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Originally published In Press as doi:10.1074/jbc.M907862199 on May 2, 2000

J. Biol. Chem., Vol. 275, Issue 28, 21185-21191, July 14, 2000
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Identity of Urinary Trypsin Inhibitor-binding Protein to Link Protein*

Hiroshi KobayashiDagger §, Yasuyuki HirashimaDagger , Guang Wei SunDagger , Michio Fujie, Takashi Nishida||, Masaharu Takigawa||, and Toshihiko TeraoDagger

From the Dagger  Department of Obstetrics and Gynecology and the  Equipment Center, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, Shizuoka 431-3192 and the || Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, 2-5-1 Shikata-cho, Okayama 700-8525, Japan

Received for publication, September 27, 1999, and in revised form, April 18, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, directly binds to some types of cells via cell-associated UTI-binding proteins (UTI-BPs). Here we report that the 40-kDa protein (UTI-BP40) was purified from the cultured human chondrosarcoma cell line HCS-2/8 by UTI affinity chromatography. Purified UTI-BP40 was digested with trypsin, and the amino acid sequences of the peptide fragments were determined. The sequences of six tryptic fragments of UTI-BP40 were identical to subsequences present in human link protein (LP). Authentic bovine LP and UTI-BP40 displayed identical electrophoretic and chromatographic behavior. The UTI-binding properties of UTI-BP40 and LP were indistinguishable. Direct binding and competition studies strongly demonstrated that the NH2-terminal fragment is the UTI-binding part of the LP molecule, that the COOH-terminal UTI fragment (HI-8) failed to bind the NH2-terminal subdomain of the LP molecule, and that LP and UTI-BP40 exhibited significant hyaluronic acid binding. These results demonstrate that UTI-BP40 is identical to LP and that the NH2-terminal domain of UTI is involved in the interaction with the NH2-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Urinary trypsin inhibitor (UTI)1 is a Kunitz-type protease inhibitor that is responsible for the inhibition of several proteases in serum and urine as well as in amniotic fluid (1). We (2-4) and others (5, 6) have found that UTI can directly bind to neoplastic cells as well as to non-neoplastic cells via cell-associated UTI-binding proteins (UTI-BPs) or specific UTI receptors. We recently reported that one of the proteins of the UTI-BP family is a pericellular matrix-associated glycoprotein of ~40 kDa (UTI-BP40) that is thought to be very similar to human link protein (LP) (4). Our previous finding demonstrated that UTI may be able to bind hyaluronic acid via the LP molecule since UTI fails to directly bind hyaluronic acid (7). Link protein is present on a wide variety of cells, including skin and fibroblasts (8), chondrocytes (9), chondrosarcoma cells (10-13), synovial cells (14), aorta (15), trachea (16), and hepatocytes (17-19). Characteristics of the LP molecule have been studied by a number of different laboratories (20, 21), and it has been shown to mediate the interaction between proteoglycans and hyaluronic acid (22, 23), a characteristic that may allow it to demonstrate pericellular matrix formation and stabilization (hyaluronic acid-rich matrix formation) (17). Several studies have suggested that a proteoglycan tandem repeat (PTR), found in most of the hyaluronic acid-binding molecules including LP and aggrecan, acts as a functional site of interaction with hyaluronic acid (24-26).

This study was undertaken to define more clearly the relationship between proteins of the UTI-BP family and the LP molecule in the human chondrosarcoma cell line HCS-2/8. For this, we first purified proteins of the UTI-BP family from HCS-2/8 cells on a large scale. Sequencing of tryptic fragments of UTI-BP40, chromatographic and electrophoretic examination, and comparison of UTI-binding properties have revealed the identity of UTI-BP40 to LP. We then tested a variety of antibodies raised against LP and the hyaluronic acid-binding region (HA-BR) of aggrecan proteoglycan to determine whether anti-LP and anti-HA-BR antibodies cross-react with proteins of the UTI-BP family. In addition, the domain-specific antibodies to LP synthetic peptides were used as probes in structural analyses of the LP molecule. Finally, we studied the binding and competition effect of UTI fragments or LP subdomains on the solid-phase binding in attempts to localize ligand sites in the UTI structure and the binding part of the LP molecule.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cells and Culture Conditions-- The human chondrosarcoma cell line HCS-2/8 (provided by one of us (M. T.)) (27, 28) was grown and cultured as described previously (29-31). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 25 mM HEPES buffer (Life Technologies, Inc.), 2.5 mM glucosamine, 3 mM glutamine (Life Technologies, Inc.), 0.03 mM sodium pyruvate (Life Technologies, Inc.), 2.5 mM sodium lactate, 5 mM glucose (Yoneyama Chemical Co., Tokyo), 100 units/ml penicillin, and 100 µg/ml streptomycin in an atmosphere of 5% CO2 and 95% air. For immunohistochemistry, ~5 × 104 cells were seeded on chamber slides and cultured.

