From Cytel Corporation and the Department of Chemistry and
Molecular Biology, Scripps Research Institute,
San Diego, California 92121
Protein sequence analysis of the cloned
sialyltransferase gene family has revealed the presence of two
conserved protein motifs in the middle of the lumenal catalytic domain,
termed L-sialylmotif and S-sialylmotif. In our previous study (Datta,
A. K., and Paulson, J. C. (1995) J. Biol. Chem.
270, 1497-1500) the larger L-sialylmotif of ST6Gal I was
analyzed by site-directed mutagenesis, which provided evidence that it
participates in the binding of the CMP-NeuAc, a common donor substrate
for all the sialyltransferases. However, none of the mutants tested in
this motif had any significant effect on their binding affinities
toward the acceptor substrate asialo
1-acid
glycoprotein. In this study, we have investigated the role of the
S-sialylmotif of the same enzyme ST6Gal I. In total, nine mutants have
been constructed by changing the conserved amino acids of this motif to
mostly alanine by site-directed mutagenesis. Kinetic analysis for the
mutants which retained sialyltransferase activity showed that the
mutations in the S-sialylmotif caused a change of
Km values for both the donor and the acceptor substrates. Our results indicated that this motif participates in the
binding of both the substrates. A sequence homology search also
supported this finding, which showed that the downstream amino acid
sequence of the S-sialylmotif is conserved for each subgroup of this
enzyme family, indicating its association with the acceptor
substrate.
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INTRODUCTION |
The transfer of sialic acid from its nucleotide donor
CMP-Sia1 to the nonreducing
terminus of oligosaccharyl structures of various glycoproteins and
glycolipids is mediated by sialyltransferases, a group of about 15 enzymes belonging to the family of glycosyltransferases (1). Cloning of
the first three sialyltransferases, namely ST6Gal I (see Ref. 2 for
nomenclature), ST3Gal I (3), and ST3Gal III (4), and analysis of their
protein sequences deduced from the respective cDNAs revealed that
all of these gene products have the type II membrane protein topology
common among all the members of the glycosyltransferases examined to
date. This topology is characterized by the presence of a short
N-terminal cytoplasmic domain, an uncleavable hydrophobic signal-anchor
sequence that serves as the membrane-spanning domain, a proteolytically
sensitive "stem" region, and a large catalytic domain that projects
into the lumen of the Golgi apparatus. In contrast to other
glycosyltransferases, however, the sequences derived from the
sialyltransferase gene family revealed the presence of two conserved
protein motifs in the lumenal catalytic domain, termed as L- and
S-sialylmotifs, consisting of about 48 and 23 amino acids, respectively
(5-7). This unique feature was exploited to clone new genes of this
family by a PCR-based strategy using degenerated primers designed based on the invariant amino acids present at either end of these two motifs.
This strategy was highly successful in cloning nine additional sialyltransferases (5, 7-14). The cloning of these new genes by PCR
confirmed that the presence of sialylmotifs in any new gene is, in
fact, the "cardinal" feature of the sialyltransferase gene family.
By now, in total 13 genes have been cloned (2). Analysis of their
protein sequences revealed that each of these enzymes shares this
common structural feature: the presence of L-sialylmotif and
S-sialylmotif. Comparison among the members showed that the larger
L-sialylmotif consisting of 48-49 amino acids in the center of each
molecule exhibits >70% identity among homologous groups, 40-60%
among heterologous groups, and has eight invariant amino acids
including one invariant cysteine. The S-sialylmotif, on the other hand,
has two invariant amino acids in a stretch of 23 residues.
Interestingly, one of these is also cysteine. These two cysteines are
predicted to be involved in disulfide linkage formation (6).
Such a unique feature of the presence of these two sialylmotifs in all
the cloned sialyltransferases suggests that these motifs might
contribute to a structural feature related to the common function of
these enzymes. For example, each enzyme transfers sialic acid from the
common donor substrate, CMP-NeuAc, to an oligosaccharide acceptor
substrate. Thus, these motifs could form part of the binding sites for
either the donor or acceptor substrates, or both. Nevertheless, their
role in the formation of a specific conformation required for its
enzymatic activity may not be ruled out.
