From the Glycobiology Research and Training Center, Departments of Medicine and Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093-0687
Received for publication, October 15, 2002, and in revised form, December 17, 2002
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ABSTRACT |
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9-O-Acetylation is a
common sialic acid modification, expressed in a developmentally
regulated and tissue/cell type-specific manner. The relevant
9-O-acetyltransferase(s) have not been isolated or cloned;
nor have mechanisms for their regulation been elucidated. We previously
showed that transfection of the GD3 synthase (ST8Sia-I) gene into
Chinese hamster ovary (CHO)-K1 cells gave expression of not only the
disialoganglioside GD3 but also 9-O-acetyl-GD3. We now use
differential display PCR between wild type CHO-K1 cells and
clones stably expressing GD3 synthase (CHO-GD3 cells) to detect any
increased expression of other genes and explore the possible induction
of a 9-O-acetyltransferase. The four CHO mRNAs showing major up-regulation were homologous to VCAM-1, Tis21, the
KC-protein-like protein, and a functionally unknown type II
transmembrane protein. A moderate increase in expression of the FxC1
and SPR-1 genes was also seen. Interestingly, these are different from
genes observed by others to be up-regulated after transfection of
GD3 synthase into a neuroblastoma cell line. We also isolated a
CHO-GD3 mutant lacking 9-O-acetyl-GD3 following chemical
mutagenesis (CHO-GD3-OAc Sialic acids are a family of 9-carbon carboxylated monosaccharides
typically located at the termini of mammalian cell surface sugar chains
on both glycoproteins and glycolipids. N-acetylneuraminic acid, the most common sialic acid, is subjected to various
modifications in vivo (1-5). One of the most prevalent
modifications is O-acetylation of the hydroxyl group at the
9-carbon position. This modification is known to reduce or abolish the
recognition of sialic acid residues by sialidases (2, 4, 6, 7), by
certain sialic acid-binding lectins like Siglecs (4, 5, 8-10), and by
several viral recognition proteins (4, 5, 11, 12). Conversely, other viruses require 9-O-acetylation for recognition of their
target cells (4, 5, 13-16). It is also known that
9-O-acetylation is regulated during development and
aberrantly expressed in melanomas and basal cell carcinomas (17-20).
These findings indicate diverse physiological and pathological roles
for 9-O-acetylation of sialic acid residues.
O-Acetyl groups can be added to the 7- and/or 9-position of
sialic acids, with the former migrating to the 9-position, either spontaneously under physiological conditions (21, 22) or under the
influence of a specific migrase enzyme (23). In the previous studies,
we and others showed that 9(7)-O-acetylation is an
acetyl-CoA-dependent enzymatic reaction (24-28) that
appears to be localized in the trans-Golgi apparatus (26,
27, 29, 30) and is presumably catalyzed by a sialic acid-specific
9(7)-O-acetyltransferase. Available evidence also suggests
that there are multiple distinct O-acetyltransferases
responsible for O-acetylating sialic acids attached to
glycans in different linkages and possibly for different classes of
glycan chains. For example, transfection of a cDNA encoding
CMP-Sia:Gal Many groups have attempted to isolate or clone the
9-O-acetyltransferases responsible for these phenomena, with
no success so far. The 9-O-acetyltransferase activity in
most systems is very sensitive to solubilization, and thus, direct
purification has proven difficult (27, 28, 34). Heterologous
cell-cDNA library pairs were applied by several investigators to
the attempted expression cloning of a 9-O-acetyltransferase
cDNA in COS cells (35-37). However, whereas several candidate
genes were isolated, neither a 9-O-acetyltransferase nor a
biologically significant inducer for 9-O-acetylation was
eventually defined. This suggests that the
9-O-acetyltransferase could be a complex of multiple gene
products, not amenable to standard expression cloning. This would fit
with our proposal in rat liver Golgi that the acetyl group is
transferred to luminal sialic acids via a transmembrane acetyl transfer
reaction (26, 34).
The induction of 9-O-acetyl sialic acid by transfection of
ST8Sia-I (GD3 synthase) into wild CHO-K1 cells described above can be
explained either by the up-regulation of the
9-O-acetyltransferase gene in the presence of this
sialyltransferase or by the preexisting expression of an Materials--
Unless otherwise stated, reagents were purchased
from Fisher or Sigma, and all oligonucleotides were from Invitrogen.
Cell Culture--
CHO-K1 cells were obtained from the American
Type Culture Collection (ATCC CCL61). All CHO cell lines were grown
under 5% CO2 and 100% relative humidity in Antibodies and Probes--
The mouse IgG3 anti-GD3 monoclonal
antibody R24 (57) was purified from hybridoma supernatant using Protein
A-Sepharose as previously described (58). Biotinylated R24 was
generated by incubating 20 µg of the antibody with 4 µg of EZ-link
sulfo-N-hydroxysuccinimide-biotin (Pierce) in 0.1 M NaHCO3 (pH 8.3) for 2 h at 4 °C,
followed by dialysis against phosphate-buffered saline to remove the
excess reagent. The mouse IgG3 anti-9-O-acetyl-GD3
monoclonal antibody 27A (59) was kindly provided by Dr. M. Farquhar
(University of California, San Diego) and was used as hybridoma
supernatant, or it was purified from supernatant using Protein
A-Sepharose (Amersham Biosciences) following the manufacturer's
instructions. FITC-conjugated 27A was generated using a Fluorotag FITC
Conjugation Kit (Sigma) following the manufacturer's instructions. A
recombinant soluble chimera of influenza C hemagglutinin-esterase fused
to the Fc region of human IgG1 (CHE-Fc) (60) was produced by stably transfected HEK-293 cells. These cells were also adapted to a protein-free medium, CHO-S-SFM II, and generously provided by Dr.
