2Department of Biochemistry, Osaka University Medical School, 22 Yamadaoka, Suita, Osaka 5650871, Japan, 3Department of Surgical Oncology, Osaka University Medical School, 22 Yamadaoka, Suita, Osaka 5650871, Japan, and 4Peptide Institute, Protein Research Foundation, 412 Ina, Minoh, Osaka 5620015, Japan
Received on April 4, 2000; revised on June 2, 2000; accepted on June 2, 2000.
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Abstract |
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Key words: annexin V/N-glycan/bisecting GlcNAc/lectin/N-acetylglucosaminyltransferase III
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Introduction |
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It has been proposed that a bisected sugar chain, a unique structure found in N-linked oligosaccharides, is responsible for a variety of biological functions. This structure is formed by the attachment of a ß14 GlcNAc residue to a core ß-mannose the reaction of which is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III). This specific transferred GlcNAc residue is referred to as a "bisecting GlcNAc." It has been suggested that GnT-III plays a regulatory role in the biosynthesis of N-linked oligosaccharides because the addition of the bisecting GlcNAc by GnT-III inhibits further branching formed via the action of GnTs-IV and V (Schachter, 1986; Gu et al., 1993
). As has been shown in our previous studies, an elevation of GnT-III expression levels via the introduction of its cDNA into cells and the resulting increase in the level of bisected sugar chains lead to some significant alterations in cells, including the suppression of metastasis in melanoma cells (Yoshimura et al., 1995
), the reduction of gene expression of hepatitis B virus (Miyoshi et al., 1995
), and an altered sorting of glycoproteins in cells (Sultan et al., 1997
). In addition, it is particularly noteworthy that GnT-III-transfected K562 cells were found to be resistant to the cytotoxicity of the natural killer cell and to give rise to spleen colonization in athymic mice (Yoshimura et al., 1996
). These observations suggest that bisected sugar chains are involved in a variety of cellular functions. However, it seems unlikely that all of these data can be explained exclusively by a modification in N-glycan biosynthesis, e.g., a reduction in branch formation, but, rather, mediation by a protein molecule which recognizes the bisected sugar chains may also play a role in these processes.
The aim of this study is to purify the bisecting GlcNAc-binding protein by affinity chromatography using an agalacto bisected sugar chain-immobilized column, in order to better understand the mechanism by which the bisected sugar exerts its biological function.
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Results |
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Discussion |
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In the purification, annexin V was not eluted from the bisected sugar chain-immobilized Sepharose column by 100 mM GlcNAc, and in the flow cytometric analysis, N-acetylchitobiose did not significantly affect the binding of annexin V to the GnT-III-transfected cells (data not shown). Therefore, it seems unlikely that the protein recognizes only a ß-GlcNAc residue. Since agalacto biantennae were used to establish the requirement of the bisecting GlcNAc for annexin V to bind to N-linked oligosaccharide in the surface plasmon resonance analysis, the effects of Gal, sialic acid, or other sugar residues in the antennae on the binding of annexin V is not clear at present. However, it is known that most of the complex type N-glycans produced by K562 cells are sialylated or sulfated but not the agalactoforms (Yoshima et al., 1982). Considering the fact that annexin V abolished the E-PHA binding to the GnT-III-transfected cells by 7080%, it would be reasonable that a major fraction of the sugar chains associated with the inhibition might contain various terminal sequences including ß-galactosylation, sialylation, and sulfation. As suggested by these and the comparison of the interactions with oligosaccharides from
-globulin and transferrin (Figure 6C), it seems certain that the bisecting GlcNAc is critical for the binding of annexin V to N-glycans while it appears that peripheral parts of the antennae are not absolutely required. Therefore, it is more likely that the binding of annexin V is dependent on the region around the core structure which contains the bisecting GlcNAc rather than the peripheral structures of the sugar chains. However, the issue of whether annexin V recognizes an overall conformation of the bisected sugar chains (Narasimhan, 1982
; Brisson and Carver, 1983
; Taniguchi et al., 1996
) or only a particular monosaccharide, the bisecting GlcNAc remains to be explored.
