From Molecular Glycobiology, Frontier Research
Program, Institute of Physical and Chemical Research (RIKEN), Wako,
Saitama 351-0198, Japan and the ¶ Department of Immunology,
Institute of Medical Microbiology and Hygiene, University of Freiburg,
Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany
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ABSTRACT |
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Two cDNA clones encoding
NeuAc Sialic acids are key determinants of carbohydrate structures that
play important roles in a variety of biological functions, like
cell-cell communication, cell-substrate interaction, adhesion, and
protein targeting. The transfer of sialic acids from
CMP-Sia1 to the terminal
positions of the carbohydrate groups of glycoproteins and glycolipids
is catalyzed by a sialyltransferase. Although roles of sialic acids
have been proposed in the regulation of many biological phenomena, the
purpose of this structural diversity remains largely obscure. To
determine the meaning of the diversity of and the regulatory mechanism
for the sialylation of glycoconjugates, it is necessary to obtain
information on the enzymes themselves and the gene structure of
sialyltransferases. Each sialyltransferase exhibits strict specificity
for acceptor substrates and linkages (3-6). Although three linkages,
Sia So far, the cloning of three members of the As far as seen with the expressed recombinant enzymes, the substrate
specificity of chick ST6GalNAc I is almost the same as that of the
mouse one, and also chick ST6GalNAc II exhibits similar substrate
specificity to the mouse enzyme. ST6GalNAc I exhibits the broadest
substrate specificity for the following structures: GalNAc-O-Ser/Thr, Gal As a result, we have cloned two members of the ST6GalNAc family from
mouse, which synthesize NeuAc Materials--
Fetuin, asialofetuin, bovine submaxillary mucin,
PCR Cloning with Degenerate Oligonucleotides--
A mouse
ST6GalNAc III cDNA fragment was prepared by PCR
amplification. The primers used were rat ST6GalNAc III cDNA,
5'-ATCCATAGCGCCATGGCCTGCATC-3' (nucleotides
To isolate the new sialyltransferases, PCR was performed as described
previously (11) with two primers (5'-primer ST-107, 5'-TGGGCCTTGG(Ino)2(A/C)AGGTGTGCTGTTG-3', and 3'-primer
ST-205, 5'-AGGCGAATGGTAGTTTTTG(A/T)GCCCACATC-3') deduced from the
conserved region in mouse ST3Gal I and II. The standard molecular
cloning techniques described by Ausubel et al. were used
(24).
PCR RACE--
Amplification of the 5'-end of mouse ST6GalNAc III
cDNA was performed as described previously (13). cDNA was
synthesized by reverse transcription (Superscript II, Life
Technologies, Inc.) of 5 µg of mouse brain poly(A)+ RNA
and NB41A3 poly(A)+ RNA using primer RT-181,
5'-TTAGGCTGTCCAAAGCAGTTAGG-3' (complementary to the ST6GalNAc III
coding strand, nucleotides 181-204), and was A-tailed. Two consecutive
PCRs were performed with two nested sets of primers,
NotI-d(T)18 (Amersham Pharmacia Biotech) and RT-181, and then following primers, 5'-AACTGGAAGAATTCGCGGCCGCAGGAA-3', and RT-91, 5' -CTTGAGGATGCAGGCCATGGCGCTATG-3' (complementary to the
ST6GalNAc III coding strand, nucleotides Preparation of Soluble ST6GalNAc Proteins Fused with Protein
A--
A truncated form of ST6GalNAc III and IV, lacking the first 28 and 36 amino acids of the open reading frame, respectively, was
prepared by PCR amplification using 5'-primers containing an
EcoRI site and 3'-primers containing a XhoI site,
i.e. 5'-primer, 5'-CGCCTTGAATTCAATGATGCGACTTTC-3'
(ST6GalNAc III Sec; nucleotides 76-99), and 3'-primer,
CCACAGCTCGAGTGTCATGGTCGG-3', (ST6GalNAc III Tail;
complementary to the coding strand, nucleotides 930-953) for ST6GalNAc
III, and 5'-primer, 5'-CGCGAATTCTGCCTGGACCGCCACCTCCC-3' (R1-Sec; nucleotides 109-128), and 3'-primer
5'-GCGCTCGAGAACTACTTGGCCCTCCAGG-3' (R1-Tail; complementary
to the coding strand, nucleotides 893-911) for ST6GalNAc IV. The
resulting amplified 878- and 803-bp fragments were subcloned into the
EcoRV sites of pBluescript SK(+). The inserted fragments
were cut out by digestion with EcoRI and XhoI, and then inserted into the EcoRI and XhoI sites
of expression vector pcDSA (25).
The insert junctions were confirmed by restriction enzyme and DNA
sequencing. The resulting plasmids consisted of the IgM signal peptide
sequence, the protein A IgG binding domain, and a truncated form of
ST6GalNAc III (pCDB8ST) and IV(pCDR1ST), respectively. Each expression
plasmid (pCDB8ST and pCDR1ST: 20 µg) was transiently transfected into
COS-7 cells on a 150-mm plate using LipofectAMINETM reagent
(Life Technologies, Inc.). Each protein A-fused ST6GalNAc III and IV
expressed in the medium was absorbed to an IgG-Sepharose gel (Amersham
Pharmacia Biotech; 50 µl of resin/50 ml of culture medium; Ref. 25)
and used as the enzyme source.