Pascal Crottet (University of Basel) (61). CHE-FcD, the esterase-inactive form of CHE-Fc (60), was prepared by treatment with
diisopropyl fluorophosphate as previously described (60). Conditioned
medium was collected and concentrated to 4 µg/ml protein concentration. For each staining, 150 µl of CHE-FcD-containing medium
was precomplexed with phycoerythrin-conjugated anti-human IgG1 at a
1:15 dilution (preoptimized for this batch of antibody) in the dark for
2 h at 4 °C. The antibodies were then preabsorbed with CHO-K1
wild type cells to eliminate nonspecific cell surface binding. Samples
were stained with these preabsorbed CHE-FcD preparations for 2 h
at 4 °C and analyzed by flow cytometry. CD22-Fc, the soluble chimera
of the extracellular domain of CD22 fused to the human IgG1 Fc domain,
was purified and used at 5 µg/ml concentration for cell staining, as
previously described (62).
Construction of CHO-K1 Cells Stably Expressing GD3 and
9-O-Acetyl-GD3--
The expression vector carrying ST8Sia-I for
transient transfection has been reported (31). Stable expression of the
same cDNA was obtained by transfecting a pcDNA3-based construct
into parental CHO-K1 cells using the LipofectAMINE reagent (Invitrogen) and selected with G418. After 68 h, cells were stained with
antibody 27A as described below, and individual positive cells were
sorted into 96-well plates by fluorescence-activated cell sorting
(FACS) using a FACStar unit (Becton Dickinson). The levels of
9-O-acetyl-GD3 expression of individual clones were analyzed
by flow-cytometric analysis as described below. One clone that showed
high and stable expression of 9-O-acetyl-GD3 (named CHO-GD3)
was used for further experiments.
Generation and Isolation of a Mutant CHO-GD3 Clone Lacking
9-O-Acetylation on GD3--
The cloned CHO-GD3 cell line (stably
transfected with ST8Sia-I) was incubated in DD-PCR--
Total RNA was extracted from CHO-K1 wild type and
CHO-GD3, respectively, using RNeasy minikits (Qiagen) and
reverse-transcribed using Moloney murine leukemia virus reverse
transcriptase and oligo(dT11N) (where N represents
A, C, or G) reverse primers (Genhunter) according to the
manufacturer's instructions. The resultant first strand cDNA was
amplified by PCR using 240 primer pairs of 80 arbitrary forward primers
and three oligo(dT11N) primer sets (where N
represents A, C, or G; Genhunter) and Amplitaq polymerase
(PerkinElmer Life Sciences). PCR products were labeled by
[33P]dATP (PerkinElmer Life Sciences) contained in the
PCR mixture. The 33P-labeled PCR products were resolved on
6% acrylamide gels followed by extraction of the products that were
specifically enhanced in CHO-GD3 cells. The isolated products were
subcloned into a pCR2.1 TOPO vector (Invitrogen) and sequenced on both
strands using M13 primers and an automated DNA sequencer (model 373A; PerkinElmer Life Sciences).
Reverse Transcriptase-mediated PCR (RT-PCR)--
First strand
cDNA was synthesized from total RNA of CHO-K1 wild type, CHO-GD3,
and CHO-GD3-OAc Plasmid Construction--
A mouse Tis21 open reading frame was
prepared by PCR from a mouse 5'-expressed sequence tag clone
(accession number BG973699) using a forward primer
(5'-CTGTAGAATTCGCCATGAGCCACGGGAAGAGAAC-3') and a reverse primer
(5'-ATAAGAATGCGGCCGCCAGCTGGAGACGGCCATCACAT-3'). The PCR product was
digested with EcoRI and NotI followed by ligation into a EcoRI/NotI site of an expression vector,
pcDNA3.1myc-His A (Invitrogen). The sequence of the subcloned
insert was confirmed by sequencing of PCR products obtained using T7
forward and BGH reverse primers.
