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Am J Physiol Gastrointest Liver Physiol 292: G818-G828, 2007. First published November 2, 2006; doi:10.1152/ajpgi.00415.2006
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LIVER AND BILIARY TRACT

Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

Kaori Mochizuki,1 Tatehiro Kagawa,1 Asano Numari,1 Matthew J. Harris,3 Johbu Itoh,2 Norihito Watanabe,1 Tetsuya Mine,1 and Irwin M. Arias3,4

1Division of Gastroenterology, Department of Internal Medicine, 2Laboratories for Structure and Function Research, Tokai University School of Medicine, Kanagawa, Japan; 3Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts; and 4Unit on Cellular Polarity, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

Submitted 8 September 2006 ; accepted in final form 1 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn109, Asn116, Asn122, and Asn125) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A1 extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.

cholestasis; bile acid; bile secretion

BILE ACIDS are amphipathic steroidal compounds produced from cholesterol in hepatocytes and are secreted into bile across the canalicular membrane as glyco- or tauroconjugates. Bile acids are required for the intestinal absorption of dietary fat and hydrophobic vitamins and predominantly return to the liver through the enterohepatic circulation. The bile salt export pump [Bsep; ATP-binding cassette (ABC) B11] is the major canalicular transporter of bile acids (5), and its plasma membrane distribution is restricted to the canalicular domain of hepatocytes (5, 10, 32, 33). Stimulation with taurocholate (TC) (11, 16, 18) or cAMP (9, 11, 18) increases bile acid secretion in part by the recruitment of Bsep to the canalicular membrane in a phosphoinositide 3-kinase-dependent manner (17). However, the effects of posttranslational modifications on Bsep function are unknown.

Many plasma membrane proteins undergo N-linked glycosylation in the endoplasmic reticulum (ER) and are further glycosylated in the Golgi before trafficking to the cell surface. Carbohydrates on membrane proteins affect their folding, stability, intracellular trafficking, and/or function. Carbohydrate deficiency sometimes causes life-threatening syndromes, which highlight the fact that proper glycosylation is essential for normal development and health (4).

Bsep belongs to the ABC transporter superfamily and harbors two sets of Walker A and B motifs and 12 putative transmembrane domains. A mutation in progressive familial cholestasis type 2 (PFIC-2) causes unstable Bsep protein due to the lack of glycosylation (21). Bsep has four putative N-linked glycosylation sites in the first extracellular loop; however, the role of these carbohydrates in Bsep function remains unknown. The aims of this study were to determine whether each putative glycosylation site is glycosylated and to examine the role of these carbohydrates in the folding, stability, and intracellular trafficking of Bsep using mutants in which the Asn residues of Asn-X-Ser/Thr sequence were replaced with Gln. We also determined the effect of N-linked glycosylation on Bsep-mediated TC transport using Madin-Darby canine kidney II (MDCK II) cell monolayers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [3H]TC was purchased from Perkin-Elmer Life Sciences (Boston, MA). TC and MG-132 were from Calbiochem (San Diego, CA). Cycloheximide, tunicamycin, and bafilomycin A1 were from Sigma (St. Louis, MO).

cDNA and antibodies. pBK-Bsep vector expressing rat Bsep (GenBank Accession No. U69487) (5) was kindly provided by P. J. Meier (University Hospital, Zurich, Switzerland). pBsep-EYFP vector expressing Bsep fused with enhanced yellow fluorescent protein (EYFP) was constructed by subcloning full-length Bsep into the KpnI/ApaI site of pEYFP-N1 (BD Biosciences Clontech, Palo Alto, CA) after the stop codon had been removed by PCR-based mutagenesis. To obtain Bsep mutants lacking N-linked glycans, Asn residues of the Asn-X-Ser/Thr sequence were replaced with Gln by site-directed mutagenesis (QuikChange XL site-directed mutagenesis kit, Stratagene, La Jolla, CA). Mutagenesis was verified by DNA sequence analysis. The antibody against rat Bsep was raised using its linker region as an antigen (10). Rat Na+-TC cotransporting polypeptide (Ntcp)-expressing vector (26), enhanced green fluorescent protein (EGFP)-fused Ntcp chimeric vector (Ntcp-EGFP) (27), and the antibody against rat Ntcp (1) were kindly provided by F. J. Suchy (Mount Sinai School of Medicine, New York, NY). beta-Galactosidase (beta-gal)-expressing vector (pCMV.SPORT-beta-gal) was purchased from Invitrogen (Gaithersburg, MD). Experiments were performed according to the guidelines for animal experimentation in Tokai University School of Medicine.

Cell culture and transfection. MDCK II cells were maintained in a humidified atmosphere at 37°C with 5% CO2 in complete minimum essential medium (Life Technologies, Rockville, MD) supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.

TC transport assay. MDCK II cells were replated on Transwell membrane inserts (no. 3470, diameter: 6.5 mm, pore size: 0.4 µm, Corning Costar, Cambridge, MA) at a concentration of 5 x 104 cells/membrane. After 24 h, cells were transfected with vectors expressing Ntcp, Bsep, or both using Lipofectamine 2000 (Invitrogen). To evaluate the TC transport activity of Bsep mutants lacking N-glycans, mutants were used instead of the wild type (WT). The total amount of DNA used for transfection was adjusted to the same by adding beta-gal-expressing vector. In 2 days, cells were incubated with complete medium (0.15 ml) in the apical compartment and with uptake buffer [containing (in mM) 116 NaCl, 5.3 KCl, 1.1 KH2PO4, 0.8 MgSO4, 1.8 CaCl2, 11 D-glucose, and 10 HEPES; pH 7.4] and 10 µM TC (containing 1 µM [3H]TC) in the basal compartment. Preliminary experiments revealed that an incubation period of 2 days after transfection was optimal for the transport activity assay. After an incubation for the indicated time periods at 37°C, radioactivity in the apical medium (0.1 ml) was determined by a scintillation counter (model LS 1801, Beckman Coulter). After the completion of the incubation period, Transwell membranes were washed by being successively dipped into ice-cold PBS, followed by the determination of radioactivity. Transcellular TC flux and intracellular TC accumulation were calculated from the radioactivity in the apical medium and cells, respectively. The permeability-surface area product across the apical membrane (PSapical) was determined by dividing the rate of transcellular TC flux by intracellular TC concentration. Cellular protein concentrations were determined by the method of Lowry et al. (13).

