From the Glycobiology Research Group, Frontier
Research Program, Institute of Physical and Chemical Research (RIKEN),
Wako, Saitama 351-0198 and ¶ Department of Food and Nutrition,
Faculty of Home Economics, Tokyo Kasei University, Tokyo
173-0003, Japan
Received for publication, November 15, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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ST8Sia II (STX) and ST8Sia IV (PST) are
polysialic acid (polySia) synthases that catalyze polySia formation of
neural cell adhesion molecule (NCAM) in vivo and in
vitro. It still remains unclear how these structurally similar
enzymes act differently in vivo. In the present study, we
performed the enzymatic characterization of ST8Sia II and IV; both
ST8Sia II and IV have pH optima of 5.8-6.1 and have no requirement of
metal ions. Because the pH dependence of ST8Sia II and IV enzyme
activities and the pK profile of His residues are similar,
we hypothesized that a histidine residue would be involved in their
catalytic activity. There is a conserved His residue (cf.
His348 in ST8Sia II and His331 in ST8Sia IV,
respectively) within the sialyl motif VS in all sialyltransferase genes
cloned to date. Mutant ST8Sia II and IV enzymes in which this His
residue was changed to Lys showed no detectable enzyme activity, even
though they were folded correctly and could bind to CDP-hexanolamine,
suggesting the importance of the His residue for their catalytic
activity. Next, the degrees of polymerization of polySia in NCAM
catalyzed by ST8Sia II and IV were compared. ST8Sia IV catalyzed larger
polySia formation of NCAM than ST8Sia II. We also analyzed the
(auto)polysialylated enzymes themselves. Interestingly, when ST8Sia II
or IV itself was sialylated under conditions for polysialylation, the
disialylated compound was the major product, even though polysialylated
compounds were also observed. These results suggested that both ST8Sia
II and IV catalyze polySia synthesis toward preferred acceptor
substrates such as NCAM, whereas they mainly catalyze disialylation,
similarly to ST8Sia III, toward unfavorable substrates such as enzyme themselves.
Polysialic acid
(polySia)1 is a unique
carbohydrate polymer in which sialic acid (Sia) residues have internal
ketosidic linkages at Recently, two cDNAs encoding polysialyltransferases have been
cloned. These enzymes, ST8Sia II (STX) and ST8Sia IV (PST), showed 59%
sequence identity at the amino acid level (20-24). Of particular
interest, both ST8Sia II and IV could synthesize polySia on the
Both ST8Sia II and IV enzymes are type II membrane proteins and have
conserved sequence motifs, namely sialyl motif L, S, and VS, which are
present in all eukaryotic sialyltransferases characterized to date (20,
33, 34). Mutagenesis analysis within sialyl motif L of ST6Gal I showed
that this region is involved in the binding of donor substrate, CMP-Sia
(35). A recent study showing that mutation of sialyl motif S of the
same enzyme caused changes in Km values for both the
donor and acceptor substrates suggested that sialyl motif S is involved
in the binding of both substrates (36). However, neither of these
motifs seems to contain a catalytic amino acid that could be involved
in the catalytic inverting mechanism (34). Furthermore, there have been
no mutagenesis studies within sialyl motif VS.
A recent study using Fc-NCAM mutants lacking the fourth plus fifth or
sixth N-glycosylation sites clearly showed that ST8Sia IV
formed larger polySia chains than ST8Sia II (31). We still cannot
exclude the possibility that the lack some N-glycosylation sites Fc-NCAM caused conformational changes that could affect the
polysialylation rate by ST8Sia II or/and IV. Unfortunately, the polySia
chains of wild-type Fc-NCAMs polysialylated by ST8Sia II and IV have
not yet been characterized in detail. The ST8Sia IV enzyme itself was
shown to be polysialylated in vitro, and this modification
seems to be important for the enzyme activity (37). Subsequently, it
was shown that both ST8Sia II and IV underwent polysialylation in
vivo and that polysialylated ST8Sia II and IV were localized to
the Golgi and cell surface, respectively (38, 39). These polysialylated
enzymes on the cell surface could modulate cell adhesion as suggested
by Colley and colleagues (38). However, there have been no reports
concerning the actual lengths of the polySia chains of these enzymes.
In this study we compared the enzymatic properties of ST8Sia II and IV.