Transfection of the Mouse Tis21 Gene into
CHO-GD3-OAc Transfection of the ST6Gal-I Gene into Cells--
Human
ST6Gal-I-containing pcDNA 3.1 plasmid or empty vector was
transfected into wild-type, CHO-GD3, or CHO-GD3-OAc Flow Cytometric Analysis--
Various types of CHO cells (1 × 106) were trypsinized to expose gangliosides (or
released with EDTA only when studying glycoproteins) and washed with
phosphate-buffered saline containing 1% bovine serum albumin. The
cells were then incubated with either R24 antibody (for detecting GD3),
27A antibody (for detecting 9-O-acetyl-GD3), CD22-Fc (for
detecting Isolation of a Clone of CHO-K1 Cells Stably Transfected with the
GD3 Synthase Gene--
Wild type CHO cells express predominantly the
monosialoganglioside GM3 (64-66). In our previous studies, we prepared
CHO-K1 cells stably transfected with GD3 synthase (ST8Sia-I) (31). This
cell population expressed not only disialoganglioside GD3 but also
9-O-acetyl-GD3 (detected with monoclonal antibodies R24 and
27A, respectively), neither of which are found in the wild type cells
(31). During continuous culture, we noticed that this population of
cells expressed varying levels of GD3 and 9-O-acetyl-GD3 (data not shown). We therefore used FACS to isolate clones with consistently high expression of GD3 and 9-O-acetyl-GD3 (see
example in Fig. 1). As expected, stable
clones expressing GD3 synthase (called CHO-GD3) expressed primarily
GD3, as determined by a resorcinol stain of high performance thin-layer
chromatography-separated total gangliosides (not shown), with smaller
amounts of 9-O-acetyl-GD3 (also detected in the total lipid
fraction of cells labeled with [3H]galactose and studied
by autoradiography; data not shown).
Isolation and Characterization of cDNAs Showing Enhanced
Expression in CHO-GD3 Cells--
Although the CHO-GD3 cells express
9-O-acetyl-GD3, neither these cells nor the wild-type CHO-K1
cells express 9-O-acetyl GM3. Thus, a GD3-specific
9-O-acetyltransferase must either preexist in wild-type
CHO-K1 cells or be secondarily induced by the presence of GD3. (Since
GD3 synthase is a Golgi-localized protein with a short cytoplasmic
tail, it is unlikely that such induction is a direct effect of the
synthase protein itself.) To explore these possibilities as well as to
evaluate the effects of GD3 and 9-O-acetyl-GD3 on the
expression of other genes, we performed DD-PCR between RNA preparations
from CHO-K1 wild type and CHO-GD3 cells using 80 arbitrary primers and
three oligo(dT11N) (where N represents A, C, or G) primers.
To minimize artifacts and false positive bands, DD-PCR using all primer
pairs was repeated three times. Six DD-PCR products that consistently
showed differentially enhanced expression in CHO-GD3 cells were finally
obtained. These products were extracted, reamplified, subcloned, and
sequenced. We designated them as 19C, 26C, 38G, 44G, 64C, and 66G,
according to the numbering of the arbitrary primers and
oligo(dT11N) reverse primer sets originally used for
detection of each.
To confirm that message expression of these DD-PCR products was
actually enhanced or induced in CHO-GD3, we performed RT-PCR using
gene-specific primers for each product. As shown in Fig. 2A, 19C, 38G, 64C, and 66G
were expressed in CHO-GD3 much more abundantly than in wild-type CHO-K1
cells. Expression of the other two products, 26C and 44G, was also
increased in CHO-GD3 cells (Fig. 2A). These results confirm
that expression of all six mRNAs is up-regulated by stable
transfection of the GD3 synthase gene. Similar data were obtained by
comparative RT-PCR between wild-type cells and another CHO-GD3 clone,
as well as with wild-type cells transiently transfected with GD3
synthase (Fig. 2B). These results confirmed the consistent
enhancement of expression of these genes by the presence of GD3.
Nucleotide Sequence Analysis of DD-PCR Products Enhanced by
Transfection of GD3 Synthase--
Nucleotide sequence analysis
followed by BLAST homology searching of the NCBI data bases revealed
similarities of each of the hamster DD-PCR products with other
previously reported genes (see summary in Table
I). Product 19C was most similar to the mouse Tis21/Btg2/PC3/APRO-1 gene (accession number M64292), which is a member of an anti-proliferative protein family (hereafter called the Tis21 gene) (67). The 64C sequence showed
homology with mouse vascular cell adhesion molecule-1 (VCAM-1;
accession number X67783) (68). The 38G sequence was found to be
homologous to a small type II transmembrane protein of unknown function
(accession number AB015632) (69). The 66G sequence is similar to a rat KC protein-like protein gene (accession number M86536), which is known
to be induced at early stages following cell stimulation by
platelet-derived growth factor (70). The 26C sequence showed the most
similarity to Rat FxC1 (accession number AF061242), a gene of unknown
function, which is induced at a certain stage of a healing femur
fracture (71). The 44G sequence is most similar to the rat SPR 1 protein gene (accession number X91824), a small proline-rich protein
that is thought to be associated with cell proliferation but whose
specific functions are unknown (72). Taken together, all of the above
results indicate that transfection of GD3 synthase into CHO-K1 causes a
major increase in expression of genes for VCAM-1, Tis21, the
KC-protein-like protein, and a functionally unknown type II
transmembrane protein and a small increase in expression of the FxC1
and SPR-1 genes. Combined with ganglioside profiles in the CHO-GD3
line, these data indicate that the six genes are specifically
up-regulated by the expression of GD3 and/or
9-O-acetyl-GD3.
