The Hepatitis B Virus X Protein Inhibits Secretion of Apolipoprotein B by Enhancing the Expression of N-Acetylglucosaminyltransferase III*

The X protein of hepatitis B virus (HBx) plays a major role on hepatocellular carcinoma (HCC). Apolipoprotein B (apoB) in the liver is an important glycoprotein for transportation of very low density lipoproteins and low density lipoproteins. Although lipid accumulation in the liver is known as one of the factors for the HCC, the relationship between HBx and apoB during the HCC development is poorly understood. To better understand the biological significance of HBx in HCC, liver Chang cells that specifically express HBx were established and characterized. In this study we demonstrate that overexpression of HBx significantly up-regulates the expression of UDP-N-acetylglucosamine:β-d-mannoside-1,4-N-acetylglucosaminyltransferase-III (GnT-III), an enzyme that functions as a bisecting-N-acetylglucosamine (GlcNAc) transferase in apoB, and increases GnT-III promoter activity in a chloramphenicol acetyltransferase assay. GnT-III expression levels of HBx-transfected cells appeared to be higher than that of hepatocarcinoma cells as well as GnT-III-transfected cells, indicating that HBx may has a strong GnT-III promotor-enhancing activity. Intracellular levels of apoBs, which contained the increased bisecting GlcNAc, were accumulated in HBx-transfected liver cells. These cells as well as GnT-III-transfected liver cells revealed the inhibition of apoB secretion and the increased accumulation of intracellular triglyceride and cholesterol compared with vector-transfected cells. Moreover, overexpression of GnT-III and HBx in liver cells was shown to down-regulate the transcriptional level of microsomal triglyceride transfer protein, which regulates the assembly and secretion of apoB. Therefore, our study strongly suggested that the HBx increase in intracellular accumulation of aberrantly glycosylated apoB resulted in inhibition of secretion of apoB as well as intracellular lipid accumulation by elevating the expression of GnT-III.

In Western countries 75-90% of hepatocellular carcinomas (HCCs) 1 are associated with chronic liver diseases (1). Hepatitis B virus is a major causative agent of acute and chronic hepatitis in humans (2) and is closely associated with the incidence of human liver cancer. Among the four proteins that originate from the hepatitis B virus genome, HBx protein is a 17-kDa multifunctional regulatory protein and has been detected with high frequency in liver cells from patients with chronic hepatitis, cirrhosis, and liver cancer (3). In our previous study HBx has an inhibitory effect on the p53-mediated transcription of the 3Ј-inositol phosphatase and tensin homologue deleted on chromosome 10, which is associated with tumor suppression (4). Therefore, HBx is thought to be associated with the development of HCC. However, the precise function of HBx in the tumorigenic transformation of liver cells remains unclear.
The liver is the major organism for both the production of plasma lipoproteins and their uptake from plasma and catabolism (5). The production of apolipoprotein B (apoB, a 500-kDa protein)-containing lipoproteins by the liver is required for the assembly and secretion of very low density lipoproteins and low density lipoproteins (6 -10). The assembly of apoB with lipid to form a secretion-competent particle is a complex process (11,12). It is widely accepted that hepatic lipid availability is obligatory for apoB-containing lipoprotein assembly within the liver. This finding has been supported by studies demonstrating the necessity of triglyceride (12,13) and phospholipid (14). The microsomal triglyceride transfer protein (MTP) also plays a key role in apoB secretion by catalyzing the transfer of lipids to the nascent apoB molecule as it is co-translationally translocated across the endoplasmic reticulum membrane (15,16).
GnT-III catalyzes the attachment of a GlcNAc residue to mannose in the ␤(1-4) configuration in the region of N-glycans and forms a bisecting GlcNAc (17), as shown in Scheme 1. Recent investigations revealed that the bisecting GlcNAc residue, a product of GnT-III activity, correlated with a number of biological events including the suppression of metastasis of mouse melanoma cells (18) and has been reported to be significantly elevated in the serum of human subjects with hepatomas, liver cirrhosis, as well as in HCC (19 -25). Therefore, GnT-III also could be a factor for the development of HCC.
ApoB is a glycoprotein and contains high mannose N-glycans or biantennary-type oligosaccharides in the case of human low density lipoproteins (26,27). Some investigators show that the glycosylation of proteins and lipids are associated with development, differentiation, and carcinogenesis (28 -30). Recently, aberrant glycosylation as the direct result of the formation of bisecting-GlcNAc by GnT-III has been shown to disrupt the function of apoB, leading to the generation of fatty liver (31,32). On the basis of the findings reported herein, we provide evidence of the molecular mechanism underlying the inhibition of apoB secretion and intracellular accumulation of triglyceride and cholesterol in vitro model by HBx/GnT-III expression.

