(Received for publication, August 16, 1995; and in revised form, November 6, 1995)
From the
Gangliosides of the C series such as G are
polysialylated glycosphingolipids whose synthesis is developmentally
regulated. Here we report the expression cDNA cloning and
characterization of G
synthase that adds the second
-2,8-sialic acid to G
,
NeuNAc
2
8NeuNAc
2
3Gal
1
4Glc
Cer,
thus forming G
,
NeuNAc
2
8NeuNAc
2
8NeuNAc
2
3Gal
1
4Glc
Cer. Unexpectedly, the cloned cDNA was found to be identical
to the cDNA that encodes G
synthase. The newly identified
enzyme was therefore named G
/G
synthase
(G
/G
ST). G
/G
ST
synthesized G
most efficiently when G
,
NeuNAc
2
3Gal
1
4Glc
Cer, was incubated as an
acceptor, indicating that G
/G
ST is a
polysialyltransferase that can transfer more than one sialic acid
residue via
-2,8 linkage to gangliosides. Moreover, a longer
period of incubation of G
with
G
/G
ST produced a significant amount of
G
and higher polysialogangliosides. Among various cell
lines expressing G
/G
ST, higher
polysialogangliosides including G
were detected only in
cell lines where the amount of G
/G
mRNA is
sufficiently high. The expression of G
/G
ST
mRNA among human tissues is highly restricted to fetal and adult
brains. The G
/G
ST gene was found to be
located at chromosome 12, region p12. Taken together, these results
indicate that C series polysialogangliosides are synthesized by a
ganglioside-specific polysialyltransferase,
G
/G
ST, that is specifically expressed in
neural tissues.
Glycoconjugates are major components of the plasma membrane of mammalian cells, and their carbohydrate structures change dramatically during development. Specific sets of carbohydrates are expressed in different stages of differentiation, and many of those carbohydrates are recognized by specific antibodies, thus providing differentiation antigens (Feizi, 1985; Fukuda, 1985). During the course of development, expression of distinct carbohydrates is eventually restricted to specific cell types, and aberrations in these cell surface carbohydrates are frequently observed in malignant cells (Hakomori, 1984). The functional significance of these cell type-specific carbohydrates and their alterations in malignancy is not well understood, although various reports suggest that some of these carbohydrates are involved in cell adhesion processes (Fukuda, 1992; Lowe, 1994).
Among glycosphingolipids, gangliosides comprise a
structurally diverse set of sialylated species and are enriched in
nervous tissues. Gangliosides have been found to act as receptors for
growth factors, toxins, and viruses and are apparently involved in cell
adhesion. For example, cholera toxin binds to G, (
)Gal
1
3GalNAc
1
4(NeuNAc
2
3)Gal
1
4Glc
1
Cer,
before its entry into cells (Spiegel and Fishman, 1987). Influenza A
virus binds to sialylparagloboside,
NeuNAc
2
3Gal
1
4GlcNAc
1
3Gal
1
4Glc
1
Cer
(Higa et al., 1985; Suzuki et al., 1986). In
addition, there have been reports suggesting that gangliosides,
G
in particular (see Fig. 1for its structure) may
play roles in cell-cell interaction. Cheresh et al.(1986)
found that G
and G
facilitate the attachment
of human melanoma and neuroblastoma cells to extracellular matrix
proteins. Epithelial-mesenchymal interactions in embryonic kidney
formation were perturbed by anti-G
antibody, which reacted
with G
on the mesenchymal cells (Sariola et al.,
1988). Gangliosides also modulate enzymatic activities. For example,
G
was found to inhibit epidermal growth factor
receptor-mediated phosphorylation (Bremer et al., 1986), and
G
was shown to inhibit ADP-ribosyltransferases
(Hara-Yokoyama et al., 1995).
