Overexpression of N-Acetylglucosaminyltransferase III Disrupts the Tyrosine Phosphorylation of Trk with Resultant Signaling Dysfunction in PC12 Cells Treated with Nerve Growth Factor*

(Received for publication, November 11, 1996, and in revised form, January 27, 1997)

Yoshito Ihara , Yoshihiro Sakamoto , Masahito Mihara , Kentaro Shimizu and Naoyuki Taniguchi Dagger

From the Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

beta -1,4-N-Acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144) is a pivotal glycosyltransferase which participates in branch formation by catalysis of the synthesis of a bisecting GlcNAc structure in N-glycans. These structures are thought to be one of the unique features of the N-glycans of neural tissues. To examine the intracellullar role of GnT-III expression and its product in neural cells, its gene was overexpressed in rat pheochromocytoma PC12 cells which normally express a low level of GnT-III. In the GnT-III gene-transfected cells, lectin blot analysis showed that some glycoproteins showed increased levels of bisecting GlcNAc structures. Following treatment with nerve growth factor (NGF) the control cells showed neurite outgrowth for differentiation whereas the transfectants showed no morphological response or change in the rate of cell growth. Transient tyrosine phosphorylation of the Trk/NGF receptor was detected at 5-15 min after NGF treatment in control cells, but not detected in the GnT-III gene-transfected cells despite the intact binding of NGF to the cells. Moreover the dimerization of Trk with NGF treatment was not induced in the GnT-III transfectant as compared with the dimerization seen in control cells. These results indicate that overexpression of GnT-III gene in PC12 cells affects some functions of glycoprotein receptors such as Trk by alteration of N-glycan structures, and results in changes in the intracellular signaling pathway of tyrosine phosphorylation modified by NGF.


INTRODUCTION

The marked changes in the sugar chain structures of cell surface membrane occurring during ontogenesis and oncogenesis suggest that they play pivotal roles in cell differentiation and proliferation (1). In the case of glycoproteins, N- and O-glycans are expressed in the majority of cell surface and secreted proteins (2). A gene for beta -1,2-N-acetylglucosaminyltransferase I (GnT-I),1 the enzyme catalyzing the formation of complex type N-glycans has been obliterated in mice. The resultant pathology showed that complex type N-glycans are required for normal embryonic development, especially of neural tissues (3, 4).

In such tissues, it is known that the level of polysialyl N-glycans decreases in the neural cell adhesion molecule as these cells mature. This indicates that the sugar chains of the neural cell adhesion molecule regulate the cell-cell interaction in neural tissues (5-8). Although the polysialyl sugar chain structure is one characteristic of most neural tissues, some unique differences for the N-glycans of mouse brain have been reported (9). Among them, a bisecting GlcNAc structure, which is biosynthesized by UDP-N-acetylglucosamine:beta -D-mannoside beta -1,4-N-acetylglucosaminyltransferase III (GnT-III: EC 2.4.1.144), has been found (Fig. 1).


Fig. 1. A bisecting GlcNAc chain in N-glycans is biosynthesized by GnT-III.
[View Larger Version of this Image (8K GIF file)]


GnT-III is one of the pivotal glycosyltransferases which participates in the branching of N-glycans (10), and produces an unique sugar chain structure, a bisecting GlcNAc (11). GnT-III has been purified from rat kidney and the rat (12), human (13), and mouse (14) genes have been cloned. The tissue distribution of its mRNA showed that the GnT-III transcript was particularly high in the brain and kidney of the mouse (14). Such high levels for the expression of GnT-III mRNA seem to be compatible with the existence of unique N-glycan structures in brain including bisecting GlcNAc. Several experimental approaches have been used to elucidate the role of GnT-III in cultured cells (15, 16). In mouse melanoma B16 cells, GnT-III gene induction resulted in changes in their N-glycan structures and suppressed the metastatic potential of the original cells (17). This implies that GnT-III also has important functions in neural tissues since melanoma cells are also of neural origin. In the present study, we have investigated the biological roles of GnT-III expression and its bisecting GlcNAc product in rat pheochromocytoma PC12 cells by introduction of the GnT-III gene.


MATERIALS AND METHODS

Cell Culture

Rat pheochromocytoma PC12 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo). PC12 and the GnT-III gene-transfected cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal calf serum, 5% horse serum, and 0.1 mg/ml of kanamycin under a humidified atmosphere of 95% air and 5% CO2. NGF (TaKaRa, Japan) was added to a final concentration of 50 ng/ml to induce PC12 differentiation.