We also constructed an expression vector containing the whole coding
region of ST6GalNAc III, in which a 1337-bp fragment from the
NH2 terminus was inserted into the pcDL-SR Sialyltransferase Assays and Linkage
Analysis--
Sialyltransferase assays were performed as described
previously (13). In brief, enzyme activity was measured in 50 mM MES buffer (pH 6.0), 1 mM MgCl2,
1 mM CaCl2, 0.5% Triton CF-54, 100 µM CMP-[14C]NeuAc (10.2 KBq), an acceptor
substrate, and an enzyme preparation, in a total volume of 10 µl. As
acceptor substrates, 10 µg of proteins, 5 µg of glycolipids, or 10 µg of oligosaccharides were used. The enzyme reaction was performed
at 37 °C for 2 h.
For linkage analysis of sialic acids,
[14C]NeuAc-incorporated fetuin was synthesized with
ST3Gal I (16), ST6GalNAc I (12), ST8Sia II (26), ST6GalNAc III, and IV.
The sialylated fetuin was treated with a linkage-specific sialidase,
NANase I (specific for
To obtain oligosaccharide portion of 14C-sialylrated
fetuin, the sialylation of fetuin was carried out essentially as
described, but on a 10-fold larger scale. To maximize the product
yield, the incubation period was extended to 24 h. The incubation
mixture was then treated with 0.1 N NaOH, 1 M
NaBH4 at 37 °C for 48 h, and neutralized by the
gradual addition of acetic acid in an ice bath. A sample was then
desalted by Sephadex G-25 chromatography (1.3 × 25 cm). The
14C-sialylated oligosaccharide alditol and reference
oligosaccharide NeuAc Analysis of ST6GalNAc III and IV Gene Expression--
The level
of the ST6GalNAc III transcript was determined by competitive PCR (30).
For the construction of a competitor DNA, PCR was performed with the
ST6GalNAc III gene-specific primers, 5'-ATGGATACATAAATGTGAGGACC-3'
(nucleotides 185-207) and 5'-GTGGATACTGTAGCAGGCATCCA-3' (complementary
to the ST6GalNAc III coding strand; nucleotides 677-699), with
ST6GalNAc III cDNA as the template. The amplified fragment (515 bp)
was subcloned into pKF18k (Takara, Japan), and then subjected to
site-directed mutagenesis using a mutagenic primer,
5'-TGAGGAAGATCTCGGCTACATG-3' (nucleotides 345-366; BglII linker is bold and italic) and a Mutan-Super Express
Km kit (Takara, Japan). From the mutagenized plasmid, a 204-bp
BglII fragment was deleted and the mutagenized plasmid was
self-ligated, giving rise to a plasmid harboring a 315-bp competitor
DNA fragment.
Poly(A)+ RNAs from various mouse tissues were
reverse-transcribed to cDNAs using oligo(dT) (Amersham Pharmacia
Biotech) as a primer with Superscript II (Life Technologies, Inc.).
These single-stranded cDNAs were mixed with 0.35 pg of the
competitor DNA and 40 pmol each of the above ST6GalNAc III
gene-specific primers, and then competitive PCR was performed.
To determine the level of ST6GalNAc IV, Northern blot analysis (24) was
performed using 5 µg of poly(A)+ RNAs from various mouse
tissues. The 0.8-kb fragment of the NH2-terminal truncated
form of ST6GalNAc IV (nucleotides 109-911) was used as the probe.
Identification and Sequence of ST6GalNAc III and New
Sialyltransferase cDNA Clones from Mice--
PCR with primers
based on the rat ST6GalNAc III cDNA sequence gave a 0.9-kb
cDNA. This fragment was used as the hybridization probe to screen a
mouse brain cDNA library. Some independent clones containing a
single open reading frame encoding a protein of 305 amino acids,
showing 94.4% identity with rat ST6GalNAc III, were obtained (Fig.
1A). According to the
following results, these clones encode a mouse homologue of ST6GalNAc
III.
Next, in order to obtain clones of the new members of the
sialyltransferase family, PCR with two degenerate oligonucleotides (ST-107 and ST-205), which were designed based on the 5'- and 3'-sequences of sialylmotif L, respectively, was performed with mouse
brain cDNA as a template. A fragment of the expected size of
approximately 150 bp was obtained. Among the PCR recombinants, one
clone, designated as pCRR1, has a unique amino acid sequence distinct
from that of the known sialylmotifs. The identity of the sialylmotif L
of pCRR1 with those of ST6GalNAc I, II and III is 44.4%, 46.6%, and
64.4%, respectively.
A mouse brain cDNA library was screened with the cDNA insert of
pCRR1 to isolate the complete coding sequence of the gene. The
screening of about 106 independent clones yielded several
overlapping clones, which were isolated and sequenced. The nucleotide
sequence of one cDNA clone included an open reading frame of 906 bp, coding for 302 amino acids with a molecular mass of 34.2 kDa,
starting with a methionine codon at nucleotide 1 with a conventional
initiation sequence (31). The nucleotide and deduced amino acid
sequences of the new sialyltransferase family member are shown in Fig.
1B. This protein has a type II transmembrane topology,
containing a 23-amino acid NH2-terminal hydrophobic
sequence bordered by charged residues, as has been found for all
glycosyltransferases cloned to date. Comparison of this primary
sequence with other amino acid sequences in DNA and protein data banks
revealed similarity in some regions to all cloned sialyltransferases.
One region (named sialylmotif L, residues 75-119) in the center of the
protein, consisting of a 45-amino acid stretch, shows 42.2-64.4%
sequence identity, whereas another, in the COOH-terminal portion (named sialylmotif S, residues 211-235), exhibits 20.0-60.0% identity. The
overall amino acid sequence identity of this protein is 11.9% to mouse
ST6GalNAc I (62),2 10.3% to mouse ST6GalNAc II (13), and
43.0% to mouse ST6GalNAc III, respectively (Table
I). These results suggest that the cloned gene belongs to the sialyltransferase gene family. In fact, the following results revealed that it is a member of the ST6GalNAc family,
so it was named ST6GalNAc IV.
