(Received for publication, November 3, 1994 )
From the
All members of the sialyltransferase gene family cloned to
date contain a conserved region, the ``sialylmotif,''
consisting of 48-49 amino acids in the center of the coding
sequence. To investigate the function of this motif, mutant constructs
of the Gal1,4GlcNAc
2,6-sialyltransferase were designed by
site-directed mutagenesis, replacing 11 individual conserved amino
acids with alanine. Each of the mutants was expressed in COS-1 cells,
and eight of these retained sialyltransferase activity, allowing
comparison of their enzymatic properties with that of the wild type
enzyme. Kinetic analysis showed that six of eight mutants had a
3-12-fold higher K
for the donor
substrate CMP-NeuAc relative to the wild type enzyme, while the K
values for the acceptor substrate were
within 0.5-1.2-fold of the wild type for all eight mutants
evaluated. The K
of the donor substrate
analog CDP was also evaluated for the recombinant sialyltransferase
with the Val to Ala mutation at residue 220, which produced a 6-fold
increase in K
of CMP-NeuAc. A
corresponding increase in K
of 3.4-fold
was observed for CDP, indicating a decreased affinity for the cytidine
nucleotide. Taken together, these results suggest that the conserved
sialylmotif in the sialyltransferase gene family participates in the
binding of the common donor substrate, CMP-NeuAc.
The family of sialyltransferases that transfers sialic acid from
CMP-sialic acid to carbohydrate groups of glycolipids and glycoproteins
has been predicted to consist of a group of 10-12
enzymes(1, 2) . So far cDNA clones have been obtained
for eight distinct members of this
family(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) .
Analysis of their protein sequences has revealed that all of the
sialyltransferases have structural features common to all
glycosyltransferases, which are relevant to their function as enzymes
involved in post-translational glycosylation of glycoproteins through
their transit through the Golgi apparatus. They each have a short
NH cytoplasmic domain, a hydrophobic signal-anchor sequence
that serves as the membrane-spanning domain, a luminal
``stem'' region not required for catalytic activity, and a
large luminal catalytic domain. However, apart from their common
topological features, there is little sequence homology among the
sialyltransferases except for 10-12% of the sequence in the
middle of the catalytic domain and a smaller sequence of 23 residues at
the carboxyl end of the sequence(17, 20) . The largest
conserved region, termed the ``sialylmotif''(12) ,
consists of 48 or 49 amino acids in the center of each molecule, which
exhibits 40-60% identity between any two enzymes and contains 8
invariant residues among all the cDNAs cloned to date.
The sialylmotif is presumed to contribute to a structural feature related to the common function of the sialyltransferases. Each enzyme transfers sialic acid from the common donor substrate CMP-NeuAc to an oligosaccharide acceptor substrate. Thus, the sialylmotif could form part of the binding sites for either the donor or acceptor substrates, or both. Alternatively, it could be a structural nucleus that maintains the conformation and spatial relationship of variable substrate binding pockets in the sialyltransferase family.
To explore the function of
the sialylmotif, we prepared single point mutants of the
Gal1,4GlcNAc
2,6-sialyltransferase (EC 2.4.99.1) as a model
in this study. This enzyme forms the NeuAc
2,6Gal
1,4GlcNAc-
sequence common to many Asn-linked
oligosaccharides(21, 22) . The cDNA encoding this
enzyme was cloned from rat liver(3) , and its kinetic
properties have been established (21, 22, 23) . In this study, we constructed
11 recombinant enzymes by site-directed mutagenesis, changing selected
conserved amino acids in the sialylmotif to alanine, and evaluated the
expressed enzymes for changes in their enzymatic properties. Most of
the mutants retained sialyltransferase activity and had increases in
the K
of CMP-NeuAc without corresponding
changes in the K
of the acceptor
substrate. Thus, the results suggest that the sialylmotif of the
Gal
1,4GlcNAc
2,6-sialyltransferase participates in binding
the donor substrate, CMP-NeuAc.
Figure 1:
spST-2 construct and its mutants in
relation to the domain structure of sialyltransferase. The cleavable
signal anchor domain from dog pancreatic insulin is fused with the
catalytic domain of Gal1,4GlcNAc
2,6-sialyltransferase
converting this membrane-bound enzyme into the soluble
form(32) . The ``sialylmotif'' in this enzyme spans
from amino acid 178 to 225 and contains eight invariant amino acids,
which are indicated by boldfaceletters. The underlined amino acid residues were changed to alanine by
single point mutagenesis.
, cleavable signal sequence; &cjs2113;,
sialyl motif;
, catalytic domain.
Expression vectors containing mutant
sialyltransferase constructs were transfected into COS-1 cells and
evaluated for expression of the recombinant proteins and for expressed
sialyltransferase activity. Immunoprecipitation of radiolabeled
sialyltransferases using the anti-sialyltransferase antibody, followed
by analysis by SDS-polyacrylamide gel electrophoresis, indicated that
all mutant sialyltransferases were expressed and exhibited similar
molecular weight to the wild type sialyltransferase (not shown). To
determine the enzymatic properties of the mutant sialyltransferases,
concentrated media from the transfected cells were used as a source of
crude enzyme. Media from COS-1 cells ``mock'' transfected
using the expression vector (pSVL) only contained low levels of
endogenous sialyltransferase, which were typically less than 2% that of
media containing the expressed soluble sialyltransferases. All the
mutants showed Gal1,4GlcNAc
2,6-sialyltransferase activities,
which varied from 25 to 85% that of the wild type enzyme except for the
mutants, C181A, R207A, and S222A, which showed very low activity (5% or
less).