Liu et al. (73) showed that transfection of GD3 synthase
gene into mouse neuroblastoma Neuro2a cells resulted in induction of
several differentiation-associated genes. Notably, these genes are
completely different from those that we have found to be up-regulated in CHO-GD3 cells. Thus, the genes secondarily induced by GD3 synthase are quite different between cell types and/or status (note that, unlike
Neuro2a cells, CHO-K1 cells are devoid of any differentiation ability).
The other possibility is that the difference of gene induction is due
to an overall difference of ganglioside expression profile. Whereas
CHO-GD3 cells express mainly GM3, GD3, and 9-O-acetyl-GD3, transfection of Neuro2a cells with the GD3 synthase gene induces not
only GD3 but also other larger b-series gangliosides (74). Similar
findings were observed in rat pheochromocytoma PC12 cells, in which
transfection of the GD3 synthase gene resulted in drastic increase of
GD1b and GT1b (75). Thus, the outcome of GD3 synthase expression could
depend not only upon the cell type but also upon the preexisting
ganglioside profile in the cells.
Construction of 9-O-Acetyl-GD3-deficient but GD3-positive CHO
Cells--
We also isolated mutant cells (called
CHO-GD3-OAc Expression of the Tis21 Gene Is Markedly Reduced in
CHO-GD3-OAc Transfection of the Tis21 Gene Restores 9-O-Acetyl-GD3 Expression
in CHO-GD3-OAc 9-O-Acetylation of Sialic Acid on N-Glycans Is Not Affected by
Tis21--
Previously, we also showed that transfection of ST6Gal-I
into wild-type CHO-K1 cells induced 9-O-acetylation
exclusively on newly synthesized
The Tis21 gene is a widely expressed but highly inducible
member of an "anti-proliferative" protein gene family. This
soluble intracellular protein was first identified as one of the
immediate early genes activated by nerve growth factor-treated PC12
cells and by 12-O-tetradecanoylphorbol-13-acetate
ester-treated NIH3T3 cells (77, 78). Tis21 was also shown to
be up-regulated by the tumor suppressor gene p53 after DNA damage
induced by genotoxic reagents (78-80) or during the embryogenesis of
neurons when the cells differentiated or ceased to proliferate (67, 81,
82). Such findings have led to the suggestion that Tis21
serves as an anti-proliferative factor through the regulation of cell
cycle, causing cell arrest, which then allows either cell
differentiation in response to nerve growth factor or cell repair in
response to DNA damage. Indeed, stable transfection of the
Tis21 gene into NIH3T3 cells caused impaired transition from
G1 to S phase (81, 83). However, the anti-proliferative
property is not consistent in all cases. Whereas Tis21
expression is induced by nerve growth factor, fetal growth factor,
interleukin-6, 12-O-tetradecanoylphorbol-13-acetate, serum,
and epidermal growth factor, the first three stimuli induce neuronal
differentiation, whereas the latter stimuli induce proliferation (67,
80). It has been suggested that response of a particular cell depends
on the duration of activation of the Ras/MEK/mitogen-activated protein
kinase pathway (80).
As mentioned above, Tis21 is also a marker of neurogenesis.
It is transiently expressed in neuroepithelial cells in the ventricular zone preceding neural differentiation into mature neurons (82). Interestingly, this is also the originally described distribution of
9-O-acetyl GD3 in the developing nervous system (84).
GD3 itself is also expressed embryonically in neuronal cells, and the
level of GD3 decreases during neural differentiation (85). Furthermore,
the involvement of 9-O-acetyl-GD3 in differentiation, development, and migration of some central neuronal cells has been
suggested (46, 86-89). These findings imply that 9-O-acetyl GD3 and GD3 are functionally linked to the Tis21 gene at
least in some tissues. However, the generality of induction of
9-O-acetylation via Tis21 gene up-regulation by
GD3 synthesis and the functional correlates of this process remain to
be explored. Since 9-O-acetyl-GD3 is a well known
tumor-associated antigen of human melanoma (17, 90-92) and basal cell
carcinoma (20, 93), studies of Tis21 in these tumors are
also warranted.