Then we examined the importance of the His residue in sialyl motif VS
of these enzymes for their catalytic activity. Next, the DPs of polySia
in NCAM catalyzed by ST8Sia II and IV were analyzed. Furthermore, we
re-examined the actions of both ST8Sia II and IV on themselves, and we
found that in this case NeuAc Materials
Tissue culture media and reagents were purchased from Life
Technologies, Inc. Nitrocellulose membranes and protein molecular weight standards were from Bio-Rad. CMP-[14C]NeuAc,
IgG-Sepharose, and protein A-Sepharose were purchased from Amersham
Pharmacia Biotech. Qiagen columns for DNA purification were obtained
from Qiagen Inc. (Chatsworth, CA). 35S-Express protein
labeling mix was purchased from PerkinElmer Life Sciences.
Peptide-N-glycosidase F was purchased from Roche Molecular
Biochemicals. A vector plasmid containing human Fc-NCAM cDNA was a
gift from Drs. P. Crocker and D. L. Simmons, Oxford University,
Oxford, UK (40). CDP-hexanolamine-Sepharose was a gift from Dr. K. J. Colley, the University of Illinois, Chicago. All other chemicals
were purchased from Sigma.
Methods
ST8Sia II-H348K and ST8Sia IV-H331K Proteins--
ST8Sia
II-H348K-bs+ and ST8Sia IV-H331K-bs+ were
generated using a Quick Change Site-directed Mutagenesis kit
(Stratagene). Primer 1 (TCCCAGGCCAGCCCCAAAACCATGCCCTTGGAA) and primer 2 (TTCCAAGGGCATGGTTTTGGGGCTGGCCTGGGA) were used to mutate the His residue
at position 348 of ST8Sia II to a Lys residue. Primer 3 (TCCAACGCCAGTCCTAAAAGGATGCCATTAGAA) and primer 4 (TTCTAATGGCATCCTTTTAGGACTGGCGTTGGA) were used to mutate the His residue
at position 331 of ST8Sia IV to a Lys residue. ST8Sia II-348K and
ST8Sia IV-H331K constructs were then cloned into the pCDSA expression
vector at the XhoI site.
Preparation of Recombinant Proteins--
COS-7 cells were grown
on 150-mm tissue culture dishes and transiently transfected using
Lipofectin with the vector plasmids designated as pCDSA-ST8Sia II,
pCDSA-ST8Sia II-H348K, pCDSA-ST8Sia IV, and pcDSA-ST8Sia IV-H331K,
containing cDNAs encoding IgG binding domain of protein A-fused
soluble mouse ST8SiaII, ST8Sia II-H348K, ST8SiaIV, and ST8Sia IV-H331K,
respectively (24, 30). Aliquots of 20 ml of the medium were collected
48 h after transfection, and the soluble enzymes were adsorbed to
20 µl of IgG-Sepharose resin (50% suspension in PBS), and then used
as enzyme preparation as described previously (30). Soluble enzymes
adsorbed to IgG-Sepharose resin were analyzed by SDS-polyacrylamide gel
electrophoresis and silver staining. Aliquots of 2 µl of each enzyme
preparation used for sialyltransferase assay were calculated to contain
3.0 pmol of the enzyme.
Fc-NCAM protein was prepared as described previously (40). Briefly,
COS-7 cells were transfected with a vector plasmid containing a
cDNA encoding human NCAM fused with the Fc region of human IgG1. After 72 h, the medium was collected and applied to a protein A-Sepharose column (1 × 1.2 cm). After the column was washed with 10 volumes of PBS, Fc-NCAM was eluted with 0.1 M sodium
citrate buffer (pH 3.0), and then neutralized immediately with 1 M Tris-HCl (pH 9.0). Fc-NCAM protein was precipitated with
3 volumes of ice-cold ethanol, suspended with water, and used for
sialyltransferase assay. Protein concentration was determined by BCA
assay (Pierce).
Enzyme Assay--
Enzyme assays were carried out in the presence
of 50 mM MES buffer (pH 6.0), 0.5% Triton CF-54, 100 µM CMP-[14C]NeuAc, 0.6 µg of Fc-NCAM, and
2 µl of enzyme preparation, in a total volume of 10 µl, as
described previously (30), unless described otherwise. For ST6GalNAc IV
enzyme assay, 10 µg of asialofetuin was used as an acceptor
substrate. After 2 h (for SDS-polyacrylamide gel analysis) or
16 h (for peptide N-glycanase digestion and for mild
acid hydrolysis) of incubation at 37 °C, the reaction mixture was
terminated by the addition of Laemmli sample buffer (for
SDS-polyacrylamide gel analysis) (41) or by quick freezing at
Product Analysis--
For product identification by
SDS-polyacrylamide gel analysis, the reaction mixture containing
Laemmli sample buffer was denatured at 65 °C for 20 min, loaded onto
the gel, and analyzed with a BAS2000 radioimage analyzer (Fuji).