Recombinant DNA Constructs

Rat GnT-III cDNA clone C4 (12) was deleted at its 5' non-coding region and the cDNA fragment containing the entire coding sequence was inserted into the pMEP4 (Invitrogen) EcoRI site to obtain the final construct, pMEPrIII. The pMEP4 is a mammalian expression vector with the metallothionein IIa gene enhancer/promoter (18) also includes the hygromycin-resistant gene.

Gene Transfection and Selection of Cells

PC12 cells used for transfection of cDNA were plated in a 10-cm plastic culture dish coated with collagen to a density of 1 × 106/ml cells. After 24 h the medium was removed and the cells washed twice with cold phosphate-buffered saline (PBS), pH 7.2, and changed to serum-free DMEM. The pMEPrIII vector (20 µg) was mixed with Lipofectamine (Life Technologies, Inc.) and 100 µl of this solution was added to PC12 cells. After 5 h incubation the medium was changed to the original as described above. Stable transfectants were screened with 0.5 mg/ml hygromycin.

Glycosyltransferase Activity

Cell pellets were homogenized in PBS containing protease inhibitors, and the supernatant after removal of the nucleus fraction by centrifugating for 20 min at 900 × g was used for the assays. GnT-III, GnT-V, and beta -1,4-galactosyltransferase activities were assayed by high performance liquid chromatography methods described previously (19, 20) using the fluorescence-labeled sugar chain (GlcNAcbeta -1,2-Manalpha -1,6-[GlcNAcbeta -1,2-Manalpha -1,3-] Manbeta -1,4-GlcNAcbeta -1,4-GlcNAc-pyridylamino) as the substrate.

Lectin Blot Analysis

Samples of wild type and gene transfectant cell extracts containing 5 µg of protein were electrophoresed on 8-12% SDS-polyacrylamide gels under reducing conditions and then transferred to nitrocellulose membranes (Schleicher & Schuell) as described previously (21). The membrane was blocked with 3% bovine serum albumin in PBS and then incubated with biotin-conjugated lectins (2 µg/ml biotinylated E-PHA or 2 µg/ml biotinylated L-PHA; Honen Corp. Tokyo) using the buffer systems as described (21). Lectin reactive proteins were detected using a Vectastain ABC kit (Vector Laboratories) and the blots were developed using the ECL chemiluminescence detection kit (Amersham) according to the manufacturer's instructions.

Characterization of Trk in Cultured Cells Treated with NGF

The GnT-III gene-transfected PC12 cells and their controls were cultured with or without 50 ng/ml NGF and then disrupted in the lysis buffer (20 mM Tris, pH 7.2, 1% Nonidet P-40, 10% glycerol, 1 mM APMSF, 5 mM aprotinin, 0.4 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM iodoacetamide). The cell-free lysates were adjusted to the same protein concentration and immunoprecipitated with the C-14 Trk antibody (Santa Cruz Biotechnology Inc.) according to the manufacturer's instructions. The precipitate was subjected to 8% SDS-PAGE, and electroblotted samples were then characterized by immunoblot or lectin blot analyses as described above. For the detection of phosphorylated tyrosine residues, a monoclonal phosphotyrosine antibody (PY20, Transduction Laboratories) was used according to the manufacturer's protocol after blocking with 5% skim milk in PBS. The blots were detected by peroxidase-conjugated rabbit anti-mouse IgG (Cappel) and developed with the ECL kit.

Phosphorylation of Trk with or without NGF was estimated by immunoprecipitation of 32P-labeled Trk as described previously (22). In brief, cells were preincubated in phosphate-free DMEM for 1 h. [32P]Orthophosphate (0.1 mCi/ml) was then added and incubated with the cells for 1 h. After 5 min of NGF (50 ng/ml) treatment, cells were lysed and Trk was immunoprecipitated as described above. The precipitates were subjected to 8% SDS-PAGE followed by autoradiography using a Fuji Film imaging plate for the BAS2000 Bioimage analyzer (Fuji Photo Film, Japan).

Growth Assays

Growth of GnT-III gene transfectants and control PC12 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (23).

DNA Synthesis

Plates of 96 wells, each containing 2 × 104 cells in 200 µl, were cultured with or without 50 ng/ml NGF for 12 h and [3H]thymidine (1 µCi/well) was added to each well. After 6 h of incubation at 37 °C the cells were harvested and [3H]thymidine incorporated into DNA was measured with the Betaplates system (Pharmacia Biotech Inc.).