One of our ultimate goals in these studies is to isolate and
characterize the specific 9-O-acetyltransferase involved in
generating 9-O-acetyl-GD3. Since many biochemical studies
indicate that such 9-O-acetyltransferases are localized in
the Golgi apparatus (26, 27, 29, 30), Tis21 (a cytosolic
protein involved in suppression of cell proliferation) (78, 79) is not
a likely candidate to be a physical part of
9-O-acetyltransferase itself. Others have also attempted to
isolate the 9-O-acetyltransferase without success. In one
study, a putative acetyl-CoA transporter that induced
9-O-acetylation of GD3 was isolated through expression cloning (36). However, this protein has an ortholog in yeasts (which do
not express sialic acids) and is predominantly expressed in the
endoplasmic reticulum. Another group attempted expression cloning of
this 9-O-acetyltransferase but instead isolated a secreted mucin-like molecule similar to the milk fat globule protein (35). Our
similar attempts at expression cloning only yielded a contaminating bacterial transcriptional repressor gene that induced
9-O-acetylation of GD3 in COS cells transfected with GD3
synthase (37). Similar problems have plagued attempts to
expression-clone the 9-O-acetyltransferases involved in
9-O-acetylation of glycoproteins, yielding candidates such
as a truncated form of a vitamin D-binding protein (37), which again could not be a component of the Golgi
9-O-acetyltransferase. All of these studies were also
confounded by the fact that GD3 synthase-expressing COS cells already
have a low level of
9-O-acetyl-GD3.2
Recently, Huang et al. (94) showed that
9-O-acetylation of GD3 was up-regulated by interleukin-4 and
-13 and down-regulated by interferon-
We also attempted to determine whether any of the same genes are
up-regulated in cells transfected with ST6Gal-I, in which selective
9-O-acetylation of N-glycans is seen. However, we
have been unable to generate the cell line required to do these
studies. Whereas the primary data in this paper are based on a stable
clone overexpressing GD3, it has been not been possible for us (for unknown reasons) to obtain a stable ST6Gal-I-expressing clone despite
many attempts; in particular, the level of ST6Gal-I expression and
9-O-acetylation of N-glycans varies and
disappears with time in culture (data not shown). On the other hand, we
felt that direct comparisons among transiently transfected cells would
not be valid, since the efficiency of transient transfection cannot be
controlled, and unrelated genes are likely to be temporarily
up-regulated during such a process. We are, however, able to say that
Tis21 up-regulation was not detected by PCR studies of CHO
cells transiently transfected with
ST6Gal-I,3 further indicating
that Tis21 induction by GD3 synthase is indeed a very
specific process.
In our present study, we employed DD-PCR and mutagenesis analysis using
some cell variants that originated from the single parental cells
(i.e. wild type CHO-K1, GD3- and
9-O-acetyl-GD3-stably expressing CHO-cells (CHO-GD3), and
9-O-acetyl-GD3-deficient CHO-GD3 mutant
(CHO-GD3-OAc
Definition of this pathway also provides useful new tools
for the characterization of GD3-9-O-acetyltransferase. For
example, comparison of gene expression profiles between
CHO-GD3-OAc). Analysis of the above
differential display PCR-derived genes in these cells showed that
expression of Tis21 was selectively reduced. Transfection of a mouse
Tis21 cDNA into the CHO-GD3-OAc
mutant
cells restored 9-O-acetyl-GD3 expression. Since the only major gangliosides expressed by CHO-GD3 cells are GD3 and
9-O-acetyl-GD3 (in addition to GM3, the predominant
ganglioside type in wild-type CHO-K1 cells), we conclude that GD3
enhances its own 9-O-acetylation via induction of Tis21.
This is the first known nuclear inducible factor for
9-O-acetylation and also the first proof that
9-O-acetylation can be directly regulated by GD3 synthase.
Finally, transfection of CHO-GD3-OAc
mutant cells with
ST6Gal-I induced 9-O-acetylation specifically on sialylated
N-glycans, in a manner similar to wild-type cells. This
indicates separate machineries for 9-O-acetylation on
2-8-linked sialic acids of gangliosides and on
2-6-linked
sialic acids on N-glycans.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-4GlcNAc
2-6-sialyltransferase
(ST6Gal-I)1 into Chinese
hamster ovary (CHO)-K1 cells (which normally express only
2-3-linked sialic acids) induced the expression of
9-O-acetyl groups only on the newly appearing
2-6-linked
sialic acids of N-glycans (31). Likewise, transfection of
GD3 synthase (CMP-Sia:GM3
2-8-sialyltransferase (ST8Sia-I)) into
CHO-K1 cells resulted in generation of 9-O-acetyl groups
only on
2-8-linked sialic acids of the newly synthesized
disialoganglioside GD3 (31). In contrast, no 9-O-acetyl
sialic acids were detected upon transfection of
CMP-Sia:Gal
1-3(4)GlcNAc
2-3 sialyltransferase into
CHO-K1 cells (31). On the other hand, regulated
9-O-acetylation on murine erythroleukemia cells and on
murine T cells appears to be on mucin-like molecules, presumably
carried on O-glycans (32, 33).
2-8
linkage-specific 9-O-acetyltransferase gene in CHO-K1 cells.
If the former is true, analysis of gene expression differences between
wild-type CHO-K1 and sialyltransferase gene-transfected CHO cells
offers the possibility of detecting the putative
9-O-acetyltransferase gene and/or biologically significant
inducers of 9-O-acetylation. We have therefore performed
differential display PCR (DD-PCR) between wild type CHO-K1 cells and
CHO-K1 cells stably transfected with ST8Sia-I (CHO-GD3 cells). In doing
so, we also intended to detect other interesting genes up-regulated by
the expression of ST8Sia-I. Such genes might be of interest regarding
the induction of 9-O-acetylation or the involvement of GD3
and/or GD3 synthase in biological functions such as differentiation,
tissue organization, and regeneration (38-47), tumor biology (19, 20,
48, 49), and apoptosis (50-56). Using this approach and a
9-O-acetyl-GD3-deficient CHO-GD3 mutant cell
(CHO-GD3-OAc
), we present evidence for a novel pathway in
which GD3 induces its own 9-O-acetylation via up-regulation
of Tis21, a member of an antiproliferative protein family.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-minimum
essential medium supplemented with 10% (v/v) fetal bovine serum.