Preparation of sinusoidal and canalicular membrane vesicles. Sinusoidal membrane vesicles (SMVs) and canalicular membrane vesicles (CMVs) were isolated from rats as previously reported (10).

Immunoblot analysis. MDCK II cells grown on 100-mm plates were transfected as described above. After 48 h, cells were washed three times with PBS, incubated for 20 min at 4°C in 200 µl of lysis buffer [50 mM Tris (pH 7.5), 2 mM CaCl2, and 1% Triton X-100] containing protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics, Indianapolis, IN), and harvested by scraping with a rubber policeman. The incubation time period of 48 h was chosen because the same condition was adopted for the transport activity assay. Cell lysates were transferred to 1.5-ml tubes and centrifuged for 10 min (1,500 g, 4°C). Supernatants were transferred to new tubes, and protein concentrations were determined. Laemmli buffer was added to a final concentration of 1x, and samples were incubated for 30 min at room temperature. Proteins were separated by SDS-PAGE on 7.5% gels, transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA), and probed with the corresponding antibodies against rat Ntcp, Bsep, GFP (Invitrogen), or beta-actin (Sigma). The antibody against GFP recognizes EYFP. Membranes were incubated with the appropriate horseradish peroxidase-coupled secondary antibodies (Cell Signaling Technology, Beverly, MA) followed by an ECL reaction (Perkin-Elmer Life Sciences). Blot images were captured by an ATTO LightCapture System (ATTO, Tokyo, Japan), and the band density was quantified by a CS Analyzer (ATTO). SMVs and CMVs were used as a positive control for Ntcp and Bsep, respectively.

Analysis of glycosylation. CMVs or cell lysates prepared from MDCK II cells transfected with WT or mutant Bsep-EYFP (5–10 µg) were digested with 20 milliunits of peptide N-glycosidase F (PNGase F; Glyko, San Leandro, CA) or 0.3 milliunits of endoglycosidase H (Glyko) for 1 h at room temperature in the corresponding incubation buffer supplied by the manufacturer. After an incubation with sample buffer for a further 30 min at room temperature, samples were subjected to SDS-PAGE and immunoblot analysis.

Determination of protein degradation half-life. MDCK II cells were transiently transfected with WT or mutant Bsep-EYFP. After 24 h, cells were incubated with cyclohemixide (20 µg/ml) to inhibit further protein synthesis. MG-132 (10 µM) or bafilomycin A1 (1 µM) was added along with cycloheximide when necessary. After an incubation for the indicated time periods, cells were harvested, lysed, and subjected to SDS-PAGE and immunoblot analysis. The band density was quantified as described above and normalized so that the density at time 0 was 100%. The log10 of the percentage of density was plotted versus time, and the half-life was calculated from the log10 of 50% for the protein.

Quantification of Bsep mRNA expression in transiently transfected MDCK II cells. MDCK II cells were transiently transfected with empty vector or WT or mutant Bsep-EYFP, and total RNA was extracted 24 h after transfection. After being digested with DNase I, cDNA was obtained by reverse transcription with SuperScript III and random hexamers (Invitrogen). Quantitative real-time PCR was performed using TaqMan technology on an ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA). The primer and FAM dye-labeled probe for rat Bsep were purchased from Applied Biosystems. The change in threshold cycle ({Delta}CT) value was obtained by normalization to an endogenous reference (rRNA), and the abundance of Bsep mRNA relative to WT mRNA was determined by calculating the value of 2–{Delta}{Delta}CT.

Confocal laser scanning microscopy. MDCK II cells grown on the Transwell membrane inserts (no. 3460, diameter: 12 mm, pore size: 0.4 µm, Corning) were transfected with Ntcp-EGFP or Bsep-EYFP expression vectors as described above. After 48 h, membranes were cut, washed twice with PBS, fixed in 4% paraformaldehyde, and mounted with 1,4-diazabicyclo-(2.2.2)octane-triethylenediamine (Sigma). Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; D8417, Sigma). After 1 h of incubation at room temperature, the localization of Ntcp-EGFP or Bsep-EYFP was analyzed with confocal laser scanning microscopy (LSM-410, Carl Zeiss, Jena, Germany) with projection and three-dimensional orthogonal reconstruction modes. The system included a 488-nm argon laser (for Ntcp-EGFP and Bsep-EYFP) and a 351/364-nm argon laser (for DAPI). Optical fluorescent signals were observed using a dichroic beam splitter (NT 80/20/543, Carl Zeiss) and the corresponding emission filters (BP510–525 for Ntcp-EGFP, LP570 for Bsep-EYFP, and LP397 for nuclei, Carl Zeiss). Double-labeled volumetric data sets of these images were digitized and subjected to image-analysis manipulation and computer-assisted three-dimensional reconstructions using LSM software (version 3.98, Carl Zeiss). The optical tomographic imaging (z-images) for three-dimensional reconstruction was performed with a C-apochromat objective lens (x63, water immersion, numerical aperture 1.2, Carl Zeiss). The z-directional movement for the optical sectioning of entire specimens was controlled by a stepping motor unit for axial scanning at 0.5-µm focus steps. The image resolution was 512 x 512 and/or 1,024 x 1,024 pixels (8 bits, 256 gray levels). Computer-assisted three-dimensional images were stored on hard disk memory and/or magnetic optical disk (EDM-230C, SONY, Tokyo, Japan).

