the Interactions between Versican and L-selectin, P-selectin, and CD44-- We next examined the effects of various GAGs on the binding of versican to L- and P-selectin- and CD44-Igs (Fig. 5). Binding of versican to the L- and P-selectin-Igs was strongly inhibited by specific GAGs, i.e. CS B, CS E, and heparan sulfate (HS) but not by any other GAGs examined (Fig. 5, A and B). CS E was particularly potent in that it inhibited the binding of versican to the L- and P-selectin-Igs by about 50% at a concentration as low as 15 ng/ml (approximately 1 nM) (data not shown). Binding of versican to the L- and P-selectin-Igs was also inhibited by sLeX-BSA, in agreement with the notion that versican binds the lectin domain of L- and P-selectin that is similar to sLeX. Conversely, the binding of the sLeX-BP probe to the L- and P-selectin-Igs was inhibited by versican (data not shown). In contrast, the binding of versican to CD44-Ig was inhibited by all of the CS chains examined, as well as by CH and HA, but not by sLeX-BSA, HS, or keratan sulfate (KS) (Fig. 5C). The binding of the biotinylated HA to the CD44-Ig was inhibited by versican, CH, and all the CS chains examined (data not shown), suggesting that versican, CH, and CS all bind the link module of CD44, similar to HA. HA inhibited the binding of versican to CD44-Ig by 50% at a concentration of 10 ng/ml (data not shown), whereas CH or CS chains inhibited the binding by 50% at about 100 to 3000 times higher concentrations (Fig. 5C), suggesting that HA has substantially higher affinity for CD44 than other GAGs. Inhibitory activity of CH and CS chains on the binding of versican to CD44-Ig appears not due to minor contamination of HA, because CH and CS preparations treated with hyaluronidase (S. hyalurolyticus) retained the same inhibitory activity (data not shown).


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  		Fig. 5.   Binding of biotinylated versican to L-selectin-, P-selectin-, and CD44-Igs in the presence or absence of increasing concentrations of various GAGs or sLeX-BSA. Biotinylated versican (1 µg/ml in A, 0.5 µg/ml in B, 0.25 µg/ml in C) was added to wells coated with L-selectin-Ig (3 µg/ml, A), P-selectin-Ig (4 µg/ml, B), or CD44-Ig (0.5 µg/ml, C) in the presence of increasing concentrations (1.5, 5, 15, 50, 150 µg/ml) of sLeX-BSA (triangle , dashed line) or GAGs: CH (open circle , solid line), CS A (, solid line), CS B (black-triangle, dashed line), CS C (triangle , solid line), CS D (, solid line), CS E (black-square, solid line), HS (open circle , dashed line), KS (, dashed line), or HA (black-triangle, solid line). After incubation, the plate was washed and the binding was determined by ELISA (Method 1) as described under "Experimental Procedures." The solid bars on the right show the binding of biotinylated versican to the wells coated with the Ig chimeras or the control human IgG1 in the absence of GAGs.

L- and P-selectin Bind to the Lipid-derivatized CS B, CS E, and HS, whereas CD44 Binds to the Lipid-derivatized CH, CS A, CS B, CS C, CS D, CS E, and HA-- To determine whether L- and P-selectin and CD44 can directly recognize GAGs, we next prepared lipid-derivatized GAGs, immobilized them on ELISA plates, and performed a direct binding assay using soluble L- and P-selectin- and CD44-Igs (Fig. 6). The efficiencies for immobilizing each lipid-derivatized GAG onto the plastic surface were comparable, and ranged from 44% to 68% as assessed by the m-hydroxydiphenyl chromogenic reaction (41) (data not shown). As shown in Fig. 6, L- and P-selectin-Igs bound to the lipid-derivatized CS B, CS E, HS, and sLeX, but not to CH, CS A, CS C, CS D, KS, or HA. On the other hand, CD44-Ig bound to the lipid-derivatized CH, all the CS chains examined, and HA but not to HS, KS, or sLeX. These results are in agreement with those obtained in the preceding section, demonstrating a direct interaction between certain GAG chains with L- and P-selectin and with CD44. The results also indicate that the GAG binding specificities of L- and P-selectin are similar to each other but different from that of CD44.