Previously, we examined the role of the L-sialylmotif using
site-specific mutants of the enzyme ST6Gal I (EC 2.4.99.1) as a model.
This enzyme, which forms the Neu5Ac
2,6Gal
1, 4GlcNAc sequence
common to many Asn-linked oligosaccharides, was cloned from rat liver
(15). Our results showed that the alanine mutation of some of the
invariant amino acids in the L-sialylmotif of this enzyme resulted in
an increased Km toward the donor substrate CMP-NeuAc
without significant effect on the acceptor binding affinity, suggesting
that this motif contributes to the binding of the common donor
substrate (16, 17). Here, we have examined the role of the
S-sialylmotif of the same enzyme ST6Gal I and show that the single
point mutation of its conserved amino acids affected the
Km values of the mutant enzymes for both the donor and acceptor substrates.
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EXPERIMENTAL PROCEDURES |
Construction of Mutants--
The single point mutants were
constructed following the megaprimer method described earlier (16, 18).
The cDNA for the ST6Gal I (GenBank accession no. M18769),
previously subcloned in pBluescript KS+ plasmid (19), was used as a
template. The nomenclature for the mutant sialyltransferases has been
assigned the number that reflects the amino acid residue number for
which alanine has been substituted; e.g. for P318A, proline
318 is replaced by alanine. The mutagenic antisense oligonucleotides
(the substituted nucleotides are underlined) used for construction of
the corresponding cDNAs were as follows (5'-3'): P318A,
GCCGGAGGATGCGGGATTTGGCTGA (nt 969-939); S319A,
CCAGCATGCCGGAGGCTGGGGGATT (nt 970-946); S320A, CCAGCATGCCGGCGGATGGGGGATT (nt 970-946); G321A,
GATACCCAGCATGGCGGAGGATGG (nt 975-952); C332A,
CCTGGTCAGCCAGCGTCATC (nt 984-1003); V335A, CGTAAATATCTGCCTGGTCACAC (nt 1015-993); V335L,
CGTAAATATCTAGCTGGTCACAC (nt 1015-993); E339A,
GGGAGGAACGCGTAAATATCTA (nt 1025-1004); F340A, GGATGGGAGGGCCTCGTAAATA (nt 1029-1008). As described
earlier (16, 18), in the first step of PCR, the 5'-end oligonucleotide
primer, GCTCTAGAATTCCAATCCTCAGTTACCACAG (5'-3', nt 214-236), and
the mutant antisense oligonucleotide (25-50 pmol each) were used to
introduce the desired mutation using Pfu DNA polymerase
(Stratagene); the conditions used were: 94 °C, 30 s; 56 °C,
1 min; 73 °C, 2 min for 20 cycles. The gel analysis showed the
generation of a single band for about 780 base pairs depending on the
position of the oligomer. This double-stranded DNA product from each
reaction was purified using Geneclean II (Bio101, San Diego, CA) and
used as a megaprimer in the second step of PCR. Our earlier results showed that the yield of the final desired product is improved by 5 cycles of linear amplification using only one primer, the megaprimer.