Both the Cloned DNAs Encode GalNAc
The truncated form of ST6GalNAc III exhibited enzyme activity that was
too low (18 fmol/h/ml of medium) to determine kinetic parameters.
Therefore, the full-length ST6GalNAc III was also subcloned into
expression vector pCDL-SR Linkage Specificity of the Two Sialyltransferases--
For linkage
analysis, [14C]NeuAc-incorporated fetuin, which was
synthesized with ST3Gal I (16), ST6GalNAc I (12), ST8Sia II (26), and
ST6GalNAc III and IV, respectively, and each sialylated fetuin was
treated with linkage-specific sialidases, i.e., NANase I
(specific for
To confirm the linkage specificity of ST6GalNAc III and IV, the
following experiments were performed. Although we report here the
results of ST6GalNAc IV, the results for ST6GalNAc III is the same as
those for IV. The 14C-sialylated oligosaccharide alditol
was prepared by Expression of the ST6GalNAc III and IV Genes in Mouse
Tissues--
To examine the expression of the mouse ST6GalNAc III gene
in various tissues, Northern blot hybridization was performed using 5 µg of poly(A)+ RNA from various adult mouse tissues,
which gave only faint signals and could not be used to examine the
specific gene expression of the ST6GalNAc III gene. Therefore, we
performed competitive reverse transcription-PCR to determine in which
tissues the ST6GalNAc III gene is expressed. In adult tissues,
significant levels of expression that could not be detected on Northern
blot analysis but could be detected on reverse transcription-PCR were
observed in brain, lung, and heart, followed by lower levels of
expression in kidney, mammary gland, spleen, thymus, and testis (Fig.
5A). We also estimated the
amount of the ST6GalNAc III transcript in brain during development
(Fig. 5B). The amount of the ST6GalNAc III transcript was
highest at E12, although whole embryos at E7, E11, and E12 were used to
isolate poly(A)+ RNA. The amount of the ST6GalNAc III
transcript in brain was lower at E16 than at embryonal stages (E11 and
E12), and then kept almost similar levels during mouse development,
suggesting that the mouse ST6GalNAc III gene may be highly transcribed
in tissues other than brain at embryonic stages.
The mRNA size and distribution of the ST6GalNAc IV gene were
determined by Northern blot analysis (Fig. 5, C and
D). Three transcripts (1.9, 2.2, and 3.6 kb) were observed
in ICR mouse tissues (8-week-old mice). Strong signals were observed in
brain and colon, and moderate ones in lung, heart, thymus, and spleen (Fig. 5C). The expression in brain was developmentally
regulated. Analysis of RNAs from embryonal stage (E12), and 1-day,
3-week, and 8-week brain revealed three RNA species of 1.9, 2.2, and
3.6 kb. The 1.9- and 2.2-kb mRNA were abundantly expressed.
In this study, we have described the isolation and
characterization of cDNAs for encoding third and fourth types of
mouse GalNAc Based on the following observations, we concluded that the cDNAs we
isolated were indeed for ST6GalNAc III and IV, which transfer CMP-NeuAc with an Similar to other glycosyltransferases, ST6GalNAc IV has a type II
membrane protein topology, a short NH2-terminal cytoplasmic tail, a hydrophobic signal-anchor domain, a proteolytically sensitive stem region, and a large COOH-terminal active domain (6). The location
of the transmembrane domain was determined by hydropathy plot according
to the Kyte and Doolittle method (34). The transmembrane domain was 23 amino acids long from position 14 to 36. We also noticed that ST6GalNAc
IV was the smallest protein (302 amino acid) among all cloned
sialyltransferases. This was mainly because of the very short stem
region of ST6GalNAc IV. The size of stem regions among members of the
ST6GalNAc family varies to a great degree. ST6GalNAc IV had only 38 amino acid residues between the transmembrane region and sialylmotif L,
while ST6GalNAc I, II, and III have 261, 123, and 53 amino acid
residues (13, 62),2 respectively. These differences may
have important implications for the in vivo functions of
individual enzymes, although this remains to be clarified.
The four members belonging to ST6GalNAc family can be classified into
two subfamilies according to the sequence similarity and substrate
specificity differences (Fig. 6 and
Tables I and III). ST6GalNAc I and II belong to one subfamily, and III
and IV belong to the other. A dendrogram constructed by the method of Higgins and Sharp (35) suggested that one subfamily (III and IV) is
separated from other sialyltransferase families, suggesting a great
difference in domain structure (Fig. 6). The dendrogram also showed
that the other ST6GalNAc subfamily (I and II) was closely related to
the group of ST3Gal families. As reported previously (13, 15-17,
36-43),4 mouse ST3Gal family
contains four cysteine residues, which are conserved in both chick and
mouse ST6GalNAc I and II. This structure is the so-called Kurosawa
motif,
Cys-Xaa75-82-Cys-Xaa1-2-Cys-Ala-Xaa-Val-Xaa150-160-Cys
(Xaa denotes any amino acid residue). However, mouse and rat ST6GalNAc
III, and mouse ST6GalNAc IV do not contain this motif, nor do any
members of the ST8Sia family (21, 25, 26, 37,
44-54)5 or ST6Gal I
(55-59).6 This indicates
that one ST6GalNAc subfamily (I and II) and the ST3Gal family, but not
another one (III and IV), share common domain structures.