NGF Binding

Cells were plated in 24-well culture plates at a density of 2 × 104 cells/ml and cultured for 12 h. Cells were washed with ice-cold binding buffer (PBS(-) with 0.1 mM CaCl2 and 1 mM MgCl2) and incubated with 500 µl of the binding buffer containing various concentrations (0.37-10 ng/ml) of 125I-labeled NGF at 4 °C for 2 h. Cells were washed four times with ice-cold PBS(-) and lysed with 1 M NaOH, and the radioactivity was counted in a gamma -counter. To estimate the specific binding of NGF to the cells, NGF binding assay using 125I-labeled NGF was also performed in the presence of various amounts of cold NGF (0.02-500 ng).

Chemical Cross-linking of Trk

Cells (2 × 106) were harvested, pelleted, and resuspended in the binding buffer as described under the method for NGF binding. NGF was added to a final concentration of 100 ng/ml and the cells were incubated at 4 °C for 1 h. The chemical cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) (Pierce) was added to a final concentration 0.5 mg/ml. The reaction was incubated at room temperature for 30 min and quenched by washing with Tris-buffered saline (pH 7.4). Cross-linked cells were lysed and subjected to the immunoblot analysis of Trk as described above.


RESULTS

Establishment of GnT-III Gene Transfectants Expressing High Levels of GnT-III Activity

To investigate the functional role of GnT-III gene transfection and its bisecting GlcNAc sugar chain product, during neural cell differentiation and development, a GnT-III gene expression vector, MEPrIII, was employed. This inserted rat GnT-III cDNA downstream of the metallothionein IIa promoter, which was then transfected into PC12 cells using a LipofectAMINE complex. The hygromycin resistant transfectants were screened as described under "Materials and Methods." The specific activity of GnT-III for each clone was assayed and then three high activity transfectants (PC12-III-1, -2, and -3) were randomly taken and used in succeeding experiments. As shown in Table I, GnT-III activity was elevated about 4-6 times over the wild type or mock transfectant (PC12-hyg). GnT-V and beta -1,4-galactosyltransferase activities assayed as the control glycosyltransferases showed no significant changes.

Table I.

Glycosyltransferase activities of control and GnT-III gene transfected PC12 cells

Enzyme activities of GnT-III, GnT-V, and beta -1,4-galactosyltransferase (GT) were measured using the fluorescence-labeled sugar chain as substrate. Each value represents the mean of three experiments, and the standard deviation is always within 6% of the mean.


GnT-III GnT-V  beta -1,4-GT

pmol/h/mg protein
PC12 220 120 1530
PC12-hyg 190 140 1390
PC12-III-1 1270 110 1350
PC12-III-2 970 114 1420
PC12-III-3 810 120 1300

Effect of Overexpression of GnT-III Activity in Cell Differentiation by NGF in PC12 Cells

PC12 cell differentiates and forms neurites under NGF treatments (24, 25). To test the role of overexpressed GnT-III and its bisecting GlcNAc sugar chain product on NGF induced differentiation, parental, mock transfectants, and GnT-III gene transfected PC12 cells were cultured with or without 50 ng/ml NGF for 5 days and cell morphologies were compared. Fig. 2 shows the morphology of cells cultured with NGF. After 5 days of treatment, cell proliferation was reduced and neurite formation was observed in the case of PC12 and the PC12-hyg, a mock transfectant. However, the GnT-III gene transfectants showed no neurite formation, and cell proliferation was not affected (PC12-III-1 and -2 representing the transfectants were shown).


Fig. 2. Morphological change of control and GnT-III gene-transfected PC12 cells on NGF treatment. Cells (2 × 104/ml) were seeded and cultured in DMEM supplemented with 10% fetal calf serum and 5% horse serum with or without 50 ng/ml NGF for 5 days. The results were reproducible in four independent experiments.
[View Larger Version of this Image (107K GIF file)]


Effect of GnT-III Gene Expression on Cell Proliferation under NGF Treatment

To test the effect of GnT-III expression on proliferation of PC12 cells, their growth rate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. As shown in Fig. 3A, part a, the cell growth was suppressed in the case of control cells under NGF treatment, compared with the cells without NGF treatment. But no suppression of cell growth was observed in the case of GnT-III gene-transfected cells with NGF treatment (Fig. 3A, part b). DNA synthesis was also evaluated by measuring the incorporation of [3H]thymidine into DNA. As shown in Fig. 3B, the thymidine incorporation was decreased in the case of control cells under NGF treatment compared with the cells without NGF. But the decrease was not observed in the GnT-III gene-transfected cells with NGF treatment.