The reaction mixture for the linear amplification contained 10 ng of
template, 80 µM dNTPs in the 1 × Pfu DNA
polymerase buffer and ~50 ng of megaprimer (estimated from the
agarose gel) and started with the addition of 2.5 units of
Pfu DNA polymerase following these conditions: 94 °C, 1 min; 73 °C, 3 min for 5 cycles. The 3'-end antisense oligonucleotide
primer, CCAGGAGAGGATCCATAAAATGAC (5'-3', nt 1270-1247), was then
added, and the reaction was continued as follows: 94 °C, 1 min;
68 °C, 1 min; 73 °C, 3 min for 20 cycles. The products were
analyzed by agarose gel electrophoresis, which showed the generation of
a major single band of 1.05 kilobases for the specific product. This
band was purified by agarose gel electrophoresis followed by Geneclean
to separate from the megaprimer. The gel-purified product for the
mutants was digested with BstBI (at nt 824) and
BspE1 (at nt 1194). The 370-base pair fragment containing
the mutation was purified for each mutant and subcloned into a
similarly digested and purified larger fragment of spST-2 (20)
following a standard procedure (21). The ligation mixture was used to
transform competent cells of Escherichia coli. The colonies
were isolated, and the plasmids were obtained using Promega's plasmid
miniprep kit (Promega). The mutation was confirmed by dideoxy
double-stranded sequencing (22) of the entire fragment that has been
subcloned, including the restriction sites used.
Expression of the Wild-type and Mutant
Sialyltransferases--
For the analysis, the wild-type ST6Gal I and
its mutants were transiently expressed in COS-1 cells as described
earlier (16). Cells (1-2 × 106 cells/100-mm dish)
were transfected using 2.0 µg of plasmid DNA using LipofectAMINE
reagent according to the supplier (Life Technologies, Inc.). Expression
of transfected proteins was typically allowed to continue for 36-48 h
post-transfection before harvesting the cells. The culture medium was
then collected and concentrated 10-fold by ultrafiltration using
micro-concentrators (MWCO 10; Amicon Inc., Beverly, MA). The
concentrated medium containing the soluble-expressed sialyltransferase
was used directly for analysis of the enzymatic activity as described
earlier (16). These transfected cells were also used for radiolabeling
of the expressed proteins. Transfection was repeated at least three
times for each mutant with plasmid DNAs from different
preparations.
Pulse-chase Labeling of Transfected COS-1 Cells and Analysis of
the Transiently Expressed Proteins--
Metabolic labeling of cells
using Trans 35S-Express protein label (NEN Life Science
Products; 100 µCi/ml) was carried out essentially as described
elsewhere (23). The radiolabeled medium from the transfected COS-1
cells was used for immunoprecipitation as follows: the radiolabeled
medium (500 µl) was incubated with 10 µl of pre-immune rabbit serum
for 20 min at room temperature. 25 µl of protein A-Sepharose
(Amersham Pharmacia Biotech) was added and incubated with rotation at
room temperature for 30 min. These were centrifuged, and the
supernatants were collected. 10 µl of affinity-purified rabbit
anti-rat ST6Gal I was added and incubated at 4 °C for overnight. 20 µl of protein A-Sepharose was added and incubated at room temperature for 30 min. The precipitated immune complex obtained by centrifugation was washed three times with immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1% SDS) and once with 10 mM Tris-HCl, pH 7.5, 0.1% SDS. Proteins were eluted from
the pellet by boiling for 5 min in Laemmli gel sample buffer containing
10%
-mercaptoethanol. Immunoprecipitated proteins were
electrophoresed on 10% SDS-polyacrylamide gels according to the method
of Laemmli (24). The gel was then soaked for fixing once in 10% acetic
acid, 30% ethanol for 40 min and then in Enlightning rapid
autoradiography enhancer as instructed by the supplier (NEN Life
Science Products) before exposed to Kodak Biomax-MR film at
80 °C.
Other Methods--
The Western blot, protein determination,
sialyltransferase assay, and the enzyme assay for kinetic analysis were
done as described earlier (16).
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RESULTS |
Expression of the S-sialylmotif Mutants in COS-1 Cells and
Comparison of Their Enzyme Activities--
For expression of the
wild-type ST6Gal I and its mutants, the mammalian expression vector
construct used was based on pSVL (Pharmacia), which contained the
soluble form of the cDNA for rat ST6Gal I as mentioned earlier
(16). The mutants were derived from this expression vector of the
wild-type enzyme spST-2, in which the N-terminal cytoplasmic tail,
signal anchor domain, and the stem regions (first 71 amino acids) were
replaced with a cleavable signal peptide of dog pancreas insulin (20).