2,3Gal
1,3GalNAc GalNAc
2,6-sialyltransferase have been
isolated from mouse brain cDNA libraries. One of the cDNA
clones is a homologue of previously reported rat ST6GalNAc III
according to the amino acid sequence identity (94.4%) and the
substrate specificity of the expressed recombinant enzyme, while the
other cDNA clone includes an open reading frame coding for 302 amino acids. The deduced amino acid sequence is not identical to those
of other cloned mouse sialyltransferases, although it shows the highest
sequence similarity with mouse ST6GalNAc III (43.0%). The expressed
soluble recombinant enzyme exhibited activity toward
NeuAc
2, 3Gal
1,3GalNAc, fetuin, and GM1b, while no significant activity was detected toward Gal
1,3GalNAc or asialofetuin, or the
other glycoprotein substrates tested. The sialidase sensitivity of the
14C-sialylated residue of fetuin, which was
sialylated by this enzyme with CMP-[14C]NeuAc, was the
same as that of ST6GalNAc III. These results indicate that the
expressed enzyme is a new type of GalNAc
2,6-sialyltransferase, which
requires sialic acid residues linked to Gal
1,3GalNAc residues for
its activity; therefore, we designated it mouse ST6GalNAc IV. Although
the substrate specificity of this enzyme is similar to that of
ST6GalNAc III, ST6GalNAc IV prefers O-glycans to
glycolipids. Glycolipids, however, are better substrates for ST6GalNAc III.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,6Gal, Sia
2,3Gal, and Sia
2,6GalNAc, are commonly found in
glycoproteins (7), and two, Sia
2,3Gal and Sia
2,8Sia, occur
frequently in gangliosides (8), each of these linkages has been found
in both gangliosides and glycoproteins (8-10).
2,6-sialyltransferase
family (ST6GalNAc I, II and III) has been reported (11-14). The
cDNAs of ST6GalNAc I and II were cloned from both chick (11, 12)
and mouse (13, 62).2 The
overall amino acid sequence identity of chick ST6GalNAc I is 30.5% to
chick ST6GalNAc II, 43.2% to mouse ST6GalNAc I, and 33.6% to mouse
ST6GalNAc II, and that of mouse ST6GalNAc I is 29.6% to mouse
ST6GalNAc II and 28.3% to chick ST6GalNAc II, and that of chick
ST6GalNAc II is 57.3% to mouse ST6GalNAc II. ST6GalNAc III has been
cloned from rat (14), and exhibits very low amino acid sequence
identity (8.2-9.8%) to mouse and chick ST6GalNAc I and II.
1,3GalNAc-O-Ser/Thr, and
NeuAc
2,3Gal
1,3GalNAc-O-Ser/Thr (11). On the other
hand, ST6GalNAc II exhibits a narrower substrate specificity, requiring
-galactosides linked to GalNAc residues, whereas sialic acid
residues linked to galactose residues are not essential for its
activity, i.e. this enzyme exhibits activity toward
Gal
1,3GalNAc-O-Ser/Thr and
NeuAc
2,3Gal
1,3GalNAc-O-Ser/Thr (12, 13). Both genes
are expressed in secretory organs, such as the submaxillary and mammary
glands, so these enzymes are considered to be involved in the
biosynthesis of O-glycans of mucin (11-13). On the other
hand, rat ST6GalNAc III exhibits the most restricted substrate
specificity, only utilizing the NeuAc
2,3Gal
1,3GalNAc sequence as
an acceptor (14). This enzyme can transfer sialic acid to both
NeuAc
2,3Gal
1,3GalNAc-O-Ser/Thr and ganglioside GM1b, suggesting that it cannot discriminate between
- and
-linked GalNAc (14). Incidentally, two types of
2,3-sialyltransferases (ST3Gal I and II) have been cloned that
exhibit activity toward Gal
1,3GalNAc but which have different
substrate preferences for glycoproteins and glycolipids,
i.e. ST3Gal I prefers glycoproteins to glycolipids, but II
prefers glycolipids (15-18). According to these observations, there
may be two scenarios; one is that ST6GalNAc III synthesizes almost all
the NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc residues, and the other
is that there may be another member of the ST6GalNAc family that has a
different substrate preference from that of ST6GalNAc III. To solve
this problem, we have extensively performed polymerase chain reaction
(PCR) cloning. Comparison of sialyltransferases cloned thus far has
revealed highly conserved regions, named sialylmotifs L, S, and VS (11,
19-21), not found in other glycosyltransferases. From the conservation
of these sialylmotifs, it was expected that other members of the
sialyltransferase gene family have the same motifs. The PCR-based
approach with degenerate primers deduced on the conserved sequence in
the sialylmotif has resulted in the isolation of several new members of
the sialyltransferase gene family (22).
2,3Gal
1,3(NeuAc
2,6)GalNAc residues. A new member, named ST6GalNAc IV, exhibits strong activity toward NeuAc
2,3Gal
1,3GalNAc of O-glycans. The other is
a homologue of rat ST6GalNAc III. Here, we report the cloning of the
cDNAs encoding the two NeuAc
2,3Gal
1,3GalNAc
GalNAc
2,6-sialyltransferases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-acid glycoprotein, CMP-NeuAc, Gal
1,3GalNAc
1-benzyl,
Gal
1,3GalNAc, Gal
1,3GlcNAc, Gal
1,4GlcNAc,
lacto-N-tetraose, benzyl-GalNAc,
N-acetyllactosamine and Triton CF-54 were from Sigma.
CMP-[14C]NeuAc (11GBq/mmol) was from Amersham
Pharmacia Biotech. NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc was from
Seikagaku Co. NANase I, Newcastle disease virus sialidase, and
sialidase from Vibrio cholerae were from Oxford Glycosystems and Roche Molecular Biochemicals, respectively. Synthetic primers were
obtained from Espec Oligo Service (Japan). The restriction endonucleases were from Takara (Japan) and Toyobo (Japan).