Statistical evaluation. Significance of differences was evaluated by repeated-measures one-way ANOVA or a paired t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mapping of the N-linked glycosylation site. We used rat CMVs to determine whether Bsep has N-linked glycans. Rat Bsep migrated at ~150 kDa (Fig. 1). Pretreatment with PNGase F shifted the molecular size to ~120 kDa, indicating that Bsep contains N-linked glycans with a molecular size of 30 kDa. Seven potential glycosylation consensus sites (Asn-X-Ser/Thr) were found in the primary amino acid sequence of rat Bsep (GenBank Accession No. U69487). Four sites were expected to face the extracellular space as deduced from Bsep transmembrane topology (5). These four sites reside in the first extracellular loop (Asn109, Asn116, Asn122, and Asn125; Fig. 2A) and are highly conserved among rat, mouse, and human orthologs (Fig. 2B). To determine whether these sites are, in fact, glycosylated, constructs were made in which Asn109, Asn116, Asn122, and Asn125 were sequentially replaced with Gln, which is structurally very similar to Asn. The following mutants were constructed: single, N109Q (QNNN); double, N109Q + N116Q (QQNN); triple, N109Q + N116Q + N122Q (QQQN); and quadruple, N109Q + N116Q + N122Q + N125Q (QQQQ) (Fig. 2C).


Figure 1
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  		Fig. 1. N-linked glycans of the bile salt export pump (Bsep) from rat canalicular membrane vesicles (CMVs). CMVs (5 µg) were incubated for 1 h at room temperature in the absence (–) or presence (+) of peptide N-glycosidase F (PNGase F), separated by SDS-PAGE, and subjected to immunoblot analysis using the antibody to rat Bsep.

 

Figure 2
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  		Fig. 2. Generation of rat Bsep mutants lacking N-linked glycosylation. A: rat Bsep is believed to have 12 transmembrane domains and two sets of Walker A and B motifs with an ATP-binding cassette (ABC) signature sequence. Four putative N-linked glycosylation sites (Asn109, Asn116, Asn122, and Asn125) reside in the first extracellular loop. B: the Asn residues to which N-glycans are expected to attach are indicated in bold and their flanking amino acid sequence is aligned with mouse and human orthologs. C: mutants in which these Asn residues were sequentially replaced with Gln are depicted. The mutants are as follows: single, N109Q; double, N109Q + N116Q; triple, N109Q + N116Q + N122Q; and quadruple, N109Q + N116Q + N122Q + N125Q. WT, wild type.

 
MDCK II cells were transfected with WT or single-, double-, triple-, or quadruple-mutant Bsep-EYFP. Immunoblot analysis of cell lysates revealed that WT Bsep consisted of a main band migrating at ~170 kDa and an extra band at ~145 kDa (Fig. 3A). The molecular size of the main band was larger than the 150-kDa Bsep band from CMVs (Fig. 1). This difference was attributable to the molecular mass of EYFP. Mutants lacking N-glycans migrated faster than did the WT in the descending order of quadruple, triple, double, and single. The molecular size of the quadruple mutant was ~140 kDa. A difference in molecular size (30 kDa) between WT and quadruple mutant Bsep-EYFP was similar to the difference between PNGase F-treated and untreated Bsep from rat CMVs (Fig. 1). Treatment with PNGase F shifted the molecular size of the WT from 170 to 140 kDa, which was similar to the lower band of PNGase F-treated and untreated quadruple mutants (Fig. 3B). The effect of an N-linked glycosylation inhibitor, tunicamycin, on the attachment of sugar moieties was also studied. MDCK II cells expressing WT or quadruple-mutant Bsep-EYFP were incubated with 10 µg/ml tunicamycin for 24 h. The presence of tunicamycin shifted both WT bands to ~140 kDa, which was identical to the bands of tunicamycin-treated and -untreated quadruple mutants (Fig. 3C). These observations suggest that each putative N-linked glycosylation site carries a carbohydrate and that no other glycosylation sites exist on Bsep. To elucidate the characteristics of the two bands observed in the WT, cell lysates were incubated with endoglycosidase H followed by SDS-PAGE and immunoblot analysis. Endoglycosidase H cleaved the extra band (~145 kDa) into a ~140-kDa protein, in which the molecular size was identical to that of endoglycosidase H-treated and -untreated quadruple mutants (unglycosylated form) (Fig. 3D). In contrast, the main band was resistant to this enzyme. The fact that endoglycosidase H cleaves core oligosaccharides but not complex-type glycans indicates that the 170- and ~145-kDa proteins represent mature (complex type) and core glycosylated forms of Bsep, respectively. The presence of similar two bands is well known for CFTR protein, and each are designated as band C and band B, respectively.