Thus, when the cDNAs for the wild-type ST6Gal I enzyme and its
mutants were transfected into COS-1 cells, the proteins were expressed
as a soluble form and were detected in the culture medium. For this
experiment, we designed the single point mutants, changing the
corresponding amino acids to alanine. A total of eight such mutants for
the S-sialylmotif were constructed (Fig.
1). In addition, we also constructed
mutant V335L by replacing Val335 with Leu.

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Fig. 1.
spST-2 construct for the soluble form of
ST6Gal I and its mutants in relation to the domain structure of
sialyltransferase. The spST-2 was constructed by replacing the
first 71 amino acids from the N-terminal of ST6Gal I with the cleavable
signal anchor sequence from the dog pancreatic proinsulin (20). The
S-sialylmotif in this enzyme spans from amino acids 318 to 340 and
contains two invariants, indicated by boldfaced letters. The
underlined amino acid residues were changed to alanine by
single point mutagenesis. , cleavable signal sequence; ,
sialylmotifs; , catalytic domain.
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For rapid evaluation of the effect of mutation of the conserved amino
acids, we used COS-1 because of its low endogenous sialyltransferase activity. Medium from COS-1 cells mock-transfected using only the
vector (pSVL) showed typically less than 2% activity compared with
that of expressed soluble sialyltransferases. Freshly grown COS-1 cells
were transfected with the cDNAs for both wild-type ST6Gal I and its
mutants. The expression of the desired sialyltransferase proteins were
checked by immunoprecipitation of the metabolically labeled proteins.
Immunoprecipitation of 35S-radiolabeled proteins using the
anti-ST6Gal I sialyltransferase antibody, followed by
SDS-polyacrylamide gel electrophoresis, indicated that all mutant
sialyltransferases were expressed and exhibited similar molecular
weight to the wild-type sialyltransferase (Fig.
2). Their similar migration in the
SDS-polyacrylamide gel electrophoresis indicated that there may not be
a gross rearrangement in their native structure because of the
introduced point mutation. To determine the enzymatic properties of the
mutant sialyltransferases, media from the transfected cells were
concentrated (Amicon Inc., Beverly, MA) and used as a source of crude
enzyme. By Western blot, we estimated the relative amount of proteins
that is used for sialyltransferase activity. By comparative analysis,
it was noted that, although the expression levels were apparently
similar to that of the wild-type enzyme for S320A, G321A, V335A, V335L, E339A, and F340A, the enzyme activities obtained were 41, 75, 83, 79, 35, and 15%, respectively. On the other hand, although the protein
expression for S319A and C332A seemed to be within 2-5-fold, the
sialyltransferase activity measured was less than 5%. For P318A, the
expression achieved was, however, very low, if any. Repeated
experiments of transfection for the expression of P318A failed to
achieve detectable expression levels for this mutant protein. This
proline is conserved in most of the sialyltransferases cloned. Proline
is known to confer unique conformational constraints on the peptide
chain of many biologically important proteins. This conformational
restriction seems to be important for the biological functions of these
proteins, as has been evidenced by a site-directed mutagenesis study
(for a review, see Ref. 25). It is possible that the alanine mutation
of this Pro318 induces some conformational change of ST6Gal
I that affected its expression.

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Fig. 2.
Immunoprecipitation of wild-type ST6Gal I
(spST-2) and its mutants from 35S-labeled medium of
transfected COS-1 cells. Metabolic labeling of transfected COS-1
cells using Trans 35S-Express protein label (DuPont NEN;
100 µCi/ml) and immunoprecipitation of expressed proteins with rabbit
anti-rat ST6Gal I was performed using medium from transfected cells of
a 48-60-h post-transfection, essentially as described earlier (16).
The fluorogram was shown only for F340A as a representative of mutants.
As shown, the mutant sialyltransferases moved similarly with the
wild-type enzyme (spST-2) in the SDS-polyacrylamide gel, indicating no
gross rearrangement because of the mutation.
