From the Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada, K1A 0R6
Received for publication, December 14, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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The L1 immunotype strain 126E of Neisseria
meningitidis has been shown to have an
N-acetyl-neuraminic acid-containing lipooligosaccharide in
which an Mucosal pathogens in the genera Neisseria,
Campylobacter, and Haemophilus all possess a major cell
surface lipooligosaccharide (LOS)1 containing
N-acetylneuraminic acid (Neu5Ac) (2-4). These carbohydrate structures are not a static feature of the bacterium's outer surface but are produced with significant variation depending on the genetic makeup of the strain and the environment in which it is growing (5-7).
This phenomenon is thought to play an important role in the
pathogenesis of these organisms (8) (9), relating as it does to the
structural similarity of the terminal oligosaccharide portion of the
LOS to those found on human glycolipids. In Neisseria a
major structure is a terminal
-linked galactose from a Pk epitope is
substituted at the O6 position (Wakarchuk, W. W., Gilbert, M.,
Martin, A., Wu, Y., Brisson, J. R., Thibault, P., and Richards,
J. C. (1998) Eur. J. Biochem. 254, 626-633).
Using a synthetic Pk-epitope containing acceptor in
glycosyltransferase reactions, we were able to show by NMR analysis of
the reaction product that the 126E(L1)-derived sialyltransferase can
make both
-2,3 and
-2,6 linkages to the terminal galactose. Gene
disruption experiments showed that the lst gene in 126E(L1)
was responsible for the in vivo addition of the
-2,6-linked N-acetyl-neuraminic acid residue. By
site-directed mutagenesis it was possible to change the
MC58(L3)-derived enzyme into a bifunctional enzyme with a single amino
acid change at position 168, where a glycine was changed to an
isoleucine. We performed a gene replacement experiment where the
126E(L1)
-2,3/6-sialyltransferase was replaced by allelic exchange
with the monofunctional MC58(L3)
-2,3-sialyltransferase and with the mutant MC58(L3) allele G168I. We observed that the level of LOS sialylation with the G168I allele was very similar to that of the wild
type 126E(L1), indicating that residue 168 is the critical residue for
the
-2,6-sialyltransferase activity in vitro as well as
in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-2,3-sialyllacto-N-neotetraose (Fig.
1), in Haemophilus influenzae
strain RM118, the LOS structures include
-2,3-sialyllactose (7), and
in the NTHi 375 strain there is a simple disialic acid structure
(perhaps related to the ganglioside GD3) (4). In Campylobacter
jejuni there are structures similar to various gangliosides, from
GD3 to GT1a (10). Such oligosaccharides provide these pathogens with a
means of evading the host immune response through molecular mimicry as well as providing ligands for binding to receptors on human cells (11),
and they are therefore potent virulence factors (12).
View larger version (10K):
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Fig. 1.
Outer core oligosaccharides from N. meningitidis MC58(L3) and 126E(L1). The genes for the
glycosyltransferases used for each addition have been identified, and
their names are shown above each sugar residue (14, 18, 23).
The genetic analysis of the biosynthesis of LOS has shown that variation in the oligosaccharide portion of the LOS can arise in several ways. In H. influenzae, some LOS biosynthesis genes including glycosyltransferases are inactivated through a mechanism involving a variable number of tandem tetranucleotide repeats, which produces a phase-variable LOS phenotype (13). A related phase variation mechanism is used in Neisseria meningitidis, Neisseria gonorrhoeae, and C. jejuni, where changes in the length of polynucleotide tracts in certain glycosyltransferase genes leads to gene inactivation, which in turn allows alternate oligosaccharides to be produced (14-16). In addition to these mechanisms, a new source of structural diversity exists in C. jejuni, where the sialyltransferase Cst-II from the OH4384 strain has been shown to be bi-functional, using two different acceptor oligosaccharides and has been shown to be mono-functional in the 0:19 serostrain (17). The existence of these different mechanisms of structural variation makes it impossible to predict the LOS structure based on the gene complement alone.
