Glycobiology Advance Access originally published online on March 10, 2006
Glycobiology 2006 16(6):564-571; doi:10.1093/glycob/cwj100
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ß1,4-N-Acetylglucosaminyltransferase III potentiates ß1 integrin-mediated neuritogenesis induced by serum deprivation in Neuro2a cells
2 Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; and 3 Division of Regulatory Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsusima, Aobaku, Sendai, Miyagi 981-8558, Japan
1 To whom correspondence should be addressed; e-mail: jgu{at}biochem.med.osaka-u.ac.jp or proftani{at}biochem.med.osaka-u.ac.jp
Received on January 13, 2006; revised on February 27, 2006; accepted on March 2, 2006
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Abstract |
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Aspects of the biological significance of the bisecting N-acetylglucosamine (GlcNAc) structure on N-glycans introduced by ß1,4-N-acetylglucosaminyltransferase III (GnT-III) in Neuro2a cell differentiation are demonstrated. The overexpression of GnT-III in the cells led to the induction of axon-like processes with numerous neurites and swellings, in which ß1 integrin was localized, under conditions of serum starvation. This enhancement in neuritogenesis was suppressed by either the addition of a bisecting GlcNAc-containing N-glycan or erythroagglutinating phytohemagglutinin (E4-PHA), which preferentially recognizes the bisecting GlcNAc. GnT-III-promoted neuritogenesis was also significantly perturbed by treatment with a functional blocking anti-ß1 integrin antibody. In fact, ß1 integrin was found to be one of the target proteins of GnT-III, as confirmed by a pull-down assay with E4-PHA. These data suggest that N-glycans with a bisecting GlcNAc on target molecules, such as ß1 integrin, play important roles in the regulation of neuritogenesis.
Key words: bisecting GlcNAc / glycosyltransferase / GnT-III / integrin / neurite formation
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Introduction |
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N-Glycosylation is one of the most common post-translational modifications of secretory or membrane proteins in eukaryotes, and a growing body of evidence suggests that N-linked glycans play important roles in protein folding, intra-and/or intercellular trafficking of N-glycosylated proteins (Varki, 1993
; Dwek, 1995; Saxon and Bertozzi, 2001). Metazoans produce various glycosyltransferases/glycosidases in the Golgi that remodel the structure of N-glycans, forming high mannose-, hybrid-, and complex-type N-glycans. The biological significance of N-glycans in the nervous system has been demonstrated in GnT-I-null mice and in neuron-specific GnT-I-deficient mice because they showed defects in neural tube formation, severe locomotor deficits, paralysis, tremors, and early postnatal death (Metzler et al., 1994; Ye and Marth, 2004). In most tissues, the outer N-acetylglucosamine (GlcNAc) residues of biantennary N-glycans (Figure 1A) are present in galactosylated or sialylated form, whereas it is noteworthy that the ratio of the GlcNAc-terminated N-glycans, GlcNAcß1-2Man
1-6 (GlcNAcß1-4)(Man1-3)Manß1-4GlcNAcß1-4(Fuc1-6) GlcNAc (BA-1) and GlcNAcß1-2Man1-6(GlcNAcß1-4) (GlcNAcß1-2Man1-3)Manß1-4GlcNAcß1-4(Fuc1-6) GlcNAc (BA-2), is abundant in the brain (Shimizu et al., 1993). This feature may be an important clue to our understanding of the roles of sugar chains in the brain because nonreducing sugars are generally thought to be significant for sugar-dependent molecular recognition events in which a variety of carbohydrate-binding proteins such as siglec or galectin are involved (Varki, 1992; Barondes et al., 1994).
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One of the interesting features of two brain-enriched N-glycans (BA-1 and BA-2) is that they contain the bisecting-GlcNAc residue, which is introduced by catalysis by ß1,4-N-acetylglucosaminyltransferase III (GnT-III) (Narasimhan, 1982; Nishikawa et al., 1992; Ihara et al., 1993). GnT-III is a unique glycosyltransferase, since the introduction of the bisecting GlcNAc leads to a drastic change in the conformation of N-glycans (Gu and Taniguchi, 2004). As a consequence, other glycosyltransferases such as GnT-V are no longer able to act on sugar chains (Schachter, 1986). Consistent with this finding, the ectopic expression of GnT-III in various types of cells results in a significant reduction in multiantennary oligosaccharides, which are regulated by other GlcNAc transferases (GnT-IV/V) (Yoshimura et al., 1995; Koyota et al., 2001). In addition, it has been shown that galactosylation in N-glycans is significantly impaired in GnT-III-overexpressed endothelial cells (Koyota et al., 2001).
