2 Natural Products Research Institute, College of Pharmacy, Seoul National University, 28 Yeonkun-Dong, Jongno-Ku, Seoul 110-460, Korea; 3 Department of Biochemistry, NCMLS, UMC Nijmegen, 6500 HB Nijmegen, The Netherlands; 4 Graduate School of Pharmaceutical Science, Chiba University, Chiba 263-8522, Japan; and 5 Department of Chemistry and Chemical Biology, Biology and Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180
Received on March 25, 2004; revised on July 19, 2004; accepted on July 22, 2004
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Abstract |
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Key words: AS-binding protein / biotinylation / Lewis lung carcinoma / nucleolin
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Introduction |
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GAGs such as heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronic acid serve as key biological response modifiers by acting as (1) stabilizers, cofactors, and/or coreceptors for growth factors, cytokines, and chemokines; (2) regulators of enzyme activity; (3) signaling molecules in response to cellular damages, such as wound healing, infection, and tumorigenesis; and (4) targets for bacterial, viral, and parasitic virulence factors for attachment, invasion, and immune system (Schmidtchen et al., 2001).
A novel GAG, acharan sulfate (AS), isolated from the body of the giant African snail Achatina fulica, has a primary structure4)-2-acetamido-2-deoxy-
-D-glucopyranose(1
4)-2-sulfo-
-L-idopyranosyluronic acid (1
(GlcNAc-IdoA2SO3) (Kim et al., 1996
). It is related to the heparin and heparan sulfate families of GAGs but is distinctly different from all known members of these classes of GAGs. In previous studies, AS showed antiangiogenic activity in inflammation models (Ghosh et al., 2002
), in vivo anticoagulant activity, antimitogenic activity on heparin-mediated basic fibroblast growth factor (FGF-2) (Wang et al., 1997
), and immunomodulating action (Shim et al., 2002
). AS showed no cytotoxicity (0200 µg/ml) on tumor cells but rather inhibited tumor growth in vivo through an antiangiogeic mechanism (Lee et al., 2003
). A cell binding and immunofluorescence assay showed that AS bound to cell surface molecules in a time- and concentration-dependent fashion. To answer the question of whether any specific binding proteins on the surface of cancer cells can be ascribed to the antitumor activity of AS, the cell surface proteins of Lewis lung carcinoma (LLC) cells were biotinylated, and the cell lysates were fractionated based on AS affinity chromatography. Characterization by mass spectrometry and western blotting suggested that nucleolin corresponded to the AS-binding protein, responsible for AS inhibition of tumor growth.
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Results |
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Cell adhesion to AS
One milliliter of AS at concentrations 0200 µg/ml was coated on each well of a 24-well plate and incubated for 07 h with LLCs. The extent of adhesion of LLCs to AS-coated plates was determined. Little adhesion of LLCs was detected when cells were incubated in wells for 03 h at all concentrations tested. When an AS concentration of at least 50 µg/ml was coated in a well and incubated with LLCs for 5 or 7 h, significant adhesion of LLCs was detected (see Figure 1). Heparin-coated wells were similarly prepared at 50 µg/ml, and again only wells incubated with LLCs for 5 or 7 h showed cell adhesion. In these experiments it was critical to use the proper number of cells (1.8 x 105) to obtain repeatable results (data not shown).
