From the
While investigating the glycosylation of nuclear envelope
proteins of neuroblastoma cells, we found several proteins that bound
the sialic acid-specific Sambucus nigra agglutinin. The
strongest signals were obtained for proteins with apparent molecular
masses of 66 and 180 kDa. The specificity of the lectin binding was
checked by acylneuraminyl hydrolase treatment of nuclear envelope
proteins, which prohibited S. nigra agglutinin binding.
Digestion of nuclear envelope proteins with the N-glycosidase
F revealed that sialic acid was N-glycosidically linked to the
180-kDa protein and very probably O-glycosidically linked to
the 66-kDa protein. Upon extraction, the latter behaved like the
nucleoporin p62 in that it was partly extracted by high ionic strength
buffers, could not be solubilized by nonionic detergent, and was
completely removed from the nuclear envelope with urea. Two-dimensional
gel electrophoretic comparison showed that the S. nigra agglutinin-binding protein and p62 have an identical isoelectric
point of about 5.0 and an identical apparent molecular mass of 66 kDa.
This, together with the binding of the anti-nucleoporin antibody,
demonstrated the identity of the 66-kDa sialoprotein and p62. S.
nigra agglutinin inhibits nuclear protein transport in
neuroblastoma cells, strongly suggesting a functional significance of
sialylation of p62.
The nuclear envelope is more than a simple barrier between the
cytoplasm and the nucleus. It controls the selective and continuous
exchange of macromolecules between nucleus and cytoplasm and, as has
become clear only recently, also carries components that are involved
in signal transduction
(1, 2) . The nuclear envelope is
composed of inner and outer nuclear membranes, nuclear pores, and
nuclear lamina (3). The nuclear lamina forms a network of intermediate
filament lamin proteins that line the inner nuclear membrane. The outer
nuclear membrane is continuous with the endoplasmic reticulum and
shares structural and functional characteristics with it
(4) .
The inner and outer nuclear membranes enclose the perinuclear cisternae
and are joined at the nuclear pores. These are formed by the nuclear
pore complexes, large (about 120 MDa) supramolecular assemblies that
mediate molecular trafficking between nucleus and cytoplasm
(5) .
A number of nuclear envelope proteins, in particular nuclear pore
complex proteins, are glycosylated in an unconventional way: they
contain single N-acetylglucosamine residues attached in
O-linkage directly to serine and threonine
residues
(6, 7, 8, 9) . The most abundant
member of these glycosylated nucleoporins is a protein of 62 kDa
(calculated from cDNA), named p62
(10, 11) . Since most
of the O-GlcNAc residues are added in the cytoplasm within 5
min of synthesis, when p62 is soluble and cytosolic, the glycosylation
mechanism is clearly distinct from the conventional N- and
O-linked glycosylation pathways in the endoplasmic reticulum
and the Golgi complex
(12) . The functional importance of the
carbohydrate moieties linked to the nucleoporins has been demonstrated
by the finding that WGA,
In our investigation on glycosylated nuclear
envelope proteins of Neuro-2a cells we used specific lectins and
glycosidases to identify proteins bearing sialic acid residues. We
found several sialylated proteins and characterized the two major
sialoproteins with apparent molecular masses of 66 and 180 kDa. We
showed the identity of the 66-kDa sialoprotein with the
O-GlcNAc-bearing nucleoporin p62. The potential functional
significance of sialylation for nuclear transport was analyzed using an
in vitro transport assay. We found that adding SNA inhibits
the nuclear import of proteins in vitro.
Unlabeled lectins were from
Vector Laboratories, Inc. (Burlingame, CA). WGA (Triticum vulgaris agglutinin), biotin-labeled, and the streptavidin alkaline
phosphatase conjugate were from Amersham Corp. N-glycosidase F
from Flavobacterium menigosepticum and sialidase from
Arthrobacter ureafaciens were purchased from Boehringer
Mannheim. DNase I was from Boehringer Mannheim, Benzonase
Nuclear envelopes were prepared by a modification of the method
described by Otto et al.(2) . Briefly, the nuclei
(2.8-3.9 mg of protein) were suspended in 24 ml of ice-cold TP
buffer (10 mM Tris/HCl, pH 8.0, 10 mM
Na
Integrity and purity of the nuclei were judged
by phase contrast microscopy. Purity of the nuclear envelopes were
judged by electron microscopy, marker enzyme activity, and Western
blotting with antibodies against marker proteins.
Protein
determination was performed according to Bradford
(17) using
bovine serum albumin as a standard.