Fig. 3. Cell growth and thymidine incorporation of control and GnT-III gene-transfected PC12 cells. A, cells (2 × 104/ml) were seeded and cultured in DMEM supplemented with 10% fetal calf serum and 5% horse serum. Cell growth of control, mock, and GnT-III gene-transfected PC12 cells with or without NGF was evaluated at indicated days using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as described under "Materials and Methods." a, control and mock gene-transfected cells; b, GnT-III gene transfectants. Each value represents the mean of three independent experiments, and the standard deviation is always within 10% of the mean. B, cells (2 × 104) were seeded into 96-well culture plates and cultured with or without NGF for 12 h. After an incubation with [3H]thymidine (1 µCi/well) for 6 h, [3H]thymidine incorporation into DNA was evaluated as described under "Materials and Methods." Each value represents the mean ± S.D. of three independent experiments.
[View Larger Version of this Image (25K GIF file)]


Lectin Blots of Glycoproteins of Control and GnT-III Gene-transfected PC12 Cells

To investigate the change of sugar component of glycoproteins from cell lysates between control and GnT-III gene-transfected cells, lectin blot analyses were performed using E- and L-PHA. E-PHA preferentially binds to sugar chains containing a bisecting GlcNAc structure which is the product of GnT-III (26). In contrast L-PHA binds to structures of complex type N-glycans containing 3 or 4 branches including GlcNAcbeta -1,6, which is the product of GnT-V (27). In the case of control cells, after 5 days of treatment with NGF, E-PHA reactivity showed no significant change but L-PHA reactivity was slightly increased in the glycoproteins of 80-95 kDa (Fig. 4, lane 1 of L-PHA). In the case of GnT-III gene-transfected cells, E-PHA reactivity to glycoproteins of about 98 kDa showed a marked increase compared with control cells without NGF treatment (Fig. 4, lanes 2 and 3 of E-PHA, arrow). However, after 5 days of treatment with NGF, the L-PHA reactivity to glycoproteins showed no significant change in contrast to the case for control cells (Fig. 4, lanes 2 and 3 of L-PHA). These data showed that some glycoproteins were modified by overexpression of GnT-III activity and E-PHA reactive glycoproteins increased. These E-PHA reactive glycoproteins produced by overexpression of GnT-III activity may suppress the appearance of L-PHA reactive glycoproteins noted in the differentiated PC12 cells after 5 days treatment with NGF. During this period, no significant change was found in the enzyme activity levels of glycosyltransferases GnT-III, -V, and beta -1,4-galactosyltransferase as the result of treatment with NGF (data not shown).


Fig. 4. Lectin blot analysis of glycoproteins from cell lysates of control and GnT-III gene-transfected PC12 cells. 5 µg of proteins from each cell lysate of mock and GnT-III transfected cells with (+) or without (-) NGF were subjected to 12% SDS-PAGE, and lectin blot analysis was performed using E- and L-PHA as described under "Materials and Methods." Lane 1, PC12-hyg; lane 2, PC12-III-1; lane 3, PC12-III-2. The results were reproducible in two independent experiments.
[View Larger Version of this Image (58K GIF file)]


Tyrosine Phosphorylation of NGF Receptor/Trk

To determine the effect of NGF signaling in GnT-III gene-transfected cells, the time course of tyrosine phosphorylation of Trk was investigated. In Fig. 5A, control and GnT-III gene-transfected PC12 cells that had been cultured with NGF were harvested at the indicated time (0, 5, 15, and 30 min) after treatment and lysed. From each cell lysate sample, Trk was immunoprecipitated with anti-Trk antibody and subjected to blotting analyses with anti-phosphotyrosine antibody and anti-Trk antibody. In Fig. 5A, tyrosine phosphorylation of Trk was detected after 5-15 min of NGF treatment in control cells. However, no significant tyrosine phosphorylation was detected in the case of Trk from GnT-III gene-transfected cells. In Fig. 5B, phosphorylation of Trk with NGF treatment was also estimated using [32P]orthophosphate-labeled cells. Despite an apparent signal of 32P-labeled Trk in control cells, no signal was detected in the GnT-III gene-transfected cells under NGF treatment. Similar results were obtained in other GnT-III gene transfectants (data not shown). These results suggest that the function of Trk may be abolished by changes in its sugar chain structure and then lead to disruption of tyrosine phosphorylation. Such an effect is consistent with failure to respond to NGF as found in the GnT-III gene-transfected PC12 cells (Fig. 2).