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Characterization of AS-binding proteins using SDSPAGE and western blotting
Recently the application of biotinylation to identify proteins on the cell surface has begun to replace the use of radioisotopes (Cole et al., 1987). In our experiments, we used water-soluble sulfo-NHS-LC-biotin as a reagent to label LLC membrane proteins. Because biotin binds with a high affinity to streptavidin, biotinylated can be sensitively detected by enzymes or fluorescent tags conjugated to streptavidin. Cells that had been biotinylated at the surface were lysed with lysis buffer containing a 1% NP-40 detergent, and then the lysate was subjected to AS affinity column chromatography to identify AS-binding proteins. The bound proteins were eluted with a stepwise salt gradient (Figure 3A) as described in Materials and methods. The eluted fractions were collected, concentrated, and analyzed using 8% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) and visualized by silver staining. Detection of biotinylated proteins on the cell surface by western blotting relied on horseradish peroxidase (HRP)-conjugated streptavidin and o-phenylenediamine (Figure 3B). Each lane in SDSPAGE contained many protein bands visualized with silver staining, but western blotting showed a smaller number of bands that reacted to HRP-streptavidin. One prominent band of molecular weight
110 kDa in the high-affinity fraction eluting at 1.0 M NaCl showed a strong response to HRP-streptavidin. Visualization of the blotted membrane with antinucleolin antibody immunologically confirmed the identified protein was nucleolin. Bands at 110 kDa in both 0.7 M and 1.0 M NaCl fractions were also reactive to antinucleolin antibody (Figure 3C). These results suggest that nucleolin is eluted from the immobilized AS column with 0.7 to 1.0 M NaCl.
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Discussion |
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The structure of AS is composed of IdoA2SO3GlcNAc and structurally resembles the heparin/heparan sulfate family of GAGs based on its alternating 1
4 linkage and its IdoA2SO3 residue. It is also similar to hyaluronan in the aspect of containing a GlcNAc residue and having a relatively low charge density. IdoA is a uniquely conformationally flexible saccharide residue and appears to play a key role in binding of IdoA containing GAGs to a variety of proteins in cells (Westergren-Thorsson et al., 1991
). Although AS is obtained from an invertebrate, its diverse pharmacological activities in mammalian systems may result from its interactions with a wide variety of proteins under physiological conditions. AS shows no cytotoxicity toward various cell types and in vivo, whereas it inhibits the growth of tumor-induced LLC-bearing C57BL/6 mice.
Cell-binding and immunofluorescence assays demonstrate that AS adheres to cell surface proteins in a dose- and time-dependent fashion. Biotinylation of tumor cell surface and AS affinity fractionation of cell lysates demonstrate that although many proteins interact with AS as detected on SDSPAGE with silver staining, only two biotinylated proteins (52 and 110 kDa) were detected in the high (1.0 M) salt fraction by western blotting. ESI Q-TOF MS demonstrated that the larger one of these two proteins was nucleolin. The use of antinucleolin antibody confirmed its identity.
Nucleolin was first described by Orrick et al. (1973), and the same protein was then identified from Chinese hamster ovary cells and several other eukaryotic cells, including human, rat, mouse, and chicken. Nucleolin has been described as a major nuclear protein, having an apparent molecular mass of 100110 kDa as determined by SDSPAGE and a calculated molecular mass of 7677 kDa as predicted by the amino acid sequence (Lapeyre et al., 1987
). The difference between the calculated and observed molecular masses is most likely due to posttranslational modifications and a high content of negatively charged amino acids in the N-terminal part of nucleolin (Harms et al., 2001
). The functions of nucleolin have been studied according to the location of the nucleus, cytoplasm, and cell surface of several cell lines (Srivastava and Pollard, 1999
).
Although nucleolin plays an important role in ribosome biogenesis in the nucleolus, it also has been involved as the adhesion receptor L-selectin, which is expressed on leucocytes and hemotopoietic progenitor cells (Harms et al., 2001). It specifically binds apo B and apo E containing lipoprotein to the surface of the HepG2 cells (Semenkovich et al., 1990
) and serves as a substrate for an ecto-protein kinase on the cell surface of HeLa cells (Jordan et al., 1994
). The neurite-promoting IKVAV site of laminin-1 binds to nucleolin on the cell surface and has been found to promote the differentiation of primary neurons and a variety of neural cell lines (Kleinman et al., 1991
). Recently, nucleolin has been shown to interact with the amino-terminal domain of hepatitis
antigens and modulate hepatitis
virus replications (Lee et al., 1998
). Nucleolin is also associated with the actin cytoskeleton (Hovanessian et al., 2000
) and related to inhibit HIV infection by the cytokine midkine (Callebaut et al., 2001
). It is very meaningful that nucleolin exists on the cell surface and binds to midkine. Midkine is a 13-kDa heparin-binding growth/differentiation factor structurally unrelated to FGFs and is rich in basic amino acids. Midkine has been reported to promote angiogenesis (Choudhuri et al., 1997
), neurite outgrowth, survival of neurons, fibrinolysis and cell growth (Muramatsu et al., 1993
), and migration (Maeda et al., 1999
) and is overexpressed in a number of human carcinomas, such as colon, breast, lung, bladder, and neuroblastoma. This enhanced expression of midkine implies that it is beneficial to tumor growth and GAG synthesis of endothelial cells (Sumi et al., 2002
).