Antibodies were
diluted with 5% (w/v) skimmed milk powder in TBS (anti-ribophorin,
1:20,000; mAb 414, 1:1000; all secondary antibodies, 1:2000) or 5%
bovine serum albumin in TBS containing 0.02% (w/v) NaN
SNA (2 µg/ml) and MAA (5 µg/ml) were dissolved in TBS
containing 1 mM MgCl
For digestion with N-glycosidase F
500 microunits were dissolved in 1.5 ml of buffer containing 50
mM sodium phosphate, pH 8.2, 5 mM EDTA, 10 µg/ml
leupeptin, 10 µg/ml aprotinin. All control samples were incubated
under the same conditions without glycosidase.
Aliquots of 100 µg
of protein of the nuclear envelope fraction were preincubated for 15
min at room temperature with Benzonase to remove residual DNA and RNA.
After sedimentation of the membranes at 10,000
For highsalt
extraction nuclear envelopes were incubated in salt buffer
containing 10 mM Tris/HCl, pH 7.4, 0.25 M sucrose,
0.1 mM MgCl
For detergentextraction the envelopes
were incubated in a buffer containing 20 mM Tris/HCl, pH 7.4,
0.25 M sucrose, 5 mM MgCl
For
ureaextraction the envelopes were incubated in 20
mM Tris/HCl, pH 7.4, 0.25 M sucrose, 0.1 mM
MgCl
All samples were boiled for 3
min and incubated overnight at 4 °C.
The incubation of the cells under transport conditions was
carried out at 30 °C for 30 min. The cells were fixed in 3.7%
paraformaldehyde in PBS for 15 min at room temperature, rinsed in PBS,
stained with DAPI for 2 min and washed with PBS. The cells were
embedded in Mowiol 4.88 (Polyscience) and examined with a Zeiss
Axiophot microscope.
Comparison of the NE fraction with the
other subcellular fractions showed for each of them an individual,
specific, and reproducible protein pattern after SDS-PAGE and staining
with Coomassie Blue (data not shown). For further characterization, all
fractions were probed with antibodies against lamin B
The specificity of the lectin binding was confirmed by sialidase
digestion. Strips of nitrocellulose with the transferred nuclear
envelope proteins were treated with sialidase, which removes terminal
sialic acid residues from oligosaccharides. After sialidase digestion
the proteins were probed with SNA and WGA. Whereas the SNA reactivity
of all proteins (including the NE-specific proteins sp66 and sp180)
disappeared, none of the WGA signals were altered (Fig. 4). The
specificity of the sialidase reaction was checked by addition of a
specific inhibitor (1 mM
2,3-dehydro-2-deoxy-N-acetylneuraminic acid) or 1 mM
substrate N-acetylneuramin-lactose. In both cases removal of
sialic acid from the blotted proteins by sialidase (2 milliunits of
sialidase, 2 h at room temperature) was completely inhibited (data not
shown).
In the present work we can show the presence of sialylated
proteins at the nuclear envelope. We obtained strong and highly
specific signals for NE proteins by probing with SNA, a lectin specific
for
The SNA signals are the
consequence of a specific binding, as could be confirmed by the
complete disappearance of the signals after sialidase treatment. The
high digestion efficiency may have been furthered by carrying out the
treatment on nitrocellulose-blots. Proteins are easily accessible
because of the absence of interfering lipids, and due to the
denaturated state, the hydrophilic carbohydrate moieties should be
exposed.
The specificity of the sialidase reaction was also checked.
A contaminating protease activity as a cause for the disappearance of
the signal could be excluded, because the signals obtained with WGA
were not influenced by sialidase treatment. Furthermore, in the case of
the control protein fetuin, the previously masked GalNAc residues
became detectable only after sialidase treatment (data not shown).
Digestion of blotted NE proteins with N-glycosidase F led
to the disappearance of the SNA signal only in the case of sp180,
strongly indicating that sp180 is modified N-glycosidically,
whereas sp66 is modified in an O-glycosidic manner.
The
sp180 may be identical with the known NE-glycoprotein gp210, which has
an apparent molecular mass of 180 kDa in SDS-PAGE. Furthermore, sp180
apparently is modified N-glycosidically, does not bind WGA,
and is not extractable with detergent in low ionic strength buffer,
characteristics also applying to gp210
(27) .
The extraction
experiments revealed that sp66 behaved in a way very similar to
nucleoporins. Very strong evidence for the hypothesis that sp66 is
identical with p62 was achieved by two-dimensional gel electrophoresis,
since after probing with SNA and mAb 414, one protein could be detected
in each case. These proteins have identical apparent molecular masses
and isoelectric points of about 5. These values are in good accordance
with reported values for murine p62
(11, 21) .
Furthermore, chromatography on a WGA-sepharose column led to a
similar enrichment of both p62 and sp66 (data not shown). An attempt to
determine amino-terminal sequences after electrophoresis and subsequent
blotting of the enriched sp66/p62 on PVDF membrane failed, probably due
to N-terminal blockage.
The possibility
cannot entirely excluded that SNA binds to a protein other than p62,
but this appears rather unlikely in view of the many characteristics
(molecular mass, isoelectric point, solubility characteristics in high
salt buffer, detergent, and 7 M urea, enrichment by
WGA-sepharose chromatography) shared by sp66 and p62.