For product analysis by Mono-Q HPLC following PNGase F digestion or
mild acid hydrolysis, enzyme reaction was carried out essentially as
described above, but on a 5- or 10-fold larger scale. The reaction
mixtures containing 14C-polysialylated Fc-NCAM were diluted
with 0.5 ml of PBS, and then 50 µl of protein A-Sepharose (50%
suspension in PBS) was added to adsorb 14C-polysialylated
Fc-NCAM. For PNGase digestion, the resin was washed with PBS five times
and was suspended with 100 µl of 10 mM Tris-HCl (pH 7.4),
50 mM EDTA, 0.2% SDS, 1% Nonidet P-40, and 20 mM
14C-Polysialylated ST8Sia II adsorbed to IgG-Sepharose was
subjected to controlled mild acid hydrolysis in 50 mM
sodium acetate buffer (pH 4.8) at 37 °C for 4 days. The method has
been previously established to detect sialic acid dimer and oligomers
(43, 50). After incubation, the sample was centrifuged, and 90% of the
supernatant was used for the Mono-Q HPLC analysis which was described
as above. The remaining sample was used for TLC analysis on 0.2-mm
thick silica gel plastic plates (Kieselgel 60; Merck) in 1-propanol, 25% aqueous ammonia, water, 6:1:2.5 by volume (43). Reference oligosaccharides were visualized using the resorcinol spray reagent after heating at 80 °C. 14C-Sialylated product was
visualized with BAS2000 radioimage analyzer (Fuji).
Determination of Optimum pH and Metal Ion Requirements--
For
determining optimum pH, the enzyme activities were measured in one of
the following buffers: 50 mM MES, pH 5.5, 5.8, 6.1, 6.4, or
6.7; or 50 mM PIPES, pH 6.7, 7.0, 7.3, or 7.6. To determine metal ion requirements, the enzyme activities were determined by
addition of 1 mM EDTA or 10 mM
MnCl2, MgCl2, or CaCl2.
Kinetic Analysis--
To determine the Km
values of ST8Sia II and IV for N-CAM substrate, various concentrations
of Fc-NCAM (0.1-15 µM) and a fixed concentration of
CMP-NeuAc (100 µM) were used for sialyltransferase
assays. Kinetic constants were obtained from Lineweaver-Burk plots. In
all cases, assays were performed in triplicate.
CDP-hexanolamine-Sepharose Affinity Chromatography--
COS-7
cells transiently expressing ST8Sia II, ST8Sia II-H348K, ST8Sia IV, or
ST8Sia IV-H331K cDNA were labeled with 35S-Express
protein labeling mix for 1 h in methionine- and cysteine-free DMEM
and then chased for 6 h in DMEM, 10% fetal bovine serum. Each
medium fraction was collected and applied to a 0.5 ml of CDP-hexanolamine-agarose column. The column was washed with buffers E
(10 mM sodium cacodylate, pH 6.5, 0.1% Triton CF-54, 0.15 M NaCl) and H (20 mM sodium cacodylate, pH 5.3, 0.1% Triton CF-54, 0.15 M NaCl) and then eluted with
buffer H containing 5 mM CDP (45, 46). IgG-Sepharose resin
(20 µl) was added to each fraction to pull down protein A-fused
enzymes and washed with buffer H. The bead fractions were analyzed by
SDS-polyacrylamide gel electrophoresis and with a radioimage analyzer.
Discrimination of Polysialylated Fc-NCAM from Polysialylated ST8Sia
II and IV Themselves by SDS-Polyacrylamide Gel
Electrophoresis--
When we used Fc-NCAM as an acceptor substrate and
protein A-tagged ST8Sia II or IV as the enzyme for enzymatic
characterization, it was necessary to discriminate between the two
kinds of reaction products, i.e. polysialylated Fc-NCAM and
ST8Sia II or IV themselves. The reaction products analyzed by
SDS-polyacrylamide gel electrophoresis and radioimage analysis are
shown in Fig. 1. When we used ST8Sia II
for enzyme assay in the absence of Fc-NCAM, we observed a broad band of
radioactivity extending from 60 to 90 kDa (Fig. 1A). In the
presence of Fc-NCAM, we observed an additional band of >220 kDa that
was thought to be polysialylated Fc-NCAM. When we used ST8Sia IV,
heterogeneous bands were also observed from 100 to 240 kDa (Fig.
1B). On addition of Fc-NCAM as a substrate, we observed a
polysialylated Fc-NCAM at >300 kDa. Migration patterns of the polydisperse bands in the absence of Fc-NCAM were similar with those of
polysialylated ST8Sia II (70-131 kDa) and ST8Sia IV (105-190 kDa)
reported previously in Chinese hamster ovary cells (38). Taken
together, these polydisperse bands were most likely polysialylated ST8Sia II and IV, respectively. In the present study, we discriminated between 14C-polysialylated Fc-NCAM and
14C-polysialylated ST8Sia II and IV by their migration
rates on SDS-polyacrylamide gels.