Fig. 5. A, the time course of tyrosine phosphorylation of immunoprecipitated Trk from control and GnT-III gene-transfected cells with NGF. Cells were harvested at the indicated time after NGF treatment. Trk was immunoprecipitated from the cell lysates of control and GnT-III gene-transfected cells. Results of immunoblot analysis are using anti-phosphotyrosine (upper panel) or Trk antibody (lower panel). B, immunoprecipitation of 32P-labeled Trk from control and GnT-III gene-transfected cells with NGF. Cells were preincubated in phosphate-free DMEM for 1 h. [32P]Orthophosphate was then added and incubated with the cells for 1 h. After 5 min of NGF (50 ng/ml) treatment, cells were lysed and Trk was immunoprecipitated as described under "Materials and Methods." The precipitates were subjected to 8% SDS-PAGE followed by autoradiography using the BAS2000 Bioimage analyzer. An arrow shows Trk of 140 kDa.
[View Larger Version of this Image (36K GIF file)]


Lectin Blots of Trk from Control and GnT-III Gene-transfected PC12 Cells

Trk was immunoprecipitated and subjected to lectin blot analyses. In Fig. 6, although the expression level of Trk protein showed no significant difference between control and the GnT-III gene-transfected cells, the results showed that reactivity to E-PHA increased in the GnT-III gene-transfected cells (lane 2 of E-PHA) compared with controls (lane 1 of E-PHA). A faint broad band reactive to L-PHA was detected in the control cells (lane 1 of L-PHA) but decreased in the transfected cells (lane 2 of L-PHA). Concanavalin A, which prefers to bind high mannose-type glycans, showed no reactivity to both of the immunoprecipitated Trks from control and GnT-III gene-transfected cells (data not shown). This indicates that Trk is also one of the glycoproteins affected by GnT-III overexpression.


Fig. 6. Lectin blot analysis of immunoprecipitated Trk from control and GnT-III gene-transfected cells. Trk was immunoprecipitated from the cell lysates of control and GnT-III gene-transfected cells. Immunoprecipitates were subjected to 8% SDS-PAGE, and then lectin blot analysis was performed using E- and L-PHA as described under "Materials and Methods." Lane 1, PC12-hyg; lane 2, PC12-III-1. An arrowhead shows Trk of 140 kDa. The result was reproducible in three independent experiments.
[View Larger Version of this Image (45K GIF file)]


NGF Binding to Control and GnT-III Gene-transfected PC12 Cells

Using 125I-labeled NGF, NGF binding to control and GnT-III gene-transfected cells was investigated as described under "Materials and Methods." As shown in Fig. 7, A and B, there is no significant difference in the binding between control and the gene-transfected cells.


Fig. 7. NGF binding to control and GnT-III gene-transfected cells. A, NGF binding to the cells was evaluated by gamma -counter after 2 h incubation of cells at 4 °C with 125I-NGF as described under "Materials and Methods." B, specific binding of NGF to the cells was estimated in the presence of cold NGF as described under "Materials and Methods." Each value represents the mean of four independent experiments, and the standard deviation is always within 10% of the mean.
[View Larger Version of this Image (24K GIF file)]


Effect of GnT-III Gene Expression on Dimerization of Trk under NGF Treatment

To clarify the precise mechanism of disruption of Trk function in the GnT-III gene transfectants under NGF treatment, Trk dimerization by NGF was investigated. The cell surface proteins were cross-linked with 3,3'-dithiobis(sulfosuccinimidylpropionate) under NGF treatment as described under "Materials and Methods." After cross-linking, cell lysates were subjected to 8% SDS-PAGE followed by immunoblotting using anti-Trk antibody. In Fig. 8, control cells showed a typical homodimer form of Trk (about 300 kDa) after 5 min treatment of NGF. But the dimerization of Trk was not observed in the gene-transfected cells.


Fig. 8. Trk dimerization by NGF treatment. The cell surface proteins were cross-linked with 3,3'-dithiobis(sulfosuccinimidylpropionate) under NGF treatment as described under "Materials and Methods." After cross-linking, Trk was characterized by immunoblotting using an anti-Trk antibody as described under "Materials and Methods." The results were reproducible in two independent experiments. Arrow, Trk monomer; arrowhead, Trk dimer.
[View Larger Version of this Image (49K GIF file)]


In conclusion, these results indicate that the change of sugar chain structure of Trk by GnT-III causes the functional change in Trk, and disrupts the dimerization of Trk leading to non-autophosphorylation in the gene transfectants under NGF treatment, despite the intact binding of NGF to the cells.