Our
observation that the nucleoporin p62 is sialylated is not in conflict
with earlier results. Whereas several fluorescein
isothiocyanate-labeled lectins specific for other sugar residues were
tested for binding to nuclei and yielded no or only faint nuclear
staining
(24) , no study is known to us in which a sialic
acid-specific lectin was used to investigate glycan structures of
nuclear envelope proteins.
Although the complete structure of the
sialic acid-containing carbohydrate moiety of p62 remains to be
clarified, some features can be determined from our experiments. p62
apparently contains carbohydrate moieties with terminal sialic acid
residues that are
p62 has been reported to
bear single O-glycosidic linked GlcNAc residues
(12) .
It is not clear whether additional GlcNAc residues are present with
sialic acid residues attached to them. Such a linkage is known from
complex glycans
(28, 29) . However, this would mean that
the lectin SNA can recognize sialic acid
The mechanism of sialic acid
transfer to p62 is unclear. As the GlcNAc residues are added to p62
within 5 min of synthesis, p62 apparently does not pass through the
Golgi apparatus
(12) . Therefore, it is unlikely that p62 is
sialylated in the conventional way in the Golgi apparatus. Instead of
this one may postulate a sialic acid transfer to p62 by a thus far
unknown sialylation mechanism outside the Golgi apparatus. At least the
substrate for sialic acid transfer, CMP-sialic acid, is present in the
nucleus
(16, 31) . CMP-sialic acid is synthesized by
CMP-sialic acid synthetase, an enzyme located in the cell
nucleus
(31) . The presence of the activated precursor would be
in correspondence with a sialic acid transfer in the nucleus or at the
nuclear envelope, but nothing is known about a nuclear transferase that
could catalyze this transfer.
Whether sialic acid residues attached
to p62 and other nuclear envelope proteins are of functional
significance is not yet clear, but this is strongly suggested by the
finding that SNA inhibits nuclear transport. SNA appears to be less
effective than WGA. This difference is reduced when the molar
concentrations rather than the concentrations in mg/ml are considered,
since SNA has a molar mass four times higher than WGA (140,000 Da
versus 36,000 Da). The remaining difference in the inhibition
efficiency may be due to a lower affinity of SNA to sialic acid than
that of WGA to GlcNAc. This is also indicated by the different
concentrations needed in the overlay assays.
Immunodepletion
experiments demonstrated that p62 is essential for nuclear protein
import and NPC
assembly
(13, 32, 33, 34) . The role of
the O-linked glycans in these processes is not understood.
WGA, binding to the O-linked glycans of p62 and other
nucleoporins, blocked the ATP-dependent translocation step but did not
interfere with the binding of nucleus targeted proteins to the pore
complexes
(35) . In a recent study it was observed that
modifications of the O-glycans attached to nucleoporins with
galactose did not result in an altered NPC structure or nuclear protein
import
(36) . Since Gal modifications had been expected to
interfere with any biological function mediated by the recognition of a
specific carbohydrate structure required for intermolecular
interactions (37-39), this finding suggests that the
galactosylatable sugar residues of NPC glycoproteins may not play a
direct role in these processes. However, this finding is not in
contradiction to the assumption that sialic acid residues play a role
in NPC assembly or nuclear transport, since sialic acid residues are
not galactosylatable carbohydrates and therefore would not have been
affected in the approach of Miller and Hanover
(36) .
In
conclusion we found sialylation of nuclear envelope proteins and
identified p62 as one of these sialoproteins. Surely, further studies
are needed to elucidate the mechanism of this sialylation and the
functional implications of this hitherto unknown glycosylation.
We are grateful to F. Hucho for continuous
encouragement and support and for critically reading the manuscript. We
thank W. Reutter for helpful discussions. Expert technical assistance
from D. Krück is gratefully acknowledged.
e 15, 14195 Berlin, Federal
Republic of Germany
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
a GlcNAc-binding
lectin, blocks the active transport of proteins through nuclear pore
complexes
(13) . In view of their potential functional
importance, a further analysis of nuclear envelope glycoproteins
appears to be essential for the understanding of the nuclear envelope.
Besides the O-GlcNAc-modified nucleoporins, an integral
membrane protein containing N-linked high mannose type
carbohydrate, named gp210
(3) , and an O-GlcNAc-bearing
integral membrane protein, POM 121
(14) , were described. Several
findings indicated that sialic acid residues bound to proteins can also
be found in the nucleus and at the nuclear envelope
(15) . The
significance of these findings, however, was not unambiguously
clarified
(16) .
Cells
Mouse neuroblastoma (Neuro-2a) cells were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal
calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin at
37 °C in a humidified atmosphere with 5% CO.