Purification of UTI-BPs-- UTI-BPs were purified by UTI-coupled Sepharose 4B and molecular sieve chromatography as described previously (4). Briefly, purified human UTI (50 mg) was coupled to CNBr-activated Sepharose 4B (15 g (dry weight) = 50-ml bed volume; Amersham Pharmacia Biotech, Uppsala) according to the manufacturer's recommendations. HCS-2/8 cells (~1 × 108 cells) were lysed in 5 ml of extraction buffer (20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 1% (w/v) Triton X-100, and 1 µg/ml Streptomyces hyaluronidase; Seikagaku Kogyo, Co., Ltd., Tokyo) and incubated at 23 °C for 30 min. The resulting extract was centrifuged (5000 × g, 30 min, 4 °C), and the supernatant was dialyzed and mixed with bovine serum albumin (BSA)-Sepharose beads previously equilibrated with 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.2% Triton X-100, 10 mM benzamidine, 1 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml ovalbumin, and 0.1 unit/ml aprotinin (all reagents from Sigma) using end-to-end rocking for 16 h at 4 °C. Unbound materials were again mixed with UTI-Sepharose beads using end-to-end rocking for 16 h at 4 °C. The affinity gel was then washed 10 times with 20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and twice with H2O. Eluted materials were dialyzed and concentrated by ultrafiltration (Amicon, Tokyo) for analysis by molecular sieve chromatography with a HPLC system (Kanto Kagaku, Tokyo). The column was equilibrated with 4 M guanidine HCl and 50 mM Tris-HCl (pH 7.4) at a flow rate of 0.5 ml/min. The fraction size was 0.5 ml. Calibration of the gel filtration column was with high and low molecular mass standards (Cosmo Bio Co., Ltd., Tokyo). Eluent was monitored at 280 nm. The eluted fractions were assayed by dot-blot analysis using anti-LP, anti-LPpep-N, anti-LPpep-C, HABR-1, and HABR-2 antibodies (see below). The fractions containing ~100-kDa UTI-BP (UTI-BP100) were separated from the mixture of UTI-BP45 and UTI-BP40. The mixture of UTI-BP45 and UTI-BP40 was further separated by reverse-phase HPLC. This sample was applied to a 4.6 × 250-mm Vydac C18 column (Kanto Kagaku) (2). The C18 columns were packed for high performance and equilibrated with 5% acetonitrile and 0.1% trifluoroacetic acid before loading. The material was pumped directly onto the column. The column was eluted at 1.0 ml/min with a gradient from 5% to 50% acetonitrile over 90 min. Eluent was monitored at 214 and 280 nm. The fractions eluting between 26 and 29% acetonitrile (UTI-BP45) and between 32 and 34% acetonitrile (UTI-BP40) were pooled, dialyzed, and concentrated. The amount of protein in the soluble fraction was quantified by the Bradford assay (Bio-Rad) using BSA as a standard (32).

Purification of Bovine Link Protein and HA-BR in Aggrecan-- The isolation of hyaluronic acid-binding protein (HA-BP) derived from bovine nasal cartilage has been described in detail elsewhere (22, 33). HA-BP was purified by affinity chromatography on hyaluronic acid covalently coupled to Sepharose. A purified preparation of HA-BP was supplied by Chugai Pharmaceutical Co., Ltd. (Tokyo) and Seikagaku Kogyo, Co., Ltd. Five mg of HA-BP was concentrated using a Centricon 10 ultrafiltration tube by centrifugation at 200 × g at 4 °C and then further purified by gel filtration chromatography on a column of Sepharose CL-6B (2.5 × 175 cm) equilibrated with 4 M guanidine HCl and 50 mM Tris-HCl (pH 7.4) as described by Tang et al. (34). The crude HA-BR and LP peaks were fractionated by gel filtration HPLC using an SW3000 column (Kanto Kagaku). Aliquots of each fraction were tested for their immunoreactivity by a specific dot-blot assay, and a HA-BR peak (>100-kDa polydisperse band by Western blotting) and a LP peak (~40-kDa band) were obtained. LP purified from HA-BP does not contain HA-BR in aggrecan, which was confirmed by Western blot analysis with specific monoclonal antibodies raised against HA-BR in aggrecan (mAbs HABR-1 and HABR-2).

Preparations of Polyclonal Antibodies Raised against UTI and Its Derivative as Well as against LP and Its Synthetic Peptides-- A highly purified preparation of UTI was supplied by Mochida Pharmaceutical Co. (Tokyo). Chondroitinase ABC (Sigma) was used for enzymatic deglycosylation. Briefly, 1 mg of purified UTI was incubated with 1.0 µg of chondroitinase ABC for 24 h at 37 °C. The COOH-terminal fragment of UTI (HI-8, 8 kDa) prepared by trypsin digestion was a gift from Dr. Dan Sugino (Nissin Food Products, Shiga, Japan). Polyclonal antibodies against UTI and HI-8 were prepared by intradermal injection of rabbits with 0.1 mg of purified proteins emulsified in Freund's adjuvant. The antiserum was specific for UTI and had a 50% maximal binding at a dilution of 1:10,000 in an ELISA. The antisera to UTI and HI-8 were reactive with the 240-kDa inter-alpha -inhibitor, the 120-kDa pre-alpha -inhibitor, 40-kDa UTI, and 8-kDa HI-8 in ELISA or Western blot assays. Affinity-purified IgG was prepared by mixing 3 ml of antiserum with 1 ml of UTI (or HI-8)-Sepharose overnight at 4 °C. Following washing, the IgG was eluted with 0.1 M glycine HCl (pH 2.5). The pH of the eluted fractions was immediately raised, and the IgG was stored at -20 °C.