DISCUSSION

Certain N-glycan structures of glycoproteins appear to play an important role in neural cell development (3, 4). GnT-III is one of the pivotal enzymes which regulates N-glycan branch structure, and the enzyme activity is high in such mammalian tissues as brain and kidney (14, 28). In the present study we have investigated some of the biological functions of GnT-III expression and its bisecting GlcNAc product in rat pheochromocytoma PC12 cells. These cells have very low GnT-III activities despite their neural origin. A GnT-III expression vector, whose expression was controlled by the metallothionein IIa promoter, was transfected into PC12 cells using a lipid complex, and the positive clones were screened by hygromycin resistance. In the case of GnT-III gene transfectants, the sugar chain structure of N-glycans was modified and especially some specific 98-kDa proteins showed increases in their level of bisecting GlcNAc as resulted by their reactivity with E-PHA. At present, it is not clear why some specific glycoproteins are susceptible to GnT-III overexpression. Conformation of N-glycosylation sites in each glycoprotein may affect the susceptibility to GnT-III action, and determine the sugar chain structure of each glycoprotein in the cells. In the case of GnT-III gene-transfected mouse melanoma B16 cells, we also experienced that specific 80- and 95-kDa proteins were enhanced in E-PHA reactivity (17). But in both of PC12 and B16 transfectants, these specific target glycoproteins could not be identified, and further investigations will be required.

The PC12 cells were found to be a most useful and popular model for the study of the actions of NGF and cell differentiation (24, 29). We have investigated the biological effect of GnT-III gene transfection into PC12 cells and found that they were not responsive to NGF as indicated by the rate of cell growth and morphological changes. These results indicated that such regulation and differentiation of PC12 cells were markedly modified by modulation of the sugar chain structure of several glycoproteins regulating their cellular differentiation.

In the mechanism of NGF action, it appears to relate to the phosphorylation of a number of cellular proteins (30-32). The NGF signaling pathway is known to be initiated by the direct binding of NGF to the high affinity NGF receptor/Trk proto-oncogene. This receptor is a protein tyrosine kinase and its activity and autophosphorylation are activated in response to NGF (33, 34). In attempts to clarify the molecular mechanism of the non-responsiveness to NGF of GnT-III gene-transfected PC12 cells, the role of Trk was investigated. The Trk receptor has 13 potential N-glycosylation sites in its molecule (35), and any of these potential sugar chain attachments might be modified. To test the function of Trk, after treatment of the cells with NGF, Trk was immunoprecipitated and then the tyrosine phosphorylation level was estimated by immunoblot analysis using a phosphotyrosine antibody. Surprisingly, in the case of GnT-III gene transfectant, the tyrosine phosphorylation level of Trk was not increased compared with the increase seen in the control PC12 cells after adding NGF (Fig. 5). The tyrosine phosphorylation of Trk is known to be accompanied with dimerization of Trk under NGF treatment (36). In the case of GnT-III gene transfectant, this dimerization did not occur even under NGF treatment as compared with the control, despite the intact binding of NGF to the cells. These data suggest that the change of sugar chain structure of Trk by overexpressed GnT-III activity affects its conformation of protein and causes disturbance of the dimerization, and disrupts its signal transduction under NGF treatment.

Laconte et al. (37) have previously shown that the N-glycans of the insulin beta  subunit receptor were essential for transmembrane signaling in Chinese hamster ovary cells by studies in which this receptor had been modified by site-directed mutagenesis of its N-glycosylation sites. Our present study also supports the importance of N-glycans in the cellular signaling systems, and provides further mechanical evidence of N-glycan function in the cellular signaling.

It has been recently reported that the GM1 ganglioside can bind to Trk and activate its autophosphorylation (38) and prevent apoptosis of PC12 cells (39). Mutoh et al. (38) also reported that the binding of GM1 to Trk was inhibited by blocking of N-glycosylation using tunicamycin, and this suggests the importance of N-glycosylation on Trk in its biological function. The relationship of sugar structures of glycolipid GM1 and glycoprotein in the mechanism of the NGF signaling pathway is not clear, but appears to relate to the existence of a novel regulation of Trk activation. In the case of neuroblastoma GOTO cells, the GnT-III activity level is high in the subconfluent state, but drastically decreases in the confluent state of the cells. This suggests that the regulation of GnT-III activity relates to the regulational system of cell proliferation (21). Similar phenomena were also observed in the case of sialyltransferase activity during cell growth in HepG2 cells (40). If cell proliferation is partially controlled by modulation of intracellular growth signaling due to autonomous regulation of sugar chain biosynthesis, cell growth associated changes of glycosyltransferase levels could relate to a feed-back like regulatory function. Although the present study has been concerned in part with the NGF-Trk signaling pathway in PC12 cells, investigations of other pathways offer attractive areas of investigation.