Antibodies
The monoclonal antibody against lamin B was a
gift from H. Henneckes (Epalinges). The polyclonal antibody against
ribophorin was a gift from T. Rapoport (Berlin). The monoclonal
antibody against p62 (mAb 414) was purchased from BAbCO (Richmond, CA).
All secondary antibodies were from Sigma.
Lectins and Glycosidases
Sambucus nigra agglutinin (SNA) and Maackia
amurensis agglutinin (MAA), digoxigenin-labeled, and the
anti-digoxigenin alkaline phosphatase-conjugated antibody were
purchased from Boehringer Mannheim.
(Benzon Nuclease) was from Merck, heparin was from Sigma, and the
other chemicals were purchased from Sigma and Bio-Rad in the highest
quality available.
Subcellular Fractionation
Nuclear Envelopes
For isolation of nuclei and
nuclear envelopes Neuro-2a cells from 12-24 154-cm culture dishes were washed twice with ice-cold PBS (137
mM NaCl, 2.7 mM KCl, 1.5 mM
KH
PO
, 8.0 mM
Na
HPO
) and scraped off in PBS. After
centrifugation at 500
g for 5 min the cells were
resuspended in 18 ml of STM 0.25 (50 mM Tris/HCl, pH 7.4, 0.25
M sucrose, 5 mM MgSO
, 2 mM
dithioerythritol, 10 µg/ml leupeptin, 1 mM PMSF). The
following manipulations were performed either with ice-cold reagents or
at 4 °C unless noted. A solution of 10% Nonidet P-40 was added to a
final concentration of 0.025%. The pellets were homogenized by 30
strokes in a glass-glass Dounce homogenizer (S-type). The homogenizer
was rinsed with 1 ml of STM 0.25 buffer, and the homogenate was
adjusted to 1.4 M sucrose by addition of an appropriate volume
of STM 2.1 (50 mM Tris/HCl, pH 7.4, 2.1 M sucrose, 5
mM MgSO
, 2 mM dithioerythritol, 10
µg/ml leupeptin, 1 mM PMSF). 10 ml of this suspension were
transferred to each centrifuge tube and laid between a 1-ml STM 2.1
cushion and 2 ml of STM 0.8 (50 mM Tris/HCl, pH 7.4, 0.8
M sucrose, 5 mM MgSO
, 2 mM
dithioerythritol, 10 µg/ml leupeptin, 1 mM PMSF). The
tubes were filled up to 14 ml with STM 0.25 and centrifuged at 100,000
g for 65 min in a swinging bucket rotor. The pellets
containing the nuclei were resuspended in a small volume of STM 0.25.
HPO
, 1 mM PMSF) containing 7.2 mg
heparin and 440 µg of DNase I (about 866 units). The suspension was
gently stirred on a magnetic stirrer for 60 min at 4 °C and 15 min
at room temperature. Nuclear envelopes were sedimented at 10,000
g for 30 min at 4 °C and washed once in a small
volume of STM 0.25.
Plasma Membrane
For preparation of the plasma
membrane fraction the interface between 1.4 and 0.8 M sucrose
of the gradient described above was carefully removed and diluted with
4 volumes of STM 0.25. The membranes were sedimented at 5000
g for 20 min, and the pellets were resuspended in STM 0.25.
This procedure yielded a plasma membrane fraction with a specific
activity of 5`-nucleotidase 2.5 times higher than in the homogenate.
For comparison, a higher than 8-fold enrichment was achieved when
plasma membranes were prepared by a different procedure from bovine
brain
(18) .
Mitochondria, Microsomes, and Cytosol
The
homogenate of cells from 12 culture dishes, prepared as described
above, was centrifuged at 170 g for 15 min at 4
°C. The supernatant was centrifuged at 300
g for 5
min at 4 °C and sedimented at 15,000
g for 10 min
at 4 °C. The pellet was resuspended in STM 0.25 and designated as
crude mitochondria fraction. The supernatant was diluted with 1 volume
of STM 0.0 (STM 0.25 without sucrose) and centrifuged at 105,000
g overnight at 4 °C. The resulting supernatant and
pellet yielded the cytosolic fraction and the microsomal fraction,
respectively.
Electron Microscopy
Isolated nuclear envelopes were centrifuged (10,000
g, 5 min), resuspended in PBS containing 2 mM
MgCl
, and centrifuged again. The resulting pellet was fixed
in 1% glutaraldehyde, 1% tannic acid in 0.1 M sodium phosphate
buffer, pH 7.4. Subsequently, it was postfixed in a 2% OsO
solution. After rinsing and dehydration in the ascending alcohol
series, the preparations were embedded in Epon. They were cut with an
Ultracut E (Reichert), and the ultrasections were contrasted with 2%
uranyl acetate and lead citrate and examined with a Zeiss EM10
transmission electron microscope.