Antibodies against LP (pAb LP) purified from bovine cartilage were prepared in a similar manner. In addition, to generate anti-LP peptide antibodies, two synthetic oligopeptide sequences, 112VFLKGGSDSDAS123 (NH2-terminal fragment of LP) and 231TVPGVRNYGFWDKDKS246 (COOH-terminal fragment of LP), corresponding to the NH2- and COOH-terminal domains of the human LP molecule, respectively, were selected. We searched for possible antigenic amino acid sequences on the LP molecule according to their predicted secondary structures and hydrophobicity. Each peptide was chosen based on its theoretical antigenic index and for specificity to the molecules. Antisera against LP synthetic oligopeptides were obtained from rabbits immunized four times with 0.2 mg of peptide conjugated with keyhole limpet hemocyanin together with Freund's adjuvant. Titration of antisera was performed by an ELISA with peptides used for immunization as antigen. When the antibody titer reached a plateau, blood was totally collected, and the serum was separated. Polyclonal antibodies against the NH2-terminal (anti-LPpep-N) and COOH-terminal (anti-LPpep-C) fragments of LP were prepared in a similar manner using the eluate from protein A-Sepharose (HiTrap, Amersham Pharmacia Biotech).

Production of Monoclonal Antibodies Raised against UTI and the Hyaluronic Acid-binding Region in Aggrecan-- Male Balb/c mice were immunized at 14-day intervals by intraperitoneal injection of 20 µg of affinity-purified UTI. Three days after the last booster, spleen cells (1 × 108) were fused with the mouse myeloma cell line NS-1 and seeded according to standard procedures (35). The antibodies were designated 2A6, 5C12, 4D1, and 8H11. mAb 8H11 showed the strongest reactivity for UTI and reacted with the NH2-terminal domain of UTI. mAb 8H11 was isolated from ascites fluid by chromatography on a protein A-Sepharose column and used for ELISA.

Monoclonal antibodies raised against HA-BR in aggrecan were prepared in a similar manner. Two antibodies were selected and designated HABR-1 and HABR-2. These mAbs were found to react with HA-BR in aggrecan, but not with LP. A list (antibody specificities and characterization) of the various mAbs and pAbs used in this study is shown in Table I. A purified preparation of each antibody was biotinylated according to the method of Guesdon (36) using N-hydroxysuccinimidyl biotinamidocaproate (Sigma) following the manufacturer's suggested procedures.

                              
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Table I
Summary of the antibodies used in this study and their characteristic features

Purification of UTI-BP40 Tryptic Fragments-- Electrophoretically homogeneous UTI-BP40 in phosphate-buffered saline (pH 7.3) was treated with bovine pancreatic trypsin at 37 °C for 3 h at a 1:100 enzyme/substrate molar ratio. Peptides were separated by 17% SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The resulting gel was stained with Coomassie Blue and electrophoretically blotted onto polyvinylidene difluoride membrane (Bio-Rad). In a parallel experiment, tryptic fragments were analyzed by reverse-phase HPLC. Sequencing of the peptides was by Edman degradation using an automated sequencer (Applied Biosystems Model 477A) with on-line phenylthiohydantoin-derivative detection.

Trypsin Treatment of TLP·HA and TLP·HA-BR Complexes-- Two major protein fractions have been isolated from a tryptic digest of the bovine proteoglycan complex (37-39): one of them, HA-BR, derives from the proteoglycan subunit and is located at its NH2 terminus, whereas the other (TLP) is a common trypsin fragment from LP; their homogeneity was assessed by SDS-PAGE. TLP differs from native LP by the removal of a short amino-terminal peptide from native LP. Tryptic digestion of TLP·HA and TLP·HA-BR complexes was carried out according to previously reported experiments (24). The 22-kDa fragment, isolated from a tryptic digest of the TLP·HA complex, corresponds to the COOH-terminal region of bovine TLP (termed LP-C in this study). On the other hand, the 20-kDa fragment arising from the TLP·HA-BR digest is the NH2-terminal fragment of TLP (termed LP-N).