Previously, we reported that AS suppresses tumor growth by the inhibition of angiogenesis. Very recently, it has been reported that nucleolin on the cell surface is a marker of endothelial cells in angiogenic blood vessels (Christian et al., 2003) and participates in both the binding and endocytosis of lactoferrin in target cells (Legrand et al., 2004
). From these previous reports, we speculate that nucleolin is one of the proteins on the cell surface that binds AS and its complex might block the growth of cells induced by midkine. Furthermore, we consider that AS and nucleolin can be translocated into cytoplasm by endocytosis and trigger the intracellular communications. A better understanding of these interactions is likely a key in solving the inhibitory mechanism of AS on the tumor growth.
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Materials and methods |
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Preparation of AS
AS was isolated from the soft body of the giant African snail by proteolysis of defatted tissue and purified by fractional precipitation and ion-exchange chromatography as previously described (Jeong et al., 2001; Kim et al., 1996
).
Measurement of solid tumors induced by LLC in C57BL/6 mice
All animal experiments were carried out in the specific pathogenic-free barrier zone of Clinical Research Institute, Seoul National University Hospital, in accordance with the procedure outlined in the Guide for the Care and Use of Laboratory Animals. Five-week-old, specific pathogen-free male C57BL/6NTac mice were purchased from Samtako Bio (Osan, Korea). LLCs were adjusted to 1 x 106 cells/0.1 ml cold PBS (Lee et al., 2003), and 0.1 ml cell suspension was injected to C57BL/6 mice subcutaneously. After tumor volume was at least 7590 mm3, mice were randomized and received the samples (saline and AS) subcutaneously around the tumor mass once daily for 24 days (Lee et al., 2003
). During the administration period, tumor volumes were measured in two dimensions by caliper every 3 days. Animals were sacrificed by cervical dislocation, and the tumor tissues were fixed in a 40% formaldehyde solution over 24 h.
Alcian bluePAS staining of tumor tissue sections
The fixed tissues were embedded in paraffin and sectioned with thickness of 5 µm on glass slides. The slides were dried at room temperature overnight, and paraffin sections were stained with alcian blue (pH 2.5)PAS (Jeong et al., 2001).
Cell culture
Mouse LLC cells were purchased from American Type Culture Collection (Rockville, MD). A549 (human lung adenocarcinoma cell), KM 1214 (human colon carcinoma cell), and Caki-1 (human kidney carcinoma cell) were obtained from KCLB (Korea Cell Line Bank, Seoul). These cell lines were cultured in DMEM containing 10% fetal bovine serum at 37°C in a humidified, 5% CO2 atmosphere.
Cytotoxicity assay
Cytotoxicity of AS was measured by the MTT method. Briefly, cultured LLC, A549, Caki-1, and KM 1214 cells were trypsinized and spread on 96-well plates at a density of 3 x 104 cells/well. After incubation for 24 h, the various concentrations of diluted AS (0200 µg/ml) were treated to the culture plate. The plates were incubated at 37°C for 24 h in 5% CO2. Cell viability was determined based on mitochondrial conversion of MTT to formazan. The amount of MTT reduced to formazan is indicative of the number of viable cells (Pumphrey et al., 2002). Each well was supplemented with 200 µl 0.5 mg/ml MTT solution for 3 h. The solution was carefully removed from each well and 200 µl dimethyl sulfoxide was added. The plates were gently agitated until the formazan (precipitate) was dissolved. The extent was measured by absorbance at 540 nm using a microplate reader.
