(Received for publication, November 11, 1996, and in revised form, January 27, 1997)
From the Department of Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan
-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.
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 -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:-D-mannoside
-1,4-N-acetylglucosaminyltransferase III (GnT-III:
EC 2.4.1.144), has been found (Fig. 1).
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.
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 ConstructsRat 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.
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 ActivityCell 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
-1,4-galactosyltransferase activities were assayed by high
performance liquid chromatography methods described previously (19, 20)
using the fluorescence-labeled sugar chain (GlcNAc
-1,2-Man
-1,6-[GlcNAc
-1,2-Man
-1,3-]
Man
-1,4-GlcNAc
-1,4-GlcNAc-pyridylamino) as the substrate.
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 NGFThe 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 AssaysGrowth of GnT-III gene transfectants and control PC12 cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (23).
DNA SynthesisPlates 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 BindingCells 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
-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).
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.
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 -1,4-galactosyltransferase activities assayed
as the control glycosyltransferases showed no significant changes.
|
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).
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.
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
GlcNAc-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
-1,4-galactosyltransferase as the result of treatment with
NGF (data not shown).
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).
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.
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.
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.
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.
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 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.
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.