Effects of pH and Divalent Cations on ST8Sia II and IV Enzyme
Activities--
Optimal pH for ST8Sia II and IV activity was
determined over the pH range of 5.5-7.6 (Fig.
2 and Table
I). Maximum enzyme activities of ST8Sia
II and IV were observed at almost the same pH range, pH 5.8-6.1, with
Fc-NCAM as an exogenous acceptor. Next, the effects of 1 or 10 mM Mn2+, Ca2+, or Mg2+
ions on ST8Sia II and IV activities were tested (Table I). At 1 mM, none of these ions showed any significant effects on
enzyme activity. ST8Sia II activity was inhibited slightly by the
addition of 10 mM MnCl2, CaCl2, or
MgCl2, whereas these ions showed lesser effects on ST8Sia
IV activity. It was also shown that 1 mM EDTA had little
effect on ST8Sia II or IV activity (data not shown).
Kinetic Analysis of ST8Sia II and IV--
Although it is widely
known that both ST8Sia II and IV can polysialylate NCAM in
vitro and in vivo, no quantitative data concerning the
affinity of these enzymes with NCAM have been reported. Next, protein
A-fused soluble enzymes, ST8Sia II and IV, were used to determine the
Km values of ST8Sia II and IV for Fc-NCAM. Enzyme
assays were performed using various concentrations of Fc-NCAM (0.1-15
µM) as described under "Experimental Procedures." The Km value of ST8Sia II for Fc-NCAM was 3.2 µM, which was almost one-third of that of ST8Sia IV (8.9 µM) (Fig. 3 and Table I).
These values were significantly lower than those for any other
glycosyltransferases for their acceptor substrates reported previously.
The His Residue in the Sialyl Motif VS Is Critical for ST8Sia
II/ST8Sia IV Catalytic Activity--
Based on our observation that the
pH dependence of ST8Sia II and IV activities and the pK
profile of His are similar, we hypothesized that a His residue
conserved in these enzymes may be involved in their catalytic activity.
All sialyltransferase genes cloned to date have three consensus motifs,
i.e. sialyl motif L, S, and VS. Previous reports suggested
that sialyl motif L is involved in binding of CMP-NeuAc and that sialyl
motif S is related to the binding of both donor and acceptor
substrates. By using degenerate primers based on highly conserved
sialyl motif L and S sequences, many sialyltransferase genes have been
cloned (48). In contrast, sialyl motif VS has been poorly
characterized. Interestingly there is a conserved His residue in sialyl
motif VS of all sialyltransferase genes cloned to date (Fig.
4). To determine whether the His residue in this motif is critical for the catalytic activity, we constructed mutant ST8Sia II and IV in which the His residue of sialyl motif VS was
changed to a Lys residue. As shown in Fig.
5A, both ST8Sia II and IV
mutant proteins, ST8Sia II-H348K and ST8Sia IV-H331K, were secreted
into the medium from COS cell transfectants, suggesting that the
mutants were folded correctly. Each enzyme protein fused with protein A
was prepared using IgG-Sepharose from medium and used for enzyme assay
as described under "Experimental Procedures." Interestingly, we
found no detectable enzyme activity for ST8Sia II-H348K or ST8Sia
IV-H331K mutants (Fig. 5B). Not only polysialyltransferases but also other types of sialyltransferase such as ST6GalNAc IV lost
enzyme activities by the replacement of His residue in sialyl motif VS
with Lys residue, as shown in Fig. 5C. The result suggested that the mutation in His residue of sialyl motif VS is not specific for
polysialyltransferases but is specific for sialyltransferases in
general. As ST8Sia II-H348K and ST8Sia IV-H331K mutants were shown to
be inactive, we next examined whether these mutant enzymes still have
binding affinity with donor substrate, CMP-NeuAc. CDP-hexanolamine, which is chemically similar to CMP-NeuAc, has been used extensively to
purify sialyltransferase (46) and has also been used to determine the
affinity of ST6Gal I dimer for CMP-NeuAc (45). We used a CDP-hexanolamine-Sepharose column to determine whether these mutants differed in their affinity for this matrix. The medium containing ST8Sia II-H348K and ST8Sia IV-H331K protein from COS cells was applied
to the column, and unbound material was removed by extensive washing.