Solid-phase Binding and Competition Assays-- The solid-phase binding assay has been described in detail elsewhere (26). 96-Well microtiter plates were coated with 100 µl of HA-BR (5 µg/ml), LP (2 µg/ml), LP-N (1 µg/ml), LP-C (1 µg/ml), or UTI-BP40 (1 µg/ml) in bicarbonate buffer (pH 9.5) at 4 °C overnight. One-hundred µl of biotinylated UTI (0-10 µg/ml) was added to each well and incubated for 2 h at room temperature. In the case of the competition assay, studies on the binding of biotinylated UTI (10 nM) to immobilized UTI-BP40 or LP were performed in the presence of unlabeled competitors (UTI, HI-8, UTI-BP40, LP, LP-N, LP-C, alpha 1-antitrypsin, alpha 2-antiplasmin, and plasminogen activator inhibitor type-1; reagents from Cosmo Bio Co., Ltd.) for 2 h at 23 °C.

In a parallel experiment, 96-well microtiter plates were coated with 50 µl of hyaluronic acid (100 µg/ml) conjugated to dipalmitoylphosphatidylethanolamine (HA-PE; a gift from Seikagaku Kogyo, Co., Ltd.) in phosphate-buffered saline at 4 °C overnight as described previously (26). For studies of specificity, the same amount of chondroitin sulfate (CS)-PE or heparan sulfate (HS)-PE (a gift from Seikagaku Kogyo, Co., Ltd.) as HA-PE was used. After the wells were blocked with Tris-buffered saline containing 1% BSA (1 h, 23 °C), 50 µl of UTI-BP40 or LP (0.5 µg/ml) was added to some of the plates (2 h, 23 °C). Fifty µl of biotinylated UTI (0.1 µmol/liter) was added to each well in the absence or presence of CS (50 µl, 100 µg/ml) or HS (50 µl, 100 µg/ml) and incubated for 2 h at 23 °C. Horseradish peroxidase-conjugated avidin was used as the detection probe.

SDS-PAGE and Western Blotting-- The cell extracts, purified proteins, or tryptic fragments were dissolved in sample buffer. The sample (20 µg of protein/lane for cell extracts and 0.1~0.5 µg of protein/lane for purified proteins) was processed for electrophoresis using a SDS-polyacrylamide gel under nonreducing conditions. The resulting gel was electrophoretically blotted onto polyvinylidene difluoride membrane, which was blocked with Tris-buffered saline containing 2% BSA, and then immunoblotted. The blot was subsequently processed by the biotin/avidin/peroxidase method (40). Bands were visualized with the ECL detection system (Amersham Pharmacia Biotech, Tokyo). The membranes were then placed between two transparencies and exposed to Kodak film. In all experiments, some strips were incubated with nonimmune rabbit or mouse IgG as a negative control.

Statistical Analysis-- The data presented are the means of triplicate determinations in one representative experiment unless stated otherwise. Data are presented as means ± S.D. All statistical analyses were performed using StatView for Macintosh. The Mann-Whitney U test was used for the comparisons between different groups. p < 0.05 was considered significant.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Determination of Antibody Specificity

Characterization of mAb 8H11-- Clone 8H11 was produced by somatic cell fusion. The interaction of mAb 8H11 with UTI was evaluated by immunoblotting with UTI, chondroitinase ABC-treated UTI (deglycosylated UTI), HI-8, and UTI reduced with 2-mercaptoethanol (Fig. 1). mAb 8H11 reacted with UTI and deglycosylated UTI, but not with HI-8 or UTI reduced with 2-mercaptoethanol (Fig. 1, right panel). This suggests that the epitope resides in the NH2-terminal domain of UTI (since it is missing in HI-8) and is destroyed by reduction of the disulfide bonds. In contrast, Western blot analysis indicated that polyclonal antibodies raised against UTI (pAb UTI) recognized a determinant present on a wide variety of UTI preparations (UTI, deglycosylated UTI, HI-8, and UTI reduced with 2-mercaptoethanol) (Fig. 1, left panel). The ELISA data also confirmed that the 8H11 determinant was sequestered in the NH2-terminal structure of UTI (data not shown).


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  		Fig. 1.   Characterization of antibodies raised against UTI. Shown are the results from SDS-PAGE followed by Western blotting of purified UTI and its derivatives under nonreducing and reducing conditions. The samples (0.1 µg) were analyzed by 5-18% gradient SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blot analyses of UTI and its derivatives were conducted for pAb UTI (left panel) and mAb 8H11 (right panel) reactivities. First lanes, UTI; second lanes, chondroitinase ABC-treated UTI; third lanes, HI-8; fourth lanes, UTI treated with 2-mercaptoethanol (2ME). The molecular masses (in kilodaltons) and positions of marker standards are indicated to the left.