Figure 3
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  		Fig. 3. Glycosylation profile of Bsep. A: cell lysates prepared from Madin-Darby canine kidney II (MDCK II) cells transfected or untransfected with WT or single-, double-, triple-, or quadruple-mutant Bsep-enhanced yellow fluorescent protein (EYFP) were separated by SDS-PAGE and subjected to immunoblot analysis using the antibody to green fluorescent protein (GFP). The following protein amounts were loaded: MDCK II, 20 µg; WT, 5 µg; single mutant, 5 µg; double mutant, 5 µg; triple mutant, 20 µg; and quadruple mutant, 20 µg. B: cell lysates prepared from MDCK II cells transiently expressing WT or quadruple-mutant Bsep-EFYP were incubated in the absence (–) or presence (+) of PNGase F for 1 h at room temperature, separated by SDS-PAGE, and subjected to immunoblot analysis using the antibody to GFP. The following protein amounts were loaded: WT, 2 µg; and quadruple mutant, 20 µg. C: MDCK II cells transiently expressing WT or quadruple-mutant Bsep-EFYP were incubated in the absence (–) or presence (+) of tunicamycin (10 µg/ml) for 24 h at 37°C. Cell lysates were collected, separated by SDS-PAGE, and subjected to immunoblot analysis using the antibody to GFP. The following protein amounts were loaded: WT tunicamycin (–), 2 µg; WT tunicamycin (+), 44 µg; quadruple mutant tunicamycin (–): 58 µg; and quadruple mutant tunicamycin (+), 52 µg. D: cell lysates prepared from MDCK II cells transiently expressing WT or quadruple-mutant Bsep-EFYP were incubated in the absence (–) or presence (+) of endoglycosidase H (Endo H) for 1 h at room temperature, separated by SDS-PAGE, and subjected to immunoblot analysis using the antibody to GFP. The following protein amounts were loaded: WT, 2 µg; and quadruple mutant, 20 µg. Different amounts of proteins were loaded so that all bands were visible in a blot.

 
Establishment of an MDCK II monolayer system to evaluate bile acid transport activity. To quantify the bile acid transport activity of Bsep, MDCK II cells in monolayers were transfected with beta-gal, Ntcp + beta-gal, Bsep + beta-gal, or Ntcp + Bsep. Cell lysates were subjected to SDS-PAGE and immunoblot analysis using the corresponding antibodies (Fig. 4A). In cells transfected with beta-gal or Bsep + beta-gal, Ntcp expression was not detected, whereas in cells transfected with Ntcp + beta-gal or Ntcp + Bsep, it was expressed with the same molecular size (~49 kDa) as in the SMV preparation, as previously reported (26). Expression of Bsep protein was observed only in cells transfected with Bsep + beta-gal or Ntcp + Bsep. The molecular size (~150 kDa) was identical to that detected in the CMV preparation. The intracellular distribution of Ntcp and Bsep was analyzed in MDCK II cells transfected with Ntcp-EGFP + beta-gal and Bsep-EYFP + beta-gal, respectively, using confocal laser scanning microscopy (Fig. 4B). Ntcp was predominantly distributed in the basolateral membrane. Only faint fluorescent signals were detected in the upper x-y plane image. In contrast, Bsep was exclusively localized along the apical plasma membrane; no fluorescent signals were detected in the middle and lower x-y plane images. Previous studies have demonstrated that Ntcp was localized along the sinusoidal (basolateral) membrane in hepatocytes (1, 24) and transfected MDCK II cells (26) and that Bsep was distributed along the apical membrane in hepatocytes (5, 10, 11) and in transfected WIF-B9 cells (33). Therefore, both molecules trafficked correctly in the transfected MDCK II monolayer. Approximately 5–7% of cells were successfully transfected.


Figure 4
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  		Fig. 4. Characterization of bile acid transport activity assay using polarized MDCK II cells. A: MDCK II cells were transfected with beta-galactosidase (beta-gal), Na+-taurocholate (TC) cotransporting peptide (Ntcp) + beta-gal, Bsep + beta-gal, or Ntcp + Bsep and lysed 48 h after transfection. Cell lysates (20 µg) were separated by SDS-PAGE and subjected to immunoblot analysis using antibodies to rat Ntcp or Bsep. Sinusoidal membrane vesicles (SMVs) and CMVs were isolated from rats and used as positive controls for Ntcp and Bsep, respectively. B: MDCK II cells were transfected with Ntcp-enhanced GFP (EGFP) + beta-gal or Bsep-EYFP + beta-gal and imaged by confocal laser scanning microscopy. Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI). Fluorescent signals of Ntcp-EGFP and Bsep-EYFP are shown in green and yellow, respectively, and nuclei are blue. Ntcp was predominantly distributed along the basolateral plasma membrane, whereas Bsep was exclusively localized along the apical membrane. Scale bars = 10 µm. C and D: MDCK II cells grown on Transwell membrane inserts were either untransfected ({triangledown}) or transfected with beta-gal ({square}), Ntcp + beta-gal ({blacktriangleup}), Bsep + beta-gal ({circ}), or Ntcp + Bsep (bullet). After an incubation for the indicated time period at 37°C with [3H]TC (1 µM) and cold TC (10 µM) in the basal compartment, apical media and cells were collected. The intracellular accumulation (C) and transcellular flux (D) of TC were calculated based on the radioactivity in the apical medium and total cells, respectively. Each point and vertical bar represents the mean ± SE of 6 independent experiments performed in duplicate. E and F: MDCK II cells transfected with Ntcp + Bsep ({circ}) or Ntcp + beta-gal (bullet) were incubated at 37°C for 60 min in uptake buffer containing 1 µM [3H]TC and various concentrations of nonlabeled TC in the basal compartment, followed by the collection of apical media and cells. The intracellular accumulation (E) and transcellular flux (F) of TC were calculated based on the radioactivity in the apical medium and total cells, respectively. A representative result from 4 independent experiments performed in duplicate is shown. The fitted lines are drawn in solid lines. G: concentration dependence of the permeability-surface area product for TC transport across the apical membrane (PSapical). PSapical was calculated by dividing the rate of transcellular TC flux by the intracellular TC concentration. Each point and vertical bar represents the mean ± SE of 4 independent experiments performed in duplicate. NS, not significant.