Specifically bound material was then eluted by 5 mM CDP. IgG-Sepharose resin was added to both wash-through and eluted fractions
to pull down enzyme proteins, and then each bead suspension was washed
and analyzed by SDS-polyacrylamide gel electrophoresis and with a
radioimage analyzer. Both ST8Sia II-H348K and ST8Sia IV-H331K mutants
were detected in the eluted fraction as well as ST8Sia II and IV,
suggesting that these mutant proteins have affinity for
CDP-hexanolamine (Fig. 6). Therefore, it
seems that the lack of catalytic activity of ST8Sia II-H348K and ST8Sia
IV-H331K mutants was not due to their inability to bind CMP-NeuAc. Due to technical problems, it was not possible to determine whether these
mutants could bind to the acceptor substrate, NCAM. However, the
mutations did not appear to affect binding to NCAM for the following
reasons. (i) A recent report suggested that the amino-terminal region
of the active domain of ST8Sia IV is involved in its NCAM recognition
(49). (ii) Mutation in sialyl motif VS of ST6GalNAc, whose substrate
specificity is different from that of polysialyltransferases, also
caused the loss of catalytic activity at all as shown in Fig.
5C.
Analysis of 14C-Polysialylated Reaction Products by
ST8Sia II and IV--
It has been reported that
[14C]polysialic acid residues on NCAM synthesized by
ST8Sia IV are longer than those synthesized by ST8Sia II as determined
from the migration patterns of 14C-polysialylated Fc-NCAM
on SDS-polyacrylamide gels (cf. Fig. 1). To obtain more
direct evidence, we performed PNGase F digestion of wild-type Fc-NCAM
products 14C-polysialylated by ST8Sia II and IV to obtain
free N-linked glycan chains. These samples were then
subjected to Mono-Q anion exchange HPLC analysis. As shown in Fig.
7, A and B,
[14C]polySia chains derived from Fc-NCAM product
synthesized by ST8Sia II were indeed shorter than those by ST8Sia IV as
reported previously (31). Nevertheless, both [14C]polySia
chains synthesized by ST8Sia II and IV were much longer than those
previously reported for mutated Fc-NCAM, and these [14C]polySia chains eluted by Mono-Q HPLC chromatography
had peaks at DP 40 for ST8Sia II and 100 for ST8Sia IV, respectively.
Fractions released from protein A-Sepharose by PNGase F digestion did
not contain any detectable amounts of glycopeptides due to possible contaminating protease activities in PNGase enzyme preparations (data
not shown). Unlike polysialylated NCAM, polysialylated ST8Sia II and IV
have not yet been characterized biochemically. Next, using the same
procedure, we analyzed the length of polySia of ST8Sia II and IV
themselves. Even though previous reports showed that the presence of
polysialylation of ST8Sia II and IV themselves (37-39), our
biochemical analysis indicated that 14C-disialylated
component was the major product for both 14C-sialylated
ST8Sia II and IV, even though 14C-polysialylated components
were also observed (Fig. 7, C and D). We
performed further analysis to detect [14C]disialic and
[14C]oligosialic acid directly from the
14C-sialylated ST8Sia II product.
14C-Sialylated ST8Sia II adsorbed to IgG-Sepharose was
subjected to controlled mild acid hydrolysis in 50 mM
sodium acetate buffer (pH 4.8) at 37 °C for 4 days. The method has
previously been established to detect sialic acid dimer and oligomers
(43, 50). After the treatment, more than 85% of
14C-radioactive components were released from Sepharose
beads, and these were identified as [14C]sialic acid
dimer as major component, and [14C]sialic acid oligomers
(DP > 2) by thin layer chromatography (Fig.
8A) and HPLC analysis (Fig.
8B). Small amounts of [14C]sialic acid monomer
were detected as minor components (4.7%). These results confirmed that
the frontal peaks in Fig. 7, C and D, which
represent sialyl oligosaccharide released by PNGase F, are indeed
disialylated and polysialylated polysaccharides. Our results suggested
that ST8Sia II and IV themselves are not preferential acceptor
substrates as compared with NCAM and that both enzymes behaved like
ST8Sia III to such substrates. A possible mechanism for disialylation
of the enzyme itself instead of polysialylation is discussed below.
Again, [14C]polySia chains in ST8Sia IV were found to be
somewhat longer than those in ST8Sia II.
The present study demonstrated similar enzymatic properties
between ST8Sia II and IV. Both enzymes showed similar pH dependence for
their activities and similar Km values for NCAM
substrate. Furthermore, we showed that both enzymes required no metal
ions for their activities as shown in Table I. Even though our results were different from those reported previously showing that ST8Sia II
was activated by Mn2+ and Mg2+, whereas ST8Sia
IV was activated by Ca2+ (26), another result in which 1 mM EDTA showed little effect on ST8Sia II or IV activities
confirmed the lack of a metal ion requirement for these enzymes.
