From the
A cDNA encoding a new
Sialic acids are ubiquitous in the oligosaccharide side chains
of glycoconjugates of a wide variety of animals(1) .
Sia
Comparison of so far cloned
sialyltransferases has revealed highly conserved regions (7, 16, 21) named sialyl motifs L and S, which
are not found in other glycosyltransferases such as
fucosyltransferases, galactosyltransferases, or N-acetylglucosaminyltransferases. From the conservation of the
sialyl motifs, it was expected that other members of the
sialyltransferase gene family might have the same motifs. A PCR-based
approach involving degenerate primers based on the conserved sequences
of the sialyl motifs has resulted in the isolation of several new
members of the sialyltransferase gene
family(8, 13, 14, 15, 16) .
Here, we report the cloning of a cDNA encoding a new type of
Approximately 10
For linkage analysis of sialic acids, fetuin sialylated with the
enzyme was precipitated with 70% ethanol, washed three times with 70%
ethanol, dissolved in water, and then digested with a linkage-specific
recombinant sialidase, NANase I (specific for
For
preparation of desialylated or de-N-glycosylated fetuin,
fetuin (100 µg) was digested with NANAase I (0.1 units/ml), NANAase
II (0.5 units/ml), or NANAase III (0.35 units/ml) in a total volume of
20 µl for 24 h at 37 °C or with N-glycanase (1.5
units; Genzyme, Cambridge, MA) in a total volume of 20 µl at 37
°C for 36 h. After inactivation of the enzyme by boiling for 1 min,
the resulting desialylated or de-N-glycosylated glycoproteins
were used as acceptors.
To isolate the complete
coding sequence of the gene containing the 0.5-kb fragment, we screened
a mouse brain cDNA library using the pCRO3 probe. Sequence analysis of
the largest clone (1.7 kb,
The product
synthesized from G
We cloned a new cDNA encoding an
It has been shown that poly-
We previously
reported that ST8Sia II (STX), which is highly regulated during
development of the brain, also only exhibits activity toward N-linked oligosaccharides of glycoproteins, and it was
suggested to be involved in polysialic acid chain
biosynthesis(8) . The reason why two different types of
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We are grateful to Dr. Masao Iwamori (Tokyo
University) for providing the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
2,8-sialyltransferase (ST8Sia III),
which exhibits activity toward the Sia
2,3Gal
1,4GlcNAc
sequences of N-linked oligosaccharides, was cloned from mouse
brain by means of the polymerase chain reaction-based approach. The
predicted amino acid sequence of ST8Sia III showed 27.6 and 34.4%
identity with those of so far cloned mouse
2,8-sialyltransferases, i.e. G
synthase (ST8Sia I) and STX (ST8Sia II),
respectively. Transfection of the protein A-fused ST8Sia III gene into
COS-7 cells led to
2,8-sialyltransferase activity toward
sialylated glycoproteins and
2,3-sialylated glycosphingolipids,
such as
2,3-sialylparagloboside and G
. However, the
kinetic properties of ST8Sia III revealed that it is much more specific
to N-linked oligosaccharides of glycoproteins than
glycosphingolipids. The expression pattern of the ST8Sia III gene was
clearly different from those of other
2,8-sialyltransferase genes.
The expression of the ST8Sia III gene was tissue and stage specific.
The ST8Sia III gene was expressed only in brain and testis, and it
appeared first in 20 postcoitum embryonal brain and then decreased.
Therefore, the new
2,8-sialyltransferase is closely involved in
brain development.