Figure 1:
Synthetic pathways of C
series polysialogangliosides. -2,8-sialic acid residues are shown
in boldface type. The rest of the sialic acid residues are
-2,3-linked. In MeWo cells, the synthesis of G
and
G
does not take place, since
-1,4-N-acetylgalactosaminyltransferase is absent.
G
and STVI are newly proposed in the present study. STII
and STIII were found to be the same enzyme in the present study, and
STVI is probably the same enzyme as STII.
Among gangliosides,
increasing attention has been directed to the so-called C series
polysialogangliosides, which have unique trisialosyl residues,
NeuNAc2
8NeuNAc
2
8NeuNAc
2
3Gal
R (Fig. 1). C series polysialogangliosides were found to be major
constituents in adult fish brain. In higher vertebrates the C series
polysialogangliosides comprise a minor proportion of total gangliosides
present in the brain (Ando and Yu, 1979). However, a substantial amount
of C series polysialogangliosides are present in fetal brain of higher
vertebrates including human. They are also found in various
neuroectodermal tumors, such as melanoma and glioma (Yates, 1988;
Nakayama et al., 1993). In the early stages of neural
development, G
is predominantly expressed in the neural
tube that consists of progenitor cells for neurons and macroglial
cells. During the later stage of development, progenitor cells migrate
and extend processes and finally differentiate to postmitotic neurons.
In this developmental period, G
decreases, and C series
polysialogangliosides, such as G
, increase
(Rösner et al., 1985).
It has been
generally accepted that each glycosyltransferase involved in the
synthesis of gangliosides transfers only one sugar residue to form a
specific linkage (Pohlentz et al., 1988) (Fig. 1).
Until recently, the studies of C series polysialogangliosides have been
limited to their structural analysis, since the enzyme responsible for
G synthesis (STIII) was not purified or cloned. In order
to understand the roles and synthesis of C series
polysialogangliosides, it is critical to isolate a cDNA clone of STIII
that forms G
.
In this report, we describe the cloning
of cDNA encoding G synthase, STIII, using a mammalian
expression cloning with a newly devised modification. Surprisingly, the
newly isolated cDNA was found to be identical to that of G
synthase, STII. By transfecting the newly isolated cDNA into HeLa
and MeWo cells and assaying the activity of the soluble form of the
enzyme, we demonstrated that a single enzyme encoded by the isolated
cDNA forms both G
and G
. We also found that
G
synthase transcripts are expressed exclusively in neural
tissues and that its gene is located at chromosome 12, region p12.
These results, taken together, indicate that polysialogangliosides are
synthesized by a single enzyme, G
/G
synthase,
which is specifically expressed in neural tissues.
Isolation of
gangliosides from cells and TLC-immunostaining were performed as
reported previously (Hirabayashi et al., 1988). The purified
gangliosides were applied onto a plastic plate (Poligram Sil G, Nagel,
Doren, Germany) and developed under the same conditions as described
above. The plate was subjected to immunostaining with R24 or M6703
antibody, followed by peroxidase-conjugated goat anti-mouse IgG
antibody (Cappel). The peroxidase activity was visualized with
4-chloro-1-naphthol/HO
.
Sialyltransferase activity was measured as described
previously (Sasaki et al., 1994b). Briefly, after a 1-min
sonication of 25 µl of 0.1 M sodium cacodylate buffer (pH
6.0) containing 20 mM MgCl, 1% Triton CF-54, 2.4
nmol of CMP-[
C]NeuNAc, and 10 µg of a
substrate with or without a competing substrate, 25 µl of the
enzyme solution was added and incubated for 4, 12, or 24 h at 37
°C. At the end of the incubation period, 200 µl of
phosphate-buffered saline was added to the incubation mixture, and the
contents were applied to an Aspec Pak tC18 cartridge (M & S, Tokyo,
Japan), according to the procedure described (Williams and McCluer,
1980). After washing the column with water, glycosphingolipids were
eluted with 3 ml of chloroform-methanol (2:1 by volume). The sample was
dried under nitrogen stream and then subjected to chromatography using
an HPTLC plate under the same conditions as described above.