The N. meningitidis -2,3-sialyltransferase, Lst, has a
relaxed acceptor specificity, being able to use synthetic acceptors that present terminal N-acetyllactosamine, lactose, or
galactose (18). Furthermore, the Lst from N. meningitidis
strains MC58(L3) and 126E(L1) can also use a terminal
-D-galactose to make an
-2,3-sialyl-Pk
epitope in vitro with a synthetic acceptor molecule (19). We have previously demonstrated that the LOS of the N. meningitidis 126E(L1) was also sialylated but at the O6 position
of the terminal
-D-galactose (1). To our knowledge, this
structure has not been reported in any mammalian glycolipid (nor in any
other LOS); hence, we cannot conclude that it is used as a form of
molecular mimicry as is the case with the
sialyllacto-N-neotetraose found in the strains with the L3
immunotype. The L1 immunotype has been isolated from disease outbreaks
(20), and therefore, this unique LOS structure may play a role in
meningococcal disease. We therefore sought to determine the enzyme
responsible for its biosynthesis.
In this paper we demonstrate the that the Lst of 126E(L1) is also
responsible for the addition of the Neu5Ac at O6 of the -galactose
in the 126E(L1) LOS. Additionally, the bi-functional nature of the
enzyme from the 126E(L1) can be introduced into the mono-functional
MC58(L3) enzyme by site-directed mutagenesis of a single residue.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Plasmids-- The following strains of N. meningitidis were used: immunotype L1 strain 126E (NRCC 4010); immunotype L3 strain MC58 (NRCC 4728). The cloning vector pCW was used for expression of the lst genes and has been described previously (21). Plasmids were propagated in Escherichia coli strain AD202 (CGSC 7297).
Preparation and Analysis of Lipooligosaccharide-- N. meningitidis 126E(L1) was grown and harvested, and the LOS was extracted by the hot phenol-water method as previously described (1), except that the plates were supplemented with 25 µg/ml CMP-Neu5Ac. Small scale isolations were performed as described previously (22). Capillary electrophoresis-mass spectrometry of LOS was performed as previously described (1, 22). Methylation analysis to determine linkage positions between residues was performed as previously described (1).
Measurement of Sialyltransferase Activity--
For detection of
activity in N. meningitidis strains, cells were scraped off
freshly grown Columbia blood agar plates that had been supplemented
with 25 µg of CMP-Neu5Ac/ml. The cell pellet was then extracted with
0.2% Triton X-100 in 20 mM Tris HCl, pH 8.0. The cell-free
extract was then assayed as previously described (19) with acceptor
molecules derived from aminophenylglycosides labeled either with FCHASE
or FEX fluorophors. Analysis of all of the site-directed mutants was
performed with a construct that contained only the transferase gene.
The recombinant proteins were assayed in sonicated extracts. The thin
layer chromatography conditions for separation of the
-2,3/2,6-sialylated Pk-FCHASE were: isopropanol,
n-butyl alcohol, 0.1 M HCl (2:1:1) or ethyl
acetate, methanol, water, acetic acid (4:2:1:0.1). High performance TLC plates (Whatman) were used for the separations.
Genetic Manipulations of the 126E(L1) and MC58(L3)
-2,3-Sialyltransferase--
The construction of the allelic
replacement was performed essentially as described previously (23).