Western Blotting
Subcellular fractions or extracts were subjected to 10% (w/v)
SDS-PAGE
(19) and transferred to nitrocellulose membranes
(Hybond, Amersham Corp.). Proteins were stained with Ponceau red to
control the efficiency of blotting and the appropriate loading of the
lanes. The membranes were then cut into strips of one lane or a half
lane each and blocked overnight at 4 °C with a solution of 5% (w/v)
skimmed milk powder in TBS-T (20 mM Tris/HCl, pH 7.4, 150
mM NaCl, 0.1% (v/v) Tween 20). Incubation with the primary
antibody was performed for 3 h at room temperature, followed by three
washes in TBS (20 mM Tris/HCl, pH 7.4, 150 mM NaCl)
and incubation with the secondary antibody for 1 h. After three washes
in TBS and an additional wash in staining buffer (0.1 M
NaHCO/NaOH, pH 9.8, 1 mM MgCl
) color
development was performed using nitro blue tetrazolium and
5-bromo-4-chloro-3-indoyl-phosphate (p-toluidine salt).
Staining was stopped with 10 mM EDTA.
(anti-lamin B
, 1:1000).
Detection of Glycosylated Proteins
Aliquots of prepared subcellular fractions were loaded on a
10% (w/v) SDS-polyacrylamide gel, and the separated proteins were
transferred to sheets of nitrocellulose (Hybond, Amersham Corp.) and
visualized with Ponceau. The blot membrane was cut into strips of one
lane or a half lane each and blocked overnight with 0.5% casein in TBS
at 4 °C. The incubation with the digoxigenin- or biotin-labeled
lectins was performed for 1 h at room temperature, followed by three
washes in TBS and incubation for 1 h with the alkaline
phosphatase-conjugated antibodies (1:2000, in TBS) or alkaline
phosphatase-conjugated streptavidin (1:2000, in TBS). After three
washes in TBS color development was performed as described above.
, 1 mM
MnCl
, 1 mM CaCl
. WGA (5 µg/ml) was
dissolved in TBS-T.
Deglycosylation of Glycoproteins
After blocking the nitrocellulose strips overnight, proteins
on the membrane were incubated with glycosidase for 24 h at room
temperature under gentle agitation. For digestion with sialidase 25
milliunits were dissolved in 1.5 ml of buffer containing 50 mM
sodium acetate, pH 5.5, 5 mM EDTA, 10 µg/ml leupeptin, and
10 µg/ml aprotinin.
Two-dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis was performed exactly as
described by Jungblut and Seifert
(20) . First dimension was a
nonequilibrium pH gel electrophoresis (1841 volt-hours) in 4% rod gels,
and the second dimension was an SDS-PAGE in 10% slab gels.
Extraction
Extraction of nuclear envelope proteins was performed
according to Snow et al.(21) and Gerace et
al.(27) with some modifications.
g the
pellets were resuspended and diluted to a final concentration of 1
mg/ml protein in the extraction buffers.
, 1 M NaCl, 1 mM
dithioerythritol, 10 µg/ml leupeptin, and 1 mM PMSF for 30
min on ice. Samples were centrifuged at 10,000
g for
30 min at 4 °C. The resulting pellet was resuspended in SDS sample
buffer (62.5 mM Tris/HCl, pH 6.8, 2% (w/v) SDS, 20% (w/w)
glycerol, 5% (v/v) mercaptoethanol, 0.1% (w/v) bromphenol blue). The
supernatant was concentrated in a Microcon
microconcentrator (Amicon, Witten) and mixed with SDS sample
buffer.
, 20 mM
KCl, 2% (w/v) Triton X-100, 1 mM dithioerythritol, 10
µg/ml leupeptin, 1 mM PMSF for 30 min on ice. After
centrifugation at 10,000
g for 30 min at 4 °C the
resulting pellet was resuspended in sample buffer. The proteins of the
supernatant were precipitated with trichloroacetic acid, and the washed
pellets were sonicated and resuspended in sample buffer.
, 7 M urea, 1 mM dithioerythritol, 10
µg/ml leupeptin, 1 mM PMSF for 30 min at room temperature.
Samples were centrifuged at 105,000
g for 60 min, and
the pellets were resuspended in sample buffer. The supernatant was
concentrated with a Microcon
microconcentrator (Amicon,
Witten) and mixed with sample buffer.
