N-Linked Glycosylation Is Critical for the Plasma Membrane Localization of Nephrin

Construction of Full-Length Nephrin cDNA

A full-length cDNA clone encoding human nephrin was cloned into the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, CA). Briefly, a λgt10 clone containing a full-length human nephrin cDNA (4) was used as template in a PCR reaction to introduce a HindIII site into the 5′-end of the clone. The upstream primer, UPHindIII 5′-ACCCTAAGCTTGCCACCATGGCCCTGGGGA-3′, containing the HindIII site (underlined), a short Kozak sequence (italic) followed by the ATG starting codon (underlined and italic), and a downstream primer, DP1 5′-GGGTCAGCAGGAGCAGCTT-3′, were used to amplify a 5′-PCR fragment. The fragment was cleaved with HindIII and EcoRI to generate a HindIII/EcoRI 5′-end fragment of the full-length clone. This 5′-fragment, together with the rest of the λgt10 full-length nephrin clone, were cleaved with EcoRI and KpnI and used to construct a full-length clone that was ligated into a HindIII/KpnI-cleaved pSL1180 vector (Amersham Pharmacia Biotech, Uppsala, Sweden). This construct was cleaved with HindIII and SalI (from the vector) and the nephrin HindIII/SalI-fragment was subcloned into a HindIII/XhoI-cleaved pcDNA3 vector to generate the final full-length nephrin construct pcDNA3NPH1. The final construct was sequenced to ensure that no mutations had been introduced during PCR and cloning.

Cell Lines and DNA Transfection

A conditionally immortalized mouse podocyte cell line (IMP) was kindly provided by Dr. Peter Mundel (Albert Einstein College of Medicine, NY). The cells were maintained and propagated in RPMI-1640 medium (Life Technologies Inc., Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) as described (8). HEK-293 cells were maintained in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 10% FBS. Transfections of both cell lines were carried out by FuGENE (Roche, Indianapolis, IN) according to the manufacturer’s protocol. Stable clones were selected in medium containing 1 mg/ml Geneticin (Life Technologies).

Antibodies

For immunofluorescence microscopy, we used a mouse monoclonal antibody (mAb2) raised against the eighth Ig domain of human nephrin, as described previously (9). Rabbit polyclonal antibodies (pAb2) against the intracellular part of human nephrin was used for immunoblotting (9). Rabbit anti-calreticulin antibody was purchased from Affinity Bioreagents, Inc. Cyanine (Cy2, Cy3)–conjugated anti-mouse and anti-rabbit antibodies and horseradish peroxidase (HRP)–conjugated goat anti-rabbit antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA) and DAKO (Glostrup, Denmark), respectively.

Tunicamycin Treatment and Immunoblot Analyses

Nephrin-transfected immortalized mouse podocytes (IMP-NPH) or HEK-293 cells (293-NPH) were cultured to almost confluent condition before the tunicamycin treatment. The cells were then washed once with phosphate-buffered saline (PBS) and cultivated for 20 h in fresh culture media in the absence or presence of tunicamycin in a dose-dependent manner. Cells were then washed twice before addition of hot sodium dodecyl sulfate (SDS) sample buffer (63 mM Tris-HCl, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, pH 6.8). The cell lysates were collected with a rubber scraper and passed through 26-gauge needles. The whole cell lysates were transferred into Eppendorf tubes, boiled, and centrifuged for 10 min before loading. The proteins of human isolated glomeruli were extracted by SDS sample buffer. All samples were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) (7.5% gels), and the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated with affinity-purified pAb2 antibodies (0.2 μg/ml), washed, and incubated with HRP-conjugated goat anti-rabbit antibodies. The immunoreactivity was detected by a chemiluminescent kit (Life Science Products, Boston, MA) according to the manufacturer’s instructions. The prestained molecular standard used in the SDS-PAGE was from Bio-Rad (Precision Protein Standards, catalog number 161–0372; Bio-Rad, Hercules, CA).