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  		Fig. 6.   Direct binding of L-selectin-, P-selectin-, and CD44-Igs to the lipid-derivatized GAGs. Human IgG1 (5 µ g/ml), L-selectin-Ig (5 µg/ml), P-selectin-Ig (3 µg/ml), or CD44-Ig (1 µg/ml) was added to wells coated with or without the lipid-derivatized GAGs or sLeX-sphingolipid (100 µg/ml), and the binding was determined by ELISA (Method 2) as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations.

Close or Overlapping Sites on Versican Are Recognized by L- and P-selectin, whereas Separate Sites Are Recognized by CD44-- To compare the binding sites on versican for L- and P-selectin and CD44, we next performed cross-blocking experiments (Fig. 7). Binding of versican to immobilized L-selectin-Ig was inhibited with soluble L- or P-selectin-Ig but not with soluble CD44-Ig. Similarly, binding of versican to immobilized P-selectin-Ig was inhibited with L- or P-selectin-Ig but not with CD44-Ig. In contrast, binding of versican to immobilized CD44-Ig was inhibited with CD44-Ig but not with the selectin-Igs. These results suggest that close or overlapping sites on versican are recognized by L- and P-selectin, whereas separate sites are recognized by CD44.


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  		Fig. 7.   Competitive binding assay. Biotinylated versican (0.1 µg/ml) was added to wells coated with human IgG1 (1 µ g/ml), L-selectin-Ig (1 µg/ml), P-selectin-Ig (0.5 µg/ml), or CD44-Ig (0.1 µg/ml) in the presence or absence of 5 µg/ml soluble Ig chimeras used as competitors, washed, and the binding was determined by ELISA (Method 1) as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations.

Versican Is Modified with At Least CS B and CS C-- We next attempted to characterize the GAGs in the versican molecule by FACE (fluorophore-assisted carbohydrate electrophoresis) analysis (Fig. 8A). After treatment with a mixture of chondroitinase ABC and ACII, Delta Di-0S, Delta Di-6S, and Delta Di-4S were detected at a molar ratio of 1:24:19. A trace amount of disulfated disaccharide was also detected. These results suggest that versican contains CS A and/or CS B and CS C, and a trace amount of CH and disulfated units.


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  		Fig. 8.   Analyses of the GAG moiety of versican. A, FACE analysis. Versican was digested with a mixture of chondroitinase ABC and ACII (0.5 unit/ml each), fluorotagged, and analyzed by FACE. B, mAbs CS-56, 2-B-6, 3-B-3, or 1-B-5 (5 µg/ml) were added to wells coated with versican (2 µg/ml) treated with or without 100 milliunits/ml chondroitinase ABC (CHase ABC), chondroitinase ACII (CHase ACII), or chondroitinase B (CHase B) at 37 °C for 2 h. The binding was determined by ELISA (Method 3) as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations.

To further characterize the GAG moiety of versican, we used various mAbs and chondroitinases with defined specificities (Fig. 8B). The mAb CS-56, which is specific for intact CS chains (42), reacted with versican; this reaction was abrogated by chondroitinase ABC treatment, as expected. The mAb 2-B-6, which recognizes stubs with the Delta Di-4S terminal structure exposed by chondroitinase digestion (43), reacted with chondroitinase B-treated versican. Because this enzyme selectively cleaves CS B (Ref. 44 and data not shown), this result indicated that versican contains CS B. This mAb also reacted with chondroitinase ABC-treated versican but not with chondroitinase ACII-treated versican. This finding supports the evidence that CS B is present on versican, because chondroitinase ABC cleaves all types of CS chains, whereas chondroitinase ACII cleaves all CS chains except CS B (Ref. 44 and data not shown). The mAb 3-B-3, which recognizes stubs with the Delta Di-6S terminal structure exposed by chondroitinase digestion (45), reacted strongly with chondroitinase ABC-treated versican, indicating that versican contains CS C as well. The mAb 1-B-5, which recognizes stubs with the Delta Di-0S terminal structure (45), reacted with chondroitinase ABC-treated versican only very weakly, if at all. Together, these results suggest that versican is modified with at least CS B and CS C.