Gal
1,3GalNAc was sialylated using the secreted form of ST3Gal I
expressed in COS-7 cells (16). NeuAc
2,3Gal
1,3GalNAc was
purified by preparative TLC
(ethanol/1-butanol/pyridine/water/acetate = 100/10/10/30/3) and
subsequent DEAE-Sephadex A-25 anionic exchange
chromatography. NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc-ol was
prepared by the reduction of
NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc (23).
12 to 12), and
5'-TCACGGTCAGGAAGCACAGCATCA-3' (complementary to the rat ST6GalNAc III
coding strand; nucleotides 916-939). The amplified 940-bp cDNA was
subcloned into the EcoRV site of a pBluescript SK(+) vector
(Stratagene). A mouse brain cDNA library was constructed and
screened using the PCR-amplified mouse ST6GalNAc III cDNA as
described previously (11), and full-length mouse ST6GalNAc III cDNA
was isolated by rapid amplification of 5'-cDNA ends (RACE)-PCR.
9 to 18). The cDNA was
amplified through 35 cycles of a step program (94 °C, 30 s; 55 °C, 30 s; 72 °C, 40 s). The amplified products were
subcloned and sequenced.
vector, named pcDL-SR
B8ST for kinetic analysis. The vector was transiently transfected into COS-7 cells as described above. After 5 h of transfection, the culture medium was changed to Dulbecco's modified Eagle's medium containing 10% fetal calf serum. After 48 h, the COS-7 cells were collected and the membrane-bound proteins were extracted by sonication in 20 mM MES buffer (pH 6.4)
containing 0.3% Triton CF-54. After centrifugation of the cell lysate
at 10,000 × g for 15 min, the resultant supernatant
was used as the enzyme source. In this case, equivalent amounts of
protein from COS cells stably transfected with pcDL-SR
were assayed
in parallel as control experiment, and then the values obtained were
subtracted from those obtained with cells expressing the full-length enzyme.
2,3-linked sialic acids), NANase III
(specific for
2,3-,
2,6-. and
2,8-linked sialic acids), or
Newcastle disease virus sialidase (specific for
2,3- and
2,8-linked sialic acids)(27). After the sialidase treatment, the
desialylated glycoprotein was subjected to SDS-polyacrylamide gel
electrophoresis (gradient gel, 5-20%).
2, 3Gal
1,3(NeuAc
2,6)GalNAc-ol were
treated with various kinds of sialidases. A radioactive sample
containing at least 10,000 cpm or a sialylated sample containing at
least 5 µg of sialic acid was spotted onto a TLC plate (Merck,
Darmstadt, Germany) and then developed with
ethanol/1-butanol/pyridine/water/acetic acid = 100/10/10/30/3 (21)
or 1-propanol/aqueous ammonia/water = 6/1/2.5 (28). The
chromatogram was visualized with a BAS2000 radio image analyzer for
14C-sialylated sample (Fuji Film) or the resorcinol method
for nonradioactive sample (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide and deduced amino acid sequences
of mouse ST6GalNAc III and IV. The nucleotide and amino acid
sequences of mouse ST6GalNAc III (A) and IV (B)
are numbered from the presumed start codon and initiation
methionine, respectively. The double underlined
amino acids correspond to a putative transmembrane domain. Sialylmotifs
L and S are boxed by solid and dashed
lines, respectively. The positions of the PCR primers are
indicated by arrows.
Amino acid-identity (%) of members of the ST6GalNAc family
2,6-Sialyltransferase--
To facilitate functional analysis of the
enzyme, it was desirable to produce a soluble and condensable form of
enzyme that could be secreted from the cells. First of all, sequences
corresponding to the putative stem and active domains of ST6GalNAc III
and IV were respectively fused to the immunoglobulin signal peptide
sequence followed by the IgG binding domain of protein A (pCDB8ST and
pCDR1ST). After transfecting each of the constructs into COS-7 cells,
the enzyme secreted into the medium was condensed with IgG-Sepharose and used for further experiments. As shown in Table
II, among the glycolipids examined in
this study, only GM1b, i.e. not asialoGM1, served as an
acceptor substrate for ST6GalNAc III and IV. ST6GalNAc III showed
higher activity toward GM1b, of which the product comigrated with
authentic GD1
in two different solvent systems (data not shown) than
ST6GalNAc IV. ST6GalNAc III and IV also exhibited activity toward
fetuin, but very low activity toward asialofetuin (Table II). These
results suggested that the new sialyltransferase (ST6GalNAc IV)
requires the NeuAc
2,3Gal
1,3GalNAc residue in fetuin and GM1b just
like ST6GalNAc III (Fig. 2). On the other hand, GD1a, which has the NeuAc
2,3Gal
1,3GalNAc sequence and an
additional NeuAc residue at the internal galactose, did not serve as an
acceptor substrate (Fig. 2). It should be noted that the
oligosaccharide, NeuAc
2,3Gal
1,3GalNAc was a good acceptor substrate for ST6GalNAc IV (Table III),
while such an oligosaccharide was a poor acceptor substrate for
ST6GalNAc III. When the relative enzyme activities of ST6GalNAc III and
IV toward fetuin were, respectively, set to 100%, the activity to
oligosaccharide NeuAc
2,3Gal
1,3GalNAc was 290% in case of
ST6GalNAc IV, while only 0.5% of the activity was detected in
case of ST6GalNAc III. ST6GalNAc IV enzyme activities toward
nonsialylated Gal
1,3GalNAc and disialylated
NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc were almost negligible
(Table III).
Acceptor specificities of mouse ST6GalNAc III and ST6GalNAc IV
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Fig. 2.
