A novel carbohydrate binding activity of annexin V toward a bisecting N-acetylglucosamine

Cong-Xiao Gao-Uozumi2, Naofumi Uozumi2,3, Eiji Miyoshi2, Kaoru Nagai2, Yoshitaka Ikeda2, Tadashi Teshima4, Katsuhisa Noda2, Tetsuo Shiba4, Koichi Honke2 and Naoyuki Taniguchi1,2

2Department of Biochemistry, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan, 3Department of Surgical Oncology, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565–0871, Japan, and 4Peptide Institute, Protein Research Foundation, 4–1–2 Ina, Minoh, Osaka 562–0015, Japan

Received on April 4, 2000; revised on June 2, 2000; accepted on June 2, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
A bisecting GlcNAc-binding protein was purified from a Triton X-100 extract of a porcine spleen microsomal fraction using affinity chromatography, in conjunction with an agalacto bisected biantennary sugar chain-immobilized Sepharose. Since the erythroagglutinating phytohemagglutinin (E-PHA) lectin preferentially binds to sugar chains which contain the bisecting GlcNAc, during purification the binding activity of the protein was evaluated by monitoring the inhibition of lectin binding to the N-acetylglucosaminyltransferase III (GnT-III)-transfected K562 cells which express high levels of the bisecting GlcNAc. The molecular mass of the purified protein was found to be 33 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By sequencing analysis, the isolated protein was identified as annexin V. Flow cytometric analysis showed that fluorescein-labeled annexin V binds to the GnT-III-transfected cells but not to mock cells, and that the binding was not affected by the addition of phospholipids. Furthermore, surface plasmon resonance measurements indicated that annexin V binds to the agalacto bisected biantennary sugar chain with a Kd of 200 µM while essentially no binding was observed in the case of the corresponding non-bisected sample. These results suggest that annexin V has a novel carbohydrate binding activity and may serve as an endogenous lectin for mediating possible signals of bisecting GlcNAc, which have been implicated in a variety of biological functions.

Key words: annexin V/N-glycan/bisecting GlcNAc/lectin/N-acetylglucosaminyltransferase III


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
It is a well-known fact that oligosaccharide moieties in glycoproteins play roles in cell adhesion, intracellular glycoprotein sorting, development, differentiation, and carcinogenesis (Rademacher et al., 1988Go). It is important to note that it is possible to obtain structural information on oligosaccharides, i.e., that they can be recognized or "decoded" through the binding of lectins. The list of lectin-mediated processes includes diverse biological phenomena, which includes intracellular routing of glycoproteins, cell–cell adhesion and phagocytosis (Inohara et al., 1996Go; Kappler et al., 1997Go; Ferguson et al., 1999Go). In fact, a number of animal lectins have been found in various animal cells, tissues, and body fluids (Weis et al., 1998Go). These molecules recognize specific oligosaccharide structures, and appear to play a key role in the transduction of information or signals which are carried by the oligosaccharides.

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 ß1–4 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, 1986Go; Gu et al., 1993Go). 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., 1995Go), the reduction of gene expression of hepatitis B virus (Miyoshi et al., 1995Go), and an altered sorting of glycoproteins in cells (Sultan et al., 1997Go). 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., 1996Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Purification of a bisecting GlcNAc-binding protein
A bisecting GlcNAc-binding protein was purified from porcine spleen extracts by ion-exchange chromatography and successive affinity chromatographies. To assess bisected sugar chain-binding activity, we employed an inhibition assay in which the protein competes with E-PHA lectin for binding to the GnT-III-transfected K562 cells. The E-PHA binds strongly to the GnT-III-transfected cells but not to mock cells (Yoshimura et al., 1996Go), suggesting that the lectin binds to the bisected sugar chains on the cell surface of the GnT-III-transfected cells (Cummings and Kornfeld, 1982Go; Narasimhan et al., 1986Go). The putative bisecting GlcNAc-binding protein was eluted from the DEAE-ion exchange column in a range of 150–200 mM NaCl (Figure 1A). These active fractions were then subjected to an agalacto non-bisected oligosaccharide-Asn-coupled Sepharose column (Figure 1B), which was employed to remove proteins which are nonspecifically bound to irrelevant sugar chains or the matrix. A protein fraction which had passed through the column was subsequently applied to a bisected sugar chain Gn(Gn)Gn(F)-Asn-coupled Sepharose column, and the protein which bound to the latter column was purified as being the presumable bisecting GlcNAc-binding protein. As shown in Figure 1C, the fractions which adsorbed to the Gn(Gn)Gn(F)-Asn column had a high specific activity. SDS-PAGE analysis demonstrated that a protein with a molecular mass of 33 kDa exhibits the binding activity (Figure 2). When one unit of inhibitory activity against the E-PHA binding was tentatively defined as the amount of protein required for a 10% decrease in the mean fluorescence intensity, purification and yield from Triton X-100 extracts were roughly estimated to be about 7000 fold and 10%, respectively. By this purification procedure, 80 µg of the purified protein was obtained from 250 g of spleen. Thus, successive affinity chromatographies involving the combined use of nonspecific ligand-immobilized and specific ligand-immobilized columns enabled us to purify reasonable quantities of the protein which specifically binds to the bisected sugar chains.