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Kinetic Analysis of the Mutant Enzymes--
The mutants, which
retained ST6Gal I sialyltransferase activity, were used for the
comparative kinetic analysis. Analysis of the kinetics of the wild-type
and each mutant sialyltransferase expressed in COS-1 cells was
performed as described under "Experimental Procedures," except for
F340A, which had low activity and was not evaluated. The
Km values for each sialyltransferase were determined
for the donor substrate, CMP-NeuAc, and the best glycoprotein acceptor
substrate of the wild-type enzyme, asialo
1-acid
glycoprotein (ASGP). This glycoprotein acceptor contains five
N-linked oligosaccharides that are predominately bi- and tetra-antennary structures, each branch of which is terminated with the
acceptor sequence Gal
1,4GlcNAc (26). Data obtained for each of the
enzymes produced Lineweaver-Burke plots with correlation coefficients
of 0.95 or greater (e.g. Fig.
3).

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Fig. 3.
Double reciprocal plots of initial rate data
with CMP-NeuAc (top) or asialo 1-acid
glycoprotein (bottom) as the varied substrate.
Top, rate data with CMP-NeuAc as the donor (26.5-201
µM) were determined at fixed concentration of the
acceptor asialo 1-acid glycoprotein, 50 µg (equivalent
to 20.75 µmol of Gal acceptor unit). Bottom, the
concentration of the acceptor asialo 1-acid glycoprotein
was varied (0.04-0.4 mM) at a fixed concentration of the
donor, 0.15 mM. The plot was shown for E339A ( ) as a
representative of the mutants and compared with that of the wild type
(spST-2) of sialyltransferase ( ). The Km
(apparent) values were determined from the X-intercept
( 1/Km (apparent)) (61) using the Cricket Graph
program (Cricket Software, Malvern, PA).
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Table I summarizes the
Km values obtained for both the substrates. This
shows that the Km values for the donor substrate
CMP-NeuAc were significantly altered, particularly for S320A and E339A
with increases in Km (apparent) of about 6-fold. The
Km values for asialo
1-acid
glycoprotein were also altered by about 2-fold for these two mutants.
Interestingly, Val335 to Ala showed increased binding
affinity toward the acceptor substrate (about 5-fold decreased
Km), which remained unchanged when mutated to Leu.
Thus, the interaction of the enzyme with the acceptor substrate is more
favored when Ala was introduced. The mutation of Gly321 to
Ala, on the other hand, increased the Km values by about 3-fold for both the donor and the acceptor substrates. It is
possible that this invariant amino acid is essential for the hydrogen
bond formation through the interaction with the OH group of both the
donor and the acceptor substrates. Therefore, even a conserved mutation
of Gly to Ala affected the binding affinities for both the
substrates.
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Table I
Kinetic constants for the wild-type ST6Gal I and its S-sialylmotif
mutants
Kinetic analysis was performed using concentrated medium of COS-1 cells
transfected with cDNAs of wild-type secretory sialyltransferase
(spST-2) and mutants for S-sialylmotif. Methods have been described
under "Experimental Procedures" and also in the text.
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Increase in Ki of CDP for S320A--
Cytidine analog CDP
is known to be a potent inhibitor of sialyltransferases, exhibiting
equilibrium dissociation constants similar to that of CMP-NeuAc (27).
Earlier, it was shown that the mutants of the L-sialylmotif, which had
increased Km values toward the CMP-NeuAc, also had
increased Ki for CDP compared with that of the
wild-type enzyme, as expected (16). To determine if this is also true
for the mutants of S-sialylmotif, CDP was tested as a competitive
inhibitor of CMP-NeuAc for mutant S320A as a representative (Fig.
4). The Ki for S320A was determined to be 50 µM, representing about a 10-fold
increase over the wild-type enzyme (4.7 µM). This
increase in Ki for CDP is comparable in magnitude
with the increase in Km (which is about 6-fold) for
CMP-NeuAc, suggesting that Ser320 participates in the
binding of the common cytidine moiety in these two ligands.