We previously reported that GnT-III down-regulates neurite outgrowth induced by stimulation by epidermal growth factor (EGF) through the Ras/extracellular signal-regulated kinase (ERK) signaling pathway or nerve growth factor (NGF) through the down-regulation of the dimerization of TrkA in PC12 cells (Ihara et al., 1997; Gu et al., 2004). Therefore, the question arises as to why higher levels of bisected N-glycans are expressed in the developing brain. To understand the biological significance of bisected N-glycans in cell differentiation, a simple neuronal differentiation model cell, Neuro2a cells, was selected, since it has the capacity to differentiate upon activation of the ERK cascade when grown under conditions of serum deprivation (Lopez-Maderuelo et al., 2001).
In the present study, the effects of the introduction of the bisecting GlcNAc on neuritogenesis induced by serum deprivation were investigated in Neuro2a cells. The findings show that the overexpression of GnT-III resulted in a significant enhancement in neuritogenesis as well as in swelling formation, which could be significantly suppressed by the exogenous addition of bisecting GlcNAc-containing biantennary N-glycans or erythroagglutinating phytohemagglutinin (E4-PHA) lectin, which preferentially recognizes bisecting GlcNAc structures. These results, for the first time, demonstrate a significant involvement of the bisecting GlcNAc on target proteins, such as ß1 integrin, in the regulation of serum deprivation-induced neuritogenesis.
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Results |
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Overexpression of GnT-III in Neuro2a cells potentiates serum deprivation-induced neuritogenesis as well as swelling formation
To investigate the biological significance of the bisecting GlcNAc in neural cells, stable GnT-III transfectants were generated in a mouse neuroblastoma cell line, Neuro2a cells, which is a neuronal differentiation model cell line. The levels of expression of GnT-III were verified by immunoblot analysis and were found to be similar among the established clones containing an enzymatic inactive GnT-III, the D321A mutant (data not shown). The GnT-III enzymatic activity in each transfectant was measured using a fluorescence-labeled agalactobiantennary glycan as an acceptor, and product formation was determined by high-performance liquid chromatography (HPLC). As shown in Figure 1B, the enzymatic activity in GnT-III transfectants was increased by
10-fold, while the activity in D321A mutants was 1/4-fold, compared with that in the mock transfectants, suggesting that the D321A mutants serve as a dominant negative (DN) molecule involved in competition with the corresponding endogenous wildtype GnT-III, as described previously (Ihara et al., 2002).
Neuro2a cells exhibit neurite outgrowth in response to serum deprivation (Wu et al., 1998; Cortes-Canteli et al., 2002). The cells were round in shape in the presence of serum but became spread and extended neurites after 24 h of serum starvation (Figure 1C). Interestingly, the overexpression of GnT-III in Neuro2a cells resulted in an enhancement of neuritogenesis, concomitant with an increase of swelling (Figure 1C) which is morphologically similar to changes that had been previously observed in hippocampal, cortical cultures (Ahmari et al., 2000; Washbourne et al., 2002). To further understand morphological changes in GnT-III-promoted neuritogenesis, two major cytoskeletal components, F-actin (red) and phosphorylated high-molecular-weight neurofilament protein (NF-H) (green) in these transfectants, were detected by fluorescent staining. F-actin plays a mechanical role in cellular morphogenesis, cell motility or adhesion and is a major composition of neurites observed often in neuronal cells, while NF-H is one of structural components of axons and used as an immunocytochemical marker of axons. As shown in Figure 2, the NF-H-positive processes (axon-like processes) were the green relatively thick threads moving out from the green neuron, whereas the neurites were the red thin threads that start on the green axon-like processes. We found that neurite formation was dramatically increased in the GnT-III transfectants (Figure 2B), compared with that in mock transfectants. Interestingly, the overexpression of GnT-III in Neuro2a cells resulted in an increase in the number of cells bearing multiple axon-like processes, while overexpression of the D321A mutant decreased neurite formation (Figure 2D). Generally, neurons initially generate several equivalent neurites, and then neurons begin to polarize when one neurite becomes an axon; the other neurites then become dendrites (Dotti et al., 1988). This seems to partially reflect in Neuro2a cell system; thus, a significant increase of neurite formation by GnT-III overexpression may result in an elevation of induction rate of axon-like process.