In Vitro Transport Assay
The nuclear transport assay was performed as described by
Adam et al.(22) . In brief, Neuro-2a cells, grown on
coverslips, were rinsed briefly in import buffer (50 mM Hepes,
pH 7.3, 110 mM potassium acetate, 2 mM magnesium
acetate, 5 mM sodium acetate, 1 mM EGTA, 2
mM dithiothreitol, 1 µg/ml leupeptin, 1 µg/ml
aprotinin) and permeabilized with 50 µg/ml digitonin in import
buffer for 5 min at 4 °C. Coverslips were rinsed in cold import
buffer. As reporter protein, TRITC-labeled bovine serum albumin was
used, coupled to a 12-residue synthetic peptide corresponding to the
SV-40 large T antigen nuclear localization signal as described (22). A
standard transport mixture contained reporter protein, 1% (w/v) bovine
serum albumin, an ATP-regenerating system (1.5 mM ATP, 10
mM creatine phosphate, and 65 units/ml creatine phosphokinase,
final concentrations), and reticulocyte lysate in a final volume of 15
µl. As control, a reporter protein without nuclear localization
signal was used or an energy depletion system (50 mM glucose,
100 units/ml hexokinase) was added instead of the ATP-regenerating
system.
Effects of Lectins on Nuclear Transport
To assay the effect of lectin addition on transport,
permeabilized cells were preincubated with WGA (0.1 mg/ml) or SNA
(1.5-3.5 mg/ml) diluted in import buffer containing 1% bovine
serum albumin for 30 min at 30 °C. The protein transport assay was
performed as described above, in the presence of 20 µg/ml WGA or
150 µg/ml SNA added to the transport mixture.
Characterization of the Nuclear Envelope
Preparation
The nuclei, isolated from Neuro-2a cells, were
checked by phase contrast microscopy and appear to be intact and devoid
of visible cytoplasmic structures attached to them
(Fig. 1A). Nuclei were incubated in a low ionic strength
buffer and treated with heparin and DNase to release all nuclear
contents. Nuclear envelope preparations were sedimented and, as
revealed by electron microscopy, consisted of the typical membranous
structures with almost no chromatin material present
(Fig. 1B).
Figure 1:
Nuclei and nuclear membranes from
Neuro-2a cells. A, phase-contrast picture of isolated nuclei.
Calibrationbar, 30 µm. B, electron
micrograph of a nuclear envelope fraction. Calibrationbar, 0.3 µm.
This observation corresponded to the low
DNA content of the NE preparation. Less than 1% of the total DNA in the
nuclei was found in the NE preparations. Lactate dehydrogenase activity
was not detectable in the NE preparation, indicating the absence of
cytosolic contamination.
, a
component of the nuclear lamina, against nucleoporins, part of the
nuclear pore complex, and against ribophorin, a constituent of the
endoplasmic reticulum membrane (Fig. 2). As expected, ribophorin
was found in both the microsomal and the NE fraction, since the outer
nuclear membrane shares some characteristics with the endoplasmic
reticulum membrane (Fig. 2). To detect nuclear pore complex
proteins we used the monoclonal antibody mAb 414. This well described
monoclonal antibody (10) preferentially binds to p62 and also
recognizes a few members of the nucleoporin family that share epitopes
with p62. The nucleoporins detected with the antibody mAb 414 were
highly enriched in the NE fraction and also detectable in small amounts
in the cytosol (Fig. 2) according to their cellular distribution
as described by Davis and Blobel
(12) . The high degree of
nuclear envelope enrichment during the preparation was demonstrated by
a strong lamin B
signal found almost exclusively in the NE
fraction (Fig. 2).
Figure 2:
Characterization of nuclear envelope
preparations. Subcellular fractions (50 µg each) were separated by
SDS-PAGE and immunoblotted with antibodies against ribophorin,
nucleoporins (mAb 414), and lamin B. To the left,
molecular masses (in kDa) are indicated. H, homogenate;
C, cytosol; NE, nuclear envelopes; NP,
nucleoplasm; PM, plasma membrane; MS, microsomes;
MI, mitochondria. The arrowheads mark the
nucleoporins nup153 and p62.
Glycoproteins at the Nuclear Envelope
Nuclear
envelopes from yeast and rat liver cells are known to contain
glycoproteins reacting with Canavalia ensiformis agglutinin
(gp210) or WGA (nucleoporins). We probed NE proteins from neuroblastoma
cells with WGA and obtained a variety of WGA-binding proteins
(Fig. 3). Comparison with molecular masses of the proteins
detected with the mAb 414 (Fig. 2) strongly suggests that two of
the WGA-binding proteins are identical with the
O-GlcNAc-bearing nucleoporins p62 and nup 153 (apparent
molecular mass 185 kDa; Ref. 23).
Figure 3:
Detection of glycoproteins containing
GlcNAc or sialic acid. Subcellular fractions (50 µg) were separated
by SDS-PAGE and blotted on nitrocellulose. Left, WGA-binding
proteins of the nuclear envelope. Among others, strong signals were
obtained for proteins with apparent molecular masses of 66 and 185 kDa,
very probably reflecting the O-glycosylated nucleoporins p62
and nup153, which were detected by mAb 414 (Fig. 2). Right,
SNA-binding proteins of subcellular fractions. After probing with the
sialic acid-specific lectin SNA two major proteins with molecular
masses of 66 and about 180 kDa, specific for the nuclear envelope
fraction, were detected
(arrowheads).