Radioactive materials were visualized by fluorography after spraying an
autoradiography enhancer (DuPont NEN). Standard and acceptor
gangliosides were visualized by the resorcinol/HCl method.
Purified DNA from one of the isolated P1 clones, clone 5459, was
labeled with digoxigenin-dUTP by nick translation. Labeled probe was
combined with sheared human DNA and hybridized to normal metaphase
chromosomes derived from phytohemagglutinin-stimulated peripheral blood
lymphocytes in a solution containing 50% formamide, 10% dextran sulfate
and 2 SSC. Specific hybridization signals were detected by
incubating the hybridized slides in FITC-labeled anti-digoxigenin
antibody followed by counterstaining with propidium iodide for one
color experiment. Probe detection for two-color experiments was
accomplished by cohybridizing the slides with a biotin-labeled probe,
D12Z1-specific for centromere of chromosome 12 and the
digoxigenin-labeled clone 5459. After incubating these slides with
Texas Red-labeled avidin and FITC-labeled anti-digoxigenin antibody,
they were counterstained with 4`,6-diamidino-2-phenylindole (Rouquier et al., 1995).
When the
COS-1G
cells were tested for the presence of
G
by M6703 antibody, however, 3.5% of COS-1
G
cells showed a strong positive signal for G
judged
in FACS analysis. We thus isolated COS-1
G
cells,
which barely reacted with M6703 antibody by FACS. The freshly sorted
COS-1
G
cells expressed only G
and were
expanded once up to 1.2
10
cells in culture.
Although a few of them still expressed G
(Fig. 2C), they were used as recipient cells for
expression cloning of G
synthase.
Figure 2:
Expression of polysialylated gangliosides
by pcDNAI-GST. COS-1
G
cells (A-D) and HeLa cells (E-J) were
transfected with pcDNAI-G
ST (B, D, F, H, J) or with control pcDNAI vector (A, C, E, G, I). Sixty-two
h after transfection, the cells were fixed and stained with monoclonal
antibodies R24 (A, B, E, F), M6703 (C, D, I, J), and KM641 (G, H), followed by FITC-conjugated (Fab`)
fragment of goat anti-mouse IgG antibody. COS-1
G
cells are positive for the M6703 antibody before transfection
with pcDNAI-G
ST (C), while HeLa cells were
negative for the M6703 antibody before transfection with the same
vector (I). Bar, 50
µm.
COS-1G
cells were transfected with the SK-MEL-28 cDNA library in pcDNAI.
Sixty-two h after transfection, COS-1
G
cells
expressing G
were enriched by FACS using M6703 antibody
under the conditions where only highly positive cells were selected.
From 2.6
10
COS-1
G
cells
applied, 4,044 cells were sorted. Plasmid DNAs were rescued from these
M6703-positive cells.
When COS-1G
cells were
transiently transfected with a mixture of the above isolated plasmids,
it was not possible to distinguish the cells that newly became
G
-positive from the cells that were endogenously
G
-positive by immunofluorescent staining with M6703
antibody. We reasoned that this failure was due to the high background
expression of G
in COS-1
G
cells (see Fig. 2C). In order to overcome this problem, the
plasmid DNAs were transfected into HeLa cells. The wild-type HeLa cells
expressed detectable amounts of G
as judged by
immunofluorescent staining using another anti-G
antibody,
KM641 (Ohta et al., 1993) but were completely negative for
G
(Fig. 2, G and I).
The
transformed bacteria obtained after the Hirt procedure were thus
divided into 20 pools, and the plasmid DNA from each plate was
transfected separately into HeLa cells. The transfectants were screened
by immunofluorescent staining using antibody M6703. Because of no
background staining for M6703 in HeLa cells, we could identify two out
of 20 plasmid pools that directed the expression of G in
HeLa cells. One of the plasmid pools, which produced strongly positive
cell staining by M6703, was selected, and subsequent rounds of sibling
selection with sequentially smaller, active pools identified a single
plasmid, pcDNAI-G
ST, that directed the expression of
G
at the cell surface of HeLa cells.