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To determine whether bisecting GlcNAc-containing N-glycans are directly involved in the GnT-III-promoted neuritogenesis, we prepared the bisected N-glycan as shown in Figure 1A and used it to examine its effects on neuritogenesis. As shown in Figure 2C, the exogenous addition of the bisected N-glycan significantly suppressed neurite formation that was enhanced by GnT-III overexpression. Simultaneously, supernumerous formation of axon-like process was greatly blocked by the treatment (Figure 2G), supporting that an increase of neurite formation by GnT-III overexpression correlates with supernumerous induction of axon-like processes. It was noteworthy that other biantennary N-glycans such as the asialobiantennary structure potently suppressed the induction of the axon-like process itself without inhibiting neurite formation (data not shown). Collectively, these data suggest that bisected N-glycan–mediated interactions and/ or complex formation play a role in GnT-III-promoted neuritogenesis.
ß1 integrin is involved in the GnT-III-enhanced neuritogenesis
Neurite outgrowth in response to differentiation stimulation is strongly promoted by extracellular matrix (ECM) ligands such as laminin, fibronectin, and collagen (Turner et al., 1989), indicating that integrins, their major receptors, play critical roles in neuritogenesis. It is well known that ß1 integrin undergoes carbohydrate remodeling depending on changes in cellular events or phenotype (Bellis, 2004). Therefore, it is possible that ß1 integrin could be one of the major target molecules for GnT-III and could play an important role in GnT-III-promoted neuritogenesis. To examine whether ß1 integrin is involved in GnT-III-enhanced neuritogenesis, treatment with anti-ß1 integrin functional blocking antibody was performed. As shown in Figure 3A, the enhanced neurite formation of GnT-III transfectants was significantly inhibited by treatment with the anti-ß1 integrin functional monoclonal antibody (mAb) (clone DF5), which had been used for blocking ß1 integrin-mediated neurite outgrowth in another neuroblastoma cell line, N1E-115 (Sarner et al., 2000; Ishii et al., 2001). On the other hand, it has been reported that integrin plays important roles in the activation of phosphatidylinositol 3-kinase (PI3-K) and ERK (Giancotti and Ruoslahti, 1999; Danen and Yamada, 2001; Schwartz and Assoian, 2001), which are required for neural differentiation (Turner et al., 1989; Sarner et al., 2000; Ishii et al., 2001). To determine whether PI3-K or ERK activation is also required for GnT-III-associated neuritogenesis, the cells were treated with the PI3-K inhibitor wortmannin and the mitogen- or extracellular signal-regulated kinase (MEK) inhibitor PD98059. As predicted, these treatments completely blocked neuritogenesis caused by serum starvation in the mock and GnT-III transfectants (Figure 3A). Taken together, these results suggest that ß1 integrin is involved in the GnT-III-enhanced neuritogenesis.
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We next examined the issue of whether the ß1 integrin is an actual target protein of GnT-III. The glycoproteins modified by the bisecting GlcNAc in each transfectant were collected by a pull-down experiment with E4-PHA. As expected, we found that the amount of ß1 integrin in E4-PHA-associated complexes was significantly increased in GnT-III transfectants, compared with that in mock transfectants (Figure 3B). Consistent with the results in given Figure 1, the amount of ß1 integrin in E4-PHA-associated complexes was significantly decreased in the D321A transfectant. Overexpression of GnT-III or D321A did not affect the levels of expression of ß1 integrin (data not shown). The mobility of ß1 integrin on SDS–PAGE was faster for the GnT-III transfectants than for that in mock cells (Figure 3B), which is a unique feature of the introduction of the bisecting GlcNAc into N-glycans on glycoproteins. This can be attributed to a significant reduction in multiantennary oligosaccharides (Yoshimura et al., 1995; Koyota et al., 2001), since the addition of bisecting GlcNAc to N-glycans can interfere with other glycosyltransferases such as GnT-V acting on the sugar chains (Schachter, 1986; Gu et al., 1993). Interestingly, the levels of the bisecting GlcNAc-bearing ß1 integrin seemed to be increased in the serum-deprived culture, compared with those in the presence of fetal bovine serum (FBS). However, the increased expression levels of bisecting GlcNAc in ß1 integrin did not induce neuritogenesis in the presence of serum, suggesting that other factors are also required for the GnT-III-induced neuritogenesis under the condition of serum deprivation.