2,8Sia sequences are widely observed in various gangliosides,
such as G
, G
, and b- and c-series
gangliosides,
(
)and are found more specifically
in glycoproteins of mammals(2) . It has been reported that
Sia
2,8Sia sequences are associated with only two proteins, the
neural cell adhesion molecule (N-CAM)
(
)(3-5) and the
subunit of the voltage-gated
sodium channels in rat brain(6) . Recently, we cloned an
2,8-sialyltransferase, namely G
synthase (ST8Sia
I)(7) , and showed that a developmentally regulated
sialyltransferase (STX, ST8Sia II) exhibited N-glycan
2,8-sialyltransferase activity(8) . Many sialyltransferase
genes have been recently
cloned(7, 8, 9, 10, 11, 12, 13, 14, 15) ,
but only two of them were confirmed to be genes encoding
2,8-sialyltransferases. However, the substrate specificities of
these two
2,8-sialyltransferases cannot fully explain how all the
known Sia
2,8Sia sequences in glycolipids and glycoproteins of
mammals are synthesized. For example, G
synthase was
reported only to synthesize G
, i.e. not
G
, G
, or other b-series
gangliosides(7) . Even if the cloned N-glycan
2,8-sialyltransferase (STX) is involved in the polysialic acid
synthesis in the case of N-CAM, it must not be the sole enzyme
synthesizing polysialic acids of N-CAM because STX itself does not
exhibit polymerization activity as to sialic acids(8) . In
addition, STX was shown to be expressed within limited neural
developmental stages(8, 16, 17) , while
polysialylated N-CAM was reported to exist at almost all
stages(6, 18) . The expression of polysialic acids is
developmentally regulated, the embryonic forms, having a high sialic
acid content, undergoing postnatal conversion to the adult forms with a
low sialic acid content(6, 19, 20) . This
evidence strongly suggests that there must be other types of
2,8-sialyltransferase.
2,8-sialyltransferase using a PCR-based approach.
Materials
Unless otherwise indicated,
the materials used in this study were essentially the same as in
previous studies(7, 13, 14, 15) .
Lactosylceramide, G, G
, G
,
G
, and G
were purchased from Sigma;
G
and paragloboside were from IATRON (Tokyo).
2,3-
and
2,6-sialylparaglobosides (SPGs) were kind gifts from Dr.
Iwamori (Tokyo University). Glycoproteins (fetuin, asialofetuin,
1-acid glycoprotein, ovomucoid, transferrin, and bovine
submaxillary mucin) were from Sigma. Asialo-
1-acid glycoprotein
and asialo-ovomucoid were prepared by mild acid hydrolysis of
glycoproteins (0.02 N HCl, 80 °C, 1 h). Protein
A-Sepharose was from Pharmacia Biotech Inc.
PCR Cloning with Degenerate
Oligonucleotides
PCR was performed using degenerate primers
(5`-primer OP-L,
T(G/A)(A/C)AGA(A/C)(A/T)TG(C/T)GC(G/C)(G/A)T(G/C)GTGGG(A/C)AA;
3`-primer OP-S, CA(C/A)(A/T)G(A/G)GAAGGGCCAGAAGCCATA) deduced from
conserved regions in STX (rat brain) (16) and G synthase (human melanoma cells)(7) . Total RNA from
3-day-old mouse brain was used as a template to synthesize cDNA. The
cycling parameters were 94 °C for 40 s, 37 °C for 40 s, and 72
°C for 1 min for the first 5 cycles, followed by 94 °C for 40
s, 55 °C for 40 s, and 72 °C for 1 min for 30 cycles. The
0.5-kb PCR products were blunt-ended, kinated, and then subcloned into
the SmaI site of pUC119. The subclones were characterized by
sequencing.
plaques of a 3-day-old
mouse brain cDNA library (14) were screened with the 0.5-kb PCR
fragments. Standard molecular cloning techniques, described by Maniatis
and co-workers (22), were used.
Construction and Purification of ST8Sia III Fused
with Protein A
A truncated form of ST8Sia III, lacking the
first 39 amino acids of the open reading frame, was prepared by PCR
amplification with 5`- and 3`-primers containing an XhoI site,
respectively (5`-CATCTTCTCGAGTCCCAAGTACGCCAGCCCG-3` and
5`-TTCCATCTCGAGTTCTTAGGCACAGTGTGACAG-3`). The amplified and digested
1028-bp XhoI fragment was inserted into the XhoI site
of a pcDSA vector(8) . The single insertion in the correct
orientation was finally analyzed by restriction enzyme and DNA
sequencing, and the resulting plasmid was designated as pcDSA-O3, which
consisted of an IgM signal peptide sequence, a protein A IgG binding
domain, and the truncated form of ST8Sia III. COS-7 cells were
transiently transfected with 10 µg of pcDSA-ST8Sia III using the
DEAE-dextran procedure and cultured as previously described(8) .