COS-1G
and HeLa cells were transiently
transfected with pcDNAI-G
ST, and the transfected cells
were examined for the expression of G
and G
by immunofluorescent staining. Fig. 2, D and J, show that the expression of G
, detected by
M6703, was clearly seen on both COS-1
G
and HeLa
cells after the transfection. The expression of G
was also
notably increased in some of the transfected HeLa cells (Fig. 2, F and H).
Sequencing of the isolated cDNA in
pcDNAI-GST revealed an insert of 1622 base pairs in size
encoding a single open reading frame in the sense orientation with
respect to the pcDNAI promoter. The open reading frame predicts a
protein of 356 amino acids in length with a calculated molecular mass
of 40,517. When this cDNA sequence was compared with other cDNAs in the
data base, it was found to be identical to that encoding another
-2,8-sialyltransferase, G
synthase (Haraguchi et
al., 1994; Nara et al., 1994; Sasaki et al.,
1994b) (Fig. 3A). The newly isolated sequence is, however,
shorter in the 5`-flanking sequence and starts with 11 base pairs
upstream from the initiation methionine (Fig. 3A). The
3`-flanking sequence just before poly(A) was shorter by 3 base pairs
compared with the reported G
synthase (Sasaki et
al., 1994b). The deduced amino acid sequence predicts that this
protein has a type II membrane topology, which has been found in all
mammalian glycosyltransferases cloned (Schachter, 1994). These results
indicate that G
synthase (STII) and G
synthase (STIII) are the same enzyme and suggests a possibility
that a single enzyme catalyzes the reactions for the formation of
disialosyl and trisialosyl groups. The newly identified enzyme is thus
called G
/G
synthase or
G
/G
ST hereafter.
Figure 3:
The
nucleotide sequence of GST and schematic representation of
G
/G
ST protein and its derivatives. A, the nucleotide sequence of the 5`-terminal region of the
cDNA insert of pcDNAI-G
ST is shown. The translated amino
acid sequence is shown below the nucleotide sequence. The
possible translation initiation methionines are at residues 1 and 16,
and they are shown in boldface type. The putative
transmembrane/anchor domain is underlined by a solid
line. The translation product starting from the second methionine
is underlined by a dotted line. The rest of the
sequence is shown by Sasaki et al. (1994b). B, the
schematic representation of two G
/G
ST
translation products and proA-G
/G
ST chimeric
protein are shown. G
/G
ST and
G
/G
-S start the translation in residues 1 and
16 shown in A, respectively. The cytoplasmic (Cyt),
transmembrane/anchor (Tm), tentative stem (Stem), and
catalytic (Cata) domains are shown. Sialylmotif L (residues
137-182) and S (residues 273-295) are shown by cross-hatched boxes. The signal peptide sequence (Sig) of the human granulocyte colony-stimulating factor and
IgG-binding domain of S. aureus protein A (ProA) was fused
with a catalytic domain of G
/G
ST. The
catalytic domain encompasses residue 57 (shown by the arrow in A) to the COOH terminus of the
G
/G
ST.
The above cDNA sequence
shows a second initiation methionine, which resides 16 codons from the
first initiation methionine. We synthesized the shorter cDNA encoding
nucleotides 37-1,080 of the cDNA sequence by PCR, allowing the
translation initiation only from the second initiation methionine (Fig. 3A). This truncated cDNA encoding
G/G
ST-S (Fig. 3B) was cloned
into pcDNAI and expressed in both COS-1
G
and HeLa
cells. The results obtained by immunofluorescent staining of the
transfected cells clearly indicate that this truncated cDNA is also
capable of G
and G
expression (data not
shown). In fact, the nucleotide sequence surrounding the second
initiation methionine, GCCATGG is consistent with the consensus
sequence, (A/G)CCATGG for optimal translation initiation (Kozak,
1991). In contrast, the nucleotide sequence surrounding the first
methionine GCGATGA does not conform with the Kozak sequence.