Cell Surface Biotinylation

IMP-NPH cells were cultured in the absence or presence of tunicamycin (5 μg/ml) for 20 h. The cells were then washed twice with ice-cold PBS and once with ice-cold Hepes buffer. Cell surface proteins were subsequently biotinylated with 0.5 mg/ml Sulfo-NHS-biotin (Pierce, Rockford, IL) in ice-cold Hepes buffer for 30 min. Nonbiotinylated control cells were washed with ice-cold Hepes buffer containing 50 mM glycine (pH 8.0) and then lysed in 1 ml of RIPA buffer (0.5% sodium deoxycholate, 1% Nonidet-P 40, 0.1% SDS in PBS) containing protease inhibitors, Complete (Roche). The cell lysates were centrifuged at 14.000 g for 15 min to remove insoluble materials. To bind the biotinylated proteins, 40 μl Streptavidin-agarose beads (Sigma) was added to each 600-μl supernatant lysate and incubated for 2 h at 4°C. The beads were settled by centrifugation, and the supernatants (unbound fractions) were saved. The beads were then washed four times with RIPA buffer, and bound proteins were eluted by addition of hot SDS sample buffer.

Immunofluorescence and Confocal Microscopy

Wild-type IMP and transfected IMP-NPH cells were cultured overnight on glass cover slips coated with laminin 10/11 (Life Technologies). The cells were washed and fixed in a fixing solution (3% formaldehyde, 4% sucrose in PBS) for 20 min at room temperature. After blocking (2% FBS, 3% bovine serum albumin (BSA), 0.2% fish gelatin in PBS) for 1 h, the cells were incubated with affinity-purified monoclonal antibody mAb2 (10 μg/ml) for 90 min. The cells were then washed and incubated with Cy2-conjugated anti-mouse IgG for 1 h. To determine the effect of tunicamycin on the subcellular localization of nephrin, IMP-NPH cells were cultured in medium in the presence or absence of tunicamycin (5 μg/ml) for 20 h. For double-staining, the cells were fixed and permeabilized with 0.3% Triton-X100 in PBS for 10 min. After blocking, cells were incubated with the mouse monoclonal anti-nephrin antibody (mAb2) and rabbit anti-calreticulin antibodies. The immunocomplexes were visualized with anti-mouse Cy2- and anti-rabbit Cy3-conjugated secondary antibodies, respectively. Confocal sections were analyzed by an Openlab confocal imaging software (Improvision, Coventry, England).

Localization of Human Nephrin in Transfected Podocytes

Due to low endogenous nephrin expression in the cultured immortalized wild-type podocytes, we decided to generate stable cell lines constitutively expressing a full-length human nephrin construct. Several such cell lines with similar expression level and subcellular localization were established. Surface immunofluorescence staining of a nonpermeabilized stable cell line IMP-NPH, using a mouse monoclonal antibody (mAb2) raised against the eighth-Ig domain of human nephrin (9), showed a clear plasma membrane localization of the recombinant nephrin (Figure 1A). No positive immunostaining of wild-type mouse podocytes was observed using this species-specific monoclonal antibody (Figure 1B) indicating the specificity of the antibody for the human nephrin. Transfected 293-NPH cells also showed a similar cell membrane immunolocalization of the recombinant human nephrin (Figure 1C), whereas the wild-type HEK-293 cells did not show any detectable endogenous nephrin expression (Figure 1D).

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Figure 1. Confocal microscopy of nonpermeabelized transfected and wild-type cells using a mouse monoclonal antibody (mAb2) against the eight-Ig domain of human nephrin. Surface immunostaing of the recombinant human nephrin (green fluorescence) in transfected mouse cell lines, IMP-NPH (A) and 293-NPH cells (C), shows membrane localization of the recombinant human nephrin. Red fluorescence shows phalloidin staining of the actin filaments. Wild-type mouse podocytes IMP (B) and HEK-293 cells (D) are devoid in nephrin staining. Bar, 10 μm.

N-Glycosylation of Human Nephrin

There are ten potential N-linked glycosylation sites in the extracellular part of the human nephrin (2). When analyzed by SDS-PAGE, the apparent molecular mass of nephrin exceeded that predicted by primary sequence analyses (here and in reference (6). It has also been reported that the shift in molecular migration of nephrin isolated from mouse glomeruli is indeed due to N-glycosylation of the molecule 6).