After 48 h of transfection, the culture medium was collected, and the
protein A-mouse STX expressed in the medium was adsorbed to
IgG-Sepharose (15 µl of resin/10 ml of culture medium) at 4 °C
for 16 h. The resin was collected by centrifugation, washed three times
with phosphate-buffered saline, suspended in 50 µl (final volume)
of Dulbecco's modified Eagle medium without fetal bovine serum,
and used as the soluble enzyme.
Sialyltransferase Assays and Product
Characterization
The enzyme activity was measured in the
presence of 0.1 M sodium cacodylate buffer (pH 6.0), 10 mM MgCl, 2 mM CaCl
, 0.5% Triton
CF-54, 100 µM CMP-[
C]NeuAc (0.25
µCi), 10 µg of acceptor substrate, and 2 µl of enzyme
preparation in a total volume of 10 µl, as described in the
previous paper(7) . After 4 h of incubation at 37 °C, the
reaction was terminated by the addition of SDS-PAGE loading buffer (10
µl), and the incubation mixtures were directly subjected to
SDS-PAGE (for glycoprotein acceptors). For glycolipid acceptors, the
incubation mixtures were applied on a C-18 column (Sep-Pak Vac, 100 mg;
Waters, Milford, MA), which had been washed with water. The glycolipids
were eluted from the column with methanol, dried, and then subjected to
chromatography on an HPTLC plate (Merck, Germany) with a solvent system
of chloroform, methanol, and 0.02% CaCl
(55:45:10), as
previously described(7) . Acceptor substrates were visualized by
staining with Coomassie Brilliant Blue (for glycoproteins) or by the
orcinol/H
SO
method (for glycolipids). The
radioactive materials in glycoproteins or glycolipids were visualized
with a BAS2000 radio image analyzer (Fuji Film, Japan), and the
radioactivity incorporated into acceptor glycoproteins was counted.
2,3-linked sialic
acids, 0.1 units/ml), NANase II (specific for
2,3- and
2,6-linked sialic acids, 0.5 units/ml), or NANase III (specific
for
2,3-,
2,6- and
2,8-linked sialic acids, 0.35
units/ml) (FACE, Glyko, Inc., Navato, CA) at 37 °C for 8 h.
Northern Blot Analysis
5 µg of total
RNA was fractionated on a denaturing formaldehyde-agarose gel (1%) and
then transferred onto a nylon membrane (Nytran, Schleicher &
Schuell). The full-length ST8Sia III cDNA (1205 bp) was amplified by
PCR using synthetic oligonucleotide primers
(5`-AGGCTCGAGCTCTCAATGGACCGATT-3` and
5`-TTCCATCTCGAGTTCTTAGGCACAGTGTGACAG-3`) from 3-day-old mouse brain
cDNA. The full-length mouse G synthase and mouse STX (8) fragments were prepared by PCR amplification, subcloned, and
then sequenced. These fragments were radiolabeled and used as probes.
Cloning and Nucleotide Sequencing of a New
Sialyltransferase cDNA
Last year, the gene encoding
G synthase (ST8Sia I) was cloned from human (7, 23, 24) and mouse.
(
)Recently, we identified the enzymatic activity of
mouse STX (ST8Sia II) as that of an N-glycan
2,8-sialyltransferase(8) . To obtain the new
-2,8-sialyltransferase, we conducted PCR cloning experiments
involving two degenerate oligonucleotide primers based on two highly
conserved regions, sialyl motifs L and S, of human ST8Sia I (7) and rat ST8Sia II(16) . The PCR product corresponding
to a 0.5-kb fragment was subcloned and sequenced. Among several clones,
one, pCRO3, encoded a peptide exhibiting 35.6 and 41.9% identity to the
160-amino acid region of mouse ST8Sia I
and mouse ST8Sia
II,
(
)respectively.
CRO3) revealed a continuous 380-amino
acid open reading frame, including 74 bp of a 5`- and 465 bp of a
3`-non-coding region (Fig. 1).
Figure 1:
Nucleotide and deduced amino acid
sequences of mouse ST8Sia III. The nucleotide and amino acid sequences
are numbered from the presumed start codon and initiation
methionine, respectively. The doubleunderlined amino
acids correspond to a putative transmembrane domain. The asterisks indicate potential N-glycosylation sites
(Asn-X-Ser/Thr). Sialyl motifs L and S are boxed by solid and dashedlines, respectively. The
positions of the PCR primers are indicated by arrows.