PolySia chains synthesized by ST8Sia IV were longer than those by
ST8Sia II, as suggested previously by Fukuda and colleagues (31).
Nevertheless, information about wild-type NCAM polysialylation was
lacking, because they used mutant NCAM proteins in which one or more
than one N-linked glycosylation site were mutated to examine polysialylation by ST8Sia II and ST8Sia IV. In this study, we used
wild-type NCAM as a substrate for ST8Sia II and ST8Sia IV, and then we
analyzed the polysialylated compounds of reaction products using Mono-Q
HPLC chromatography. We showed that polysialylated compounds
synthesized by ST8Sia IV had a peak at DP 100, whereas polysialylated
compounds produced by ST8Sia II had a peak at DP 40. Both of these
peaks were eluted at higher DP than those in case of mutant NCAMs.
We also analyzed the polysialylation of ST8Sia II and ST8Sia IV
themselves when NCAM was not added as a substrate. Interestingly, disialylated compound was the major product for both polysialylated enzymes. This result suggested that neither ST8Sia II nor ST8Sia IV are
preferred substrates as compared with Fc-NCAM and that they catalyze
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,5, 8, or 9 (reviewed in Ref. 1). PolySia
occurs widely in nature, from the capsular polysaccharide of
neuroinvasive Escherichia coli K1 (1), insects (2),
unfertilized rainbow trout (3), sea urchin eggs (4), to the human
brains (5, 6). PolySia expression is also observed in human tumors (1,
7-10). NCAM, which is the most extensively studied
2,8-linked
polysialylated glycoprotein, functions in embryonic development and
mediates a variety of cell-cell adhesive interactions in the nervous
system (reviewed in Refs. 11-13). PolySia in NCAM is believed to
attenuate the adhesive properties of NCAM itself and/or other adhesion
molecules and facilitates cell migration such as neurite outgrowth and
activity-induced synaptic plasticity (5, 6, 14-19).
2,3-linked Sia residues on NCAM in vivo and in
vitro without an initiator
2,8-sialyltransferase (21-23,
25-29). Increased levels of ST8Sia II expression were observed in the
embryonic brain, whereas ST8Sia IV was continuously expressed in
various tissues throughout development (19, 28, 30). The respective
roles of these enzymes in polySia biosynthesis of NCAM remain unknown (31). Detailed information on the enzymatic properties of these respective enzymes is also lacking, although there was a recent report
concerning characterization of
2,8-polysialyltransferase activity in
the embryonic chick brain (32).
2,8-NeuAc
2,3-R was the major product
instead of
2,8-polysialylation.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C (for PNGase F digestion and for mild acid hydrolysis).
-mercaptoethanol, and then 10 µl (10 unit) of
PNGase F was added to it (42). After incubation at 37 °C for 16 h with gentle shaking, the sample was centrifuged, and the supernatant containing free N-linked glycan chains was collected for
further analysis by Mono-Q HPLC (31, 43, 44). The sample was diluted to
2 ml with water, and then applied to the Mono-Q HR 5/5 column (0.5 × 5 cm, Amersham Pharmacia Biotech) pre-equilibrated with solvent A
(10 mM Tris-HCl (pH 8.0)). By using solvent A and solvent B
(1 M NaCl, 10 mM Tris-HCl (pH 8.0)), two-step
linear gradients were generated with a Tosoh HPLC system. Elution was
performed for the first 5 min with 0% of solvent B, 0-30% solvent B
for the next 20 min, and 30-55% solvent B for the next 75 min. The elution rate was 1 ml/min, and fractions were collected every minute
with a connected fraction collector. As an internal reference, mild
acid hydrolysates of colominic acid were co-injected with the sample.
The elution patterns of oligo/polysialic acid chains and
14C-polysialylated compounds were monitored by UV
absorption at 210 nm and scintillation counting, respectively. Before
the next sample was injected, the column was washed with solvent B for 10 min and then re-equilibrated with solvent A.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Polysialylation of ST8Sia II and IV
themselves in vitro. Fc-NCAM was purified using a
protein A-Sepharose column from the medium of COS-7 cells transiently
expressing this molecule. ST8Sia II and IV, which were fused with IgG
binding domain of protein A, were purified using IgG-Sepharose resin
and were incubated in the presence or absence of Fc-NCAM. ST8Sia II
(A) and ST8Sia IV (B) were incubated with (+) or
without ( ) purified Fc-NCAM and with CMP-[14C]NeuAc as
described under "Experimental Procedures." SDS- polyacrylamide
gel electrophoresis was carried out using a 5% polyacrylamide gel
followed by radioimage analysis.