Thin layer chromatography of sialylated
glycosphingolipids. A series of glycosphingolipids as acceptor
substrates (GM3 in lane 1, asialoGM1 in
lane 2, GM1b in lane 3,
GD1a in lane 4, and paragloboside in
lane 5), CMP-[14C]NeuAc as donor
substrate, and ST6GalNAc IV were used for sialyltransferase assays as
described under "Experimental Procedures." The glycosphingolipid
fractions were extracted from the reaction mixtures and separated on
TLC plates in chloroform/methanol/0.2% CaCl2 = 55/45/10,
and visualized using a BAS2000 radioimage analyzer (A) or
with orcinol-H2SO4 (B). Note that
the product of the transferase acting on GM1b is GD1 , though the
orcinol reagent does not show a spot at the GD1
position in the GM1b
lane in B.
Relative activities of mouse ST6GalNAc IV toward
oligosaccharides
to yield pcDL-SR
B8ST. The enzyme
fraction (3.6 pmol/h/µl of cell lysate) was then obtained as
described under "Experimental Procedures." The COS cell lysates intrinsically exhibit strong ST3Gal I and II, and significant ST6Gal I
activities; thus, this enzyme fraction is not suitable for analyzing
substrate specificity but for estimating Km and
relative Vmax/Km values for
acceptor substrates. The apparent Km value for GM1b
was 200 µM, which was lower than those for fetuin (8,000 µM) and NeuAc
2,3Gal
1,3GalNAc-benzyl (670 µM). The relative
Vmax/Km value for GM1b was
1.0, which was higher than those for fetuin (0.31) and
NeuAc
2,3Gal
1,3GalNAc-benzyl (0.019). These results suggested that
the expressed ST6GalNAc III prefers glycolipids to glycoproteins.
2,3-linked sialic acids), NANase III (specific for
2,3-,
2,6-, and
2,8-linked sialic acids), and Newcastle disease virus sialidase (specific for
2,3- and
2,8-linked sialic acids)(27) (Fig. 3). The resulting
patterns of the ST6GalNAc III and IV enzymes were virtually identical
to that of ST6GalNAc I. The [14C]NeuAc residue of fetuin
sialylated by both the enzymes was removed by the NANase III treatment
but not by the NANase I or Newcastle disease virus sialidase treatment
(Fig. 4). These results indicate that the
incorporated sialic acids each contain an
2,6-linkage, and the
expressed enzymes are the mouse homologue of rat ST6GalNAc III,
and a new member of the ST6GalNAc family, ST6GalNAc IV,
respectively.
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Fig. 3.
Linkage analysis of incorporated sialic
acids. [14C]Sialylfetuin sialylated with ST6GalNAc
IV (A) and III (B), ST3Gal I (C),
ST6GalNAc I (D), or ST8Sia II (E) was treated
with each sialidase. The resulting product was subjected to
SDS-polyacrylamide gel electrophoresis (gradient gel, 5-20%) and
radioactivity was detected with a BAS2000 radioimage analyzer (Fuji
Film). , without sialidase treatment; I, NANase I
treatment; III, NANase III treatment; NDV,
treatment with sialidase of Newcastle disease virus.
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Fig. 4.
Thin layer chromatography of the
oligosaccharide alditols derived from fetuin 14C-sialylated
by ST6GalNAc IV. The TLC plates were developed in
ethanol/1-butanol/pyridine/water/acetic acid = 100/10/10/30/3
(A) or 1-propanol/aqueous ammonia/water = 6/1/2.5
(B). The oligosaccharide alditols derived on 0.1 N NaOH, 1 M NaBH4 treatment of
14C-sialylated fetuin (lanes 1-4)
and NeuAc 2,3Gal
1,3(NeuAc
2,6)GalNAc-ol, as a reference
(lanes 5-8), were, respectively, treated in the
absence of sialidase (lanes 1 and 5)
or the presence of
2,3-linkage-specific NANase I (lanes
2 and 6),
2,3- and
2,8-specific Newcastle
disease virus sialidase (lanes 3 and
7), or V. cholerae sialidase (lanes
4 and 8). Each sample was then chromatographed on
a TLC plate and visualized with a BAS2000 radioimage analyzer (Fuji
Film) for 14C-sialylated fetuin products (lanes
1-4) or with resorcinol reagent for reference
oligosaccharide products (lanes 5-8).
Oligo 1,
NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc-ol; Oligo
2, Gal
1,3(NeuAc
2,6)GalNAc-ol.