The predicted amino acid
sequence encoding a protein with a type II transmembrane topology, as
found for so far cloned sialyltransferases, consisted of a
NH-terminal cytoplasmic tail, a transmembrane domain, a
proline-rich stem region, and a large COOH-terminal active domain.
Cloned Gene Encoded a New
To facilitate functional
analysis of the new sialyltransferase, expression plasmid pcDSA-O3 was
constructed and transfected into COS-7 cells, and the protein A-fused
protein adsorbed to IgG-Sepharose in the medium was used as the enzyme
source. In view of the sequence similarity of the sialyl motifs to
ST8Sia I and ST8Sia II, we examined the sialyltransferase activity
toward several gangliosides and glycoproteins as acceptor substrates.
When fetuin was used as an acceptor, we detected strong
sialyltransferase activity, as seen in the case of mouse ST8Sia II. No
activity toward fetuin was observed in the culture medium from cells
transfected with the vector alone. As shown in Fig. 2A,
sialylated glycoproteins such as 2,8-Sialyltransferase
1-acid glycoprotein, fetuin,
ovomucoid, and transferrin served as acceptors for the new
sialyltransferase. However, the new sialyltransferase did not exhibit
activity toward asialo-glycoproteins at all.
Figure 2:
Incorporation of sialic acids into various
glycoproteins and glycolipids by the cloned sialyltransferase. A, various glycoproteins were incubated with the cloned
sialyltransferase secreted by COS-7 cells transfected with pcDSA-O3 as
a protein A-fused soluble enzyme, as described under
``Experimental Procedures,'' and then the reaction mixtures
were analyzed by SDS-PAGE. Lane1, 1-acid
glycoprotein; lane2, asialo-
1-acid
glycoprotein; lane3, fetuin; lane4, asialofetuin; lane5, ovomucoid; lane6, asialo-ovomucoid; lane7,
human transferrin; lane8, bovine transferrin; lane9, bovine submaxillary mucin (BSM); lane10, no acceptor substrate glycoprotein. Lanes11 and 12 show the incorporation of
sialic acids into fetuin by the culture medium of COS-7 cells
transfected with the vector alone and pcDSA-O3, respectively. It should
be noted that aisalo-glycoproteins did not serve as acceptors at all. B, the various glycolipids indicated in the panel were incubated with the cloned sialyltransferase, and the
resulting glycolipids were analyzed by HPTLC with a solvent system of
CHCl
, CH
OH, 0.2% CaCl
(55:45:10).
It should be noted that 2,3-SPG, but not 2,6-SPG, served as an acceptor
for the new sialyltransferase.
C-Labeled
sialic acid incorporation from CMP-[
C]NeuAc was
also observed when G
was used as an acceptor substrate (Fig. 2B), as seen in the case of G
synthase (ST8Sia I). This sialyltransferase exhibits low activity
toward G
. 2,3-SPG
(Sia
2,3Gal
1,4GlcNAc
1,3Gal
1,4Glc
1,1Cer) served
as the best acceptor substrate among the glycolipids examined for this
sialyltransferase. On the other hand, 2,6-SPG did not serve as an
acceptor at all. Other gangliosides, such as G
,
G
, G
, G
, and G
as well as neutral glycosphingolipids, did not serve as acceptor
substrates for this sialyltransferase (). There was no
sialyltransferase activity toward gangliosides, including 2,3-SPG as
well as neutral glycosphingolipids, in the medium obtained from COS-7
cells transfected with the vector without the insert.
by the sialyltransfer-ase comigrated
with authentic G
(NeuAc
2,8NeuAc
2,3Gal
1,4Glc
1,1Cer) on HPTLC
with two different solvent systems. In addition, a
C-sialylated ganglioside was eluted from DEAE-Sephadex at
the position of disialylated gangliosides (data not shown). The
linkages of the incorporated sialic acids were also confirmed by
digestion of
C-sialylated fetuin with linkage-specific
sialidases. The incorporated
C-labeled sialic acids were
completely resistant to treatment with
2,3-specific or
2,6-
and
2,3-specific sialidase but almost completely disappeared on
treatment with
2,3-,
2,6-, and
2,8-specific sialidase (Fig. 3). Although it has been shown that NeuAc
2,9NeuAc and
NeuAc
2,7NeuAc linkages are present in glycosphingolipids and
glycoproteins, and we did not determine the linkages by chemical
analyses, e.g. methylation analysis, the linkage analyses
performed here indicated that the sialic acids incorporated by the new
sialyltransferase were most probably linked to terminal sialic acids
through
2,8 linkages and the synthesized Sia
2,8Sia sequences.