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Fig. 2.
ST8Sia II and ST8Sia IV enzyme activities at
different pH values. Enzyme activities of ST8Sia II (A)
and ST8Sia IV (B) are expressed as values relative to the
maximum activity obtained at pH 6.1 set to 100%. For details see
"Experimental Procedures."
Enzymatic properties of ST8Sia II and ST8Sia IV
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Fig. 3.
Lineweaver-Burk plot (1/v
versus 1/([substrate]) to determine the
Km and Vmax
values for NCAM of the ST8Sia II and ST8Sia IV activities.
Polysialyltransferase assays were carried out for ST8Sia II
(A) and ST8Sia IV (B) using different
concentrations of Fc-NCAM (0.1-15 µM).
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Fig. 4.
Conserved His and Glu residues in sialyl
motif VS regions in sialyltransferases. Sialyl motif VS regions of
18 cloned sialyltransferases are listed. The conserved amino acids are
indicated in bold characters. All of the sialyltransferase
genes are from the mouse except two genes (marked with a)
from the hamster.
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Fig. 5.
Enzyme activities of ST8Sia II-H348K, ST8Sia
IV-H331K, and ST6GalNAc IV-H332K. A, COS-7 cells
transiently expressing ST8Sia II, ST8Sia II-H348K, ST8Sia IV, or ST8Sia
IV-H331K cDNA were respectively labeled for 1 h with
35S-Express protein labeling mix in methionine- and
cysteine-free in DMEM and then chased for 6 h in DMEM, 10% fetal
bovine serum. As each enzyme protein was fused with IgG binding domain
of protein A, the enzyme was pulled down using IgG-Sepharose resin from
medium and analyzed by SDS-polyacrylamide gel electrophoresis and
radioimage analysis. B and C, sialyltransferase
assays were performed as described under "Experimental Procedures"
using wild-type polysialyltransferases, ST8Sia II, and ST8Sia IV and
their respective mutants, ST8Sia II-H348K and ST8Sia IV-H331K
(B), and using wild-type ST6GalNAc IV and ST6GalNAc IV-H332K
mutant (C).
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Fig. 6.
Binding properties of ST8Sia II and ST8Sia IV
with CMP-NeuAc. COS-7 cells transiently expressing ST8Sia II,
ST8Sia II-H348K, ST8Sia IV, or ST8Sia IV-H331K cDNAs were labeled
with 35S-Express protein labeling mix and then chased for
6 h. Each medium fraction was collected and applied to a
CDP-hexanolamine-Sepharose column. The column was washed with buffers E
and H (W fraction) and then eluted with buffer H containing
5 mM CDP (E fraction). As the enzymes were fused
with IgG binding domain of protein A, IgG-Sepharose resin (20 µl) was
added to each fraction to pull down enzymes and washed with buffer H. The beads fractions were analyzed by SDS-polyacrylamide gel
electrophoresis and radioimage analysis.
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Fig. 7.
Mono-Q HPLC analysis of polysialylated
glycans of Fc-NCAM and enzymes themselves by ST8Sia II and ST8Sia
IV. Fc-NCAM was 14C-polysialylated by ST8Sia II
(A) or ST8Sia IV (B) with
CMP-[14C]NeuAc as described under "Experimental
Procedures". C and D, the enzyme assays were
performed without addition of Fc-NCAM proteins to examine the
14C-polysialylation of ST8Sia II and ST8Sia IV themselves,
respectively. Each product was treated with PNGase F to liberate
N-linked glycan chains that were then analyzed by HPLC on a
Mono-Q column. Positions of NeuAc mono-, di-, tri- and tetramers were
indicated by arrows. E, the DPs of
[14C]polySia chains were determined by comparing their
elution positions to that of oligo(NeuAc) derived from mild acid
hydrolysates of colominic acid.
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Fig. 8.
Mono-Q HPLC analysis of the products of mild
acid hydrolysis of 14C-polysialylated ST8Sia II.
ST8Sia II that was fused with IgG binding domain of protein A was
14C-sialylated under polysialylation conditions. The
reaction product that was adsorbed to IgG-Sepharose was subjected to
controlled mild acid hydrolysis in 50 mM sodium acetate
buffer (pH 4.8) at 37 °C for 4 days. A, mild acid
hydrolysates of the sample (S) were subjected to TLC
analysis and visualized with BAS2000 radioimage analyzer. A series of
sialyl oligomers were also carried out and visualized with the
resorcinol method. Positions of NeuAc mono-, di-, and trimers were
indicated by arrows. B, the sample was also analyzed by HPLC
on a Mono-Q column. Eluted positions of NeuAc mono-, di-, tri-, and
tetramers were indicated by arrows.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,8-monosialylation of such substrates similarly to ST8Sia III. It
has been suggested that "initiase,"
2,8-sialyltransferase activity, is necessary for subsequent
2,8-polysialylation by "polymerase," which is believed to act on
2,8-linked sialic acid residues. This hypothesis was based on the finding that during trout
oogenesis, disialylated O-linked glycan chains were always detected together with polysialylated O-linked glycan chains
of polysialoglycoproteins (47). In contrast, accumulation of
disialylated structures has not been detected in polysialylated NCAM.