Neurite swelling contains ß1 integrin
Next, we examined whether the subcellular distribution of ß1 integrin in differentiated Neuro2a cells is altered by GnT-III overexpression. As shown in Figure 4A, ß1 integrin was partially co-localized with neural cell-adhesion molecule (NCAM), a marker of neurite swelling, which contains a trans-Golgi network (TGN) component (Sytnyk et al., 2002). In support of this, Golgin97, one of TGN components, was also localized in the swelling (data not shown). Importantly, we found that the number of neurite swelling was significantly increased in GnT-III transfectant (Figure 4B). It is unclear why the number of swellings in the D321 mutant was not decreased to a far greater extent than that of mock transfectant, but neurite formation was down-regulated in the mutant (Figure 2D).
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E4-PHA treatment inhibits neurite and swelling formation
As shown in Figure 2, exogenous bisected N-glycans suppressed the GnT-III-enhanced neurite formation. To confirm whether the bisected N-glycans is involved in those changes, treatment with E4-PHA was performed. We found that addition of E4-PHA not leukoagglutinating phytohemagglutinin (L4-PHA), which recognizes the ß1-6 GlcNAc branching as a negative control, completely inhibited neurite formation (Figure 5A). Moreover, when treated with E4-PHA, abnormal axonal expansion was observed in mock and GnT-III transfectants. It could be explained by the fact that the treatment with E4-PHA resulted in down-regulation of target proteins for GnT-III such as integrins, thereby inhibiting cytoskeletal F-actin formation. The extensive cell spreading observed in most D321A transfectants was also induced in mock and GnT-III transfectants when treated with a higher concentration of E4-PHA (data not shown), suggesting that the extensive cell spreading observed in D321A mutant may be ascribed to an increased sensitivity for E4-PHA treatment. Consistent with the bisected N-glycan treatment (Figure 2), NF-H remained distributed in an expanded axon-like process, though F-actin network was significantly disrupted. Furthermore, concomitant with this, swelling formation was also significantly decreased in each transfectant by treating with E4-PHA (Figure 5B). Taken together, these results suggest that the bisecting GlcNAc on glycoproteins, such as ß1 integrin, plays an important role in neuritogenesis induced by serum deprivation in Neuro2a cells.
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Discussion |
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The findings of this study indicate that GnT-III potentiates ß1 integrin-mediated neurite genesis induced by serum deprivation. Furthermore, GnT-III-promoted neuritogenesis can be blocked by the exogenous addition of the Gn(Gn) Gn-bi glycopepetide as well as E4-PHA, suggesting that the bisecting GlcNAc-containing N-glycans may play important roles in neuritogenesis in vivo.