Cell binding assay
In this binding assay, the 24-well (1.85 cm diameter) plates were coated with 0200 µg AS in 1 ml of a coating solution and dried overnight at 37°C or room temperature. These plates were blocked with 3% bovine serum albumin (BSA) in D-PBS for 1 h at 37°C and then washed twice with 0.1% BSA in D-PBS (Engbring et al., 2002). The cells, cultured in DMEM containing 0.1% BSA (starvation medium) to remove traces of serum were added to each well and incubated at 37°C for varying times up to 7 h. Each well was washed with 0.1% BSA in D-PBS, and the unbound cells were removed from the plate with trypsin-EDTA. The adherent cells were stained for 10 min with 0.2% crystal violet and washed twice with D-PBS. The cells were lysed with 250 µl 10% SDS, and the absorbance of the solubilized crystal violet was measured at 540 nm. Each assay was performed in triplicate at least four times.
ELISA
To test the stability of an anti-AS antibody, the stock solutions of several GAGs (AS, dermatan sulfate, heparosan, and de-O-sulfated AS) were diluted to proper concentrations (0100 µg/100 µl) in the coating solution. Aliquots of 100 µl diluted solutions were added to a 96-well plate and incubated to dry overnight at 4°C (Alban and Gastpar, 2001). The unbound GAGs were discarded by washing with PBS0.05% Tween 20 (PBST) three times. The plate was necessarily washed with PBST three times for 10 min every step. After blocking by 2% BSA-PBST, the plate was incubated with 100 µl anti-AS antibody tagged with the His-VSV diluted 1:10 overnight at 4°C or at 37°C for 2 h. Then, 100 µl of mouse anti-VSV antibody (clone P5D4) was added to each well and incubated at 37°C for 1 h. After washing, 100 µl anti-mouse IgG conjugated with FITC diluted by 1:100 was added to the plate and incubated at 37°C for 1 h. After last washing, 100 µl of PBST was added to the plate for fluorescence detection. The fluorescence was determined at excitation 490 nm and emission 525 nm.
Immunofluorescence staining for cultured cells
LLC cells were grown on glass coverslips in DMEM containing 10% fetal bovine serum for 24 h. After incubation, AS was added to the cells for 5 h. Each well was washed with PBS and fixed in 4% paraformaldehyde or 0.1% Triton X-100 in 4% PFA for 10 min at room temperature (Goicoechea et al., 2000). To quench autofluorescence, the cells were blocked with 2% BSA-PBS for 1 h at 37°C, followed by 30 min incubation in 0.02 M glycine (Asplund and Heldin, 1994
). Then the cells were incubated with the primary antibody-anti-AS antibody tagged with His-VSV (1:10) overnight at 4°C. After washing with 2% BSA-PBS three times, the cells were incubated with mouse anti-VSV antibody (clone P5D4) at 37°C for 1 h and washed three times. Then anti-mouse IgG-FITC conjugate (1:100 or 1:64) and propidium iodide were treated to the coverslips at 37°C for 1 h. After washing with 0.1% Triton X-100 in PBS and PBST three times, respectively, the coverslips were mounted in an antifading agent. The samples were visualized by fluorescence microscopy.
Biotinylation of cell surface proteins
LLC cells grown in culture dishes were washed with ice-cold PBS (without added serum or protein). Each 1.0 ml of cell suspension (107 cells) was added to 0.5 mg/ml EZ-Link sulfo-NHS-LC-biotin in PBS (pH 7.4) (Goicoechea et al., 2000), and the cells were incubated at 4°C for 45 min. The biotinylation reaction was terminated by addition of 50 mM TrisHCl (pH 7.5). Then the cells were washed in PBS and lysed in lysis buffer (50 mM TrisHCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% NP 40, 1 mM phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors). These lysates were centrifuged at 20,000 x g at 4°C for 30 min. The supernatant was stored at 80°C until being used for affinity chromatography.