By comparing sialyltransferase expression by Western blot to
the enzymatic activity observed it was noted that the expression level
was apparently similar to the wild type enzyme for V184A, L190A, D219A,
V220A, K223A, and T225A, while the enzyme activities obtained were 45,
28, 56, 30, 32, and 27%, respectively. While the mutation of
Cys, Arg
, and Ser
to Ala
reduced the enzyme activity to 5% or less, the protein expression
appeared to be reduced only 2-3-fold.
Figure 2:
Double-reciprocal plots of initial rate
data with CMP-NeuAc (top) or asialo -acid
glycoprotein (bottom) as the varied substrate. Top,
rate data with CMP-NeuAc as the donor (26.5-201 µM)
were determined at a fixed concentration of the acceptor asialo
-acid glycoprotein, 50 µg. Bottom, the
concentration of the acceptor asialo
-acid
glycoprotein was varied (0.04-0.4 mM) at a fixed
concentration of the donor, 0.15 mM. The plot was shown only
for T225A (
) in top and V220A (
) in bottom, as a representative of the mutants and compared with
that of the wild type (spST-2) of sialyltransferase (
). The K
(apparent) values were determined from
the x intercept (-1/K
(apparent))(35) .
As summarized in Table 1, the K values obtained for asialo
-acid glycoprotein were within 0.5-1.2-fold that
of the wild type enzyme for each of the mutant enzymes examined. In
contrast, the K
values for the CMP-NeuAc were
significantly altered, particularly for V184A, L190A, D219A, V220A,
K223A, and T225A, with increases in K
(apparent)
of 3-12-fold observed.
Figure 3:
Inhibition kinetics of sialyltransferase
by CDP. Initial rate data for the wild type enzyme (top panel) 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 V220A (bottom panel) were determined in the presence of the varied
concentrations of CMP-NeuAc (0.25-2.0 mM) and in the
absence (
) or presence (⧫) of a fixed concentration of
16.7 µM CDP. The concentration of the acceptor asialo
-acid glycoprotein was kept constant at 50 µg
throughout for both enzymes. The K
values
were extracted from the x intercept in the presence of
inhibitor (35) , where the x intercept =
-(1/K
) (1 +
[I]/K
) and where K
is obtained in the absence of the
inhibitor CDP and [I] is the concentration of CDP.
The sialylmotif of the sialyltransferase gene family has been identified as a conserved stretch of 48-49 amino acids found roughly in the center of the coding sequence of these enzymes. Based on the premise that this motif was restricted to the sialyltransferase gene family, five sialyltransferase genes have been successfully cloned using a PCR homology approach, bringing the total number of distinct members of sialyltransferases cloned to date to eight(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) . Further evidence that the sialylmotif is unique to the sialyltransferase gene family is that no other gene in GenBank, including representative cloned cDNAs from the fucosyltransferase, galactosyltransferase, N-acetylglucosaminyltransferase, or N-acetylgalactosaminyltransferase families, has been found to have a homologous sequence. These facts alone argue that the sialyltransferase genes described to date originate from a common gene and that the sialylmotif participates in a structural feature or function common to all of these enzymes.
The aim of this present
study was to evaluate the functional role of the conserved sialylmotif
of the sialyltransferase gene family. As described under
``Results,'' the Gal1,4GlcNAc
2,6-sialyltransferase
was used as a representative enzyme in these studies, substituting
alanine for conserved amino acid residues in the sialylmotif by
site-directed mutagenesis to see if the alteration had an effect on
sialyltransferase activity. The 11 mutants reported in this study
include 7 of the 8 amino acid residues found to be invariant in all the
sialyltransferase genes cloned to date (see Fig. 1). In
addition, several other highly conserved amino acids were examined.
The results suggest that the sialylmotif participates in the binding
of the donor substrate, CMP-NeuAc, and has little contribution to the
binding of the oligosaccharide acceptor substrate. Indeed, of the eight
mutant sialyltransferses that exhibited sialyltransferase activity, six
exhibited increased K values of 3-12-fold
over that of the wild type for the donor substrate, CMP-NeuAc (Table 1). In contrast, all of the mutant enzymes had K
values for the acceptor substrate
(asialo-
acid glycoprotein) that were within
0.5-1.2-fold that of the wild type sialyltransferase. The
mutations spanned the length of the sialylmotif (see Fig. 1),
and the magnitude of the change in K
for CMP-NeuAc
did not appear to be dependent on the location of the amino acid in
this conserved domain. Further examination of one of the mutants,
V220A, revealed that it exhibited a decreased affinity (increased K
) for the competitive substrate CDP relative to
the wild type enzyme.
The conclusions drawn from these studies are in keeping with observations made previously by others. Wen et al.(10) suggested that the sialylmotif may form a conserved surface feature of the sialyltransferases, consistent with a substrate binding pocket, due to the large number of conserved charged residues found in the sialylmotif. Kurosawa et al.(16) further suggested that the conserved region was less likely to be an acceptor oligosaccharide binding site considering that the cloned enzymes transferred sialic acid to galactose, N-acetylgalactosamine, and sialic acid found in a variety of glycoprotein and glycolipid carbohydrate groups. The fact that the sialylmotif appears to participate in binding the common donor substrate, CMP-NeuAc, suggests that the PCR homology approach to cloning sialyltransferase cDNAs may continue to have utility in defining this diverse gene family.