-minimum essential
medium containing 450 µg/ml ethylmethane sulfonate (EMS; Sigma) for
16 h in a 37 °C incubator. The concentration of EMS was
determined in pilot experiments in which the frequency of ouabain
resistance was monitored, exactly as previously described (63). Cells
were allowed to recover for 72 h in regular medium.
Mutagenized cells were then stained with FITC-conjugated 27A antibody
and with biotinylated R24 antibody followed by streptavidin-Cychrome
(Pharmingen). Cells that stained positive for R24 and negative for 27A
were isolated on a FACStar unit (Becton Dickinson). Individual clones
were analyzed by flow cytometry as described below. One clone that
showed stable GD3 expression (R24-positive) without detectable
9-O-acetylation (27A-negative) was expanded and named
CHO-GD3-OAc
.
using oligo(dT) primer and Superscript II
(Invitrogen). PCR was performed using Amplitaq polymerase (PerkinElmer
Life Sciences) and gene-specific primers for each DD-PCR product. In
addition, the expression of the Chinese hamster
isopentenyl-diphosphate: dimethylallyl diphosphate isomerase
gene (GenBankTM accession number AF003836) was employed as
a control. The RT-PCR products were subjected to electrophoresis on a
1.5% agarose gel.
Cells--
Mouse Tis21-containing pcDNA
3.1 plasmid or empty vector was transfected into
CHO-GD3-OAc
using LipofectAMINE Plus reagent (Invitrogen)
according to the manufacturer's instructions, and the cells were
incubated for 48 h. The cycle of transfection, 48-h incubation,
and detachment from a culture plate was carried out three times, and
re-expression of 9-O-acetyl-GD3 was monitored by flow
cytometry using 27A.
mutant cells using LipofectAMINE Plus reagent (Invitrogen), and the
cells were incubated for 48 h. Cells were detached by EDTA solution (without trypsin treatment) and subjected to flow cytometric analysis using CHE-FcD to detect 9-O-acetyl groups.
2-6-linked sialic acids without
9-O-acetylation), or CHE-FcD (for detecting
9-O-acetyl sialic acids). The reagents were either
precomplexed or complexed in situ with
phycoerythrin-conjugated goat anti-mouse secondary antibody as
previously described. After final washing, the fluorescent intensity
on the cell surface was observed on a Becton Dickinson FACScan instrument.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Ganglioside profiles in CHO cells stably
transfected with GD3 synthase (CHO-GD3 cells). Flow cytometric
analysis of CHO-GD3 cells is shown. Cells were stained with either
mouse monoclonal antibody R24 (specific to GD3; upper
panel) or 27A (specific to 9-O-acetyl-GD3;
lower panel). Antibodies were precomplexed by
anti-mouse IgG conjugated with phycoerythrin. Wild-type CHO cells
showed no staining with either antibody (data not shown).
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Fig. 2.
Comparison of expression levels of DD-PCR
product genes. A, RT-PCR of the six DD-PCR products
predicted to show selective expression in CHO-GD3 cells (see Table I),
studied in RNA from CHO-K1 wild type cells (lane
1) or the CHO-GD3 cells (lane 2).
RT-PCR was performed using gene-specific primers for each product.
B, RT-PCR for the six DD-PCR product genes was performed
with the same gene-specific primer sets as in A, using RNA
from CHO-K1 wild-type cells transiently transfected with the empty
pcDNA 3.1 (lane 1) or transiently transfected
with GD3 synthase in pcDNA 3.1 (lane 2) or
another stable CHO-GD3 clone (lane 3).
Genes similar to the DD-PCR products enhanced in CHO-GD3 cells in
comparison with CHO-K1 wild type cells
) that no longer express
9-O-acetyl-GD3 while continuing to express GD3. The cloned
CHO-GD3 cells that showed consistently high expression of both GD3
(antibody R24-positive) and 9-O-acetyl-GD3 (antibody 27A-positive) were treated with the mutagen EMS at a dose that limited
the mutation rate per cell to slightly above 2 × 10
4 (see "Experimental Procedures"). Cells were
incubated with EMS and allowed to recover for 3 days. Subsequently,
individual cells that had lost 9-O-acetyl-GD3 expression but
continued to express GD3 were screened for by double color flow
cytometric analysis using two antibodies, FITC-conjugated 27A and
biotinylated R24. A clone with such properties was isolated by FACS.
Compared with CHO-GD3 (Fig. 1A), these
CHO-GD3-OAc
cells completely lacked immunoreactivity
against antibody 27A, whereas R24 antibody staining remained unchanged
(Fig. 3).
View larger version (19K):
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Fig. 3.