Moreover, the size of the cytoplasmic sequence in this shorter
translated product is reasonably short (12 residues), which is
characteristic for all glycosyltransferases cloned to date (Schachter,
1994). These results suggest that actual initiation of translation
starts probably from this downstream methionine coding for a protein of
341 amino acids with a molecular mass of 38,901.
Figure 4:
Expression of polysialylated gangliosides
in stably transfected HeLa and MeWo cells. A-H, the
parent HeLa (A, B) and MeWo (E, F)
cells and those stably transfected HeLaG
(C, D) and MeWo
G
(G, H) cells were examined by immunofluorescent
staining using R24 (anti-G
) antibody (A, C, E, G) or M6703 (anti-G
)
antibody (B, D, F, H). Bar, 50 µm. I-K, gangliosides expressed in
HeLa
G
and MeWo
G
cells were
analyzed by TLC. Gangliosides were extracted from HeLa (lane
1), HeLa
G
(lane 2), MeWo (lane
3), and MeWo
G
(lane 4) cells and
subjected to HPTLC as described under ``Experimental
Procedures.'' Lane 5 represents ganglioside standards.
The plate was then visualized by resorcinol/HCl (I), by R24
(anti-G
) antibody (J), or by M6703
(anti-G
) antibody (K) followed by a
peroxidase-conjugated goat anti-mouse IgG. L,
G
/G
ST transcripts in various cells were
analyzed by competitive PCR analysis. Single-stranded cDNAs
reverse-transcribed from total RNA of a variety of cells labeled on the
top or standard G
/G
ST cDNAs (50, 500, 2,500,
and 5,000 fg) were mixed with 500 fg of the competitor cDNA (truncated
G
/G
ST cDNA) and subjected to 23 cycles of PCR
as described under ``Experimental Procedures.'' The amplified
products were separated by electrophoresis in 1.8% agarose gel and
visualized by staining with ethidium bromide. Size markers, from the top were 4.3, 1.8, 1.1, 0.68, 0.38, 0.25, and 0.12
kb.
In order to confirm that the
transfected cells synthesize both G and G
,
gangliosides were isolated from the parent HeLa and MeWo cells and
their stable transfectants. The thin-layer chromatogram of the
gangliosides, detected by resorcinol reaction, which reacts with sialic
acid, showed that the HeLa
G
cells contained both
G
and G
, while the parent HeLa cells
contained no G
(Fig. 4I, lanes 1 and 2). Similarly, the MeWo
G
cells
contained G
, whereas the parent MeWo cells did not contain
G
(see lanes 3 and 4 in Fig. 4I). These results were confirmed by
immunostaining of gangliosides after separation by thin-layer
chromatography. The parent HeLa cells express a very small amount of
G
, but HeLa
G
cells express a
substantially increased amount of G
, detected by R24
antibody (Fig. 4J, lanes 1 and 2).
The parent MeWo cells, on the other hand, express a significant amount
of G
(Fig. 4J, lane 3), but no
G
was detected by M6703 antibody (Fig. 4K, lane 3). In contrast, both HeLa
G
and
MeWo
G
cells express a large amount of G
(Fig. 4K, lanes 2 and 4). These
results, taken together, clearly indicate that
G
/G
ST transfers an
-2,8-linked sialic
acid to G
and G
, forming G
and
G
, respectively. The immunofluorescent stainings of
HeLa
G
and MeWo
G
cells by M6703
were completely abolished by pretreatment of chloroform-methanol (2:1)
extraction (data not shown), indicating that all of the newly formed
trisialosyl groups are attached to glycosphingolipids. If some of them
were attached to glycoproteins, some staining should remain because
M6703 also reacts with trisialosyl residues in glycoproteins (Nakayama et al., 1993).