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Fig. 1. A typical chromatographic elution pattern in the purification of the bisecting GlcNAc-binding protein from porcine spleen. Column chromatography profiles are shown for DEAE ion-exchange chromatography (A), GnGn(F)-bi-Asn-Sepharose 4B (B), and Gn(Gn)Gn(F)-Asn-Sepharose 4B (C). Solid circles represent the absorbance at 280 nm. Open circles indicate shifts in fluorescent intensity in the inhibition using flow cytometry. Bars indicate the fractions which were collected for further investigations. Details of the analysis are described under Materials and methods.

 


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Fig. 2. SDS-PAGE analysis of the purified bisecting GlcNAc-binding protein. Fractions eluted from a GnGn(F)-Asn- (A) and Gn(Gn)Gn(F)-Asn-Sepharose (B) were analyzed by SDS-PAGE. Lane numbers in these panels correspond to the fraction numbers in Figure 1B and 1C, respectively. (C) A profile of the purified bisecting GlcNAc-binding protein is shown. The molecular masses of marker proteins are indicated on the left.

 
Identification of the purified protein as annexin V
When the purified protein was subjected directly to an automated Edman degradation procedure, no PTH-amino acids were produced, indicating that the N-terminus is blocked. To obtain the internal amino acid sequence, the protein was transferred onto a PVDF (polyvinylidene difluoride) membrane and digested by lysylendopeptidase, and the resulting peptides were separated by reversed phase HPLC. Although a total of ten components were isolated and sequenced, the amino acid sequences of six peptides were unequivocally determined. As shown in Figure 3, the peptide sequences were found to be essentially identical to annexin V. Thus, it may reasonably be concluded that the bisecting GlcNAc binding-protein purified from porcine spleen is a member of annexin family, and is probably a porcine orthologue of annexin V.



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Fig. 3. Sequence alignment between human annexin V and partial peptides derived from the purified protein. Partial sequences of the purified protein are shown with the entire sequence of human annexin V. Identical amino acids are underlined.

 
The binding of annexin V to the cells expressing bisected sugar chains
To confirm the carbohydrate-binding activity of annexin V, we investigated the issue of whether human annexin V is capable of binding to the GnT-III-transfected cells. As shown in Figure 4A, preincubation of the cells with annexin V in the presence of Ca2+ led to a significant decrease in the binding of the FITC-E-PHA to the GnT-III-transfected cells. On the other hand, annexin V did not inhibit the bindings of FITC-L-PHA (Figure 4B) and FITC-DSA (data not shown), both of which have specificities unrelated to the bisecting GlcNAc. It thus appears that annexin V competes with E-PHA for binding to the oligosaccharides. Furthermore, the binding assay using FITC-labeled annexin V showed that the annexin V binds significantly to the GnT-III-transfected cells while the level of binding to mock cells was nearly negligible (Figure 4C). These results suggest that annexin V is capable of specifically binding to the bisected sugar chains that are expressed on the cell surface as the result of the introduction of the GnT-III cDNA. These results support the conclusion that annexin V acts as a bisecting GlcNAc-binding protein.