Very recently Canossa et al. (41) have reported that the low affinity NGF receptor (p75NGFR) could accelerate Trk-mediated signaling, and activate some p75NGFR-associated protein kinases. To assess the glycosylation change of p75NGFR in the GnT-III gene-transfected cells, p75NGFR was immunoprecipitated from control and GnT-III gene-transfected cells using a rabbit anti-mouse p75NGFR polyclonal antibody (Chemicon Int. Inc.) which can also react to rat p75NGFR, and subjected to the lectin blot analysis using E- and L-PHA as described in the case of Trk. Even in the GnT-III gene-transfected cells, no increase of E-PHA reactivity was observed (data not shown), and suggested that p75NGFR was not affected by GnT-III overexpression. p75NGFR has one N-glycan in its extracellular domain and the removal of the N-glycosylation site does not affect the NGF binding to the non-glycosylated p75NGFR (42). This implies that N-glycosylation in p75NGFR may be not important for its function. Taken together, in the NGF-associated signaling of PC12 cells, we conclude that GnT-III overexpression mainly affects Trk itself but not another known pathway of p75NGFR. However, to clarify the precise functional correlation between Trk and N-glycosylation, further investigations of Trk function with its modified N-glycosylation should be required using various cell models expressing Trk.

Our studies demonstrate that a specific N-glycan structural change affects some receptor glycoprotein functions, and causes modulation of a cell biological function such as cell differentiation. Until now it has been believed that a sugar chain mainly functions outside of the cell but we have found that the modulation of sugar chain structures can lead to the modulation of various biological functions by affecting intracellular events such as signal transduction pathways.


FOOTNOTES

*   This work was supported in part by Grant-in-aid 05274103 for Scientific Research on Priority Areas from the Ministry of Education, Science, Culture, and Sports, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom all correspondence should be addressed: Dept. of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, 565 Osaka, 565, Japan. Tel.: 81-6-879-3420; Fax: 81-6-879-3429; E-mail: proftani{at}biochem.med.osaka-u.ac.jp.
1   The abbreviations used are: GnT, N-acetylglucosaminyltransferase; PAGE, polyacrylamide gel electrophoresis; E-PHA, erythroagglutinating phytohemagglutinin; L-PHA, leukoagglutinating phytohemagglutinin; NGF, nerve growth factor; DMEM, Dulbecco's modified Eagle's medium.

ACKNOWLEDGEMENTS

We are grateful to Y. Hatanaka and S. Yanagidani for technical assistance, Dr. T. Nakagawa for fruitful discussions and suggestions, and Prof. H. F. Deutsch (University of Wisconsin Medical School) for editing this manuscript.