To find out how much the N-glycosylation of nephrin contributes to the relative molecular migration of the human recombinant nephrin expressed in both IMP-NPH and 293-NPH cell lines, total protein extracts of both transfected cell lines were analyzed by SDS-PAGE and Western blotting using a rabbit polyclonal antibody (pAb2) against the entire intracellular part of the human nephrin (9). As shown in Figure 2A, the recombinant nephrin in the total extract of IMP-NPH cells migrated as a double immunoband of 185 kD. The total extract of 293-NPH cells also showed a double band with about the same molecular migration. A sample of isolated human glomeruli migrated as a major immunoband of 200 kD and a weak immunoband of the same migration of the lower immunoband from transfected cells (Figure 2A). When cells were treated with tunicamycin (5 μg/ml) for 20 h, both cell extracts showed a single immunoband of 150 kD, indicating that N-linked carbohydrate moieties contribute to a molecular shift of about 35 kD (Figure 2A). No immunoband was detected when cell extracts from nontransfected cell lines cultured in the same condition were analyzed (data not shown). To investigate the sensitivity of nephrin glycosylation to tunicamycin, both transfected cells were cultured in the absence or the presence of various concentrations of tunicamycin (0.01 to 5 μg/ml). As shown in Figure 2B, extremely low concentrations of tunicamycin (0.05 to 0.1 μg/ml) could inhibit the N-glycosylation of nephrin core protein in both transfected cell lines, indicating that the N-glycosylation process in both cell lines is highly sensitive to inhibition by Tunicamycin.

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Figure 2. Relative molecular migration of nephrin isolated from human glomeruli, transfected IMP-NPH podocytes and 293-NPH cells cultured in the absence (−TM) or presence (+TM) of tunicamycin. (A) Nephrin from human glomeruli migrates as a major immunoband of 200 kD. A double immunoband of 185 kD is present in the protein extracts from both transfected cell lines in the absence of tunicamycin. When Tunicamycin (5 μg/ml) was included in the culture medium for 20 h, a single immunoband of 150 kD could be seen in both cell extracts. (B) Treatment of cell lines with tunicamycin at a dose dependent (0 to 5.0 μg/ml) manner for 20 h, showing that N-linked glycosylation of nephrin is highly sensitive to inhibition by tunicamycin. Nephrin in total cell extracts were analyzed by Western blot analyses using a rabbit polyclonal antibodies (pAb2) against the intracellular part of human nephrin. Molecular markers showing the relative molecular migration of a 250-kD and a 150-kD protein from a BioRad prestained protein standard as molecular markers.

N-Glycosylation Is Critical for Proper Folding of Nephrin and Its Localization on the Plasma Membrane

Cell surface biotinylation was used to determine the cell surface expression of nephrin in the absence or the presence of tunicamycin. As shown in Figure 3, cell surface biotinylated fraction of intact cells clearly showed two immunobands, which due to biotinylation migrated more slowly than the unbiotinylated fraction in the total cell extract. In contrast, in the surface-biotinylated fraction of the tunicamycin-treated cells, no immunoproduct was detected, indicating that nonglycosylated nephrin does not appear on the cell surface. In the tunicamycin-treated cells, nonglycosylated cytosolic fraction of nephrin was shown to migrate as a 150-kD immunoband. The lack of nephrin on the plasma membrane of tunicamycin-treated cells was further conformed by double immunostaining of nephrin and calreticulin, an endoplasmic reticulum (ER)-resident chaperon protein. As expected, untreated IMP-NPH cells showed the presence of nephrin in the ER and on the plasma membrane (Figure 4). In contrast, nephrin was localized only in the ER in the tunicamycin-treated cells (Figure 4). Altogether, the data showed that glycosylation of nephrin is a prerequisite for its proper folding and transport from the ER to the plasma membrane.

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Figure 3. Western blot analyses of cell surface biotinylated IMP-NPH cells. Protein extracts were prepared from surface biotinylated (+) or nonbiotinylated (−) cells, either in the presence (+) or absence (−) of tunicamycin. Biotinylated and nonbiotinylated protein fractions were separated by streptavidin agarose beads. The total cell extract (TCE) and biotinylated bound fractions (B) and unbound fractions (U) were analyzed by SDS-PAGE and Western blot analyses using the pAb2 rabbit polyclonal antibodies against the intracellular part of human nephrin.

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Figure 4. Double immunolocalization of nephrin and calreticulin in IMP-NPH cells incubated either in the presence (+TM) or absence (−TM) of tunicamycin (5 μg/ml). Permeabilized cells were stained for recombinant nephrin using the monoclonal antibody mAb2 (green fluorescence) and polyclonal antibodies against the endoplasmic reticulum (ER) marker calreticulin (red fluorescence). The untreated cells showed nephrin expression in the ER and plasma membrane (arrows). In contrast, nephrin was only colocalized with calreticulin in the ER in the tunicamycin-treated cells. Bars, 5 μm.