EXPERIMENTAL PROCEDURES
Cell Culture and Transfection-Chang cells (ATCC number CCL-13), a human liver cell line, were maintained using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C in a humidified 5% CO 2 incubator. The cells were used for stable transfection with HBx and GnT-III cDNA using LipofectAMINE reagent following the manufacturer's instructions. The 465-bp and 1.6-kb cDNAs encoding the open reading frames for HBx and GnT-III were inserted into pcDNA3 expression vector at the HindIII/KpnI and BamHI/EcoRI sites, respectively. Transfected cells were then selected by cell culture medium containing 600 g/ml G418 sulfate.
Northern Blot Analysis-To detect mRNA levels of HBx, GnT-III, and MTP, total RNA was prepared using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. For Northern blot analysis, 10 g of total RNA was separated by electrophoresis in a 1% formaldehyde agarose gel. After electrophoresis the gel was blotted on Hybond C membrane (Amersham Biosciences). RNA was fixed to the membrane by cross-linking for 3 min using ultraviolet (UV). Hybridization was performed in Expresshyb (Clontech) to a random prime-labeled probe (Clontech) that encompassed the partial HBx, GnT-III, and MTP genes.
Production GnT-III Antibody-Aglycosyl recombinant N-acetylglucosaminyltransferase-III protein deficient in the first 23 amino acids was expressed in Escherichia coli and purified by DEAE-Sephacel chromatography (Amersham Biosciences) followed by Sephacryl S-200 gel (Amersham Biosciences) filtration, and finally, preparative gel electrophoresis (33). The procedure employed for the production of monoclonal antibodies was based on the protocol described by Harlow and Lane (34). Balb/c mice were immunized by an intraperitoneal injection of aglycosyl recombinant N-acetylglucosaminyltransferase-III and mixed with Freund's complete adjuvant. Spleen cells from the mice were fused with the murine myeloma cell line SP2/0-Ag14. The monoclonal antibody-producing hybridomas were cloned by the limiting dilution technique and propagated by injecting them into mice. Subsequently, ascitic fluids were harvested and processed by protein G-Sepharose 4B chromatography to obtain purified monoclonal antibody as described previously (35).
Immunoprecipitation-The cell lysate (0.5 mg/ml) was precleared with 50 l of protein A-Sepharose beads at 4°C for 1 h and clarified by centrifugation at 14,000 rpm for 10 min. The precleared lysate was incubated with an anti-apoB antibody for 1 h, then 50 l of protein A-Sepharose beads were added, and the mixture was incubated for 1 h. After extensive washing with radioimmune precipitation assay buffer, the immunoprecipitated apoB were eluted from beads with 50 l of SDS sample buffer and subjected to 6% SDS-PAGE under reducing conditions. Western blot and lectin blot were performed with anti-apoB and E-PHA, respectively, as described above.

Construction of Plasmids and Transfections of Chang Cells with GnT-III Promoter-Chloramphenicol Acetyltransferase (CAT) Gene Fusion
Vector-pSV0-CAT, which expresses chloramphenicol acetyltransferase (CAT), was from the laboratory of Molecular Glycobiology, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejon, Korea. pGNT-CAT plasmid was constructed by ligating DNA fragment ranging from Ϫ1 to Ϫ1058 bp of GnT-III upstream region of promoter in pSV0-CAT. Cells were co-transfected with 10 g of pGNT-CAT plasmid and different concentration of pCDNA-HBx gene by a LipofectAMINE-based transfection method. Transfected cells were cultured at 37°C in 3% CO 2 for 24 h followed by 5% CO 2 for 24 h and used for the CAT assay.
CAT Assay-The procedure previously developed by Bullock and Gorman (36) was employed. Transfected cells from 1 plate were harvested, resuspended in 100 l of 0.25 M Tris-HCl (pH 7.8), and subjected to 3 cycles of freezing in a dry ice-ethanol bath and thawing in 37°C incubator. The cell debris was removed by centrifugation, and the supernatant was saved in a clean tube. Total cell lysates were assayed for CAT activity were mixed with 4 l of [ 14 C]chloramphenicol (54 mCi/mmol), 70 l of 1.0 M Tris-HCl (pH 7.8), 20 l of 4 mM acetyl-CoA, and distilled water to a final volume of 150 l. After incubation at 37°C for 1 h, chloramphenicol was redissolved in 30 l of ethyl acetate and applied to a TLC plate (Merck). The plate was incubated for 15 min in chloroform:methanol (95:5) and air-dried. After autoradiography, the radioactive spots on TLC plate were shown.
