©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Nuclear Pore Complex Protein p62 Is One of Several Sialic Acid-containing Proteins of the Nuclear Envelope (*)

Sonja Emig (1)(§), Dirk Schmalz (1), Mehdi Shakibaei (2), Klaus Buchner (¶)

From the (1) Arbeitsgruppe Neurochemie, Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Federal Republic of Germany and (2) Institut für Anatomie, Freie Universität Berlin, Königin-Luise-Strae 15, 14195 Berlin, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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,() 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) .

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.


MATERIALS AND METHODS

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.

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 (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 KHPO, 8.0 mM NaHPO) 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.

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 NaHPO, 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.

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.

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.

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 (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.

SNA (2 µg/ml) and MAA (5 µg/ml) were dissolved in TBS containing 1 mM MgCl, 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.

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.

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.

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 g the pellets were resuspended and diluted to a final concentration of 1 mg/ml protein in the extraction buffers.

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, 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.

For detergentextraction the envelopes were incubated in a buffer containing 20 mM Tris/HCl, pH 7.4, 0.25 M sucrose, 5 mM MgCl, 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.

For ureaextraction the envelopes were incubated in 20 mM Tris/HCl, pH 7.4, 0.25 M sucrose, 0.1 mM MgCl, 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.

All samples were boiled for 3 min and incubated overnight at 4 °C.

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.

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.

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.


RESULTS

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.

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, 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).

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).


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).




DISCUSSION

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 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.

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 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.

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 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.

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.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a scholarship from the KFN, Freie Universität Berlin, FRG.

To whom correspondence should be addressed: Arbeitsgruppe Neurochemie, Institut für Biochemie, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Federal Republic of Germany. Tel.: 49-30-838-3381; Fax: 49-30-838-3753.

The abbreviations used are: WGA, T. vulgaris agglutinin; MAA, M. amurensis agglutinin; NE, nuclear envelope; NLS, nuclear localization signal; PMSF, phenylmethylsulfonyl fluoride; N-glycosidase F, peptide-N-(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.

S. Emig, C. Weise, and K. Buchner, unpublished observations.


ACKNOWLEDGEMENTS

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.


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