In order to detect sialylated
glycoproteins at the nuclear envelope the lectins SNA (specific for
sialic acid 2-6 linked to Gal or GalNAc) and MAA (specific
to sialic acid
2-3 linked to Gal) were used. The incubation
with the lectin MAA did not show any specific binding to NE proteins
(data not shown). In contrast to this, several proteins unique to the
nuclear envelope fraction could be detected with SNA (Fig. 3).
The major signals were associated with proteins of approximately 66 kDa
(designated as sialoprotein sp66) and 180 kDa (sp180) (Fig. 3).
Figure 4:
Nuclear envelope glycoproteins after
glycosidase treatment. Nuclear envelope proteins (50 µg) blotted on
nitrocellulose were treated with sialidase or N-glycosidase F.
After this treatment, the nitrocellulose strips were probed with SNA or
WGA. Sialidase treatment led to the disappearance of the SNA binding to
both the 66- and the 180-kDa protein, whereas the WGA binding remained
unaffected. Treatment with N-glycosidase F abolished the SNA
binding of the 180-kDa protein, whereas the SNA binding of the 66-kDa
protein remained unaffected. WGA binding was not affected by
N-glycosidase F treatment.
The type of linkage between the proteins and the sialic
acid-bearing oligosaccharide was differentiated by treating blotted NE
proteins with the glycopeptidase N-glycosidase F. This
glycosidase removes N-linked oligosaccharides by cleaving the
GlcNAc linkage to asparagine. All O-glycans, including those
with the unusual linkage of single GlcNAc-residues to serine or
threonine, remain unaffected. After N-glycosidase F digestion
only the sp66 protein (but not the sp180) was still recognized by SNA
(Fig. 4). As a control the proteins were incubated with WGA. The
WGA reactivity of all NE proteins remained unaffected upon
N-glycosidase F treatment (Fig. 4). The completeness of
N-glycan cleavage was monitored by digestion of the control
protein fetuin (10 µg) under the same conditions (data not shown).
Characterization of the Extraction Behavior of Nuclear
Envelope Proteins
Nuclear envelopes were extracted under various
conditions to further characterize the sialylated proteins. To follow
the distribution of the glycoproteins and some marker proteins, blotted
proteins were probed with the lectin SNA and antibodies against lamin
B, ribophorin and the nucleoporins. When nuclear envelopes
were extracted with TX-100 in a low ionic strength buffer, only
integral membrane proteins, represented by ribophorin, were
solubilized, whereas all sialoproteins remained in the pellet
(Fig. 5). After extraction of NE with high salt concentration to
remove membrane-associated proteins, a small amount of the major
sialoprotein sp66 and the nucleoporins appeared in the supernatant,
whereas the major part remained in the pellet. Sp180 was only
detectable in the pellet (Fig. 5). Finally, extraction of nuclear
envelopes with 7 M urea leads to complete solubilization of
the nucleoporins and the sialoprotein sp66 from the nuclear envelope
(Fig. 5). Most of the lamins, demonstrated by probing with the
lamin B
-antibody (data not shown), but only a part of
ribophorin were also extractable under these conditions, whereas sp180
remained in the pellet.
Figure 5:
Extraction of nuclear envelope
glycoproteins. Nuclear envelopes (100 µg of protein) were extracted
with high salt (NaCl), 2% Triton X-100 (TX-100), or 7
M urea (urea) as described under ``Materials and
Methods.'' After SDS-PAGE and blotting, the nitrocellulose strips
were probed for SNA-binding proteins, nucleoporins (mAb 414), or
ribophorin. The sialoprotein sp66 showed the same distribution between
supernatant (S) and pellet (P) fractions as the
nucleoporin p62.
The sialoprotein sp66 showed the same
behavior upon extraction as the nucleoporins: it cannot be solubilized
with detergent, it is partly extractable with high ionic strength
buffers, and it is completely removed from the nuclear envelope with
urea. These results suggested that this sialoprotein, like the members
of the nucleoporin family, could be a constituent of the nuclear pore
complex. Furthermore, we observed that probing nuclear envelope
proteins with SNA, mAb 414, and WGA resulted in a major signal at the
same apparent molecular weight ( Fig. 3and Fig. 5).
Comparison of sp66 and p62 by Two-dimensional Gel
Electrophoresis
To obtain further evidence for the assumption
that sp66 is, in fact, identical with the nucleoporin p62, we used
two-dimensional gel electrophoresis. 100 µg of protein of isolated
nuclear envelopes were loaded on each tube gel to carry out
nonequilibrium pH gradient electrophoresis. In the second dimension
proteins were separated according to their molecular weight with
SDS-PAGE. Following transfer of the proteins to nitrocellulose,
membranes were probed with SNA or mAb 414. In both cases one protein
was detected at 66 kDa. These proteins have an identical isoelectric
point of about 5.0 and an identical apparent molecular weight
(Fig. 6), thus providing strong evidence that the sialylated
protein sp66 is identical to the nucleoporin p62.