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Fig. 4.
Inhibition kinetics of sialyltransferase by
CDP. Initial rate data for the wild-type enzyme
(bottom) were determined in the presence of varied
concentrations of the donor substrate CMP-NeuAc (0.025-0.2
mM) and in the absence ( ) and presence ( ) of a fixed
concentration of 12.5 µM CDP. Similarly, kinetics for the
mutant S320A (top) were determined in the presence of the
varied concentrations of CMP-NeuAc and in the absence ( ) or presence
( ) of a fixed concentration of 16.7 µM CDP. The
concentration of the acceptor asialo 1-acid glycoprotein
was kept constant at 50 µg throughout for both enzymes. The
Ki values were extracted from the x
intercept in the presence of inhibitor (61), where x
intercept = (1/Km)(1+[I]/Ki),
Km is obtained in the absence of the inhibitor, CDP,
and [I] is the concentration of CDP.
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Identification of a Putative Linkage-specific Sequence--
A
protein sequence homology search among the cloned sialyltransferases
showed that the downstream amino acid sequence of the S-sialylmotif is
conserved for each subgroup of this enzyme family. For example, the
sequence LYGFWPF is present in the members of the ST8Sia subfamily
only. Similarly, conserved sequence could be identified for the members
of the other subfamilies. This finding suggests that such a conserved
sequence may confer the linkage specificity for the sialic acid
transferred to the glycosyl moiety of the acceptor substrate.
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DISCUSSION |
The family of glycosyltransferase enzymes transfers respective
sugars from its activated nucleotide-sugar donor to various glycolipids
and glycoproteins. By now, a considerable number of clones have been
obtained for different members of this glycosyltransferase family (see
Ref. 29 for a review) that include sialyltransferases, fucosyltransferases, and galactosyltransferases. Analysis of the deduced protein sequence indicated that all of these members have common structural features: the presence of a short N-terminal cytoplasmic protein domain, which is not essential for the catalytic activity, followed by about a 20-amino acid transmembrane domain that
determines the retention signal sequence for these Golgi lumenal
enzymes. Apart from the stem region, the rest of the protein that
resides in the lumen confers the catalytic activity. There is evidence
to support such a model. For example, ST6Gal I sialyltransferase, the
subject of this study, transfers sialic acid from CMP-NeuAc to the
Gal
1,4GlcNAc sequence common to many Asn-linked oligosaccharides (30, 31). The analysis of its protein sequence deduced from the
cDNA cloned from rat liver (15) suggests that the primary structure
of this enzyme consists of a short N-terminal cytoplasmic domain (amino
acids 1-9), a membrane anchor and signal sequence (amino acids
10-27), a stem region (amino acids 28-62), and a large lumenal
catalytic domain (amino acids 63-403). This enzyme is found to be
active even after truncation of 71 amino acids from its N-terminal that
defines the cytosolic, transmembrane, and stem regions. Similarly,
Macher and his colleagues (32) have obtained evidence that 20% of the
N-terminal amino acid sequence of two human
1,3/4
fucosyltransferases (FucT III and V) is not required for enzyme
activity, whereas truncation of the C terminus of these enzymes results
in their inactivation. Although these studies showed that the lumenal
C-terminal part of these enzymes confers the catalytic activity,
nothing has been known about the substrate binding sites until
recently. Studies have been initiated by different groups to define the
substrate binding sites of various members of this glycosyltransferase
family using different techniques, including chemical and site-directed
mutagenesis (32-37).
We have initiated the structure-function analysis of sialyltransferases
to find out its possible substrate binding sites. This enzyme family
has a unique structural feature that is not present in other members of
the glycosyltransferases. Each of these enzymes shares the presence of
two conserved protein domains termed sialylmotifs (see Ref. 17).