Briefly, the kanamycin resistance gene from pUC-4K was liberated from
the plasmid as a SalI fragment. The 5'-upstream region and
3'-downstream regions of the
-2,3-sialyltransferase structural gene
were amplified from N. meningitidis 126E(L1) by polymerase
chain reaction with Pwo polymerase as described by the manufacturer
(Roche Molecular Biochemicals). The primers for the 126E(L1)
upstream-flanking sequence (5' end) of the coding regions were:
5'-GGGGGGGAATTCCATATTTTGGCGGCTTTGTTCGCGC-3' and 5'-GGCGGTGGGCATATGATCCCTAAAACTCCATTCCGACAAATTG-3'. The
region is 355 base pairs. The primers for the L1 downstream-flanking sequence (3' end) of the coding sequence were
5'-GGGGGGGTCGACTCGCATAGCAAATCAAAATAGAAAATGG-3' and
5'-GGGGGGAAGCTTCCGCGCACTGCCCGCCGTTTGGTCGG-3'. The region is 350 base pairs. The coding sequence from MC58 L3 was amplified with the
following primers:
5'-CTTAGGAGGTCATATGGGCTTGAAAAAGGCTTGTTTGACC-3' and
5'-GGGGGGGTCGACTTAATTTTTATCGTCAAATGTCAAAATC-3'. The
fragment was 1140 base pairs. The plasmid for the gene replacement was assembled in the following steps. pUC19 was digested with
EcoRI and HindIII and purified for use as the
cloning vector. A four-fragment ligation was performed with the
5'-flanking sequence of 126E(L1)-lst (EcoRI/NdeI-digested), the
MC58(L3)-lst structural gene
(NdeI/SalI-digested), the 3'-flanking sequence of
126E(L1)-lst (SalI/HindIII), and the pUC19 vector (EcoRI/HindIII). Once this construct
was verified, we then inserted a KanR marker between the
structural gene and the 3'- flanking sequence. The recombinant
plasmids were introduced into N. meningitidis by electroporation.
The gene disruption vector was constructed in a different fashion. The sialyltransferase gene (NdeI-SalI fragment as described above) was restricted with Sau3A-I to generate three fragments. The middle 22-base pair fragment was discarded. The two larger fragments were ligated with the kanamycin resistance marker that had been liberated from pUC-4K with BamHI. These fragments were then ligated into pUC19 which had been digested with EcoRI and SalI. These plasmids were then introduced into the N. meningitidis strain by electroporation.
Site-directed Mutagenesis-- The MC58(L3)-lst allele was mutagenized using the U-DNA method of Kunkel (24). All mutant genes were completely sequenced to ensure no other mutations were introduced.
Capillary Zone Electrophoresis-Electrospray Mass Spectrometry-- A crystal model 310 CE instrument (AYI Unicam, Madison, WI) was coupled to an API 3000 mass spectrometer (PerkinElmer Life Sciences/Sciex, Concord, Canada) via a microionspray interface. A sheath solution (isopropanol, methanol, 2:1) was delivered at a flow rate of 1 µl/min to a low dead volume tee (250 µm internal diameter, Chromatographic Specialties, Brockville, Canada). The separations were obtained on a 90-cm-length bare-fused silica capillary using 30 mM morpholine in deionized water, pH 9.0, containing 5% methanol. A voltage of 25 kV was typically applied for the separation. The outlet of the capillary was tapered to ~15 µm internal diameter using a laser puller (Sutter Instruments, Novato, CA). Mass spectra were acquired with dwell times of 3.0 ms/step of 1 m/z unit in full-mass scan mode. 20 nl of sample was typically injected by using 150 mbar for a duration of 0.1 min.
NMR Spectroscopy--
NMR experiments were performed on a Bruker
AMX-600 and Varian INOVA 600 NMR spectrometers. Experiments on
Pk-FCHASE compound were done using an inverse broadband
detection probe at 27 °C with the mono-deuterated water resonance at
4.756 ppm. Experiments on -2,6-sialyl-Pk-FCHASE compound
were done using a 5-mm Z gradient triple resonance probe at 25 °C
with the mono-deuterated water resonance at 4.774 ppm. NMR samples were
prepared by dissolving 0.1-0.3 mg of material in 600 µl of
D2O after freeze-drying. All NMR experiments were performed
as previously described (17). For the proton chemical shift reference,
the methyl resonance of internal or external acetone was set at 2.225 ppm (1H). For the 13C chemical shift
reference, the methyl resonance of internal or external acetone was set
at 31.07 ppm relative to external dioxane at 67.40 ppm.