Figure 6:
Comparison of SNA binding sp66 and p62
after two-dimensional gel electrophoresis. NE proteins were separated
by two-dimensional gel electrophoresis and blotted on nitrocellulose.
Following that the membranes were probed with SNA and mAb 414,
respectively. SNA and mAb 414 each bound to a protein with a pI of
about 5.0 and an apparent molecular mass of 66 kDa. The crosses indicate the position of a protein marked on the blot membrane
after Ponceau red staining.
Effect of SNA on Protein Transport
To investigate
the functional importance of the sialic acid residues for nuclear
transport we applied the nuclear transport assay developed by Adam
et al.(22) , to Neuro-2a cells. TRITC-labeled bovine
serum albumin, cross-linked to a synthetic nuclear localization signal
(NLS) peptide was used as a reporter protein. The transport of the
reporter protein into the nucleus depended on the presence of a NLS,
since a TRITC-bovine serum albumin conjugate lacking the NLS did not
enter the nucleus (Fig. 7A), whereas a strong
accumulation of an NLS-linked TRITC-bovine serum albumin conjugate was
observed after 30 min (Fig. 7B). This accumulation is
energy-dependent, since energy depletion by treatment with
hexokinase/glucose inhibited nuclear import (data not shown). Similar
to the observations reported earlier for other cell
types
(13, 24, 25, 26) , WGA inhibits
protein import into the nucleus in Neuro-2a cells as well.
Preincubation of these cells in import buffer containing 0.1 mg/ml WGA
caused transport inhibition (Fig. 7C). Interestingly,
preincubation of permeabilized Neuro-2a cells with transport buffer
containing 3.5 mg/ml SNA also blocked nuclear protein transport
(Fig. 7D). Partial inhibition of nuclear transport by
SNA was observed with a concentration of 1.5 mg/ml (data not shown).
Figure 7:
Inhibition of nuclear protein import by
SNA. Fluorescence micrographs show nuclear import of TRITC-labeled
reporter protein in digitonin-permeabilized Neuro-2a cells
(calibrationbar, 10 µm). The transport of the
reporter protein into the nucleus depended on the presence of an NLS,
since a TRITC-bovine serum albumin conjugate lacking the NLS did not
enter the nucleus (A), whereas a strong accumulation of an
NLS-linked TRITC-bovine serum albumin conjugate was observed after 30
min (B). Preincubation with WGA prevented nuclear accumulation
of NLS-TRITC-bovine serum albumin (C). The nuclear import of
NLS-TRITC-bovine serum albumin was blocked after preincubation of
Neuro-2a cells with the sialic acid binding lectin SNA
(D).
2-6 linked sialic acid. Strong signals were also
expected for proteins of the plasma membrane and of the Golgi
apparatus. However, the signals for NE proteins were stronger than
those for proteins of the plasma membrane and microsomal fractions.
This unexpected finding may be due to the
2-6
linkage-specificity of SNA, since MAA (
2-3-specific) bound
to many proteins of the microsomal and plasma membrane fraction but not
of the nuclear envelope fraction.
(
)
2-6 linked to Gal or GalNAc or, perhaps,
other sugars. The sialic acid-containing glycostructure is linked to
the protein most probably in an O-glycosidic manner, since the
SNA binding was not affected by N-glycosidase F. However, p62,
in contrast to sp180, might be a poor substrate for
N-glycosidase F. Another known possibility for failure of
N-glycosidase F cleavage is the linkage of the glycan to the
N-terminal amino acid. This, however, appears very unlikely, since the
N-terminal amino acid of p62 is methionine.
2-6 linked to
GlcNAc in addition to the known specificity. It seems unlikely that
GalNAc acts as an ``acceptor'' for sialic acid, since the
GalNAc-specific peanut agglutinin PNA yielded no signal with p62 after
sialidase treatment (data not shown). There is evidence indicating that
p62 also bears other sugar residues than GlcNAc; it was reported that a
lectin from the coral Gerardia savaglia bound to two proteins
of a nuclear pore complex lamina fraction with molecular masses of 62
and 190 kDa
(30) . This binding could be competed with mannose,
leading to the conclusion that these proteins are mannose-containing
glycoproteins
(30) , but the identity of these proteins with p62
and gp210 was not unequivocally shown.
-(N-acetyl-
-glucosaminyl)-asparagine-amidase
F (peptide-N-glycosidase F); sialidase, acylneuraminyl
hydrolase; SNA, S. nigra agglutinin; TRITC,
tetraethylrhodamine isothiocyanate; PAGE, polyacrylamide gel
electrophoresis; mAb, monoclonal antibody.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.