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Fig. 4. The novel carbohydrate binding activity of annexin V. (A) Inhibition of E-PHA binding to the transfected cells by annexin V. (B) Effect of annexin V on L-PHA binding. In both experiments, the cells were preincubated with (I) or without (II) preincubation of annexin V prior to incubation with FITC-conjugated E-PHA. Autofluorescence intensity of the cells is also shown (III). (C) The binding of FITC-labeled annexin V to mock cells (I) and the GnT-III transfected cells (II).

 
Since it is known that annexin V binds to apoptotic cells via cell membrane phospholipids, the effect of phospholipids on the binding of annexin V to the transfected cells was examined, in order to further confirm the interaction of annexin V with the oligosaccharides on the cell surface. While no binding of annexin V to the untreated mock cells was observed, the protein was found to strongly bind to apoptotic mock cells (Figure 5A,B). As expected, this binding was nearly completely inhibited by exogenously added phospholipids. On the other hand, the addition of phospholipids had no effect on the binding of annexin V to the GnT-III-transfected cells (Figure 5C). These results indicate that the binding of annexin V to the GnT-III-transfected cells involves the presence of bisected sugar chains but not phospholipids and, therefore, is distinct from phospholipid-mediated binding as found in the apoptotic cells.



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Fig. 5. The effect of exogenous phospholipids on the carbohydrate binding activity of annexin V. The binding of annexin V to the normal mock cells (A), apoptotic mock cells (B), and GnT-III transfected K562 cells (C). Peaks I, II, and III represent unstained cells, cells incubated without and with phospholipids, respectively.

 
Interaction of annexin V with bisected sugar chains
The binding of annexin V to bisected sugar chains was further characterized in more detail by surface plasmon resonance using oligosaccharides. In this analysis, annexin V was immobilized to a CM5 sensor chip. When a pyridylaminated agalacto bisected biantennary was used as the ligand, binding to the immobilized annexin V was observed (Figure 6A). The Kd value for the binding was determined to be ~200 µM by calculation using steady state data. On the other hand, no significant binding was detected in the case of the corresponding nonbisected sugar chain, agalacto biantennary (Figure 6B). This suggests that annexin V does not interact with N-linked oligosaccharides in which the bisecting GlcNAc is absent. In the measurements using total oligosaccharides prepared from {gamma}-globulin and transferrin, it was found that {gamma}-globulin-derived sugar chains bind to the immobilized annexin V whereas those from transferrin exhibit no binding (Figure 6C). This observation is consistent with the suggestion that annexin V specifically binds to the bisecting GlcNAc since bisected sugar chains are contained in {gamma}-globulin but not by transferrin (Spik et al., 1975Go; Takahashi and Ishii, 1987Go; Yamashita et al., 1989Go). These analyses support the view that the bisecting GlcNAc is important in the binding of annexin V to N-linked sugar chains. In addition, while these bindings were almost completely abolished by the addition of 3 mM EDTA in the assay, phospholipids (5 µM) had no effect on the binding (data not shown). These results suggest that the interaction of annexin V with the oligosaccharides requires Ca2+ and involves the binding site distinct from that for phospholipids.



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Fig. 6. Surface plasmon resonance measurements of the interaction of sugar chains with immobilized annexin V. Pyridylaminated agalacto bisected biantennary sugar chains (A) and corresponding non-bisected ones (B) were used as the ligands. The traces a–f indicate measurements at ligand concentrations of 80, 60, 40, 20, 10, and 5 µM, respectively. Total N-linked oligosaccharides from glycoproteins were used as the ligands (C). Traces (I) and (II) are measurements using sugar chains from {gamma}-globulin and transferrin, respectively. The concentrations of the ligands were 1 mM.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Annexin is a family of calcium-dependent phospholipid-binding proteins. A variety of biological activities have been reported for this family (Wallner et al., 1986Go; Creutz, 1992Go; Emans et al., 1993Go; Tressler et al., 1993Go, 1994; Chung et al., 1996Go; Lafont et al., 1998Go; Ortega et al., 1998Go; Pencil and Toth, 1998Go). Recently, it was reported that annexin V exhibits a lectin-like activity toward glycosaminoglycans in a calcium-dependent manner (Ishitsuka et al., 1998Go; Capila et al., 1999Go). In the present study, annexin V was identified as a bisecting GlcNAc-binding protein as the result of its isolation and identification of an unidentified carbohydrate binding protein. In addition, the carbohydrate binding properties were characterized by flow cytometric analysis and surface plasmon resonance analysis. The findings show that annexin V binds to both a free bisected oligosaccharide and the same bisected sugar chains on the cell surface.