In this study, when we analyzed the polysialylation of ST8Sia II and
ST8Sia IV enzyme themselves, we found large amounts of disialylated
structures in addition to small amounts of polysialylated structures.
We did not detect significant amounts of oligosialylated structures except disialylated structures. Based on this finding, we proposed the
following model, which is illustrated in Fig.
9. ST8Sia II and ST8Sia IV can form
enzyme-substrate complex with
2,3-linked N-glycosylated
proteins with a relatively broad specificity. (a) If stable
enzyme-substrate complex is formed (e.g. NCAM is utilized as
a substrate), subsequent polysialylation occurs. (b) If
substrates are not preferred by these enzymes (e.g. enzyme
themselves are utilized as substrates), enzyme-substrate complex
somehow becomes unstable after a
2,8-monosialylation, and this
complex would be easily broken resulting in termination of
polysialylation, and therefore disialylated structures would be the
major product. At present, it is not clear if the peptide moiety and/or
specific carbohydrate structures such as
1,6-linked fucose residue
attached to di-N-chitobiose of N-linked glycan
chain on NCAM are preferentially recognized by these enzymes. The
recently identified disialylated structure in the N-linked
glycoprotein of porcine embryonic brain may be
2,8-monosialylated by
ST8Sia II or IV, both of which are highly expressed in the embryonic
brain (51, 52).
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Fig. 9.
Proposed model of polysialylation or
disialylation by ST8Sia II and ST8Sia IV. Polysialic acid
synthases such as ST8Sia II and IV form enzyme-substrate complexes with
2,3-sialylated substrates. A, if acceptor substrate is
preferred by these enzymes (e.g. NCAM), stable
enzyme-substrate complex produces polysialylation. B, if the
substrate is not preferred by the enzymes (e.g. ST8Sia II
and ST8Sia IV themselves), the complex is somehow broken after
2,8-monosialylation and therefore no more polysialylation occurs. In
this case the disialylated structure is the major product.
In this study, we also demonstrated an indispensable role of the His
residue in sialyl motif VS for the catalytic activity of ST8Sia II and
ST8Sia IV. Sialyl motif VS that is located at the carboxyl terminus of
sialyltransferases has two amino acid residues, Glu and His, that are
conserved among all mammalian sialyltransferases characterized to date.
It seems that catalytic amino acid residues are located in neither
sialyl motif L nor sialyl motif S. Although Geremia et al.
(34) suggested that the conserved Glu residue in sialyl motif VS might
be involved in catalysis, we postulated that the His residue could be a
catalytic amino acid residue based on the similarity between optimum pH of ST8Sia II and ST8Sia IV enzymes and the pK value of the
His residue. When we changed the His residue to a Lys residue, no catalytic activity was observed for both ST8Sia II and ST8Sia IV,
although they were folded correctly and secreted, and they showed
affinity for CMP-NeuAc. It seems that this His residue in sialyl motif
VS has a critical role in the catalytic activity of sialyltransferases,
because other sialyltransferases such as ST6GalNAc IV also lost its
enzyme activity following mutation of this His. It is necessary to
determine three-dimensional structure of sialyltransferases by
crystallization to confirm whether the His residue within sialyl motif
VS is indeed a catalytic residue.
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FOOTNOTES |
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* This work was supported by Grant-in-aid for Encouragement of Young Scientists 11780435 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.
§ Present address: Glyco-chain Function, Frontier Research Program, Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan. To whom correspondence should be addressed. Tel.: 81-48-467-9616; Fax: 81-48-467-9617; E-mail: shinobuk@postman.riken.go.jp.
Present address: Graduate School of Biological
Sciences, Nara Institute of Science and Technology, Nara, 630-0101, Japan.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M010371200
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ABBREVIATIONS |
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The abbreviations used are: polySia, polysialic acid; Sia, sialic acid; NCAM, neural cell adhesion molecule; Fc, the hinge and constant region of IgG; Ig, immunoglobulin; DP, degree of polymerization; HPLC, high performance liquid chromatography; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PNGase F, peptide N-glycosidase F; bs+, Bluescript; MES, 4-morpholineethanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.
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