Characterization of mAbs HABR-1 and HABR-2 and pAbs Raised against LP and LP Synthetic Peptides as Well as Polyclonal Antibodies Raised against UTI-BP-- Using procedures described previously (2, 4), UTI-BP was purified from human HCS-2/8 cell lysates by UTI-coupled Sepharose 4B. As shown in Fig. 2, HCS-2/8 cell-derived UTI-BP is composed of three different molecular species: UTI-BP100, UTI-BP45, and UTI-BP40. pAb raised against UTI-BP (pAb UTI-BP) reacted with all members of the UTI-BP family (UTI-BP100, UTI-BP45, and UTI-BP40) and the purified LP molecule as well as with both the NH2-terminal immunoglobulin-like domain of LP (LP-N, ~20 kDa) and the COOH-terminal PTR domain of LP (LP-C, ~22 kDa). After immunoabsorption of anti-UTI-BP antibodies with LP, the remaining antibodies recognized UTI-BP100 and UTI-BP45 in UTI-BPs, but not the UTI-BP40 (Fig. 3). This shows that the 40-kDa band does not contain more than LP. The interaction of anti-LP antibodies (pAb LP) with UTI-BPs was also evaluated by immunoblotting with UTI-BP100, UTI-BP45, and UTI-BP40. pAb LP reacted with UTI-BP40 and the purified LP molecule as well as with both LP-N and LP-C. However, pAb LP failed to react with UTI-BP100 or UTI-BP45. pAb LPpep-N, in which the epitope presents on the NH2-terminal domain of LP, reacted with UTI-BP40, LP, and LP-N, but not with LP-C, UTI-BP100, or UTI-BP45, whereas pAb LPpep-C, in which the epitope resides in the COOH-terminal domain of LP, reacted with UTI-BP40, LP, and LP-C, but not with LP-N, UTI-BP100, or UTI-BP45. Western blot analysis thus demonstrated that pAbs raised against LP synthetic peptides exclusively recognized both their respective domains of LP and UTI-BP40. It is unlikely that UTI-BP100 and UTI-BP45 have antigenically cross-reactivity with LP. These results suggest that the UTI-binding sites purified from HCS-2/8 cells may contain other binding proteins or UTI receptors rather than LP. mAbs HABR-1 and HABR-2 reacted with UTI-BP100, but not with UTI-BP45 or UTI-BP40 (data not shown), suggesting that UTI-BP100 is composed of HA-BR from the aggrecan fragment.


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  		Fig. 2.   Characterization of antibodies raised against UTI-BP and HA-BR as well as LP and its fragments. Shown are the results from SDS-PAGE (15% gel) of purified proteins (UTI-BP (1 µg/lane) and LP, LP-N, and LP-C (0.2 µg/lane)) under nonreducing conditions. Also shown are the results from Western blotting (WB) of UTI-BP, LP and its subdomains using pAbs raised against UTI-BP (pAb UTI-BP), LP (pAb LP), LP-N (pAb LPpep-N), and LP-C (pAb LPpep-C) as well as mAb raised against HA-BR in aggrecan (mAb HABR-1). The molecular masses (in kilodaltons) and positions of marker standards are indicated to the left. The result from Western blotting using mAb HABR-2 was same as that using mAb HABR-1.


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  		Fig. 3.   Characterization of anti-UTI-BP antibodies immunoabsorbed with LP. Shown are the results from 12% SDS-PAGE and Western blotting of UTI-BP (1 µg/lane) using anti-UTI-BP antibodies (lane 1) and antibodies immunoabsorbed with LP (lane 2) under nonreducing conditions. The molecular masses (in kilodaltons) and positions of marker standards are indicated to the right.

Amino Acid Sequences of UTI-BP40 Tryptic Fragments

Purified UTI-BP40 was digested with trypsin, and the resultant peptides were purified by immunoblotting or reverse-phase HPLC and identified by NH2-terminal sequencing (Fig. 4). Aliquots of each blotting or each pool were analyzed by gas-phase sequencing. A comparison with data in the GenBankTM/EBI Data Bank (accession number 001884) showed that the six tryptic peptides were identical to subsequences found in human LP. In every case, the UTI-BP40 fragments corresponded to those expected from cleavage of LP at tryptic sites. The molecular masses of the six tryptic fragments were equivalent to 32.2% of the mass of LP (354 amino acids).


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  		Fig. 4.   Amino acid sequences of human link protein and tryptic fragments of UTI-BP40. Upper row, amino acid sequence of LP; lower row, amino acid sequences of tryptic fragments of UTI-BP40. X, unidentified residue.

Reverse-phase HPLC and SDS-Polyacrylamide Gel Electrophoresis of UTI-BP40 and LP

Authentic LP and UTI-BP40 displayed equivalent retention times (64 min) on a C18 reverse-phase HPLC column as determined by A214 detection. Similar electrophoretic migration (40 kDa) of the two proteins was observed on a 15% SDS-polyacrylamide gel under nonreducing conditions (data not shown).