 
Intracellular TC accumulation was compared in MDCK II cells transfected and untransfected with beta-gal, Ntcp + beta-gal, Bsep + beta-gal, or Ntcp + Bsep (Fig. 4C). In controls (untransfected, beta-gal, and Bsep + beta-gal), intracellular TC concentration was minimal, whereas in the Ntcp + beta-gal and Ntcp + Bsep groups, the intracellular TC concentration increased in a time-dependent manner, reaching ~150 pmol/well at 60 min, which was significantly higher than in the other groups (P < 0.001). These observations indicate that Ntcp expression is required to transport TC into the cells. The mean intracellular TC accumulation in the Ntcp + Bsep group was slightly lower than that in the Ntcp + beta-gal group; however, the difference was not significant.

The basal to apical transcellular TC flux was also determined (Fig. 4D). Cells transfected with Ntcp + beta-gal showed a transcellular flux of 19.2 ± 3.1 pmol/well (mean ± SE) at 60 min, which was significantly higher than that in untransfected, beta-gal, and Bsep + beta-gal cells (P < 0.001). Cells coexpressing Ntcp and Bsep transported 51.1 ± 11.3 pmol/well of TC at 60 min, which was 2.7-fold greater than that in cells expressing Ntcp and beta-gal (P < 0.001). These data indicate that Bsep transports TC across the apical membrane. Cellular protein concentrations were not significantly different among groups (data not shown).

Kinetic analysis was performed to characterize TC intracellular accumulation and transcellular flux in MDCK II cells transiently expressing Ntcp + beta-gal or Ntcp + Bsep. These two groups showed similar saturation curves for TC accumulation (Fig. 4E). The transcellular TC flux increased along with the substrate concentration and exhibited saturable kinetics (Fig. 4F). To evaluate TC transport across the apical membrane, PSapical was determined by dividing the rate of transcellular TC flux by the intracellular TC concentration (Fig. 4G). PSapical in MDCK II cells expressing Ntcp + Bsep was saturated and significantly larger than that in cells expressing Ntcp + beta-gal (P < 0.05).

Activity, expression, and intracellular distribution of Bsep lacking N-linked glycosylation. The TC transport activity of Bsep mutants was analyzed in MDCK II monolayers. Bsep-EYFP transported TC similarly with Bsep [105.2 ± 7.5% (mean ± SE) of Bsep, n = 5], indicating that fusion with EYFP at the COOH terminus does not disturb Bsep function. In cells expressing Bsep-EYFP lacking N-linked glycans, Bsep-mediated TC transport was reduced in proportion to the reduction in glycosylation [single mutant: 92.0 ± 6.3%, double mutant: 65.9 ± 6.2%, triple mutant: 16.0 ± 4.2%, and quadruple mutant: 9.0 ± 5.1% (means ± SE); Fig. 5A]. Bsep expression in MDCK II cells transiently transfected with WT or single-, double-, triple-, or quadruple-mutant Bsep-EYFP was also determined. No bands were observed in cells transfected with the empty vector, whereas a ~170-kDa glycoprotein appeared in cells transfected with WT Bsep (Fig. 5B). The molecular size and expression level of Bsep decreased in order of WT, single, double, and triple mutants. Prolonged exposure of the quadruple mutant revealed a ~140-kDa protein (data not shown). Single and double mutants exhibited slightly reduced expression relative to the WT (92.1 ± 13.9% and 71.9 ± 13.7%, respectively, means ± SE). Triple and quadruple mutants expressed the protein at very low levels (17.6 ± 8.3% and 4.1 ± 3.3%, respectively). A significant difference was observed between double and triple mutants (P < 0.01). The trend in protein expression levels was similar to that in TC transport activity, suggesting that impaired function of Bsep mutants in MDCK II cells is attributable to decreased protein expression. The transfection efficiency was 5–7% and did not differ among mutants. To exclude the possibility that the decrease in the protein expression level was caused by the decrease in mRNA expression, we used quantitative real-time PCR to determine mRNA expression in MDCK II cells transiently transfected with each construct. Cells transfected with the empty vector showed trace-level mRNA expression (1/16,500 of the WT; Fig. 5C). Bsep mRNA expression levels were not significantly different in cells transfected with various mutants, suggesting that Bsep transcription was unimpaired.