Measurement of Triglyceride and Cholesterol in Liver Cells-To determine triglyceride and cholesterol content in vector and GnT-III-and HBx-transfected cells, Chang cells were starved for 24 h and then incubated with Dulbecco's modified Eagle's medium supplemented with 1 M concentrations of all-trans-retinoic acids (t-RAs) for 48 h. Triglyceride and cholesterol mass content from the cells was measured enzymatically (Sigma).
Statistics-Results are expressed as the mean Ϯ S.D. and averages of three to five experiments. Means were compared by t tests to determine statistical significance. A p value of Ͻ0.05 was considered significant.

RESULTS
HBx Enhances the Expression of GnT-III by the Promoter Activity of the GnT-III Gene-Chang cells were transfected with the HBx cDNA using pcDNA expression vector. The ex-SCHEME 1. A bisecting GlcNAc chain in N-glycans in biosynthesized by GnT-III. GnT-III catalyzes the attachment of ␤-1,4-GlcNAc to the ␤-D-mannoside of the tri-mannose core structure of an N-glycan.
pression of both HBx mRNA and protein was verified before the investigation of HBx-induced effects. The expression of HBx mRNA was confirmed by Northern blot analysis, and the RNA controls of the corresponding blots are shown in Fig. 1A. Furthermore, the expression of HBx protein was confirmed by Western blot analysis using monoclonal anti-HBx antibodies (Fig. 1B).
Although we were searching for some factors that induced GnT-III, we found that HBx protein enhanced the mRNA and protein expression of GnT-III in the HBx-transfected cells, Chang-HBx. To elucidate whether the endogenous GnT-III gene is activated in the Chang-HBx, the GnT-III mRNA and protein levels were measured by Northern blot (Fig. 2A), RT-PCR (Fig. 2B), and Western blot analysis (Fig. 2C). As shown by these data, the expression of GnT-III gene and protein was significantly elevated in Chang-HBx cells compared with Chang and Chang-pcDAN3 cells.
Here, we postulate whether HBx may enhance the promoter activity of the GnT-III gene in the endogenous system. In a previous study we isolated and characterized the 5Ј-flanking region of GnT-III gene from human placental genome library (37). A schematic diagram of the GnT-III promoter from Ϫ1058 to Ϫ1 is shown in Fig. 3A. Putative promoter elements are based on sequence comparison to known motifs: GRE, halfpalindromic glucocorticoid-response element (TGTCCT), recognition sites for CREB (CGTGACGA), AP-2 (GGCCTGGGGA), and SP1 (GGGCGG). The EMBL data library accession number is L48489. To examine the effect of HBx on the promoter activity of human GnT-III gene, the plasmid pGNT-CAT containing GnT-III promoter in front of the CAT reporter gene was co-transfected in human normal liver Chang cells with an increasing amount of HBx expression vector (pcDNA3-HBx) (see "Experimental Procedures"). Indeed, as shown in Fig. 3B, the CAT activity of the GnT-III promoter was gradually elevated up to 5-fold by increasing the HBx expression vector, although detailed characterization of the promoter activity is limited due to its comprehensive properties of the GnT-III promoter region (38,39). Therefore, these results showed that the HBx protein transactivated GnT-III gene transcriptionally and translationally.
Comparison of the Levels of HBx and GnT-III mRNA and Protein in Normal Cells, Hepatocarcinoma Cells, and Our Transfected Cells-To compare the expression levels of HBx and GnT-III among the several cell lines, GnT-III cDNA was transfected into liver Chang cells, and RT-PCR and Western blot analysis were carried out using normal liver Chang cells, the hepatocarcinoma cell line (HepG2), and our transfected cells as described under "Experimental Procedures." As shown in Fig. 5, we could not detect the HBx level in HepG2 cells by RT-PCR (Fig. 4A) and Western blot (Fig. 4B) because HepG2 is well known as hepatocarcinoma cell line, which is not integrated with hepatitis B virus genome. The level of GnT-III expression in HepG2 cells was similar to that of GnT-III-transfected cells but appeared to be lower than that in HBx-transfected cells. Moreover, in densitometry analysis (Fig. 4C) based on protein levels, GnT-III levels in HBx-transfected cells was increased up to 2-fold over that in GnT-III-transfected cells.