Isolation of
9-O-acetyl-GD3-deficient CHO-GD3 mutant cells
(CHO-GD3-OAc ). CHO-GD3 cells
were treated with EMS and 9-O-acetyl-negative cells isolated
by FACS as described under "Experimental Procedures." Mutant
cells were immunostained with either R24 (A) or 27A
(B) monoclonal antibody precomplexed by the
phycoerythrin-conjugated secondary antibody, as described in the legend
to Fig. 1.
Cells--
To examine whether expression of
any of the six DD-PCR product genes up-regulated by GD3 synthase
expression (Table I) was altered in the CHO-GD3-OAc
cells, we performed comparative RT-PCR of these mRNAs. As shown in
Fig. 4, only the Tis21 gene
was found to show decreased expression in the mutant cells, whereas all
of the others were detected at levels similar to those found in the
parent line CHO-GD3. The unchanged expression levels of the other genes
also confirm that they are enhanced by GD3 and not by
9-O-acetyl-GD3.
View larger version (18K):
[in a new window]
Fig. 4.
Selective reduction of Tis21 gene (19C)
expression levels in CHO-GD3-OAc
cells. RT-PCR for the DD-PCR product genes using RNA obtained from
CHO-GD3 (lane 1) or CHO-GD3-OAc
(lane 2) cells was performed using gene-specific
primers for each DD-PCR product.
Cells--
There are three possible
explanations for the above results. First, the reduction of
Tis21 gene expression may have led to the deficiency of
9-O-acetyl-GD3. Alternatively, the decrease of
Tis21 gene expression may have resulted from the deficiency of 9-O-acetyl-GD3 (i.e. Tis21 gene
expression was originally induced in CHO-GD3 cells by
9-O-acetyl-GD3). The third possibility is that the induction
of Tis21 and 9-O-acetylation are not causally related but
rather are activated by a common upstream effector, which is
up-regulated by GD3 expression. To differentiate between these
possibilities, we transfected a cDNA for the Tis21 gene into the CHO-GD3-OAc
cells and allowed the cells to
recover for ~2 days. One round of transfection resulted in small but
significant reexpression of 9-O-acetyl-GD3 after a 2-day
incubation, in comparison with cells transfected with empty vector
alone (data not shown). Since gangliosides may have a slow
synthesis/turnover, we repeated the cycle of transient transfection and
2-day incubation. Flow cytometric analysis of the transfected cells
after additional transfections showed increasing restoration of
9-O-acetyl-GD3 (see example after three rounds in Fig.
5). These results show that
9-O-acetylation of GD3 is up-regulated by the
Tis21 gene in a linear pathway and exclude the possibility
that expression of the Tis21 gene was induced or enhanced by
9-O-acetyl-GD3. In addition, judging from the markedly
reduced Tis21 mRNA level, the mutation in the
9-O-acetyl-GD3 negative cells is likely to be in the
promoter region of the Tis21 gene or in further upstream
gene(s) responsible for the induction of Tis21 gene
expression. Taken together, these results indicate that
Tis21 is up-regulated by GD3 and is then involved in the induction of 9-O-acetylation of GD3.
View larger version (22K):
[in a new window]
Fig. 5.
Restoration of
9-O-acetyl-GD3 expression levels in
CHO-GD3-OAc cells by transient
transfection of the Tis21 gene.
CHO-GD3-OAc
cells were immunostained with the
9-O-acetyl-GD3-specific antibody 27A followed by the
phycoerythrin-conjugated secondary antibody and analyzed by flow
cytometry after transfection of the empty vector (line) or a
vector expressing Tis21 (thick line).
A progressive increase in 9-O-acetyl-GD3 expression levels
was seen with each round of transfection. An example of the result from
a third round of transfection is shown.
2-6-linked sialic acids of
endogenous N-linked glycans (31). To examine whether
reduction of Tis21 also abolished the potential to
9-O-acetylate
2-6-linked sialic acids, we transfected the ST6Gal-I gene into CHO-GD3-OAc
cells and investigated
the generation of 9-O-acetyl sialic acid residues on
glycoproteins by flow cytometry using a 9-O-acetyl sialic
acid-specific probe, CHE-FcD, which is a soluble form of influenza
virus C hemmagglutinin 9-O-acetylesterase conjugated with a
portion of the IgG1 Fc domain whose esterase activity was irreversibly inactivated by treatment with diisopropyl fluorophosphate (60, 76). As shown in Fig. 6,
9-O-acetyl sialic acids on proteins were detected by flow
cytometric analysis of EDTA-released cells (Fig. 3), despite the
continued absence of 9-O-acetylation of GD3 (detected by 27A
after tryspinization; data not shown). Thus, the loss of
Tis21 gene expression is not functionally related to
9-O-acetylation of
2-6-linked sialic acids of
N-glycans, supporting the notion that the mechanism of
9-O-acetylation of
2-8-linked sialic acids on
gangliosides is different from that of
2-6-linked sialic acid on
N-glycans. A different specific
9-O-acetyltransferase is either up-regulated by some other
pathway that is turned on by the presence of the
2-6 linked sialic
acid residues or is already present in the original CHO cells. Taken
together, these data indicate that GD3 synthase expression induces the
expression of the Tis21 gene, which in turn, leads to the
up-regulation of 9-O-acetyl-GD3 (Fig.