Figure 5
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  		Fig. 5. Activity, expression, and subcellular distribution of WT and mutant Bsep transiently expressed in MDCK II cells. A: MDCK II cells grown on Transwell membrane inserts were cotransfected with Ntcp + beta-gal or Ntcp and either WT or mutant Bsep-EYFP. In 2 days, cells were incubated for 1 h at 37°C with [3H]TC (1 µM) and cold TC (10 µM) in the basal compartment, and transcellular TC flux was determined based on the radioactivity in the apical medium. Bsep-dependent TC transport activity was calculated from the difference in transcellular TC flux between Ntcp + beta-gal and Ntcp + Bsep-EYFP and normalized to the WT (57.6 ± 10.8 pmol/well) as 100%. Each value represents the mean ± SE of 8 independent experiments performed in duplicate. B: MDCK II cells were transiently transfected with empty vector or WT or single-, double-, triple-, or quadruple-mutant Bsep-EYFP and lysed. Cell lysates (5 µg) were separated by SDS-PAGE and subjected to immunoblot analysis using the antibody to GFP. The expression of beta-actin was studied as the control. A representative result is shown in the top, and the bottom shows calculated Bsep-EYFP protein expression levels relative to those of beta-actin normalized to the WT as 100%. Each value represents the mean ± SE of 3–6 independent experiments. C: MDCK II cells were transiently transfected with empty vector, WT Bsep, or mutant Bsep-EYFP, and total RNA was extracted 24 h after transfection. cDNA was obtained by reverse transcription with SuperScript III and random hexamers, and quantitative real-time PCR was performed using TaqMan technology on an ABI 7700 sequence detection system. The change in threshold cycle ({Delta}CT) value was obtained by normalization to an endogenous reference (rRNA), and the abundance of Bsep mRNA relative to the WT was determined by calculating the value of 2–{Delta}{Delta}CT. Each value represents the mean ± SE of 3 independent experiments. D: MDCK II cells grown on Transwell membrane inserts were transfected with single (a), double (b), triple (c), or quadruple (d) mutant Bsep-EYFP. In 2 days, cells were observed with confocal laser scanning microscopy. Nuclei were counterstained with DAPI. Bsep-EYFP-derived fluorescence is shown by yellow and nuclei by blue. The top, middle, and right show x-z, x-y, and y-z plane images, respectively. Scale bars = 10 µm.

 
To determine whether Bsep lacking N-linked glycans is correctly targeted, we analyzed the intracellular localization of mutant Bsep-EYFP expressed in MDCK II cells grown on Transwell membrane inserts (Fig. 5D). Single- and double-mutant proteins were distributed along the apical membrane. In contrast, triple- and quadruple-mutant proteins remained exclusively intracellular.

Biochemical half-lives of wild-type and mutant Bsep. To determine how protein expression was attenuated in N-glycan-deficient Bsep, the biochemical half-lives of WT and mutant Bsep expressed in MDCK II cells were determined (Fig. 6A). The mature form (band C) of the WT exhibited a half-life of 3.76 h. The core glycosylated form (band B) was degraded more rapidly (half-life {approx} 1.20 h). The half-lives of the mature form were 3.65 h for the single mutant and 3.24 h for the double mutant (Fig. 6, B and C). In contrast, the mature forms of the triple and quadruple mutants were unstable (half-life {approx} 1.35 h for the triple mutant and 0.52 h for the quadruple mutant) compared with WT, single-mutant, and double-mutant Bsep (Fig. 6, C and D). The core glycosylated form of Bsep demonstrated a similar trend, and decay was generally faster than that of the mature form. Next, we studied the effect of inhibitors on protein stability to explore how Bsep protein was degraded. MG-132, a proteasome inhibitor, stabilized WT Bsep and extended the half-life of both forms (Fig. 7, A, C, and D). In contrast, bafilomycin A1, a lysosome inhibitor, did not extend the half-life in either form (Fig. 7, B–D). These results suggest that substantial amounts of WT Bsep are degraded in the proteasome. MDCK II cells expressing WT, double-mutant, or quadruple-mutant Bsep were also cultured in the absence or presence of MG-132 or bafilomycin A1. The change in the protein expression level was analyzed. The addition of MG-132 increased WT Bsep protein expression by 4.6-fold (Fig. 7, E and G) and increased double and quadruple mutant protein expression by 8.5- and 23.3-fold, respectively. On the other hand, bafilomycin A1 slightly increased the protein expression in the WT and mutants (Fig. 7, F and H). These results suggest that the lysosome plays a limited role in the degradation of mutant Bsep protein.


Figure 6
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  		Fig. 6. Biochemical half-lives (t1/2) of WT and mutant Bsep. MDCK II cells were transiently transfected with WT (A) or single (B), double (C), triple (D), or quadruple (E) mutant Bsep-EYFP. After 24 h, cells were further cultured in the presence of cycloheximide (20 µg/ml) and lysed at 0, 3, 6, and 12 h (A–C) or at 0, 1, 2, and 4 h (D and E). Cell lysates were separated by SDS-PAGE and subjected to immunoblot analysis using the antibody to GFP; 5 µg of protein were loaded. A representative blot is shown in top. The band density was measured and normalized so that the density at time 0 was 100%. The log10 of the percentage of density was plotted versus time (bottom), and t1/2 was calculated from the log10 of 50% for bands B and C. Each value represents the mean ± SE of 3–6 independent experiments performed in duplicate.

 

Figure 7
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  		Fig. 7. Effect of MG-132 on Bsep protein expression. A–D: MDCK II cells were transiently transfected with WT Bsep-EYFP. After 24 h, cells were cultured with cycloheximide (20 µg/ml) in the absence or presence of MG-132 (10 µM; A) or bafilomycin A1 (Baf; 1 µM; B) and lysed at 0, 3, 6, and 12 h. Cell lysates were separated by SDS-PAGE and subjected to immunoblot analysis using the antibody to GFP; 5 µg of protein were loaded. Representative blots are shown in A and B. The band density was measured and normalized so that the density at time 0 was 100%. C and D: log10 of the percentage of density was plotted versus time, and t1/2 was calculated from the log10 of 50% for bands B and C. Each value represents the mean ± SE of 3–6 independent experiments performed in duplicate. E–H: MDCK II cells were transiently transfected with WT, double-mutant, or quadruple-mutant Bsep-EYFP. After 24 h, cells were cultured in the absence (–) or presence (+) of MG-132 (10 µM; E and G) or bafilomycin A1 (1 µM; F and H) for further 24 h and lysed. Cell lysates were separated by SDS-PAGE and subjected to immunoblot analysis using the antibody to GFP. Representative blots are shown in E and F. The following protein amounts were loaded: WT, 5 µg; double mutant, 10 µg; and quadruple mutant, 25 µg. The fold increases in expression levels with the addition of MG-132 (G) or bafilomycin A1 (H) were calculated. Each value represents the mean ± SE of 3–6 independent experiments. The fold increase with the addition of MG-132 in the quadruple mutant was significantly greater than that in the WT and double mutant (P < 0.01). In contrast, the fold increase with the addition of bafilomycin A1 was not significantly different.