Therefore, the cloned gene encoded a new
2,8-sialyltransferase
(ST8Sia III).
Figure 3:
Linkage analysis of incorporated sialic
acids. Fetuin was C-sialylated with the cloned ST8Sia III,
and then the
C-sialylated glycoprotein (1000 cpm, 10
µg) was digested with
2,3-specific sialidase (NANase I),
2,3- and
2,6-specific sialidase (NANase II), or
2,3-,
2,6-, and
2,8-specific sialidase (NANase III).
C-Sialylated fetuin was also digested with N-glycanase (1.5 units) at 37 °C for 36 h. The resulting
glycoproteins were subjected to SDS-PAGE and visualized with a BAS2000
image analyzer (A), and then the residual radioactivity at the
position of enzyme-treated fetuin was quantified (B). C, I, II, III, and N indicate treatment with no enzyme, NANase I, NANase II, NANase
III, and N-glycanase, respectively.
In view of the fact that the new
2,8-sialyltransferase exhibits activity toward 2,3-SPG and
G
but not toward 2,6-SPG, it may exhibit activity only
toward the Sia
2,3Gal sequence. This possibility was confirmed by
measuring the activity toward desialylated fetuin. The activity of the
new
2,8-sialyltransferase toward desialylated fetuin on treatment
with
2,3-specific sialidase, as well as that on treatment with
2,3- and
2,6-specific or
2,3-,
2,6-, and
2,8-specific sialidase, was completely abolished (Fig. 4).
Under the same digestion conditions,
2,3-SPG was desialylated by
2,3-specific sialidase, but 2,6-SPG was completely resistant to
treatment with
2,3-specific sialidase.
Figure 4:
Effects of treatment with sialidase and N-glycanase of fetuin on the activity of ST8Sia III. Fetuin
was digested with NANase I, II, and III, respectively, and the
resulting desialylated glycoproteins were incubated with protein
A-fused soluble mouse ST8Sia III and subjected to SDS-PAGE; then, the
radioactivity incorporated into the desialylated glycoproteins was
visualized and quantified with a BAS2000 radio image analyzer. C, I, II, and III indicate
treatment with no enzyme, NANase I, NANase II, and NANase III,
respectively. Glycoproteins were first digested with N-glycanase. Then, the resulting de-N-glycosylated
glycoproteins were incubated with mouse ST8Sia III and
CMP-[C]NeuAc, and the incorporated sialic acids
were visualized and counted. C and N indicate
treatment with no enzyme and N-glycanase,
respectively.
To determine whether the
sialic acids are incorporated into N-linked oligosaccharides
or O-linked oligosaccharides of fetuin, C-sialylated fetuin was digested with N-glycanase. The sialic acids incorporated into fetuin were
completely released from the protein (Fig. 3). In addition, N-glycanase-treated fetuin did not serve as an acceptor (Fig. 4). Since G
, G
,
G
, and O-linked oligosaccharides in fetuin,
which contain Sia
2,3Gal
1,3GalNAc sequences, did not serve as
acceptors for this sialyltransferase, and 2,3-SPG was a good acceptor
for it, it is specific for the Sia
2,3Gal
1,4GlcNAc sequences
of N-linked oligosaccharides of glycoproteins as well as
glycolipids.