Binding and Competition Assays

Specific Binding of Biotinylated UTI to Immobilized Potential Ligands (LP, LP Subdomains, UTI-BP40, and UTI-BP100)-- Since the yields of UTI-BP45 were small, studies on the specific binding of biotinylated UTI to immobilized UTI-BP45 could not be carried out in this study. We have separated the NH2- and COOH-terminal regions of LP by subfragmentation with trypsin to determine which one of the subdomains of LP interacts with UTI. The separated subdomains have been used to investigate epitope mapping of several antibodies (Fig. 2) and have been extensively used in binding (Figs. 5 and 6) and competition assays. LP-C showed a 22-kDa single monomeric band, whereas LP-N represented a 20-kDa intense band and had additional fast migrating weak bands that appeared to be degradation products (see Fig. 2, WB (pAb LP)). The solid-phase binding assay was used to support a more extensive analysis of the LP-binding site (Fig. 5). LP, LP-N, and UTI-BP40 exhibited significant biotinylated UTI binding, whereas LP-C and UTI-BP100 showed no significant affinity for UTI, even if the concentration of biotinylated UTI was increased to 1 µmol/liter. Although UTI binding to different ligands cannot be quantitatively compared by plate binding, our results indicate that the subdomain for UTI binding is the NH2-terminal domain of the LP molecule and that UTI shows no significant affinity for HA-BR in aggrecan (UTI-BP100). Our results support the hypothesis that HA-BR itself has an ability to bind UTI via the LP molecule since HA-BR is known to directly and specifically interact with LP. To assure that the applied proteins stuck to the microtiter plates, we performed an immunodetection assay using respective antibodies. Significant signals of absorbance at A450 were obtained from these ligands compared with those from the BSA control (data not shown).


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  		Fig. 5.   Solid-phase binding assay: specific binding of biotinylated UTI to immobilized potential ligands. Data points represent the means ± S.D. for estimates done in triplicate. , LP-coated wells; black-square, LP-N-coated wells; black-diamond , UTI-BP40-coated wells; black-triangle, UTI-BP100-coated wells; diamond , LP-C-coated wells. The results are representative of four independently conducted experiments. *, p < 0.05.

Effect of Ligands on the Binding of Biotinylated UTI to Immobilized LP or UTI-BP40-- To localize and to characterize the ligand and binding structures in UTI involved in binding to UTI-BP40 or LP, studies on the binding of biotinylated UTI to immobilized UTI-BP40 or LP were performed in the absence or presence of unlabeled competitors (Fig. 6). The UTI-BP40-coated microtiter plates were incubated with biotinylated UTI at a concentration of 10 nM in the absence (Delta A450 = 0.73) or presence of each competitor for 2 h at 23 °C. The inhibition of specific binding obtained with competitors in excess was 94% for UTI (1 µM), 51% for UTI-BP40 (1 µM), 53% for LP (1 µM), and 48% for LP-N (1 µM), whereas the quenching caused by HI-8 and LP-C was insignificant (<20%). Unrelated proteins (alpha 1-antitrypsin, alpha 2-antiplasmin, and plasminogen activator inhibitor type-1) and BSA failed to inhibit biotinylated UTI binding to immobilized UTI-BP40. In a parallel experiment, potent inhibition by UTI, UTI-BP40, LP, and LP-N was also observed in LP-coated microtiter plates (data not shown).


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  		Fig. 6.   Effect of ligands on the binding of biotinylated UTI to immobilized UTI-BP40: competition inhibition of biotinylated UTI binding to immobilized UTI-BP40 by UTI, HI-8, UTI-BP40, LP-N, LP-C, alpha 1-antitrypsin (A1AT), alpha 2-antiplasmin (A2AP), and plasminogen activator inhibitor type-1 (PAI-1). Specific binding observed in the presence of increasing concentrations (10-9 to 10-6 mol/liter) of UTI, HI-8, UTI-BP40, LP-N, and LP-C is expressed. Data points represent the means ± S.D. for estimates done in triplicate. The results are representative of three independently conducted experiments. *, p < 0.05.

Proof of the identity of UTI-BP40 to LP was directly provided by the following competition assays. First, studies on the binding of anti-UTI-BP antibodies to immobilized UTI-BP40 were performed in the presence of LP (data not shown). We carried out an immunodetection assay using biotinylated anti-rabbit IgG and peroxidase-conjugated avidin. This experiment showed that LP (1 µM) was able to inhibit by ~90% anti-UTI-BP antibody binding to UTI-BP40 bound to a plate. Second, UTI-BP40 (1 µM) almost completely blocked anti-LP antibody binding to LP bound to a plate (data not shown). Thus, the antibodies were each blocked to >90% by the antigens indicated.

Biotinylated UTI Binding to LP or UTI-BP40 Anchored via Hyaluronic Acid

We studied the interaction of LP or UTI-BP40 with HA, CS, or HS. Biotinylated UTI was added to the HA-PE-, CS-PE-, or HS-PE-coated wells preincubated with or without LP or UTI-BP40 (Fig. 7). LP and UTI-BP40 exhibited significant HA binding, whereas LP and UTI-BP40 showed no significant affinity for CS or HS. These results indicate that there is no significant difference in UTI-binding activity between LP and UTI-BP40 bound to immobilized HA. In addition, we added LP or UTI-BP40 to the HA-PE-coated plates together with CS and HS to see if either could compete with LP or UTI-BP40 binding. However, neither CS nor HS could compete with LP or UTI-BP40 binding to HA. These results strongly indicated the specific interaction of both LP and UTI-BP40 with HA, but not with CS or HS. The direct binding studies strongly demonstrate that UTI does not directly and specifically interact with HA, CS, or HS (data not shown). These results support the hypothesis that UTI has an ability to bind HA via the LP molecule or UTI-BP40. We confirmed again that the applied proteins (LP and UTI-BP40) stuck to HA-coated microtiter plates (data not shown).