 
These experiments revealed that the rate of protein degradation did not increase stepwise by the removal of N-glycans but that a significant gap was present between the results with double and triple mutants, suggesting that a specific N-linked glycosylation site (i.e., Asn122 in Fig. 2C) is critical for protein stability. To study this possibility, we constructed seven new mutants in which four Asn residues were replaced with Gln in various combinations and analyzed the protein expression in MDCK II cells transfected with relevant mutants. Mutants harboring two or three glycans (NNNQ, NQQN, QNNQ, and QNQN) exhibited expression >60% of that of the WT (Fig. 8). Three mutants with one glycan (NQQQ, QNQQ, and QQNQ) were expressed below 20% of the WT. These observations suggest that the number of glycans (i.e., at least 2 of 4) rather than a specific site is important for Bsep expression.


Figure 8
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  		Fig. 8. The number of N-glycans is important for protein stability. MDCK II cells were transiently transfected with WT or mutant Bsep-EYFP and lysed in 48 h. Cell lysates (5 µg) were separated by SDS-PAGE and subjected to immunoblot analysis using the antibody to GFP. The expression of beta-actin was studied as the control. A representative result is shown in the top, and the bottom shows calculated Bsep-EYFP protein expression levels relative to those of beta-actin and normalized to the WT as 100%. Each value represents the mean ± SE of 4 independent experiments. The expression levels of Bsep with one glycan (NQQQ, QNQQ, and QQNQ) were significantly lower than those of Bsep with two or more glycans (WT, NNNQ, NQQN, QNNQ, and QNQN, P < 0.01).

 

   DISCUSSION
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We examined the role of N-linked glycans in Bsep protein stability, intracellular trafficking, and function in the apical membrane of transfected MDCK II cells. The results indicate that each of the four putative sites is glycosylated and no other glycosylated sites are present on Bsep. N-linked glycosylation is required for Bsep protein stability. At least two of the four glycans are required to maintain proper Bsep trafficking and function in the apical membrane.

TC transport activity was quantified in an MDCK II monolayer that coexpressed Ntcp and Bsep. MDCK II cells that expressed Ntcp secreted 19.2 pmol/well of TC into the apical side at 60 min, which was significantly greater than that observed in cells not expressing Ntcp (Fig. 4D). Two possibilities may explain these observations. First, diffusion out of the cells may be responsible because the TC concentration inside the cells was considerably higher than that in the apical medium. The observation that the PSapical for TC transport was not saturated in cells expressing Ntcp alone (Fig. 4G) supports this hypothesis. Second, there may be other TC transporters. Bsep is the major TC transporter expressed exclusively in hepatocytes (5) and is not expressed endogenously in MDCK II cells (Fig. 5, B and C). Multidrug resistance protein 3, which is expressed in MDCK II cells, might be involved, although this molecule is distributed in the basolateral membrane (36). PSapical for TC transport was saturated in MDCK II cells coexpressing Ntcp and Bsep (Fig. 4G), indicating that TC transport across the apical membrane depends on Bsep function. Although the intracellular TC concentration was not different, cells coexpressing Ntcp and Bsep secreted more TC into the apical medium than did cells expressing Ntcp alone (Fig. 4, C and D). These observations support the hypothesis that TC transport across the apical membrane is the rate-determining process as demonstrated in hepatocytes (30). Thus, our MDCK II monolayer system is suitable to quantify bile acid transport activity and permits the investigation of Bsep regulatory mechanism(s) and the search for choleretic or cholestatic agents.

All four of Bsep putative N-linked glycosylation sites (Asn109, Asn116, Asn122, and Asn125) contained carbohydrates (Fig. 3A). Because we did not analyze the mutant Bsep in which only Asn116 or Asn122 residues were replaced with Gln, we cannot exclude the possibility that a site unglycosylated in the basal state acquires a sugar moiety when adjacent inherent glycosylation sites are removed, as observed in organic anion transporter 1 (28). However, this assumption is unlikely because the molecular size of Bsep decreased after the stepwise removal of glycosylation sites. Because treatment with PNGase F and tunicamycin did not shift the band of the quadruple mutant (Fig. 3, B and C), we conclude that no other glycosylation sites are present on Bsep. A ~145-kDa protein was detected along with a ~170-kDa protein in WT Bsep. Its sensitivity to endoglycosidase H and PNGase F indicates a core glycosylated form (Fig. 3, B and D) that is generated in the ER and thereafter traffics to the Golgi apparatus for further trimming. This species of glycoprotein was not apparent in CMV preparations extracted from the rat liver (Fig. 1). We (10, 11) have previously reported that newly synthesized Bsep protein quickly traverses the Golgi (30–60 min) and appears on the canalicular membrane in 2 h in the rat liver. Thus, a core glycosylated form presumably residing in the ER would not be detected in a steady state. Additionally, our observations may be explained by the rapid decay of the core glycosylated form (half-time: 1.20 h; Fig. 6A) compared with the mature form (half-life: 3.76 h), which was also reported for CFTR expressed in HEK cells (34).