Accumulation of ApoB with Increased Bisecting-GlcNAc in HBx-transfected Cells-To investigate the increase of accumulation of intracellular apoB in HBx-transfected liver cells, immunoprecipitation analysis was performed using cell lysates with anti-apoB antibody as described under "Experimental Procedures." As shown in Fig. 5A, compared with pcDNA transfected cells as control, HBx-transfected cells contained higher levels of intact apoB bands, 150-and 50-kDa apoB species.
Next, to determine whether these apoB species are aberrantly glycosylated, lectin blot analysis were performed using E-PHA lectins, which are known to react preferentially with bisecting-GlcNAc (40). In Fig. 5B, immunoprecipitated apoB species showed increased bisecting-GlcNAc, which is a consistent result with Western blot analysis in HBx-transfected cells. However, although an ϳ80-kDa protein reacted with E-PHA, this band appeared to be nonspecific because it could not detect in Western blot analysis (Fig. 5A). Furthermore, the reactivity of immunoprecipitated apoB to E-PHA was blocked in the presence of an authentic inhibitor, GalNAc (41). Therefore, these results indicated that HBx-transfected cells significantly increased the intracellular accumulation of apoB species, which contained increased bisecting GlcNAc.
Enhanced Expression of GnT-III by HBx Decreases the Secretion of ApoB and Increases Accumulation of Cellular Triglyceride and Cholesterol Contents-Based on the finding that the expression level of GnT-III in HBx-transfected cell was higher than that in GnT-III-transfected cell as shown in Fig. 4, we hypothesized that the aberrant glycosylation of apoB by HBx-induced GnT-III expression may be involved in apoB secretion; the apoB protein level was measured by Western blot from culture media in the control vector, GnT-III, and HBx-transfected cells because apoB plays an important role for delivery of triglyceride from liver to peripheral tissue. Cells were treated with t-RA for 2 days in serum-free media because secretion of apoB induced by t-RA was increased in dose and time-dependent manner (data not shown). The same result was observed in treatment with oleic acid, and there was no apoptotic fragmentation in the cell (data not shown). The secretion of apoB was significantly decreased by 40 and 95% in GnT-III-and HBx-transfected cells, respectively, compared with in vector control (Fig. 6A), indicating that GnT-III expression level is an important factor for the inhibition of apoB secretion. This finding suggested that aberrant glycosylation of apoB mediated by enhanced expression of HBx-induced GnT-III inhibits the secretion of apoB and increases accumulation of intracellular apoB.
As a result of the Western blot experiment of inhibition of apoB secretion in HBx-and GnT-III-transfected cells, as shown in Fig. 6B, we postulated that cellular triglyceride and cholesterol mass may be increased in the HBx-and GnT-III-transfected cells. To test this, we measured the accumulation of triglyceride and cholesterol in the cytosolic fraction in Chang cells transfected with HBx and GnT-III. As expected, in Fig. 6C cellular triglyceride levels in GnT-III-and HBx-transfected cell were higher than vector-transfected cells up to 25% (p Ͻ 0.05) and 85% (p Ͻ 0.01), respectively. Cellular cholesterol levels in GnT-III-and HBx-transfected cells were slightly increased by 21% (p Ͻ 0.05) and 35% (p Ͻ 0.01), respectively. These results clearly indicated that increased accumulation of intracellular apoBs caused an accumulation of triglyceride and cholesterol by enhanced expression of HBx-induced GnT-III.
Expression Levels of MTP mRNA-Because MTP has been shown to play a critical role for apoB assembly and secretion, to determine whether MTP expression may be affected in the GnT-III-and HBx-transfected liver cells, MTP mRNA levels were measured by RT-PCR and Northern blot analysis (Fig. 7,  A and B). The expression of MTP was significantly decreased in GnT-III-and HBx-transfected cells compared with pcDNAtransfected cells after treatment of t-RA for 48 h in serum-free media. When the cells were treated with t-RA, however, there was no difference in apoB gene expression by RT-PCR (data not shown). This result suggested that the relationship between HBx and GnT-III may regulate MTP expression for apoB assembly and secretion. DISCUSSION Here, we provide evidence of the relationship between HBx and GnT-III responsible for inhibition of apoB secretion in vitro model. Many investigators have used HepG2 cell for studying apoB secretion. However, the HepG2 cell line is already a HCC, and increase of the expression of GnT-III gene was reported in human hepatocarcinoma tissues, fetal liver tissues, and hepatoma cell lines (42). In this study we have chosen a liver Chang cell line that is originally derived from normal liver tissue but is subsequently established via HeLa cell contamination.