-elimination of the sialylated fetuin with ST6GalNAc
IV. A desalted sample was then spotted onto a TLC plate and developed
with ethanol/1-butanol/pyridine/water/acetic acid = 100/10/10/30/3. All of the radioactive product migrated as a low
molecular compound, i.e. no radioactivity remained at the
origin, suggesting that 14C-sialylation occurred
exclusively on O-linked glycan chains of fetuin (data not
shown). Furthermore, more than 70% of the radioactivity comigrated
with the reference oligosaccharide,
NeuAc
2, 3Gal
1,3(NeuAc
2,6)-GalNAc-ol (data not shown). This
radioactive material was isolated by preparative TLC and used for
linkage analysis (Fig. 4). The reference oligosaccharide, NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc-ol, was also used for
comparison. The 14C-sialylated oligosaccharide alditols
were detected by BAS2000 radioimage analyzer (Fig. 4, lanes
1-4). In case of reference oligosaccharide, resorcinol
reagent was used for detection (Fig. 4, lanes
5-8). On NANase I or Newcastle disease virus digestion, NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc-ol (oligo 1, lane 5) was converted to
Gal
1,3(NeuAc
2,6)GalNAc-ol (oligo 2, lanes
6 and 7). With these sialidase treatments, the
radioactive band that co-migrated with oligo 1 (lane
1) migrated to the same position as oligo 2 (lanes 2 and 3). It should be noted
that the digestion with NANase I and Newcastle disease virus sialidase
was partial for some reason. When
NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc-ol (oligo 1) was treated with
V. cholerae sialidase (
2,3,
2,6, and
2,8
linkage-specific sialidase), it was converted to
Gal
1,3GalNAc-ol, which was not detectable with the resorcinol
method (lane 8). With this treatment, the
radioactive band comigrated with neither oligo 1 nor oligo 2, but with
NeuAc, indicating that the linkage type of the [14C]NeuAc
residue in the oligosaccharide alditol is
2,6 (lane
4). The reason that [14C]NeuAc band in lane 4 (Fig. 4A) was remarkably sharp could be because the prepared
14C-sialylated oligosaccharide alditol contained more
salts, which sometimes affect the migration pattern on TLC plate. Taken
together, the results strongly suggest that the
14C-sialylated oligosaccharide alditol derived from fetuin
is NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc-ol. Thus, the cloned enzyme
is a new member of the ST6GalNAc-family, named ST6GalNAc IV.
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Fig. 5.
Estimation of the amounts of the mouse
ST6GalNAc III and IV transcripts. A, competitive PCR
was performed using single-stranded cDNAs reverse-transcribed from
poly(A)+RNA derived from various tissues (ICR mouse) and
0.35 pg of the competitor DNA. In parallel, glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) cDNA was amplified to estimate the
amounts of the cDNAs used. Br, brain; SG,
salivary gland; Th, thymus; He, heart;
Lu, lung; Li, liver; Sp, spleen;
Ki, kidney; In, small intestine; Co,
colon; Te, testis; MG, mammary gland.
B, competitive PCR was performed using single-stranded
cDNAs reverse-transcribed from poly(A)+ RNA derived at
different stages of development (embryo (Emb) and brain
(Br)) and 0.35 pg of the competitor DNA. In parallel,
glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA
was amplified to estimate the amounts of the cDNAs used.
C, Northern blot analysis was performed using
poly(A)+ RNA (5 µg) derived from 8-week ICR mouse
tissues. Br, brain; SG, salivary gland;
Th, thymus; Lu, lung; He, heart;
Li, liver; Ki, kidney; Sp, spleen;
In, small intestine; Co, colon; Te,
testis; MG, mammary gland. D, Northern blot
analysis was performed using poly(A)+ RNA (5 µg) prepared
from 12-day postcoital ICR mouse embryos (E12), and 1-day
(P1), 3-week (3w), and 8-week (8w) ICR
mouse brains and livers. The hybridization probe was prepared from the
NH2-terminal truncated fragment (nucleotides 109-111) of
ST6GalNAc IV.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,6-sialyltransferase (ST6GalNAc III and IV). The mouse ST6GalNAc III shared very high sequence similarity (94.4%) at the
amino acid level with the rat one (14). The mouse ST6GalNAc IV cloned
in this study turned out to be encoded for a novel type of
sialyltransferase. The cDNAs were isolated by PCR cloning method based on the highly conserved regions from previously cloned
sialyltransferases (11, 19-21). The cDNA for ST6GalNAc IV was also
isolated independently by mRNA differential display method by
comparing the gene expressions between native and activated CD8+
cells.3
2,6-linkage to a GalNAc residue on
NeuAc
2,3Gal
1,3GalNAc of glycoproteins and glycolipids. First,
fetuin, which contains the O-glycosidically linked
NeuAc
2,3Gal
1,3GalNAc sequence (32), was shown to serve as a good
acceptor for both enzymes. However, both asialofetuin (contains the
Gal
1,3GalNAc sequence) and asialo-bovine submaxillary mucin (5% of
the total carbohydrate chains contain Gal
1,3GalNAc sequences) (33)
served as much poorer acceptors compared with fetuin. Second, the study
of the sensitivity of 14C-sialylated oligosaccharide
alditol to various sialidase including NANase I, III, V. cholerae sialidase, and Newcastle disease virus sialidase revealed
that the sialylated product was NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc and the linkage type of the [14C]NeuAc was
2,6. The
14C-sialylated oligosaccharide alditol was derived from the
fetuin sialylated by both enzymes with CMP-[14C]NeuAc.
Furthermore, GM1b could be served as an acceptor for both enzymes, and
the product was GD1
.
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Fig. 6.
Dendrogram of the cloned mouse
sialyltransferases.[enspace]After analyzing the deduced amino
acid sequences of cloned mouse sialyltransferases according to Higgins
and Sharp (35), a dendrogram was constructed. Note that one subfamily
of the ST6GalNAc family (III and IV) is separated from other
sialyltransferase families, and that the other ST6GalNAc subfamily (I
and II) is near the group of ST3Gal families. Parentheses
indicate the accession number of GenBankTM/EBI data
base.
The mouse ST6GalNAc III and IV almost have the same substrate
specificities, but they differ in substrate preference (Tables II-IV). ST6GalNAc III preferred
glycolipid as substrate over glycoprotein. On the other hand, ST6GalNAc
IV preferred glycoprotein as substrate over glycolipid. For example,
the activity of mouse ST6GalNAc III toward GM1b was stronger than that
toward fetuin, which is similar to rat ST6GalNAc III (14). However,
mouse ST6GalNAc IV exhibited stronger activity toward fetuin than GM1b.