In order to formally prove if G synthase also has G
synthase activity, a putative
catalytic domain of this protein was expressed as a protein fused with
the IgG-binding domain of Staphylococcus aureus protein A
preceded by a signal peptide sequence (Sasaki et al., 1993)
(see ProA-G
/G
ST in Fig. 3B).
The cDNA encoding this chimeric protein was cloned into pcDNAI and
expressed in COS-1 cells. The fusion protein secreted into the culture
medium was absorbed to IgG-Sepharose and then incubated with G
or G
and the donor substrate
CMP-[
C]NeuNAc. As shown in Fig. 5, lane 3, the soluble form of G
/G
ST
synthesized both G
and G
when incubated with
G
. These results establish that the newly identified
enzyme, G
/G
ST, is a polysialyltransferase
that adds more than one sialic acid residue in
-2,8-linkage.
Figure 5:
In
vitro sialyltransferase assay using the protein
A-G/G
ST chimeric protein. The chimeric
soluble G
/G
ST was incubated for 4 h with
CMP-[
C]NeuNAc and 10 µg of G
(lane 3) or 10 µg of G
(lane
5), and the reaction products were subjected to HPTLC followed by
fluorography. Lanes 4 and 6 represent the experiments
that used a control vector, pPROTA, that lacks
G
/G
ST cDNA, and G
(lane
4) or G
(lane 6). The products are shown for
those experiments after 12 h (lane 7) and 24 h (lane
8) incubation using G
as the acceptor. Lane 9 represents the experiment under the same conditions as lane 8 except that pPROTA was used. Lane 10 shows the products
when 10 µg of G
was incubated for 4 h with
CMP-[
C]NeuNAc together with 50 µg of
G
, and lane 11 shows the products when 10 µg
of G
was incubated for 24 h with
CMP-[
C]NeuNAc together with 50 µg of
G
. Lanes 1 and 2 represent the G
and G
used as acceptors, and lane 12 is a
mixture of standard gangliosides, detected by
resorcinol/HCl.
The above experiments also suggested that
G/G
ST added sialic acids much less
efficiently when G
was used as an acceptor (Fig. 5, lane 5). However, the enzyme added sialic acid residues to
G
(136 µM final concentration) after a longer
period of incubation (Fig. 5, lanes 7 and 8).
Under these conditions, G
/G
ST also
synthesized higher polysialogangliosides, which presumably have more
than three sialic acid residues, as shown in lanes 7 and 8 of Fig. 5.
We also tested if the product or an
intermediate inhibits the enzymatic reaction as competing substrates.
The results shown in Fig. 5indicate that G (680
µM final concentration) inhibits the formation of G
and G
from G
(Fig. 5, lane
10), while G
(567 µM final
concentration) inhibits the formation of G
and higher
polysialogangliosides from G
(Fig. 5, lane
11). These results taken together support the above conclusion
that G
/G
ST synthesizes G
from
G
and then G
/G
ST utilizes
G
as an acceptor to form G
.
The above
results also suggested that the amount of
G/G
ST mRNA transcript may be proportional to
the amount of polysialylated gangliosides synthesized. In order to test
this hypothesis, G
/G
ST transcript was
quantitated in the parent HeLa, HeLa
G
, parent MeWo,
and MeWo
G
cells. Fig. 4L shows that
the HeLa
G
cells express a significant amount of the
G
/G
ST transcript (330 fg), while the parent
HeLa cells scarcely express it. The MeWo
G
cells
express approximately the same amount (370 fg) of the transcript as the
HeLa
G
cells and about 15 times more than that in the
parent MeWo cells (23 fg). As shown in Fig. 4, I, J, and K, the parent MeWo cells express G
but barely express G
, while the MeWo
G
and HeLa
G
cells express both G
and G
. These results clearly indicate that G
is synthesized only when G
/G
ST is
abundantly present.