Comparison of Acceptor Substrate Specificities
between the New
The acceptor substrate
specificity of the new 2,8-Sialyltransferase and So Far Cloned
2,8-Sialyltransferases
2,8-sialyltransferase (ST8Sia III) was
compared with those of so far cloned
2,8-sialyltransferases, i.e. G
synthase (ST8Sia I) and STX (ST8Sia II),
as shown in . STX exhibited sialyltransfer activity only
toward sialylated glycoproteins such as
1-acid glycoprotein or
fetuin, with no activity being detected toward glycolipids including
G
and 2,3-SPG, while G
synthase exhibited
activity only toward G
, i.e. not toward
sialylated glycoproteins. Comparison of the substrate specificities of
these two
2,8-sialyltransferases showed that of the new
sialyltransferase was rather broad. Both sialylated glycoproteins and
glycolipids served as acceptors for this sialyltransferase. Although
the substrate specificity for glycoproteins of ST8Sia III was similar
to that of ST8Sia II, fetuin acts as a better acceptor (10-fold) than
1-acid glycoprotein in the case of ST8Sia III, whereas the
incorporation of sialic acids into fetuin was almost the same as that
into
-acid glycoprotein in the case of ST8Sia II. Thus, the
structure of oligosaccharides on glycoproteins acting as acceptors for
ST8Sia III is probably different from that in the case of ST8Sia II. On
the other hand, the substrate specificity of ST8Sia III toward
glycolipids was rather similar to that of ST8Sia I (G
synthase) because both sialyltransferases were able to synthesize
G
from G
. Surprisingly, ST8Sia III, but not
ST8Sia I, could synthesize G
from G
. The
apparent K
values of ST8Sia III for
2,3-SPG, G
, and G
were 68, 588, and 3300
µM, respectively (). The V
/K
values shown
in strongly indicate that 2,3-SPG is a much more suitable
acceptor for ST8Sia III than G
or G
. In
addition, the V
/K
values for fetuin indicate that ST8Sia III is much more
specific for complex-type N-linked oligosaccharides, which
contain Sia
2,3Gal
1,4GlcNAc sequences.
Tissue-specific and Developmentally Regulated
Expression of the New
To
evaluate the expression pattern and message size of the new
sialyltransferase gene, we isolated total RNA from several mouse
tissues (brain, heart, liver, lung, kidney, spleen, salivary gland,
thymus, testis, and placenta) and analyzed them by Northern blotting.
Three RNA species, 6.7, 2.2, and 1.7 kb, were expressed in brain (Fig. 5, A and B). Strong expression of a
3.7-kb transcript was observed in testis but not in brain (Fig. 5A). The distribution of these transcripts was
similar to that in the case of STX (ST8Sia II).
2,8-Sialyltransferase Gene
Figure 5:
Northern blot analyses of the ST8Sia III
gene. A, total RNAs (5 µg each) were prepared from various
adult mouse tissues. The hybridization probe was the 1205-bp XhoI fragment of ST8Sia III cDNA. B, total RNAs (5
µg each) were prepared from 14 and 20 postcoitum mouse embryo (E-14 and E-20) and 3-day-old, 2-week-old, and
8-week-old mouse brains. The full-length cDNAs for ST8Sia III, mouse
ST8Sia II (STX), and mouse STSia I (G synthase) were used
as probes, respectively.
We previously
reported that expression of the STX (ST8Sia II) gene was detected in
fetal and newborn mouse brain(8) . To compare the transcription
pattern of the ST8Sia III gene with those of other
2,8-sialyltransferase genes during mouse brain development, total
RNA prepared from the brains of 14 and 20 postcoitum fetal and
3-day-old, 2-week-old, and 8-week-old mice was analyzed by Northern
blot hybridization (Fig. 5B). The transcripts of ST8Sia
III appeared first in 20 postcoitum fetal brain and decreased
thereafter during development. On the other hand, a 6.0-kb transcript
of ST8Sia II was first detected in 14 postcoitum fetal brain, and then
its level increased to reach a peak in 20 postcoitum fetal brain. Then,
the ST8Sia II message decreased to an almost undetectable level by 2
weeks after birth. An approximately 9-kb transcript of ST8Sia I was
also expressed in the brain throughout development, its level being
highest in 20 postcoitum fetal brain. These results suggest that the
three enzyme genes are expressed differently during brain development.