Single and double mutants revealed slightly shorter biochemical half-lives than that of the WT, suggesting that the removal of one or two glycans can affect Bsep protein stability. However, this effect was limited, and these mutants were trafficked to the apical membranes similarly with WT Bsep (Fig. 5D). There was a significant difference in protein decay between double and triple mutants (Fig. 6). Bsep mutants with single glycan were rapidly degraded compared with Bsep mutants having two or more glycans. These observations suggest that the number of glycans rather than a specific glycosylation site is important for protein stability. At least two glycans are required for stable expression, although the reason for this phenomenon is unknown. Glucosidase I (GI), glucosidase II (GII), and ER mannosidase I play critical roles in trimming nascent glycoproteins in the ER (8). GI removes the outermost of the three glucose residues, and GII removes the middle glucose. Newly synthesized glycoproteins harboring monoglucosylated glycans are allowed to enter the calnexin (Cnx)/calreticulin (Crt) cycle where folding occurs. Recently, Deprez and coworkers (2) demonstrated that more than one glycan is needed for ER GII, which permits glycoprotein entry into the Cnx/Crt cycle. At least two glycans must be close enough to each other to permit GII to remove the middle glucose. Singly glycosylated Bsep might not be cleaved by GII and prevented entry into the Cnx/Crt cycle. Mono- and unglycosylated Bsep (quadruple mutant) would be misfolded and degraded in the ER-associated degradation system (3, 8). Bsep is the first membrane protein proven to require at least two glycans for stable expression in MDCK II cells.

MG-132 treatment increased immunodetectable WT Bsep protein expression by 4.6-fold (Fig. 7G), suggesting that significant amounts of the WT protein are misfolded and degraded. Such an inefficient intracellular processing is not unique for Bsep. For example, only ~25% of newly synthesized CFTR proteins are processed to mature glycoprotein (34). Likewise, 50% or more of WT {delta}-opioid receptors fail to mature and are degraded (20). It remains unknown why cells adopt this strategy. However, most cell surface peptides presented with major histocompatibility complex class I rise from degraded newly synthesized proteins (22). Therefore, when a cell is infected by a virus and produces viral proteins, infection can be rapidly detected by T cells.

Intracellular trafficking and bile acid transport function in the apical membrane were similarly impaired by the removal of carbohydrates when Bsep was expressed in MDCK II cells (Fig. 5, A and D). Because the stepwise reduction was similar to the decreased protein expression (Fig. 5, A and B), these events likely resulted from the increased degradation of misfolded proteins. The functional importance of attached carbohydrates on protein stability, trafficking, or function varies with different proteins. Several membrane glycoproteins such as human norepinephrine transporters (15) and voltage-gated potassium Kv1.4 channels (35) require N-linked glycosylation for correct folding. Decreased cell surface expression has been reported for hyperpolarization-activated cyclic nucleotide-gated channels (19), organic anion transporter 1 (28), and dopamine transporters (12), although the role of carbohydrates in protein stability was not investigated in these studies. In contrast, sugar attachment does not play a major role in protein stability and cell surface expression of the M2 muscarinic acetylcholine receptor (31). CFTR and multidrug resistance (MDR)1, which are structurally closely related to Bsep, show sharp contrast to Bsep. Removal of both CFTR potential glycosylation sites did not affect anion transport activity in HeLa cells (7), implying correct targeting to the plasma membrane. BRO melanoma cells stably overexpressing MDR1 with all three potential glycosylation sites removed exhibited drug resistance activity similar to that of the WT (23). The different roles of Bsep and MDR1 carbohydrates were unexpected, because these two molecules share 70% similarity in amino acid sequence.

BSEP mutations cause PFIC-2 (25), which is potentially fatal without liver transplantation (29). It is not known whether mutations of Asn residues carrying a carbohydrate occur in these patients. However, a recent study (21) demonstrated that Bsep protein with a PFIC-2 mutation in the first nucleotide-binding domain (D482G) is unstable because of lack of glycosylation, suggesting that sugar attachment may be inhibited by a mutation in a region unrelated to N-linked glycosylation. Further investigation of Bsep glycosylation should be performed in PFIC-2 mutants.

The mechanism of reduced biliary secretion (i.e., cholestasis) in acquired liver diseases is multifactorial and not fully resolved. Genetic heterogeneity giving rise to reduced N-linked glycosylation of Bsep may be a contributing factor. A carbohydrate-deficient form of transferrin has been detected in alcoholic liver cirrhosis (6, 14). Theoretically, decreased activity of N-linked glycosylation-related enzymes may give rise to insufficiently glycosylated Bsep, thereby impairing bile secretion. Current studies are being directed to elucidating the mechanisms of N-linked glycosylation in acquired liver diseases and the consequent effect on Bsep function.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54785 and DK-35652 (to I. M. Arias) and by a Japanese Grant-In-Aid for Scientific Research (to T. Kagawa).


   ACKNOWLEDGMENTS
 
We thank M. Sato, H. Tsukamoto, and H. Kamiguchi for technical support and Y. Wakabayashi for helpful advice.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Kagawa, Div. of Gastroenterology, Dept. of Internal Medicine, Tokai Univ. School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan (e-mail: kagawa{at}is.icc.u-tokai.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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T. Kagawa, N. Watanabe, K. Mochizuki, A. Numari, Y. Ikeno, J. Itoh, H. Tanaka, I. M. Arias, and T. Mine
Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells
Am J Physiol Gastrointest Liver Physiol, January 1, 2008; 294(1): G58 - G67.
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