Figure 6:
Northern blot analysis of
G/G
ST in various human tissues. Each lane contained 2 µg of poly(A)
RNA. The blot for
the first two lanes at the far left was made
separately and apparently contained less poly(A)
RNA
than the other Northern blots. The same blots were probed by
P-labeled G
/G
ST cDNA
(G
/G
ST) or
-actin cDNA
(
-actin).
In order to localize precisely the
G/G
ST gene, we utilized fluorescence in
situ hybridization (FISH) procedures. First, P1 plasmid harboring
G
/G
ST gene, named clone 5459 was isolated,
and genomic DNA was prepared from this P1 clone. Using this genomic DNA
as a probe, the initial experiment resulted in specific labeling of the
short arm of a group C chromosome. A second experiment was conducted in
which a biotin-labeled probe (D12Z1) specific for the centromere of
chromosome 12 was cohybridized with the digoxigenin-labeled clone 5459.
This experiment resulted in the specific labeling of the centromere of
chromosome 12 in red and the short arm of the same chromosome
in green (Fig. 7A). Measurements of 10 specifically
hybridized chromosome 12 demonstrated that the clone 5459 is located at
a position that is 43% of the distance from the centromere to the
telomere of short arm, an area that corresponds to 12p12 (Fig. 7B). This location is close to that of the
c-Ki-ras protooncogene (Muleris et al., 1993) and
human islet amyloid polypeptide gene (Christmanson et al.,
1990).
Figure 7:
Chromosomal localization of
G/G
ST gene as revealed by FISH. A,
two-color FISH analysis on metaphase chromosomes using a
digoxigenin-labeled P1 clone 5439 encoding G
ST (stained as green signal) and biotin-labeled D12Z1, a specific probe for
the centromere of chromosome 12 (stained as red signal). A
discrete signal is discernible on all chromosome 12 chromatids for each
probe. DNA was counterstained with 4`,6-diamidino-2-phenylindole. B, the specific hybridization occurs on chromosome 12, region
p12.
The present study describes the isolation of a cDNA clone
encoding G synthase, the key enzyme responsible for C
series polysialogangliosides using expression cloning with a newly
devised modification. We utilized COS-1 cells as recipient cells for
the enrichment of plasmids that directed the expression of
G
. Since the large T antigen is synthesized in COS-1
cells, plasmids such as pcDNAI that contain the replication origin of
SV40 are amplified in the cells. In order to test whether or not
plasmids isolated from sorted COS-1
G
cells could
convert G
to G
, COS-1
G
cells were not suited as recipient cells because of a substantial
background of G
expression. In contrast, HeLa cells do not
express G
but synthesize a small amount of G
.
Therefore HeLa cells should be able to synthesize G
once a
cDNA encoding G
synthase is introduced. By using HeLa
cells, we thus could identify a pool of plasmids that directed G
expression. This is the first report where recipient cells for
enriching plasmids differ from cells used for testing the enriched
plasmids that direct the expression of a desired gene.
It was
generally accepted that G synthase (STII) produced the
first disialosyl linkage, forming
NeuNAc
2
8NeuNAc
2
3Gal
1
4Glc
Cer, and
then another enzyme, G
synthase (STIII), added one more
sialic acid, forming NeuNAc
2
8NeuNAc
2
8NeuNAc
2
3Gal
1
4Glc
Cer (Fig. 1). The present study, however, demonstrates that the same
enzyme can form disialosyl and trisialosyl residues.