REFERENCES

  1. Varki, A. (1993) Glycobiology 3, 97-130 [Abstract]
  2. Kornfeld, S., and Kornfeld, R. (1985) Annu. Rev. Biochem. 54, 631-664 [CrossRef][Medline] [Order article via Infotrieve]
  3. Ioffe, E., and Stanley, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 728-732 [Abstract/Free Full Text]
  4. Metzler, M., Gertz, A., Sarkar, M., Schachter, H., Schrader, J. W., and Marth, J. D. (1994) EMBO J. 13, 2056-2065 [Abstract]
  5. Friedlander, D. R., Brackenbury, R., and Edelman, G. M. (1985) J. Cell Biol. 101, 412-419 [Abstract]
  6. McCoy, R. D., Vimr, E. R., and Troy, F. A. (1985) J. Biol. Chem. 260, 12695-12699 [Abstract/Free Full Text]
  7. Breen, K. C., and Regan, C. M. (1988) Development 104, 147-154 [Abstract]
  8. Kadmon, G., Kowitz, A., Altevogt, P., and Schachner, M. (1990) J. Cell Biol. 110, 209-218 [Abstract]
  9. Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. (Tokyo) 114, 334-338 [Abstract]
  10. Taniguchi, N., and Ihara, Y. (1995) Glycoconj. J. 12, 733-738 [Medline] [Order article via Infotrieve]
  11. Narasimhan, S. (1982) J. Biol. Chem. 257, 10235-10242 [Abstract/Free Full Text]
  12. Nishikawa, A., Ihara, Y., Hatakeyama, M., Kangawa, K., and Taniguchi, N. (1992) J. Biol. Chem. 267, 18199-18204 [Abstract/Free Full Text]
  13. Ihara, Y., Nishikawa, A., Tohma, T., Soejima, H., Niikawa, N., and Taniguchi, N. (1993) J. Biochem. (Tokyo) 113, 692-698 [Abstract]
  14. Bhaumik, M., Seldin, M. F., and Stanley, P. (1995) Gene (Amst.) 164, 295-300 [CrossRef][Medline] [Order article via Infotrieve]
  15. Miyoshi, E., Ihara, Y., Hayashi, N., Fusamoto, H., Kamada, T., and Taniguchi, N. (1995) J. Biol. Chem. 270, 28311-28315 [Abstract/Free Full Text]
  16. Yoshimura, M., Ihara, Y., Ohnishi, A., Ijuhin, N., Nishiura, T., Kanakura, Y., Matsuzawa, Y., and Taniguchi, N. (1996) Cancer Res. 56, 412-418 [Abstract]
  17. Yoshimura, M., Nishikawa, A., Ihara, Y., Taniguchi, S., and Taniguchi, N. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8754-8758 [Abstract]
  18. Richards, R. I., Heguy, A., and Karin, M. (1984) Cell 37, 263-272 [Medline] [Order article via Infotrieve]
  19. Taniguchi, N., Nishikawa, A., Fujii, S., and Gu, J. (1989) Methods Enzymol. 179, 397-408 [Medline] [Order article via Infotrieve]
  20. Nishiura, T., Fujii, S., Kanayama, Y., Nishikawa, A., Tomiyama, Y., Iida, M., Karasuno, T., Nakao, H., Yonezawa, T., Taniguchi, N., and Tarui, S. (1990) Cancer Res. 50, 5345-5350 [Abstract]
  21. Ihara, Y., Nishikawa, A., and Taniguchi, N. (1995) Glycoconj. J. 12, 784-794
  22. Mutoh, T., Tokuda, A., Guroff, G., and Fujiki, N. (1993) J. Neurochem. 60, 1540-1547 [Medline] [Order article via Infotrieve]
  23. Qian, F., Vaux, D. L., and Weissman, I. L. (1994) Cell 77, 335-347 [Medline] [Order article via Infotrieve]
  24. Greene, L. A., and Tischer, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2424-2428 [Abstract]
  25. Greene, L. A., and Tischer, A. S. (1980) in Advances in Cellular Neurobiology (Federoff, S., and Hertz, L., eds), pp. 373-414, Academic Press, New York
  26. Yamashita, K., Hitoi, A., and Kobata, A. (1983) J. Biol. Chem. 258, 14753-14755 [Abstract/Free Full Text]
  27. Cummings, R. D., and Kornfeld, S. (1982) J. Biol. Chem. 257, 11230-11234 [Abstract/Free Full Text]
  28. Nishikawa, A., Gu, J., Fujii, S., and Taniguchi, N. (1990) Biochim. Biophys Acta. 1035, 313-318 [Medline] [Order article via Infotrieve]
  29. Guroff, G. (1984) in Cell Culture in the Neurosciences (Bottenstein, J., and Sato, G., eds), pp. 245-271, Plenum, New York
  30. Halegoua, S., and Patrick, J. (1980) Cell 22, 571-581 [Medline] [Order article via Infotrieve]
  31. Nakanishi, N., and Guroff, G. (1985) J. Biol. Chem. 260, 7791-7799 [Abstract/Free Full Text]
  32. Landreth, G. E., and Rieser, G. D. (1985) J. Cell Biol. 100, 677-683 [Abstract]
  33. Kaplan, D. R., Hempstead, B. L., Martin-Zanca, D., Chao, M. V., and Parada, L. F. (1991) Science 252, 554-558 [Medline] [Order article via Infotrieve]
  34. Ohmichi, M., Decker, S. J., Pang, L., and Saltiel, A. R. (1991) Biochem. Biophys Res. Commun. 179, 217-233 [Medline] [Order article via Infotrieve]
  35. Martin-Zanca, D., Oskam, R., Mitra, G., Copeland, T., and Barbacid, M. (1989) Mol. Cell Biol. 9, 24-33 [Medline] [Order article via Infotrieve]
  36. Jing, S., Tapley, P., and Barbacid, M. (1992) Neuron 9, 1067-1079 [Medline] [Order article via Infotrieve]
  37. Leconte, I., Auzan, C., Debant, A., Rossi, B., and Clauser, E. (1992) J. Biol Chem. 267, 17415-17423 [Abstract/Free Full Text]
  38. Mutoh, T., Tokuda, A., Miyadai, T., Hamaguchi, M., and Fujiki, N. (1995) Proc Natl. Acad. Sci. U. S. A. 92, 5087-5091 [Abstract]
  39. Ferrari, G., Anderson, B. L., Stephens, R. M., Kaplan, D. R., and Greene, L. A. (1995) J. Biol. Chem. 270, 3074-3080 [Abstract/Free Full Text]
  40. Hahn, T. J., and Goochee, C. F. (1992) J. Biol. Chem. 267, 23982-23987 [Abstract/Free Full Text]
  41. Canossa, M., Twiss, J. L., Verity, A. N., and Shooter, E. M. (1996) EMBO J. 15, 3369-3376 [Abstract]
  42. Baldwin, A. N., and Shooter, E. M. (1995) J. Biol. Chem. 270, 4594-4602 [Abstract/Free Full Text]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.