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., 1982Go). Considering the fact that annexin V abolished the E-PHA binding to the GnT-III-transfected cells by 70–80%, 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 {gamma}-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, 1982Go; Brisson and Carver, 1983Go; Taniguchi et al., 1996Go) 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., 1987Go; Huber et al., 1990Go; Schlaepfer et al., 1992Go). 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., 1996Go, 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.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
2-Amino pyridine and neuraminidase (Clostridium perfringens ) were obtained from Wako Pure Chemicals (Osaka, Japan). ß-Galactosidase (Aspergillus sp.) was obtained from Toyobo Co. (Shiga, Japan). Toyo-pearl HW-40, TSK-gel ODS-80TM, and Amide-80 columns were purchased from Tosoh (Tokyo, Japan). Bovine {gamma}-globulin, human transferrin, human annexin V, and Coomassie brilliant blue G250 were purchased from Sigma Chemical (St. Louis, MO). Pronase (Streptomyces griseus), lysylendopeptidase, kanamycin sulfate and actinomycin D were purchased from Seikagaku Corp. (Tokyo, Japan). Annexin V fluorescein conjugate was obtained from Southern Biotechnology Associates Inc. (USA). Activated CH-Sepharose 4B and the CM5 sensor chip were purchased from Amersham Pharmacia (Buckinghamshire, UK). RPMI 1640 medium, fetal calf serum, and geneticin were obtained from GIBCO/BRL (USA). Penicillin G potassium was purchased from Banyu Pharmaceutical Co., Ltd. (Tokyo, Japan). YM30 membrane and polyvinylidene difluoride membrane were obtained from Amicon (USA) and Millipore (USA), respectively. A pHOOKTM-2 vector was obtained from Invitrogen (Groningen, Netherlands), and Apopladder ExTM kit was purchased from Takara Co. (Kyoto, Japan).

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 {gamma}-globulin as described previously (Nishikawa et al., 1992Go; Uozumi et al., 1996aGo), 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 manufacturer’s instructions. A bisected sugar chain, Gn(Gn)Gn(F)-Asn, was prepared from the purified oligosaccharides described above as previously reported (Nishikawa et al., 1990Go). 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 manufacturer’s 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., 1996Go; Sultan et al., 1997Go). 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 0–0.5 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 sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the Laemmli’s method (Laemmli, 1970Go) 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., 1996bGo).

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., 1987Go). 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 manufacturer’s protocol.

Preparations of phospholipid vesicles
Small unilamellar phospholipid vesicles were prepared as described previously (Gabriel and Roberts, 1984Go; Taits et al., 1989Go). 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 annexin–carbohydrate 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 manufacturer’s recommendations.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This study was supported, in part, by the Grant-in-Aid for Scientific Research on Priority Areas (No. 10178104) from the Ministry of Education, Science, Sports and Culture of Japan. We thank our many colleagues in our laboratory for valuable discussion, technical advice, and criticism of this work.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 

GnT-III, UDP-N-acetylglucosamine:ß-D-mannoside ß-1,4-N-acetylglucosaminyltransferase III; E-PHA, erythroagglutinating phytohemagglutinin; L-PHA, leukoagglutinating phytohaemagglutinin; DSA, Datura stramonium agglutinin; GlcNAc, N-acetylglucosamine; GnGn(F)-bi-Asn, GlcNAcß1–2Man{alpha}1–6(GlcNAcß1–2Man{alpha}1–3)Manß1–4GlcNAcß1–4(Fuc{alpha}1–6)GlcNAc-Asn; Gn(Gn)Gn(F)-Asn, GlcNAcß1–2Man{alpha}1–6(GlcNAcß1–2Man{alpha}1–3)(GlcNAcß1–4)-Manß1–4GlcNAcß1–4(Fuc{alpha}1–6)GlcNAc-Asn; HPLC, high performance liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; PTH-amino acid, phenylthiohydantoin amino acid; PVDF, polyvinylidene difluoride; PA, 2-aminopyridine; PBS, phosphate-buffered saline.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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