Preparation of AS affinity column
A diaminodipropylamine column was extensively washed by water and conjugation buffer, pH 4.7 (0.9% NaCl in 0.1 M 2-[N-morpholino]ethanesulfonic acid), respectively, five times. After washing, 250 mg of AS in 25 ml conjugation buffer was mixed with the gel. Then 750 mg EDC dissolved in conjugation buffer was added to the gel-AS slurry, and they were mixed by shaking for 3 h at room temperature. The resulting affinity column was washed with conjugation buffer containing 1.0 M NaCl. The total amount of the immobilized AS was quantified by carbazole assay (Bitter and Muir, 1962).
Purification of biotinylated surface proteins
Solubilized biotinylated surface proteins from LLC cells were purified on the AS affinity column. The column was incubated with the supernatant (cell lysate) and washed with the running buffer (50 mM TrisHCl, 1 mM EDTA). The column was eluted sequentially with 25 ml of the running buffer containing 0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M NaCl. Each fraction was concentrated by ultrafiltration (molecular weight cutoff 10,000).
SDSPAGE and western blotting
Each fraction was separated by SDSPAGE on 8% and 10% gels. After electrophoresis, gels were stained with either Coomassie blue or 0.1% silver staining solution. Alternatively, gels were electrotransferred to the PVDF membranes at 0.25 A at 4°C for 1617 h in CAPS buffer. The membranes were then blocked for 1.5 h at room temperature with 3% BSA in TBS buffer containing 0.1% Tween 20 (TBST). The blots were incubated with HRP-conjugated streptavidin (1:10,000 dilution) for 1.5 h at room temperature. The membranes were washed four times with TBST for 30 min. For visualization, HRP was reacted to o-phenylenediamine in a phosphate-citrate buffer (pH 5.0). To confirm more specific bands, the electrotransblotted membranes were blocked with 5% skim milk in TBST overnight at 4°C. Blots were incubated with mouse antinucleolin antibodyconjugated HRP (at a 1:50 dilution) for 1.5 h at room temperature. After washing, the membranes were detected with ECL plus reagent.
ESI Q-TOF MS
The each gel spot was reduced by dithiothreitol, alkylated with iodoacetic acid, and digested with trypsin. The resulting peptides were dissolved in 0.1% acetic acid for MS analysis (Wu et al., 2002). To identify proteins by ESI Q-TOF MS, all MS/MS experiments for peptide sequencing were performed using a nano-LC/MS system consisting of an Ultimate HPLC system (LC Packings, Netherlands) and a Q-TOF2 mass spectrometer (Micromass, UK) equipped with a nano-ESI source. Each sample (10 µl) was loaded by the autosampler (FAMOS, LC Packings) onto a C18 trap column (ID 300 µm, length 5 mm, particle size 5 µm; LC Packings) for desalting and concentration at a flow rate 30 µl/min. Then the peptides trapped were back-flushed and separated on a C18 nano-column (ID 75 µm, length 150 mm, particle size 5 µm; LC Packings). The gradient used was 0% acetonitrile for 10 min, followed by 0% to 50% acetonitrile over 80 min and 50% acetonitrile for 10 min at a flow rate 150 nl/min. In the nano-ESI source, the end of the capillary tubing from the nano-LC column was connected to pico-tip silica tubing (ID 5 µm; New Objectives, USA). The applied voltage to liquid junction to produce an electrospray was 1.52.0 kV, and cone voltage was set at 30 V. Argon was introduced as a collision gas at a pressure of 10 psi. MS/MS spectra were acquired in data-dependant MS/MS mode, for which collision energy was increased to 25, 30, and 35 eV. For protein identification, MS/MS spectra were searched by MASCOT (Matrix Science, UK) or manually sequenced by Masslynx software 3.5 (Micromass, UK). Proteins containing at least one significant peptide (
individual score) were selected from database search results.
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Acknowledgements |
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Abbreviations |
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References |
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