As of this writing, our previous studies have reported that the modification of membrane glycoprotein receptors by GnT-III usually had inhibitory effects on their biological function. The overexpression of GnT-III inhibited 5ß1 integrin-mediated cell spreading and migration and also the phosphorylation of the focal adhesion kinase. The affinity of the binding of integrin 5ß1 to fibronectin was significantly reduced as the result of the introduction of the bisecting GlcNAc to the 5-subunit (Isaji et al., 2004). In PC12 cells, another neural differentiation cell model, the overexpression of GnT-III resulted in the inhibition of TrkA dimerization and TrkA-mediated neurite outgrowth upon stimulation by NGF (Ihara et al., 1997) as well as the inhibition of EGF-induced neurite outgrowth through the Ras/MAP kinase activation pathway (Gu et al., 2004). In the present study, we clearly show that the overexpression of GnT-III promoted neurite formation and that this promotion was substantially blocked by the presence of the bisecting GlcNAc-containing glycopeptide. The discrepancy could be explained as follows: (1) bisected N-glycans play different roles in different types of cells; and (2) the experimental conditions used were different. In fact, growth factor stimulation is essentially required for the differentiation model of PC12 cells, whereas serum starvation leads to cell apoptosis (Batistatou and Greene, 1991; Xia et al., 1995). Moreover, in the case of PC12, the culture condition requires ECM coating such as collagen. It has been shown that the expression of integrin 1 is regulated by NGF in PC12 (Zhang et al., 1993). Furthermore, Neuro2a cells do not express NGF receptor (Matta et al., 1986). Thus, different cellular responses induced by serum deprivation between PC12 and Neuro2a cells may be ascribed to a difference of their cell-adhesion receptors such as integrins and/or growth factor receptors, which have been identified as target molecules for GnT-III.
The introduction of the bisecting GlcNAc results in the suppression of further processing and the elongation of N-glycans, catalyzed by other glycosyltransferases, since they are not able to use the bisected oligosaccharide as a substrate. Thus, GnT-III is regarded to be a key glycosyltransferase in N-glycan biosynthetic pathways. The higher expression levels of GnT-III and its products in the brain have long been postulated to have biological functions. Of particular interest is the fact that the exogenous addition of the bisected N-glycan inhibited GnT-III-promoted neuritogenesis. This is the first report, to our knowledge, which directly shows that the bisected N-glycans, in fact, have important biological functions. The specific effect could be ascribed to the terminated GlcNAc, of the bisected N-glycans, since the GlcNAc-terminated biantennary N-glycans were also shown to have some inhibitory effects (data not shown). Indeed, the importance of the terminal GlcNAc has been shown by reports that neurite outgrowth is inhibited by immobilized Psathyrella velutina lectins (PVL), which recognize the terminal GlcNAc in human neuroblastoma SH-SY5Y cells (Kitamura et al., 2004). In addition, it has been reported that some GlcNAc-binding proteins such as Na+/K+-ATPase ß1-subunit are present in the mouse brain (Kitamura et al., 2005). On the other hand, it has been reported that integrin-mediated neurite outgrowth in neuroblastoma cells is dependent on the activation of K+ channels (Arcangeli et al., 1993). It has recently been shown that ß1 integrin is physically linked to human ether-a-go-go-related gene (hERG) K+ channels (Cherubini et al., 2002, 2005). Thus, it will be interesting to test whether the interaction between ß1 integrin and molecules such as K+ channels are dependent on the presence of the bisected glycan.
It is well known that integrins, especially ß1 integrin, play crucial roles in cell growth, migration, and cell differentiation (Hynes, 1992; Sheetz et al., 1998) and are also major carriers of N-glycans. In the present study, ß1 integrin was found to be one of glycoproteins bearing bisected N-glycans in Neuro2a cells. The anti-ß1 integrin function-blocking antibody suppressed GnT-III-promoted neuritogenesis, further supporting the view that ß1 integrin is not only a real structural but also a real functional target protein of GnT-III. Interestingly, ß1 integrin was present in neurite swelling as described above, from which growth cones were regenerated in axonal-injured model using PC12 (Nakayama et al., 2001), suggesting that ß1 integrin may functionally regulate both swelling and neurite formation.
The role of bisected N-glycans appears to have also been observed in vivo. It has been reported that mice carrying the inactive GnT-III mutant have a neurological phenotype, which includes an abnormal leg clasp, altered gait, and lack of nurturing newborn pups by homozygous mothers (Bhattacharyya et al., 2002). It has recently been reported that the structure or expression of brain-enriched sugars in brains of neurological mutant mice such as staggerer or sivelerer is altered (Nakakita et al., 2005). Interestingly, staggerer mice have a defect in that Purkinje cells are unable to develop into mature dendritic trees (Bradley and Berry, 1978), suggesting that the bisecting GlcNAc plays a significant role in neurite formation. The present study not only demonstrates the role of the bisected N-glycans in neuritogenesis, but also opens the possibility that neuritogenesis and the reverse pathological condition could be controlled by using a variety of N-glycan conjugates in the nervous system.