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  		Fig. 7.   Biotinylated UTI binding to LP or UTI-BP40 anchored via hyaluronic acid. Biotinylated UTI (0.1 µg/ml) was added to HA-PE-coated wells preincubated with LP (0.5 µg/ml; stippled bars), UTI-BP40 (0.5 µg/ml; hatched bars), or BSA (1.0 µg/ml; black bars) in the absence or presence of CS (100 µg/ml) and HS (100 µg/ml). Data points represent the means ± S.D. for estimates done in triplicate. The results are representative of three independently conducted experiments. *, p < 0.05.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Extracellular proteolysis is required in inflammation and tumor processes where cell migration and invasion occur (41-45). A growing body of evidence demonstrated that UTI effectively inhibits tumor cell invasion and metastasis. Tumor cell-associated plasmin, but not urokinase activity, is efficiently inhibited by UTI (46-49). UTI interacts with a variety of cell types, including neoplastic and non-neoplastic cells. The presence of UTI at the cell surface has been explained by the demonstration of UTI-binding sites on the cell membranes. UTI-BPs and the UTI receptor have been recently detected, but only a few have been isolated or extensively characterized (2-6, 50). We initially reported the presence of proteins of the UTI-BP family on human choriocarcinoma SMT-cc1 cells and uterine fibroblasts (2, 4). Although UTI-BP40 was described as a UTI-binding site, it is able to bind hyaluronic acid as well and has been localized abundantly in cartilage and ovary in mice and rats.2 Therefore, in this study, we tried to isolate and to characterize proteins of the UTI-BP family from human chondrosarcoma HCS-2/8 cells on a large scale since these cells express cartilage proteoglycans associated with hyaluronic acid to form proteoglycan aggregates (51, 52).

Using the UTI immunoaffinity beads, several proteins of the UTI-BP family were purified from HCS-2/8 cell extracts. UTI-BP40 was the major band consistently and specifically bound to UTI. UTI-BP100 and UTI-BP45 were minor bands directly or indirectly bound to UTI. This study has extended the characterization of UTI-BP40. The purification of UTI-BP40 to apparent homogeneity gave us access to partial amino acid sequence information. First, the amino acid sequences of tryptic fragments of UTI-BP40 were identical to subsequences found in human LP. Second, UTI-BP40 was identical to LP with respect to molecular mass and behavior upon reverse-phase HPLC and SDS-PAGE. Third, a number of domain-specific anti-LP antibodies cross-reacted with UTI-BP40. Fourth, authentic LP and UTI-BP40 displayed similar UTI binding. The binding of biotinylated UTI by LP and UTI-BP40 was specific in that unrelated proteins (alpha 1-antitrypsin, plasminogen activator inhibitor type-1, and alpha 2-antiplasmin) and BSA did not inhibit it. The IC50 for unlabeled UTI was in the low nanomolar range. However, we could not explain why UTI-BP40, LP, and LP-N give only ~50% inhibition of UTI binding to UTI-BP40 bound to a plate. The NH2-terminal LP fragment (20 kDa) showed substantial UTI-binding activity, whereas the COOH-terminal LP fragment (22 kDa) did not have UTI-binding ability, indicating that the subdomain for UTI binding is the NH2-terminal domain in the LP molecule. Furthermore, LP and UTI-BP40 specifically bound hyaluronic acid. The common structural motif in LP for hyaluronic acid binding appears to be a PTR module in the COOH-terminal region of this protein. Thus, we have been able to identify the NH2-terminal fragment in the LP molecule as a probable binding domain for UTI. Fifth, the COOH-terminal UTI fragment (HI-8) failed to bind LP itself or the NH2-terminal subdomain of the LP molecule. Sixth, UTI-BP and LP exist in association with hyaluronic acid in the extracellular matrix of cultured cells. Collectively, we conclude that UTI-BP40 is LP and that the NH2-terminal domain of UTI is involved in the interaction with the NH2-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix (Fig. 8).


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  		Fig. 8.   Schematic representation of the hypothesized interaction of UTI with link protein and hyaluronic acid. Link protein lacks a transmembrane domain and contains one Ig domain and two PTR loops. Binding to hyaluronic acid is mediated through sequences at the tip of the PTR loops. The Ig loop of the link protein may permit interaction with UTI. The NH2-terminal domain of UTI is involved in the interaction with the NH2-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix.