Lymphoid Cells Can Bind to the CS Chains of Versican in a Manner Dependent on L-selectin or CD44-- We next examined whether the interaction between versican and L-selectin or CD44 can mediate cell adhesion. Mouse lymphoma EL-4 cells expressing an inactive form of CD44 and no L-selectin bound to versican-coated wells only very weakly, whereas EL-4 cells transfected with human L-selectin bound well, and this binding was inhibited by treatment with chondroitinase ABC, or the anti-human L-selectin mAbs TQ-1 or DREG-56, but not by treatment with hyaluronidase or the control mouse IgG1 (Fig. 9A). Mouse lymphoma BW5147 cells expressing an active form of CD44 (46) but not L-selectin bound well to versican- or HA-coated wells, and these bindings were inhibited by the anti-mouse CD44 mAb KM201 (47) but not by control rat IgG1 (Fig. 9B). After treatment with a lower concentration of chondroitinase ABC (5 milliunits/ml), the binding of BW5147 cells to versican- but not to HA-coated wells was specifically inhibited (Fig. 9B), indicating that CS chains of versican are involved in cell adhesion. As shown in Fig. 9C, chondroitinase ABC specifically cleaved CS at this concentration. A higher dose of chondroitinase ABC (100 milliunits/ml) inhibited the binding of BW5147 cells to both versican- and HA-coated wells, because it cleaved both CS and HA at this concentration. Hyaluronidase that specifically cleaved HA inhibited the binding of BW5147 cells to HA- but not to versican-coated wells. Taken together, these results indicate that the interactions between CS chains of versican and L-selectin or CD44 can mediate cell adhesion.


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  		Fig. 9.   Lymphoid cells bind to the CS chains of versican in an L-selectin- or CD44-dependent manner. Mouse lymphoma EL-4 cells or EL-4 cells transfected with human L-selectin cDNA (A) or mouse lymphoma BW5147 cells (B) were allowed to bind to the wells coated with versican (4 µg/ml) or HA (4 µg/ml) that had been treated with or without 100 milliunits/ml or 5 milliunits/ml of chondroitinase ABC (CHase), or 40 TRU/ml of hyaluronidase (HAase; from S. hyalurolyticus) in the presence or absence of anti-human L-selectin mAbs TQ-1 or DREG-56 (30 µg/ml), control mouse IgG1 (30 µg/ml), anti-mouse CD44 mAb KM201 (20 µg/ml), or control rat IgG1 (20 µg/ml). The binding of the cells was determined as described under "Experimental Procedures." C, mAbs 2B1 or CS-56, or biotinylated HABP (5 µg/ml), was added to the wells coated with versican (4 µg/ml) or HA (4 µg/ml) that had been treated with or without 100 milliunits/ml or 5 milliunits/ml of chondroitinase ABC (CHase) or 40 TRU/ml hyaluronidase (HAase; from S. hyalurolyticus) at 37 °C for 2 h (solid bars). Mouse IgG1 (mIgG1), mouse IgM (mIgM), or BSA was used as a control (open bars). The binding was determined by ELISA (Method 3) as described under "Experimental Procedures." Each bar represents the mean ± S.D. of triplicate determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study shows that versican, derived from a renal adenocarcinoma cell line, ACHN, binds L- and P-selectin and CD44 and this binding is mediated by an interaction between the CS chains of versican and the lectin domain of L- and P-selectin or the link module of CD44. This study also shows that, in the absence of any core protein, a specific subset of GAG chains, including CS B, CS E, and HS, bind L- and P-selectin, whereas a relatively wide range of GAG chains, including CH, CS A, CS B, CS C, CS D, CS E, and HA, bind CD44.