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Materials and Methods |
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Reagents, antibodies, and lectins
The following primary antibodies were used for immunofluorescent staining: mAb against NF-H (SMI 31, Sternberger Monoclonals, Baltimore, MD); mAbs against ß1 integrin (clone 18) and NCAM (clone 12F11) from Bioscience Pharmingen (San Diego, CA). Alexa488-, Alexa546-conjugated fluorescent secondary antibodies and Alexa488-conjugated phalloidin were obtained from Molecular Probes (Eugene, OR). E4-PHA or L4-PHA lectins were purchased from Seikagaku (Tokyo, Japan) and used at the concentration of 4 µg/mL in media. The functional blocking anti-ß1 integrin mAb (clone DF5) was from MP Biomedicals (Costa Mesa, CA). Mouse control IgG was from Santa Cruz Biotechnology (Santa Cruz, CA). PD98059 (a MEK-1 inhibitor) and wortmannin (a PI3-K inhibitor) were purchased from Sigma (St. Louis, MO).
Plasmids and selection of stable transfectants
The wildtype cDNA-encoding rat GnT-III was subcloned into the EcoRI sites of the pCXNII expression vector, which contained a neomycin-resistant gene. The D321A mutant was constructed by site-directed mutagenesis experiments according to Kunkel (1985), as described previously (Ihara et al., 2002). The fidelity of each construct was confirmed by DNA sequencing. To obtain colonies that overexpress GnT-III or D321A mutant, Neuro2a cells were transfected with pCXNII (mock), pCXNII/GnT-III (GnT-III), and pCXNII/D321A (D321A) using the lipofectAMINE reagent, following the manufacturer’s instructions. Selection was performed in 10% FBS in Dulbecco’s modified Eagle’s medium (DMEM, high glucose formulation, Sigma) containing 1 mg/mL G418. After a 2-week incubation, G418-resistant colonies were isolated and recloned by serial dilution to ensure clonality, and the expression levels and enzymatic activity of GnT-III in each of the transfectants were finally confirmed by western blotting and a GnT-III activity assay (Taniguchi et al., 1989).
GnT-III activity assay
Cells were suspended and homogenized in phosphate-buffered saline (PBS) containing protease inhibitors. The supernatants, after removal of the nucleus fraction by centrifugation for 15 min at 700x g, were used as sources for an enzymatic assay by HPLC using a pryidylaminated (PA) biantennary sugar chain as an acceptor substrate, as described previously (Taniguchi et al., 1989).
Phase-contrast and fluorescent imaging
The transfectants were initially grown in DMEM with penicillin, streptomycin, and 10% FBS, with passage every 2–3 days in a 5% CO2 and 95% air humidified atmosphere at 37°C. They were seeded in a 35-mm glass-bottomed dish (Matunami Glass Ind., Osaka, Japan) at a density of 2 x 104 cells/cm2 and cultured for 24 h. Cell differentiation was initiated by serum starvation in DMEM. After a 24 h incubation, the cells were fixed in 4% paraformaldehyde at room temperature (RT) for 10 min, followed by washing with PBS, and phase-contrast images were taken at 20x magnification (Olympus CKX41; Olympus, Tokyo, Japan). Neurite swelling formation was assessed as the percentage of the number of swellings, which is morphologically observed at the size of 1–2 µm diameter (Sytnyk et al., 2002) by phase-contrast microscopy, in total cell number in each field.
The fixed cells were subsequently permeabilized with 0.1% Triton X-100 at room temperature and then blocked with 1% bovine serum albumin in PBS for 1 h. After labeling with the first primary antibody (1 h at RT) and washing with PBS, the cultures were stained with the labeled secondary antibody (Alexa488- or Alexa546-conjugated; 1 h at RT) and then washed with PBS. F-actin was labeled with Alexa488-conjugated phalloidin. Fluorescence images were observed by confocal microscopy using a LSM5 PASCAL microscope (Carl Zeiss, Oberkochen, Germany). The results were analyzed using a PASCAL of confocal microscopic systems. All images were imported into Adobe Photoshop in TIFF format for contrast manipulation and figure assembly.