Annexin V is also known as a calcium-dependent phospholipid-binding protein and is widely used at present for the detection of apoptosis because the protein binds to the cell surface of the apoptotic cells via translocated phosphatidyl serine. Nevertheless, it was found that the nature of the annexin V binding to the cell surface of the GnT-III-transfected cells is distinct from that to the apoptotic cells, particularly with respect to the inhibition of binding by phospholipids. The nature of the binding of annexin V to the sugar chains was not affected by phospholipids, suggesting that the binding site for the bisected sugar chains in annexin V is different from that for phospholipids.
Annexin V has been studied extensively with a goal of elucidating its biological function and these studies have implicated inhibitory activities in blood coagulation and for protein kinase C (Iwasaki et al., 1987; Huber et al., 1990
; Schlaepfer et al., 1992
). These functions of annexin V could be modulated by interaction with bisected sugar chains. On the other hand, it is also possible that a variety of the biological functions of the bisecting GlcNAc, which have been suggested by our previous studies (Taniguchi et al., 1996
, 1999) are, at least in part, mediated by annexin V. Therefore, the identification of this novel carbohydrate binding activity of annexin V provides a clue for the elucidation of the mechanisms of signal transduction via the bisected sugar chains.
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Materials and methods |
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Preparation of oligosaccharides and affinity columns
In this study, two different oligosaccharide-coupled Sepharose columns were prepared for affinity chromatography. N-linked oligosaccharides were prepared from bovine -globulin as described previously (Nishikawa et al., 1992
; Uozumi et al., 1996a
), and digested with sialidase and ß-galactosidase. The agalacto non-bisected oligosaccharides were purified from the digested oligosaccharides by HPLC using a TSK-gel ODS-80 column (4.6 x 150 mm). Aliquots of the purified oligosaccharides were pyridylaminated and then subjected to structural analysis. A GnGn(F)-bi-Asn ligand column was prepared by reacting 150 µmol of the agalacto non-bisected oligosaccharides with 15 ml of activated CH-Sepharose 4B according to the manufacturers instructions. A bisected sugar chain, Gn(Gn)Gn(F)-Asn, was prepared from the purified oligosaccharides described above as previously reported (Nishikawa et al., 1990
). The structures were confirmed by reversed phase HPLC, after pyridylamination. A Gn(Gn)Gn(F)-Asn-Sepharose column was prepared by reacting 80 µmol of Gn(Gn)Gn(F)-Asn with 8 ml of activated CH-Sepharose 4B according to the manufacturers protocol. N-Linked oligosaccharides were also prepared from transferrin as described above.
Flow cytometric analysis
Flow cytometric analysis using an FITC-labeled lectin was carried out as described previously (Yoshimura et al., 1996; Sultan et al., 1997
). In brief, approximately 1 x 107 cells were washed with ice-cold PBS and resuspended in 100 µl of 10 mM HEPES/NaOH, pH7.4, 140 mM NaCl and 2.5 mM CaCl2 buffer. FITC-labeled E-PHA was added to the cell suspension to a final concentration of 5 µg/ml, and the resulting cells were subjected to flow cytometry (FACSORT). Unstained cells were used as controls. The data were processed using the Macintosh Cell Quest computer program.
When the binding activity of the protein was evaluated, based on the inhibition of E-PHA binding, the cells were preincubated with fractions from each purification step or 600 µM human annexin V for 15 min at 4°C prior to flow cytometric analysis. For the binding assay of the purified protein or human annexin V, their FITC conjugates were used for the flow cytometry, as carried out for FITC-labeled E-PHA. The final concentration of annexin V is 3 µM. When the effect of exogenous phospholipid on the binding activity of annexin V was analyzed, phospholipid vesicles were added to a final concentration of 1-palmitoyl-2-oleoyl-phosphatidyl serine of 5 µM.
Purification and column chromatography
Step 1. Porcine spleen (250 g) was homogenized in 4 volumes of 10 mM Tris-HCl, pH 7.4, containing 0.25 M sucrose, 1 mM benzamidine hydrochloride, and 0.03% sodium azide, with a Polytron homogenizer (Brinkmann Instruments).
Step 2. After centrifugation at 900 x g for 10 min, the resulting supernatant was further centrifuged at 105,000 x g for 1 hr. The pellets were resuspended in a solution of 10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 2 mM EDTA, and 0.5% Triton X-100 buffer, and the proteins were then extracted by gentle stirring for 2 days at 4°C, followed by centrifugation at 105,000 x g for 1 h. The resultant supernatants were collected and concentrated using a YM 30 membrane.