Blocking studies using various mAbs showed that versican is recognized by the lectin domain of L- and P-selectin and the link module of CD44 (Fig. 3), both of which mediate the binding of these adhesion molecules to known ligands. The inability of mAb 1.2B6 to inhibit the binding of versican to P-selectin, however, suggests that versican binds a site in the lectin domain of P-selectin that is different from that used for sLeX binding. This antibody inhibits the binding of P- and E-selectin to sLeX by recognizing a site outside the sLeX binding site within the lectin domain, and presumably inducing a conformational change in the sLeX binding site (36). Two anti-CD44 mAbs with different specificities, BRIC235 (37, 38) and RAMBM 5.5.8 (39), inhibited the binding of both versican and HA to CD44 completely when used in combination, although they could not do so on their own (Fig. 3). HA is thought to interact with multiple residues in the link module of CD44, such as Arg-41, Tyr-42, Arg-78, and Tyr-79 (31). Our result suggests that versican also interacts with multiple sites in the link module of CD44.

Our results demonstrate that versican binds L- and P- but not E-selectin (Fig. 2), although the three selectins share considerable structural similarity and all bind sLeX. This finding is reminiscent of previous reports showing that sulfation plays an important role in the interactions of L- and P- but not E-selectin with their ligands. For instance, sulfated glycoconjugates such as HNK-1-reactive sulfoglucuronyl glycolipids (48), heparin oligosaccharides (49), and HS GAGs (50) are recognized by L- and P- but not E-selectin. PSGL-1, originally identified as a ligand for P-selectin, binds all three selectins; however, sulfation on the tyrosine residues of PSGL-1 is required for L- and P-selectin binding but not for E-selectin binding (26, 27, 51). The ligands for L-selectin on the high endothelial venules bind L-selectin in a sulfation-dependent manner (23, 25, 52). Considering this, it is interesting to note that our preliminary study indicated that the sulfation of versican CS chains is indeed critical for its binding to L- and P-selectin.2 On the other hand, ligand sulfation does not seem to be essential for recognition by CD44. Although serglycin from a hematopoietic cell line (29, 53) and the invariant chains on antigen-presenting cells (30) bind CD44 through their CS chains, HA is not a sulfated GAG. Our present result showing that CH, a non-sulfated GAG, can interact with CD44 (Fig. 6) also supports the notion that sulfation is not required for recognition by CD44.

The GAG structure of versican remains to be fully characterized. However, FACE analysis and studies with anti-CS mAbs in combination with chondroitinases with defined specificities indicated that versican is modified with at least CS B and CS C (Fig. 8). Given that both L- and P-selectin bind CS B and CS E avidly (Fig. 6), we speculate that at least CS B or CS B-related structures on versican may serve as binding sites for L- and P-selectin. Due to the absence of an available CS E-specific mAb, we have been unable to verify whether or not CS E is also present in the GAG moiety of versican, although our preliminary analysis of the disaccharide composition of versican after chondroitinase ABC treatment using HPLC failed to detect CS E (data not shown). It has been reported that chondroitinase ABC digestion of CS chains containing GlcA(3-O-sulfate) residues specifically destroys the disaccharide units containing these sugar residues, and thus they cannot be detected by HPLC (54). Thus, it remains possible that disaccharide units containing GlcA(3-O-sulfate), such as GlcA(3-O-sulfate)beta 1-3GalNAc(4,6-O-disulfate), which are known to be present in the squid cartilage CS chains (55), remained undetected in our FACE and HPLC analyses. Further study is needed to determine the exact carbohydrate structures of versican that are recognized by L- and P-selectin and CD44.