However, the conclusion from the set of immunoprecipitation experiments is that anti-LP antibodies can immunoprecipitate ~60% of UTI-binding activity from the cell extracts (data not shown). Incomplete reduction after immunoprecipitation could be due to the presence of a heterogeneous population of UTI-binding proteins since some fractions (UTI-BP100 and UTI-BP45) may be unable to react with the LP-related antibodies. It is likely that UTI can bind to components rather than to members of the LP molecule family. UTI-BP100 was identified immunologically as a HA-BR in the aggrecan molecule. Therefore, UTI-BP100 and the aggrecan G1 domain share similar epitopes, or they are closely related if not identical molecules. It is unlikely that HA-BR in aggrecan is another candidate for UTI-BP since UTI does not directly bind to HA-BR. We have considered that HA-BR may bind UTI via the LP molecule. The identity of UTI-BP100 to the aggrecan G1 domain should also be established by amino acid sequencing, tryptic mapping, and specific binding and competition experiments.

In addition, the minor UTI-BP45 was also specifically isolated. It is unlikely that UTI-BP40 represents a degradation product of UTI-BP45 since anti-LP antibodies did not cross-react with UTI-BP45. This may be a novel protein or may be a subunit of the UTI-BP complex, each having the ability to bind UTI. Since the yield of UTI-BP45 was small, studies on the specific binding of UTI to UTI-BP45 could not be carried out in this work. Whether UTI-BP100 represents the aggrecan G1 domain or UTI-BP45 is a new member of the UTI-BP family remains an open question.

The molecular mass of LP produced by HCS-2/8 cells is almost the same as that of UTI-BP40.3 LP is synthesized by the chondrosarcoma cells themselves and stabilizes the binding between proteoglycan subunits and hyaluronic acid (34, 53, 54). Since LP is found in the extracellular matrix, it is thought to be involved in the organization of a hyaluronic acid-rich matrix. We reported for the first time that LP directly binds UTI, which corresponds to a light chain of the inter-alpha -inhibitor. These data strongly demonstrate that locally produced and expressed UTI-binding sites accumulate free UTI and/or the inter-alpha -inhibitor in the extracellular matrix of chondrosarcoma cells.

It is possible that UTI serves a number of different functions through the LP molecule. UTI could interact with LP anchored via the hyaluronic acid-rich matrix on the surface of tumor cells. This may result in the effective inhibition or regulation of tumor cell-associated protease activity. Furthermore, our previous studies demonstrated the specific internalization of UTI by tumor cells (2, 50, 55). The process of UTI-BP- or UTI receptor-mediated endocytosis has been the subject of extensive study. Proteins of the UTI-BP family may be involved in the active endocytosis of UTI. Further research will reveal additional characteristics for the very interesting proteins of the UTI-BP family.

In summary, this study has characterized the proteins of the UTI-BP family biochemically, immunologically, and immunohistochemically and has identified UTI-BP40, which is identical to LP. Our results strongly support that the UTI-binding site is located in the NH2-terminal region of this molecule and that the NH2-terminal domain of UTI may be involved in the interaction with the NH2-terminal fragment of LP, which is bound to hyaluronic acid in the extracellular matrix via the COOH-terminal PTR domain of the LP molecule. Several lines of evidence demonstrate that UTI-BP100 is the aggrecan G1 domain, although a definitive answer can only be found after sequencing and cloning. Whether UTI-BP45 is a novel member of the expanding UTI-BP family also remains an open question.

    ACKNOWLEDGEMENTS

We thank Drs. K. Shibata, T. Noguchi, and A. Suzuki (Equipment Center and Photo Center, Hamamatsu University School of Medicine) for help with the biochemical analysis. We are also thankful to Drs. T. Kobayashi and N. Kanayama (Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine), Drs. H. Morishita and K. Kato (BioResearch Institute, Mochida Pharmaceutical Co., Tokyo), Drs. Y. Tanaka and T. Kondo (Chugai Pharmaceutical Co., Ltd.), and Drs. S. Miyauchi and M. Ikeda (Seikagaku Kogyo Co., Ltd.) for the continuous and generous support of our work.

    FOOTNOTES

* 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.: 81-53-435-2309; Fax: 81-53-435-2308/1626.

Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M907862199

2 H. Kobayashi, Y. Hirashima, G. W. Sun, M. Fujie, T. Nishida, M. Takigawa, and T. Terao, submitted for publication.

3 M. Takigawa, unpublished data.

    ABBREVIATIONS

The abbreviations used are: UTI, urinary trypsin inhibitor; UTI-BP, urinary trypsin inhibitor-binding protein; LP, link protein; PTR, proteoglycan tandem repeat; HA, hyaluronic acid; HA-BR, hyaluronic acid-binding region; HA-BP, hyaluronic acid-binding protein; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; mAb, monoclonal antibody; pAb, polyclonal antibody; ELISA, enzyme-linked immunosorbent assay; TLP, trypsin fragment from link protein; PAGE, polyacrylamide gel electrophoresis; PE, dipalmitoylphosphatidylethanolamine; CS, chondroitin sulfate; HS, heparan sulfate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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