Pull-down assay with lectin and western blotting
Neuro2a cells were solubilized by incubation with lysis buffer (10 mM Tris–HCl, pH 7.4, 2 mM EGTA, 1 mM sodium orthovanadate, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin) for 30 min on ice. For cell-surface labeling, transfectants were washed with biotinylation buffer (0.1 M HEPES–HCl, pH 8.0, 50 mM NaCl, 1 mM PMSF, and 20 µg/mL leupeptin) and surface labeled with 1 mg/mL sulfo-NHS-LC-biotin for 20 min at RT. The cell lysates were collected in a 1.5-mL Eppendorf tube with a rubber policeman, centrifuged at 700x g for 10 min to exclude the nuclear fraction. E4-PHA-associated complexes were precipitated from the supernatant using 2 µg of E4-PHA lectin beads. The pellets were suspended in reducing sample buffer, heated at 100°C for 3 min, resolved on 10% SDS–PAGE, and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% skim milk and then incubated with anti-ß1 integrin mAb (BD Transduction Laboratories, Lexington, MN). Immunoreactive bands were visualized with the HRP-conjugated secondary antibody by means of an enhanced chemiluminescence kit from Amersham (Buckinghamshire, UK).
Preparation of biantennary N-glycans containing the bisecting GlcNAc
Sialylglycopeptides (SGP) were purified from hen eggs as described previously (Seko et al., 1997), and then hydrolyzed by treatment with 2 M CH3COOH, at 80°C for 2 h to remove sialic acid residues. The resulting products were injected into a 4.6 x 250 mm TSK-gel ODS-80 column (TOSOH, Tokyo, Japan) in a reverse-phase HPLC system (Shimazu, Kyoto, Japan), and the asialobiantennary N-glycans were separated using a gradient of solvent systems A (0.1% trifluoroacetic acid) and B (0.1% trifluoroacetic acid in 80% acetonitrile): 5% solvent system B for 5 min followed by a linear gradient to 35% solvent system B over 30 min (flow rate, 2.0 mL/min; the monitoring absorbance, 210 nm). Each peak was analyzed and confirmed by mass spectrometry (Bruker Daltonics’ Ultraflex Instrument, Bremen, Germany). For preparation of agalactobiantennary N-glycans, purified asialobiantennary N-glycans were digested with ß-galactosidase (Sigma) at 30°C for 6 h in ammonium acetate buffer (20 mM, pH 4.5). After completion of the digestion by monitoring by reverse-phase HPLC, agalactobiantennary N-glycans were purified using a cellulose cartridge Glycan preparation kit (Takara, Kyoto, Japan). The products of the bisected N-glycans were prepared by incubating agalactobiantennary N-glycans with purified GnT-III, as previously reported (Ikeda et al., 2000), and purified using a cellulose cartridge. The purity of the product was verified by reverse-phase HPLC and mass spectrometry.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of interest statementAcknowledgmentsReferences |
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We thank H. Korekane and T. Suzuki for their valuable suggestions and discussions. This work was partly supported by the 21st Century COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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Abbreviations |
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DMEM, Dulbecco’s modified Eagle’s medium; E4-PHA, erythroagglutinating phytohemagglutinin; ECM, extracellular matrix; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GnGn-bi, GlcNAcß1-2Man
1-6(GlcNAcß1-2Man1-3) Man ß1-4GlcNAcß1-4GlcNAc-biantennary; Gn(Gn)Gn-bi, GlcNAcß1-2Man1-6(GlcNAcß1-2Man1-3)(GlcNAcß1-4) Manß1-4GlcNAcß1-4GlcNAc-biantennary; GnT-III, UDP-N-acetylglucosamine:ß-D-mannoside ß-1,4-acetylglucosaminyltransferase III; HPLC, high-performance liquid chromatography; L4-PHA, leukoagglutinating phytohemagglutinin; MEK, mitogen- or extracellular signal-regulated kinase; NCAM, neural cell-adhesion molecule; NGF, nerve growth factor; NF-H, phosphorylated high-molecular-weight neurofilament protein; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PI3-K, phosphatidylinositol 3-kinase |
References |
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