Previously, our laboratory (29) and others (56, 57) reported that CD44 binds HA but not CS GAGs, which may probably be due to the differential affinity of HA and CS for CD44 (Fig. 5). However, the lipid-derivatized CS GAGs we used in this study allowed us to detect the binding of CD44, probably because of their effective immobilization and/or clustering on the plastic surface. The binding of L- and P-selectin and CD44 to certain lipid-derivatized GAGs in the absence of any core protein (Fig. 6) suggests that proteoglycans, other than versican, that are sufficiently modified with the appropriate GAG chains may also bind these adhesion molecules. We suggest that proteoglycans sufficiently modified with CS B and/or CS E should bind L- and P-selectin and CD44 and that proteoglycans sufficiently modified with HS but not other GAGs should bind L- and P-selectin, but not CD44. Similarly, proteoglycans sufficiently modified with CH, CS A, CS C, or CS D but not other GAGs may bind CD44 but not selectins. In support of this hypothesis, serglycin from a mouse T cell line, CTLL-2 (29, 53), and aggrecan from a rat chondrosarcoma,2 which is modified exclusively with CS A, can bind CD44 but not L-selectin. In addition, an L-selectin-reactive heparan sulfate proteoglycan, which we have recently described (58), binds L- and P-selectin but not CD44.2

The interaction of L- and P-selectin and CD44 with versican that has been modified with the appropriate GAG chains may have several functional consequences. First, it may promote the binding of leukocytes expressing L-selectin or CD44 to the extracellular matrix, resulting in enhanced cell migration. The fact that the interaction between L-selectin or CD44 and ACHN tumor-derived versican can mediate cell adhesion (Fig. 9) raises the possibility that this interaction plays a role in leukocyte infiltration into the extracellular matrix of the tumor. Second, the interaction of these adhesion molecules with versican may trigger signal transduction, because L-selectin (59, 60), P-selectin (61), and CD44 (29, 62) are all known to transduce signals into cells. Third, the interaction may possibly function to present chemokines or growth factors to their receptors in a role analogous to that of the heparan sulfate proteoglycans. In line with this idea, we have observed that ACHN tumor-derived versican can bind a certain type of chemokines.3 Experimental verification is now required to assess the in vivo role of versican under physiological as well as pathological conditions.

    ACKNOWLEDGEMENTS

In addition to those who provided antibodies and recombinant proteins listed under "Experimental Procedures," we thank T. Miwa for technical assistance. We also thank Drs. T. Tanaka and T. Murai for carefully reading the manuscript.

    FOOTNOTES

* This work was supported by a grant-in-aid for Center of Excellence Research and Scientific Research on Priority Areas: Sugar Remodeling and Cellular Communications from the Ministry of Education, Science and Culture, Japan, by grants from the Science and Technology Agency, Japan, and by Ono Pharmaceutical Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 81-6-6879-3970; Fax: 81-6-6879-3979; E-mail: mmiyasak@orgctl.med.osaka-u.ac.jp.

Published, JBC Papers in Press, August 18, 2000, DOI 10.1074/jbc.M003387200

2 H. Kawashima, unpublished observation.

3 J. Hirose, H. Kawashima, and M. Miyasaka, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: GAG, glycosaminoglycan; HS, heparan sulfate; CS, chondroitin sulfate; KS, keratan sulfate; HA, hyaluronic acid; EGF, epidermal growth factor; sLeX, sialyl Lewis X; CH, chondroitin; mAb, monoclonal antibody; HABP, hyaluronic acid binding protein; HRP, horseradish peroxidase; ELISA, enzyme-linked immunosorbent assay; Delta Di-0S, Delta 4,5HexAalpha 1-3GalNAc; Delta Di-4S, Delta 4,5HexAalpha 1-3GalNAc(4-sulfate); Delta Di-6S, Delta 4,5HexAalpha 1-3GalNAc(6-sulfate); BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; FACE, fluorophore-assisted carbohydrate electrophoresis; TRU, turbidity reducing unit; HPLC, high pressure liquid chromatography.

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ABSTRACT
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RESULTS
DISCUSSION
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