7). To our knowledge, this is the first
proposal of a mechanism for GD3-induced 9-O-acetylation
of GD3.
View larger version (44K):
[in a new window]
Fig. 6.
Expression of
2-6-linked 9-O-acetyl sialic
acids in CHO-GD3-OAc
cells
transiently transfected with ST6Gal-I. Wild-type CHO-K1, CHO-GD3,
or CHO-GD3-OAc
cells were transfected with
pcDNA3.1-based ST6Gal-I and immunostained with CD22-Fc (to detect
2-6-linked sialic acid without 9-O-acetyl groups;
left panels) or CHE-FcD (to detect
9-O-acetyl sialic acids; right
panels), followed by the phycoerythrin-conjugated secondary
antibody.
View larger version (21K):
[in a new window]
Fig. 7.
Model for induction of
9-O-acetyl-GD3 by GD3 via the Tis21
gene in CHO-K1 cells. The endogenously synthesized GD3
resulting from GD3 synthase expression enhances the expression of the
Tis21 gene, which in turn induces 9-O-acetylation
of GD3 (presumably via induction of a 9-O-acetyltransferase
specific for GD3). The unlikely but formal possibility that the GD3
synthase protein acts directly to induce Tis21 expression is
also indicated by the dotted line.
in basal layer keratinocytes
in chronic lesions. Such findings further indicate that the synthesis
of 9-O-acetyl-GD3, namely the expression and/or activation
of GD3 9-O-acetyltransferase, is regulated by multiple
endogenous and exogenous factors. This, together with the possibility
that the 9-O-acetyltransferase may consist of an unstable
complex of multiple subunits, may explain the many failures to
characterize the 9-O-acetyltransferase by a variety of
biochemical and molecular approaches.
)). This approach was expected to minimize
the possibility of characterizing biologically unrelated factors for
9-O-acetylation of GD3 even if they exhibited induction of
9-O-acetylation. There are multiple possible reasons for the
lack of detection of the GD3-9-O-acetyltransferase gene
itself, such as low level expression of mRNA encoding for this
Golgi enzyme or masking of the 9-O-acetyltransferase gene
DD-PCR product by other abundantly expressed gene(s). Regardless, the
DD-PCR between CHO-K1 wild type and CHO-GD3 cells showed that six genes
are specifically turned on by the presence of GD3 on CHO cells, and the
present data do demonstrate that GD3-induced 9-O-acetylation
is mediated via the Tis21 gene in CHO-K1 cell lines (Fig.
7). Evidently, the endogenously synthesized GD3 resulting from GD3
synthase expression somehow enhances the expression of the
Tis21 gene, and this in turn induces
9-O-acetylation of GD3, presumably via induction of a
9-O-acetyltransferase that is specific for GD3. Of course,
we cannot rule out the possibility that the GD3 synthase protein acts
directly in some unknown way to induce Tis21 expression
(Fig. 7). However, as stated earlier, this seems unlikely, since this
enzyme is primarily confined to the lumen of the Golgi apparatus and
has a short cytoplasmic tail.
cells and those stably or transiently
transfected with Tis21 by DD-PCR or another method such as
gene subtraction could be a more promising approach. We also need to
study some of the other DD-PCR products, such as the type 2 membrane
protein of unknown function. Such studies are in progress.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sandra Diaz, Justin Sonnenberg, and Takashi Angata for help with some experiments and for useful comments on the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
These two authors contributed equally to this work.
§ Supported by the Suntory Institute for Bioorganic Research (Osaka, Japan).
¶ Supported by United States Public Health Service Grant R01-GM32373. To whom correspondence should be addressed: CMM-E, Rm. 1065, Mail Code 0687, University of California, San Diego, La Jolla, CA 92093-0687. Tel.: 858-534-3296; Fax: 858-534-5611; E-mail: avarki@ucsd.edu.
Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.M210565200
2 H. Satake and A. Varki, unpublished observations.
3 H. Satake, H. Y. Chen, and A. Varki, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ST6Gal-I, CMP-Sia:Gal1-4GlcNAc
2-6-sialyltransferase;
CD22-Fc, soluble
chimeric CD22 conjugated with IgG1 Fc domain;
CHE-Fc, soluble chimeric
influenza C hemagglutinin esterase fused to IgG1 Fc domain;
CHE-FcD, diisopropyl fluorophosphate-treated CHE-Fc;
CHO, Chinese hamster ovary;
CHO-GD3 cells, CHO-K1 cells stably transfected with GD3 synthase gene;
CHO-GD3-OAc
, 9-O-acetyl-GD3-deficient CHO-GD3
mutant cell derived from CHO-GD3 cells;
DD-PCR, differential display
PCR;
EMS, ethylmethane sulfonate;
RT-PCR, reverse
transcriptase-mediated PCR;
ST8Sia-I, GD3 synthase or CMP-Sia:GM3
2-8-sialyltransferase;
FITC, fluorescein isothiocyanate;
FACS, fluorescence-activated cell sorting;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
VCAM-1, vascular cell adhesion molecule-1. Ganglioside nomenclature is based on
the system of Svennerholm (95).
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