(Received for publication, July 31, 1995; and in revised form, September 18, 1995)
From the
-D-mannoside
-1,4-N-acetylglucosaminyltransferase III (GnT-III)
catalyzes the addition of N-acetylglucosamine in
1-4 linkage to the
-linked mannose of the trimannosyl
core of N-linked oligosaccharides and forms a bisecting GlcNAc
structure. Although the biological meaning of the bisecting GlcNAc
structure remains unclear, it is known that the attachment of a
bisecting GlcNAc inhibits further processing of oligosaccharides by
other glycosyltransferases. To investigate whether or not structural
changes of oligosaccharides affect secretion and gene expression of
hepatitis B virus (HBV), we introduced the GnT-III gene into a human
hepatoma cell line, HB611, which secreted HBV-related proteins into the
medium. Positive transfectants were cloned by hygromycin resistant
selection. Three clones have high activities of GnT-III and secreted
lower levels of HBV-related proteins into the medium in comparison with
other clones. These clones showed marked suppression of HBV-related
mRNAs and an increased binding with E-PHA as judged by lectin blot.
Expression of
actin,
fetoprotein, albumin, and prealubmin
was not correlated with GnT-III activity in all the seven clones.
Treatment of these cells with tunicamycin or swainsonine resulted in
enhanced expression of HBV-related mRNA. These results indicate that
some glycoproteins whose oligosaccharide structures are changed by
overexpression of GnT-III suppress HBV gene expression.
Oligosaccharides in glycoproteins have various biological
functions including cell adhesion, sorting, birth, differentiation, and
carcinogenesis(1) . It is known that many viruses have specific
oligosaccharides in their structural or secreted proteins. For example,
gp120 produced by the AIDS virus has 24 asparagine-linked
oligosaccharides that are involved in attachment to CD4 in lymphocytes (2, 3) . Mouse Moloney leukemia virus has six
asparagine-linked oligosaccharides(4) , and Hepatitis B virus
(HBV) has two asparagine-linked oligosaccharides in the
surface antigen (HBsAg)(5) . Some of the oligosaccharides on
those viral proteins have functions in viral replication, transport,
and secretion(4, 6, 7, 8) . These
studies provide evidence for the importance of oligosaccharides in
certain viruses. However, they are not representative of physiological
conditions, and the inhibitory effects of tunicamycin or
deoxynojirimycin are not specific for the processing of sugar chains in
viruses.
-D-mannoside
-1,4-N-acetylglucosaminyltransferase III (GnT-III) is one
of the glycosyltransferases that catalyzes formation of
asparagine-linked oligosaccharides in glycoproteins(9) .
Although the activity of GnT-III is very high in rat kidney, brain, and
fetal liver, it is very low in normal adult rat liver(10) .
However, GnT-III is strongly expressed during hepatocarcinogenesis in a
rodent model(11) . In human liver diseases, GnT-III activity in
serum or liver is also increased with progression of the
disease(12) . The product of GnT-III, a bisecting GlcNAc
structure of N-linked oligosaccharides, inhibits further
addition of another sugar chain by other
glycosyltransferases(13) . Recently, we were able to
demonstrate suppression of lung metastasis of melanoma cells by GnT-III
gene transfection(14) , suggesting that a bisecting GlcNAc
structure has a biological meaning in vivo. Based on these
observations, there is a possibility that GnT-III induces structural
changes in viral sugar chains, thereby altering their replication.
The cell line HB611 established by transfecting the HBV genome into a human hepatoblastoma cell line Huh6 (15) produces a large amount of HBsAg, hepatitis B e antigen (HBeAg), and HBV virion into the medium. Several studies on HBV replication or expression of HBV-related proteins have been reported using the HB611 cell(16, 17, 18) . Recently, we have found that expression of GnT-III was specifically suppressed by HBV(19) . Although the biological meaning is at present unknown, this phenomenon may be concerned with the fact that HBsAg does not have a bisecting structure in its sugar chains(5) . According to our previous studies, we proposed a hypothesis that a high activity of GnT-III in host cells influences replication or sorting of HBV. To test this hypothesis, we transfected a GnT-III gene into HB611 cells and investigated the expression of HBV gene as compared with other genes of host cells. We found that overexpression of GnT-III suppressed gene expression of HBV in the HB611 cells.
Figure 1: Northern blot analysis on GnT-III mRNA in each transfectant. Total RNAs (30 µg) extracted from each transfectant were electrophoresed on a 1.0% agarose gel containing 2.2 M formaldehyde, and expression of GnT-III was analyzed by Northern blot hybridization.
Figure 2:
Northern blot analysis on HBV-related mRNA
in each transfectant. Total RNAs (10 µg) extracted from each
transfectant were electrophoresed on a 1.0% agarose gel containing 2.2 M formaldehyde and then analyzed by Northern blot
hybridization. Used probes were P-labeled HBV cDNA (top) and
actin cDNA (middle). The bottom
panel showed ethidium bromide staining comparable amounts of RNA
in each lane.