Because of this conserved nature, we have studied these sialylmotifs
for their possible role in the catalytic activity of these enzymes
using ST6Gal I as a model. Mutants have been constructed for this
purpose by site-directed mutagenesis of the conserved amino acids
present in its two sialylmotifs. Earlier, our kinetic analysis showed (see Table II) that a majority of the
alanine mutants for the L-sialylmotif had a significant effect on the
binding affinity toward the donor substrate, indicating that the
L-sialylmotif predominantly participates in binding of the common donor
substrate CMP-NeuAc (16).
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Table II
Summary of analysis for the sialylmotif mutants of ST6Gal I
Mutants were constructed by site-directed mutagenesis for the conserved
amino acids present in L- and S-sialylmotifs. For S-sialylmotif,
methods for analysis have been described under "Experimental
Procedures" and in the text. For L-sialylmotif, these were described
earlier (16).
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In the present study, similar analyses have been performed with the
single point mutants for the S-sialylmotif. Out of nine mutants, five
mutant enzymes showed enzyme activity sufficient for kinetic analysis.
The analysis showed that the alanine mutation affected the
Km of these mutants for both the donor and acceptor
substrates (Tables I and II), suggesting that the S-sialylmotif participates in binding with both substrates. This result is supported by the following observation: so far, 13 clones have been obtained for
sialyltransferases with distinct substrate specificity. Considering their strict acceptor substrate specificity, these cloned
sialyltransferases are subdivided into various groups or subfamilies
(2). Protein sequence analysis among the members of this subfamily
revealed that each of these share a common sequence near the 3'-end of the S-sialylmotif. For example, members of the ST8Sia subfamily share a
conserved amino acid sequence LYGFWPF at the 3'-end of the
S-sialylmotif (Fig. 5). The only
variation observed in this sequence is by similar amino acid residues:
I for L as in ST8Sia I and F for Y as in ST8Sia V. Nevertheless, all
the members share this conserved sequence except ST8Sia V, where Pro is
substituted by Ala. Each of these members utilizes CMP-NeuAc as a
common donor substrate and various glycolipid and glycoprotein
acceptors with restricted substrate specificity. Although ST8Sia I
transfers sialic acid to the terminal sialic acid of GM3 to
form GD3 (38-40) and GT3 (41), the other
members, ST8Sia II-IV, transfer it to the terminal sialic acid of the
N-linked glycan to form dimeric or polymeric sialyl
structures (5, 11, 12, 42-46). The recently cloned ST8Sia V, on the
other hand, utilizes GD1a and GT1b as acceptors
to form GT1a and GQ1b, respectively (14).
However, one common feature among all these members is that each of
these transfers sialic acid to the C-8 position of the sialic acid of the various oligosaccharide acceptors. This sequence, LYGFWPF, is
present only in the ST8Sia subfamily and not in others, indicating that
this motif may be serving a common purpose unique only for these
members. In the absence of any experimental data, it is difficult at
this stage to understand the functional role, if any, of this conserved
sequence. Nevertheless, considering the conserved nature of this
sequence present only among the members of the ST8Sia subfamily, it may
be proposed that this sequence may well determine the linkage
specificity, i.e. the position C-8 on the sialic acid of the
oligosaccharide acceptor, where another sialic acid is transferred from
CMP-sialic acid by these enzymes. Thus, it is not surprising that, by
using the reverse degenerated oligonucleotide primer designed based on
the sequence at the 3'-end of the S-sialylmotif of ST8Sia subfamily,
the new clones obtained were for the enzymes with
2,8-sialyltransferase activity (11, 14). Interestingly, the genomic
analysis of two recently cloned members of this enzyme subfamily,
ST8Sia II and ST8Sia III, showed that this conserved region originates
from a common exon (47, 48).

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Fig. 5.
Amino acid alignment for the S-sialylmotif of
the cloned sialyltransferases. Members of the sialyltransferase
enzyme family are grouped according to the linkage formed by these
enzymes (see Ref. 2). The sequences are obtained from rat ST6Gal I
(15), porcine ST3Gal I (3), rat ST3Gal II (9), rat ST3Gal III (4),
human ST3Gal IV (10), chicken ST6GalNAc I (8), chicken ST6GalNAc II
(7), rat ST6GalNAc III (13), mouse ST8Sia I (38), rat ST8Sia II (5),
mouse ST8Sia III (11), hamster ST8Sia IV (41), and mouse ST8Sia V (14).