Miyoshi et al. (43) observe that during hepatocarcinogenesis, GnT-III messenger RNA levels were increased in LEC rats, and Ishibashi et al. (21) also reported that GnT-III activity in human serum, and liver and hepatoma tissues were increased in liver cirrhosis and hepatoma patients. We observed that HBx increases GnT-III expression by transcription as well as translation levels. In the previous study we found that the promoter region of GnT-III has seven AP-2 sites by sequence homology search (37). Elevation of GnT-III gene expression by HBx may be modulated by AP-2 activation since HBx has been shown to activate promoters through several transcription factors such as AP-1, AP-2, NF-B, CREB, and ATF-2 (45-48), although HBx does not bind directly to DNA (49). We also showed that GnT-III expression levels in HBx-transfected cells is higher than that in hepatocarcinoma HepG2 cells as well as GnT-IIItransfected cells, indicating that HBx induces GnT-III expression with strong GnT-III promotor activity. Therefore, this finding supports several studies which show that GnT-III expression may be involved in the development of HCC (23,25). Unfortunately, this study did not determine the precise promotor region involved in the activation for GnT-III expression by HBx because of the limitation of the comprehensive properties of the GnT-III promoter region (38,39), but this is the first report that HBx transactivates GnT-III expression.
The hepatic production of apoB-containing lipoproteins is regulated largely at posttranscriptional levels, with nascent apoB molecules secreted or degraded intracellularly (11). Based on our findings that intracellular accumulation of aberrantly glycosylated apoB species, which exhibit strong reactivities to E-PHA, is increased and low molecular weight immunoreactive apoB species (especially 150 and 50 kDa) are detected in HBx-transfected cells, the present study supports our previous results (31) and those of Ihara et al. (32), who report that overexpression of GnT-III in transgenic hepatocytes induced aberrant glycosylation of apoB and disrupted apoB secretion. They also showed that the 130-and 50-kDa apoB species were immunoprecipitated and detected with lectin blot analysis. In addition, apoB mRNA levels in liver Chang cells are not affected by HBx transfection by RT-PCR (data not shown), consistent with the concept that, under most conditions of altered apoB secretion from HepG2 cells, apoB mRNA levels remain unchanged (13,51,52).
Some investigators suggest that hepatic triglyceride accumulation has a greater influence on apoB secretion (53) and cholesteryl ester is the major lipid species stimulating very low density lipoprotein secretion (54). However, in our study, even though there is intracellular accumulation of triglyceride and cholesterol in GnT-III-as well as HBx-transfected liver Chang cells, inhibition of apoB secretion occurs in these cells. Therefore, intracellular accumulation of apoB caused by the ability of HBx to induce GnT-III expression is the major determinant for the inhibition of apoB secretion and intracellular lipid accumulation.
With regard to the association of apoB with tumorigenesis, lipid accumulation in the liver resulted in the development of dysplasia and carcinoma of the liver in mice expressing aberrantly truncated apoB (55). Our study also revealed that an increase of the accumulation of triglyceride and cholesterol in liver cells is detected in GnT-III-as well as HBx-transfected cells, which is consistent with previous studies by Lee et al. (31) and Ihara et al. (32), which show that lipid accumulation in the liver in GnT-III transgenic mice leads to the generation of liver abnormality. Thus, intracellular accumulation of triglyceride and cholesterol in liver cells caused by HBx-induced GnT-III expression may give us a new insight for HBx-mediated HCC development.
In addition, MTP, an intraluminal protein in the endoplasmic reticulum plays an essential role in regulating the assembly and secretion of apoB containing lipoproteins (44,50,56). It is interesting to note that HBx-and GnT-III-transfected cells showed down-expression of MTP mRNA but not in vectortransfected cell. Because cells were treated with t-RA in our experiment, MTP expression levels by RT-PCR and secretion of apoB by Western blot were increased in dose-and time-dependent manners (data not shown). However, these data questioned whether down-regulation of MTP transcriptional levels by overexpression of GnT-III and HBx may result from the accumulation of triglyceride and cholesterol in liver cells primarily or secondarily. The contribution of down-regulation of MTP expression by GnT-III and HBx remains to be clarified.
The availability of transgenic mice aberrantly expressing the human GnT-III in the liver may help not only to elucidate the role of protein/lipid glycosylation in the development and pathological change of the liver but also to develop therapeutic agents for human diseases caused by glycosylation abnormality. We are currently investigating whether hepatocytes from these transgenic mice show an altered sensitivity to viral infection or abnormal receptor-ligand interactions as the result of aberrant glycosylation of cell surface protein and lipids. In conclusion, HBx induced GnT-III expression may disrupt lipid metabolism by abnormal glycosylation of apoB and may target MTP for assembly and secretion of apoB in the liver cells.