Figure 3:
Northern blot analysis on AFP, albumin,
and prealbumin mRNA in each transfectant. Northern blot hybridization
was performed as described in the legend to Fig. 2. Used probes
were P-labeled AFP (top), albumin (middle), and prealbumin (bottom)
cDNA.
Figure 4:
Southern blot analysis on the HBV gene in
each transfectant. DNAs (10 µg) extracted from each transfectant
were digested with EcoRI (top), BamHI (middle), and HindIII (bottom) and then
electrophoresed. After blotting onto a nylon membrane, the DNA was
hybridized with P-labeled HBV
cDNA.
Figure 5: Lectin blot analysis on each transfectant. 3 µg of proteins from each transfectant and a Huh6 cell were electrophoresed, and lectin blot analysis using E-PHA was performed as described under ``Materials and Methods.'' The numbers at left indicate the molecular mass of standards.
Figure 7: Northern blot analysis of the HB611 parent, HB611-GNT-III(1), and HB611-GNT-III(3) cells treated with swainsonine or tunicamycin. After treatment of swainsonine or tunicamycin for 14 days, total RNAs (20 µg) extracted from the HB611 parent, HB611-GNT-III(1), and HB611-GNT-III(3) were electrophoresed, and then expression of HBV mRNA was investigated by Northern blot hybridization. Lanes 1 indicate control; lanes 2, indicate swainsonine treatment; and lanes 3 indicate tunicamycin treatment in each cell.
Many viruses, including the AIDS virus, HBV, and mouse Moloney leukemia virus, have specific sugar chains in their structural proteins. Although their functions in vivo are little known, in vivo experiments suggest they may be important for infection or secretion(4, 6, 8) . Recently, Block et al.(7) reported that secretion of HBV was inhibited in HBV-infected HepG2 cells when the imino sugar N-butyldeoxynojirimycin, a potent inhibitor of oligosaccharide processing, was present. Molecules such as this result in changes to many oligosaccharides, including glycoproteins of the host cells. It is difficult to conclude, however, that the inhibition of viral secretion results solely from changes in viral sugar chains. Transfection of a specific glycosyltransferase gene into the cell may also change the sugar chain structure, but here again an explanation for the results obtained is difficult to formulate. In this regard, site-directed mutagenesis of amino acids in viral genes is a plausible argument for the functions of sugar chains. However, up-regulation of some glycosyltransferases is observed in vivo, and GnT-III activity in the liver is changed in relation to the clinical stage of liver disease(12) . This change in activity is thought to affect sugar chains of viral proteins and host cells. The levels of HBV-related proteins in serum and its distribution in patients with hepatitis are greatly changed during the different stages of hepatitis(26, 27) . Although the immune system may be involved in this phenomena, the morphology of hepatocytes change in the liver of patients with liver cirrhosis. Expression of GnT-III is very high in fetal rat liver (28) and in chronic myelogenous leukemia cells when in blast crisis(29) . These data show that GnT-III is expressed in undifferentiated cells.
All attempts to produce HBV particles by transfection of a cloned HBV DNA into non-liver cells have so far failed(5) , indicating that viral replication and gene expression are dependent on specific factors in hepatocytes. Furthermore, differentiation of hepatocytes is thought to be related to HBV gene expression. This notion is supported by some reports that only well differentiated hepatoma cell lines can produce the viral replicative intermediates and virions after transfection of a cloned HBV DNA(30, 31) . Recent work using a HBV transgenic mouse shows low expression of HBV gene in hepatoma lesion despite the high expression in the surrounding tissues(32) . These data suggest that it is possible to speculate that some inhibitory factors that can express HBV gene may exist in some kinds of hepatoma cells. The present study demonstrates that bisecting structures of N-glycans are one of the likely candidates. The mechanism by which HBV gene expression is suppressed by the oligosaccharide structure is not yet clear; a bisecting GlcNAc structure produced by ectopic expression of GnT-III may lead to changes of oligosaccharides in glycoproteins such as receptor and adhesion molecules. The second possibility is that some unidentified lectins in HB611 cells, which may directly recognize a bisecting GlcNAc may control the intracellular transport of certain proteins. For these reasons, GnT-III may change differentiation status of HB611 cells. Korczak and Dennis (33) reported that inhibition of N-linked oligosaccharide processing in tumor cells is associated with enhanced gene expression of tissue inhibitor metalloproteinase I. They argued that the activity of an autocrine factor, of its receptor, or of a glycoprotein involved in the response to an autocrine factor was possibly affected by inhibition of N-linked oligosaccharide processing.
In transgenic mice containing the GnT-III gene, ballooning of hepatocytes are observed (data not shown). This phenomenon suggests that GnT-III changes the character of hepatocytes. On the other hand, HBV suppressed GnT-III activity selectively among the various glycosyltransferases(19) , suggesting that HBV may transform host cells in a good place for viral replication. The suppression of HBV gene expression by enhancement of GnT-III might be a unique approach to prevent HBV replication.