The consensus sequence consisting of 10 conserved amino acids are
shown. The amino acid residues shown are present in more than 75% of
the cloned sialyltransferases. This consensus sequence also includes
two invariant amino acids, glycine and cysteine. The restricted
variations observed for the other conserved amino acids are also shown.
The arrow indicates the C-terminal end of the previously
described S-sialylmotif (17, 28).
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This characteristic feature of amino acid sequence homology near
the C-terminal end of S-sialylmotif is also observed among the members
of other subfamilies of sialyltransferases. For example, the sequence
YGFGADS is present among the members of the ST3Gal sialyltransferases
that transfer sialic acid to the C-3 position of the terminal
galactosamine residue of O-linked glycoproteins (3, 9,
49-52). The sequence, AGFGYD, on the other hand, is present in ST3Gal
III, which transfers sialic acid to the C-3 position of the terminal
galactose residue of the N-linked glycoproteins (4, 53). It
will be interesting to find out whether the acceptor substrate
specificity changes from O-linked to N-linked
glycoproteins by changing the Ala residue to Tyr (and vice
versa) by site-directed mutagenesis. Nevertheless, it should be
noted that the enzyme, ST3Gal IV (10, 54), which transfers sialic acid
to the C-3 position of the terminal galactose unit of both
O-linked and N-linked glycoproteins, has an
AGFGYPD sequence instead. Similarly, the sequence YGXI
(X is any amino acid) is found conserved among the members
of ST6GalNAc subfamily (7, 8, 13). These enzymes transfer sialic acid
from its nucleotide sugar to C-6 of GalNAc of O-linked
glycoproteins. Although ST6GalNAc I and -II transfer sialic acid to
asialo Gal
1,3GalNAc-Ser/Thr (7, 8), the ST6GalNAc III transfers it
to the C-6 of GalNAc of Neu5 Ac
2,3Gal
1,3GalNAc-Ser/Thr (13).
For the ST6Gal subfamily, only one enzyme has been obtained so far,
though from different species (15, 55-59). It remains to be seen
whether the sequence YEFLPSK, present in ST6Gal I, could also be found
conserved among other ST6Gal enzymes (see Ref. 60 for review) once
cloned. Thus, it seems that the sequence homology near the C-terminal
part of the S-sialylmotif determines the linkage specificity by which
the sialic acid moiety is attached to the terminal galactose,
galactosamine, or sialic acid of the oligosaccharide acceptor unit of
the glycoprotein or glycolipid.
As mentioned above, protein sequence analysis also showed that each of
these two sialylmotifs of ST6Gal I contains one cysteine residue. These
two residues are invariantly present in all the cloned
sialyltransferases (17). In our previous study (16) and in the present
experiment, we observed that the alanine mutation of these two
conserved cysteine residues, Cys181 and Cys332,
caused the mutant ST6Gal I enzymes inactive. Our preliminary result
(data not shown) also indicated that the wild-type ST6Gal I is
sensitive to dithiothreitol and other reducing agents. These findings
suggest that these two cysteine residues are important for the enzyme
activity and are involved in disulfide linkage formation. From the fact
that both the sialylmotifs participate in the binding of the donor
substrate CMP-NeuAc, it seems that these two cysteine residues bring
the two sialylmotifs closer together by forming an intra-disulfide
linkage and, thus, form the conformation required for binding of the
donor substrate. Our present result, along with the finding of a
probable linkage-specific sequence near the C-terminal end of the
S-sialylmotif, also suggests that this smaller motif is most likely
associated with the binding site of the acceptor substrate.
We thank Dr. Eric Szoberg for critical
reading of the manuscript and Dorothy Wharry for secretarial
help.