2,8-sialyltransferase
(ST8Sia III) from a mouse brain cDNA library by a PCR-based approach
based on the sialyl motif sequences of so far cloned
2,8-sialyltransferases, i.e. G
synthase
(ST8Sia I) and STX (ST8Sia II). The expression of the ST8Sia III gene
is regulated in a tissue- and developmental stage-specific manner, like
STX, i.e. it is expressed in brain but not in other tissues
except testis and strongly so in the embryonal stage. ST8Sia III
synthesizes the Sia
2,8Sia sequence through
2,8-sialyltransfer
to Sia
2,3Gal
1,4GlcNAc-R, since 2,3-SPG, not 2,6-SPG, served
as the best acceptor substrate for this enzyme among the several
glycolipids examined. Comparison of the acceptor substrate
specificities and kinetic properties revealed that ST8Sia III is
specific to complex-type N-linked oligosaccharides of
glycoproteins, which contain Sia
2,3Gal
1,4GlcNAc sequences,
even though ST8Sia III exhibits activity toward glycosphingolipids such
as 2,3-SPG. Although G
and G
serve as
acceptors for ST8Sia III in vitro, they must not be actual
acceptors in vivo because transfection of the ST8Sia III gene
into G
- or G
-expressing cells did not lead to
expression of G
or G
(data not shown).
However, it should be noted that ST8Sia III exhibits the ability to
synthesize Sia
2,8Sia
2,8Sia sequences from Sia
2,8Sia
sequences.
2,8-sialosyl
sialyltransferase activity is restricted to an early stage of
development(25) . From the results of chemical analysis (26) and overexpression of
Gal
1,4GlcNAc
2,6-sialyltransferase during Xenopus embryogenesis(27) , it was suggested that polysialic acids
are attached to the Sia
2,3-Gal residues of N-linked
oligosaccharides. The gene expression pattern and substrate specificity
of ST8Sia III suggested that it is very closely involved in the initial
step of sialic acid polymerization, i.e. the biosynthesis of
Sia
2,8Sia
2,3Gal of N-glycans.
2,8-sialyltransferase with similar substrate specificities toward N-linked oligosaccharides exist in mouse brain is not clear at
present. One possibility is that the glycoproteins, which act as
acceptor substrates for ST8Sia II (STX) and ST8Sia III, are different.
Indeed, at least two brain glycoproteins, i.e. N-CAM and the
subunit of voltage-gated sodium channels, are known to be
polysialylated(3, 4, 5, 6) . Another
possibility is that the two enzymes may exhibit activity toward almost
the same acceptor glycoproteins in vivo but are controlled
through different regulation systems. The gene expression of ST8Sia II
(STX) and that of ST8Sia III during brain development is distinct from
each other. The ST8Sia II gene appeared in fetal brain and then
completely disappeared, at least in 2-week-old mouse brain, while the
ST8Sia III gene was still expressed in 2-week-old mouse brain. It has
been reported that the expression of polysialic acids of N-CAM is
developmentally regulated, the embryonic form, having a high sialic
acid content, undergoing postnatal conversion to the adult form with a
low sialic acid content(6, 19, 20) . ST8Sia II
and ST8Sia III may be responsible for the polysialic acid chain
biosynthesis of the embryonic and postnatal forms of N-CAM,
respectively. Identification of the actual substrates for the two
sialyltransferases is required. However, the restricted and
developmentally regulated expression of two different
2,8-sialyltransferases for glycoproteins in the brain led us to
the conclusion that the synthesis of Sia
2,8Sia sequences of
glycoproteins is closely related to brain and neural development, like
the differential expression of Sia
2,8Sia-containing gangliosides.
Table: Comparison of the acceptor substrate
specificities of the three cloned 2,8-sialyltransferases
Table: Kinetic
properties of ST8Sia III
/EMBL Data Bank with accession number(s) X80502.
synthase; ST8Sia II, STX reported as a developmentally regulated
member of the sialyltransferase family by Livingston and Paulson (16);
ST8Sia III, a new member of the
2,8-sialyltransferase family
reported in this paper; PCR, polymerase chain reaction; HPTLC, high
performance thin layer chromatography; 2,3-SPG,
2,3-sialylparagloboside (IV
NeuAc
nLc
OseCer); 2,6-SPG,
2,6-sialylparagloboside
(IV
NeuAc nLc
OseCer); kb, kilobase(s); bp, base
pairs; PAGE, polyacrylamide gel electrophoresis.
/EMBL Data Bank accession no. X84235.
/EMBL Data Bank accession no. X83562.
2,3- and
2,6-sialylparaglobosides.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.