Step 3. The above extracts were applied to a DEAE ion-exchange column (16x10 cm; Pharmacia HR 16/10), pre-equilibrated with 10 mM Tris-HCl buffer, pH 7.4, 5 mM CaCl2, and 0.01% Triton X-100. The column was washed with the same buffer and then subjected to elution by a linear gradient of 00.5 M NaCl in the starting buffer. The binding activities in the collected fractions were monitored by determining the inhibition of E-PHA binding to the GnT-III-transfected K562 cells using flow cytometry, as described under Flow cytometric analysis.
Step 4. Active fractions obtained from the DEAE column were collected, and the buffer was changed to 10 mM Tris-HCl buffer, pH7.4, containing 5 mM CaCl2 using a YM30 membrane. These fractions were then applied to a GnGn(F)-bi-Asn-Sepharose 4B column (16 x 10 cm; Pharmacia HR 16/10), which had been equilibrated with 10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 5 mM CaCl2, and 0.01% Triton X-100 buffer. The unbound fractions which showed inhibition activity for E-PHA binding were collected.
Step 5. The pooled fractions were applied to a Gn(Gn)Gn(F)-Asn Sepharose 4B column (10 x 8 cm; Pharmacia HR 10/10), as described above. Proteins which bound to the column were eluted with 0.5 M NaCl in the washing buffer.
SDS-polyacrylamide gel electrophoresis
Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the Laemmlis method (Laemmli, 1970) under the reducing conditions. The gel was stained with Coomassie brilliant blue G-250.
Determination of partial amino acid sequences
The purified proteins were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane after SDS-PAGE. The protein band was excised and subjected to an Applied Biosystem 473A Protein Sequencer for the N-terminal sequencing analysis. The internal amino acid sequences were determined by sequencing the peptide fragments which were obtained from proteolytic digestion of the protein, as described previously (Uozumi et al., 1996b).
Cell culture and cell lines
K562 cells, a human erythroleukemia cell line, were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in RPMI 1640 supplemented with 100 µg/ml of kanamycin sulfate, 50 U/ml of penicillin, and 10% fetal calf serum in 5% CO2 humidified air at 37°C. They were transfected with an expression plasmid in which rat GnT-III cDNA was subcloned into a pHOOKTM-2 by electroporation (Chu et al., 1987). Mock transfectants were also prepared by introduction of only the vector. The GnT-III-transfected cells and mock cells were selected with 1 mg/ml of geneticin.
Induction and detection of apoptosis
The cells were washed several times with PBS and then cultured at 1 x 107 cells/ml in a medium which contained 1 µg/ml actinomycin D for 9 h. The DNA ladder was detected using Apopladder ExTM kit according to the manufacturers protocol.
Preparations of phospholipid vesicles
Small unilamellar phospholipid vesicles were prepared as described previously (Gabriel and Roberts, 1984; Taits et al., 1989
). Aliquots of phospholipid stock solutions in chloroform were mixed to yield the desired molar ratios (60% of 1-palmitoyl-2-oleoyl-phosphatidyl choline, 20% diheptanoyl-phosphatidyl choline, 20% 1-palmitoyl-2-oleoyl-phosphatidyl serine) and the chloroform was subsequently evaporated with nitrogen. The evaporated phospholipids were then dissolved in 0.05 M HEPES-Na, pH 7.4, 0.1 M NaCl, 3 mM NaN3 by sonication for 5 min on ice, followed by equilibration at 4°C, overnight.
Analysis of annexincarbohydrate interaction by surface plasmon resonance
Annexin V was dissolved in 10 mM acetate (pH 4.0) and immobilized on a CM5 sensor chip. In the measurement, oligosaccharides in 10 mM HEPES-Na, pH 7.4, 140 mM NaCl and 2.5 mM CaCl2 were injected at a flow rate of 10 µl/min. After a dissociation phase, the sensor surface was regenerated by a 10 µl pulse of 10 mM glycine-HCl, pH 2.0, a regenerating buffer. The flow cell to which nothing was immobilized was used to subtract the contribution of nonspecific interactions. Kinetic parameters were calculated using the BIA Evaluation 2.1 program according to the manufacturers recommendations.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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