It has been reported that GD1 is expressed highly in embryonic rat
brain, but the expression level decreases dramatically in adult brain (60). Additionally, GD1
has been assumed to be a molecular component
of a variety of important biological processes including metastasis of
highly virulent lymphomas and motor learning as elaborated by Purkinje
cells (60, 61). The spatial and temporal expression of the rat
ST6GalNAc III gene correlates well with the expression of GD1
. In
the case of mouse ST6GalNAc III, its gene expression in brain and other
tissues also seems to correlate with that of GD1
, although the
amount of the transcript was relatively low in adult tissues. Based on
these observations, ST6GalNAc III could be a strong candidate for
GD1
synthase among members of the ST6GalNAc family.
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It is known that trisaccharide, NeuAc2,3Gal
1,3GalNAc, cannot be
an acceptor substrate for mouse ST6GalNAc I, II. However, it did serve
as a substrate for ST6GalNAc III, although it was not a good one.
Similar results were reported for rat ST6GalNAc III (14). On the other
hand, mouse ST6GalNAc IV showed strong enzyme activity toward
NeuAc
2, 3Gal
1,3GalNAc. It is also interesting that the peptide
portion of an acceptor substrate is not necessary for the ST6GalNAc IV
activity. The expression levels of ST6GalNAc IV in various adult mouse
tissues were very different from those of III. The mRNA expression
of ST6GalNAc IV could be easily detected by Northern blotting using 5 µg of poly(A)+RNA in mouse brain, thymus, lung, heart,
spleen, and colon, but the RNA expression was too low to be detected in
the case of ST6GalNAc III. Because
NeuAc
2,3Gal
1,3(NeuAc
2,6)GalNAc structures are found in almost
all tissues, ST6GalNAc IV may be the main candidate for synthesizing
the NeuAc
2,3Gal
1,3(NeuAc
2, 6)GalNAc residue, which is
usually found in the O-linked glycan chains of glycoproteins.
At present, we still cannot answer the question whether or not there
are separate sialyltransferases that are responsible mainly for
glycolipid or glycoprotein synthesis. This is a important point for
understanding the nature of biosynthesis for glycolipids and
glycoproteins. Finding the second type of NeuAc2,3Gal
1,3GalNAc GalNAc
2,6-sialyltransferase may help to solve this problem. The
controversial point is how to distinguish the functions of ST6GalNAc
III and IV in vivo. Work related to this is currently in
progress. Mouse ST3Gal I and II, which both use Gal
1,3GalNAc-residue as substrate (16, 17), also have different preferences for glycolipids
and glycoproteins. The existence of these groups, or subfamilies, is
probably important for fine control of the expression of
sialylglycoconjugates, resulting in stage- and tissue-specific variety.
The study concernnig the four members of the ST6GalNAc family will help
us to understand the sialylglycoconjugates' biological functions
during the course of development.
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ACKNOWLEDGEMENTS |
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We thank Dr. James C. Paulson for the valuable discussion and for giving us the cDNA sequence of rat ST6GalNAc III prior to publication, and Dr. Yoshitaka Nagai, Director of the Glycobiology Research Group, and Dr. Tomoya Ogawa, Coordinator of the Group, Frontier Research Program of the Institute of Physical and Chemical Research (RIKEN), for their support in this work.
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FOOTNOTES |
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* This work was supported by Grants-in-aid 10152263 and 10178104 for Scientific Research on Priority Areas and Grant-in-aid C 09680639 for Scientific Research from the Ministry of Education of Japan.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y11342 (mouse ST6GalNAc III) and Y15779, Y15780, and AJ007310 (mouse ST6GalNAc IV).
§ Present address: Div. of Biotechnology, Faculty of Natural Resources and Life Science, Dong-A University, Saha-ku, Pusan 604-714, Korea.
Special postdoctoral researcher, Institute of Physical and
Chemical Research (RIKEN).
** Present address: Dept. of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
To whom correspondence should be addressed. Tel.:
81-48-467-9615; Fax: 81-48-462-4692; E-mail:
stsuji{at}postman.riken.go.jp.
2 The accession number of the cDNA sequence of mouse ST6GalNAc I is Y11274.
3 M. Kaufmann, C. Blaser, S. Takashima, S. Tsuji, and H. Pircher (1999) Int. Immunol., in press.
4 In addition to the references, the following data also support it: EMBL accession nos. X96667, AF059321, AF035250, and Y15003.
5 In addition to the references, the following data also support it: EMBL accession nos. U73176, L38677, U82762, AF004668, AF003092, AF008194, and U90215.
6 In addition to the references, the following data also support it: EMBL accession nos. A17362, 23699, and Y15111.
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ABBREVIATIONS |
---|
The abbreviations used are:
Sia, sialic acid;
NANase, N-acetylneuraninidase;
ST6GalNAc I, GalNAc
2,6-sialyltransferase (EC 2.4.99.3);
ST6GalNAc II, Gal
1,3GalNAc
GalNAc
2,6-sialyltransferase;
ST6GalNAc III, the first type of
Sia
2,3Gal
1,3GalNAc GalNAc
2,6-sialyltransferase (EC 2.4.99.7);
ST6GalNAc IV, the second type of Sia
2,3Gal
1,3GalNAc
GalNAc
2,6-sialyltransferase (EC 2.4.99.7);
NeuAc, N-acetylneuraminic acid;
CMP-NeuAc, cytidine
5'-monophospho-N-acetylneuraminic acid;
PCR, polymerase
chain reaction;
kb, kilobase(s);
bp, base pair(s);
E, embryonic day;
RACE, rapid amplification of cDNA ends;
MES, 4-morpholineethanesulfonic acid. The nomenclature for gangliosides
follows the system of Svennerholm (1). The abbreviated nomenclature for
cloned sialyltransferases follows the system of Tsuji et al.
(2).
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REFERENCES |
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