It was also
noted that the stable transfectants of both HeLa and MeWo cells
acquired gangliosides larger than G (see the arrowhead in Fig. 4K). Since MeWo cells lack
-1,4-N-acetylgalactosaminyltransferase (Yamashiro et
al., 1995), G
, or G
can not be formed
in MeWo
G
cells (see Fig. 1for the structure
of G
and G
). In addition, M6703 recognizes
not only trisialosyl residues but also tetrasialosyl residues. (
)When G
was incubated with the soluble
G
/G
ST, slow migrating gangliosides were also
produced in addition to G
(Fig. 5, lanes 7 and 8). Since this assay was performed in the absence of
other enzymes, these higher gangliosides are most likely
polysialogangliosides having more than three sialic acid residues. The
results, taken together, strongly suggest that the slow migrating band
may represent G
shown in Fig. 1. It is possible
that G
is present as a very minor component so that it has
escaped attention. Further studies are necessary to confirm the
presence of this glycosphingolipid.
The present study indicates that
the same enzyme is apparently capable of adding all of the
-2,8-linked sialic acid, forming disialosyl, trisialosyl, and
possibly tetrasialosyl residues in gangliosides. This finding is very
similar to those reported for polysialylation of N-CAM. We and others
have recently cloned a polysialyltransferase, which is responsible for
polysialylation of N-CAM (Eckhardt et al., 1995; Nakayama et al., 1995). Although it has been suggested that the first
disialosyl linkage is separately formed by an initiation enzyme (see
Kitazume et al., 1994), the results obtained on mutant Chinese
hamster ovary cells lacking polysialylation strongly suggest that
polysialyltransferase carries out all reactions that form
-2,8-linked sialic acid polymer in N-CAM (Eckhardt et
al., 1995). Moreover, it has been demonstrated recently that
polysialyltransferase can add all
-2,8-linked sialic acid residues
necessary for polysialic acid formation (Nakayama and Fukuda, 1996).
These studies, taken together, strongly suggest that the
polysialylation is catalyzed by single enzymes in both N-CAM
glycoprotein (polysialyltransferase) and glycosphingolipids
(G
/G
ST).
The present study demonstrated
that G/G
ST synthesizes G
more
efficiently from G
than from G
. It is
tempting to speculate that G
/G
ST first binds
to G
and then continuously adds sialic acid residues until
the binding of the enzyme to the product is weakened. In fact, the
excess amount of G
inhibited the formation of G
from G
, confirming that G
is an
intermediate in the polysialylation. Once the enzyme is released from
the enzyme-acceptor complex, it is likely that the enzyme has much less
affinity with the product, which would be an acceptor for another
reaction. Similarly, N-CAM-specific polysialyltransferase was shown to
scarcely add a sialic acid on the polymerized sialic acid residues such
as colomic acid (McCoy et al., 1985). These results suggest
that termination of polysialylation takes place when the enzyme no
longer binds to the polysialylated acceptor. It is apparent that
G
/G
ST terminates its reaction early, most
likely due to its inefficiency in binding to trisialosyl or
tetrasialosyl residues.
It was shown recently that Neuro2a cells
exhibited better neurite extension after the cells were stably
transfected to express G/G
ST (Kojima et
al., 1994). Although these authors thought that this effect was
due to the synthesis of G
and B series gangliosides, the
effect may be due to the synthesis of G
and C series
polysialogangliosides in the transfected Neuro2a cells. We have shown
recently that neurite outgrowth in substratum cells is enhanced by the
presence of polysialic acid in N-CAM (Nakayama et al., 1995).
Previous studies showed that the presence of polysialic acid not only
exhibits antiadhesive properties on homophilic N-CAM interaction but
also influences cell-cell interactions carried by other cell surface
receptors (Edelman, 1985; Rutishauser et al., 1988; Jessell et al., 1990). Further studies are thus needed to determine if
the presence of polysialylated gangliosides influence the cell-cell
interaction carried by other adhesive molecules.
Together with previously cloned polysialyltransferase cDNA that forms polysialic acid in glycoproteins, the cDNA cloned in the present study will be a powerful tool to dissect the intricate and complex roles of polysialic acids attached to glycoproteins and glycosphingolipids in cell-cell interactions during development.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L43494[GenBank].