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RESULTS |
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Detection of -2,6-Sialyltransferase Activity--
When we
analyzed reactions performed with the 126E(L1)-Lst and the
Pk-FCHASE acceptor, we noticed a second product peak
(slightly faster migrating) in the CE electropherograms (Fig.
2), which was sensitive to sialidase
treatment (data not shown). This second product is only seen with
Pk-FCHASE acceptor. The major product (~90% of the
product) had the same migration time as
-2,3-Neu5Ac-Pk-FCHASE that we had previously described
(19). We were able to resolve these two products by TLC, but we could
only see a second product in reactions performed with the 126E(L1)-Lst
and not MC58(L3)-Lst. We were able to obtain enough of the unique secondary product using preparative TLC, and this material was subjected to analysis by NMR spectroscopy (see below).
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NMR Analysis--
To assess the linkage specificity of the
126E(L1)-Lst, the unique product formed with Pk-FCHASE as
an acceptor was analyzed by NMR spectroscopy on nanomole amounts. We
have previously published the NMR spectrum for
-2,3-Neu5Ac-Pk made with the MC58(L3)-Lst (19), and we
found the NMR spectrum of the major sialylated product made with
126E(L1)-Lst was identical to our previously published one. We present
here only the spectra from a nonsialylated acceptor,
Pk-FCHASE = compound I, and the unique product formed
in a reaction with the 126E(L1)-Lst,
-2,6-sialyl-Pk-FEX = compound II. The comparison of
these two spectra clearly shows the site of sialylation of the terminal
-galactose in the acceptor. At the time the preparative sialylation
reaction was performed, we were using the FEX aglycon, but we have seen
that during storage that the FCHASE aglycon is more stable. The
nonsialylated Pk-FCHASE spectra was collected on material
made at a later time.
Both compounds were soluble and gave sharp resonances with line widths of a few Hz since the H-1 anomeric doublets (J1,2 = 4 and 8 Hz) are well resolved (data not shown). For compound I, the mono-deuterated water resonance was very broad and overlapped with one of the anomeric signals. In Table I, the proton assignments were obtained from standard homonuclear experiments, correlated spectroscopy (COSY), total correlation spectroscopy (TOSCY), nuclear Overhauser effect spectroscopy (NOESY), one-dimensional NOESY, one-dimensional TOCSY, and the 13C assignments from a heteronuclear multiple quantum coherence (HMQC) experiment, which detects C-H correlations (17).
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The linkage site on the aglycon was determined from a comparison of the
13C chemical shifts of the enzymatic product with those of
the precursor to determine glycosidation shifts, as done previously
(17). Although a different fluorescent tag was used, it only influenced the proton chemical shift of the Glc residue. For compound I, the
proton and 13C chemical shifts were in accord with those
reported for
Gal-1,4-Gal-
1,4-Glc-
-1-O-2-(trimethylsilyl)ethanol (25).
Compound II was identified as
Neu5Ac-
2,6-Gal-
1,4-Gal-
1,4-Glc-
-1-FEX. From a comparison of
the heteronuclear multiple quantum coherence spectra of compound I and
II, it is obvious that the linkage site is at C-6 of
Gal due to the
large downfield shift of
2.6 ppm for
Gal C-6 upon sialylation
(Fig. 3). Large proton chemical shifts
differences of 0.18 and
0.17 ppm were also observed for the H-6 and
H-6' resonances of
Gal upon sialylation. An upfield shift of 1.3 ppm
for the
Gal C-5 was also observed (Table I), typical of 2-6
sialyloligosaccharides (26). The 13C chemical shifts of
compound II are similar to those reported for the
NeuN-
2,6-Gal-
1,4-Gal-
1,4-Glc moiety present in the 126E(L1)
LOS from N. meningitidis, except for the chemical shift of
NeuN C-4, which is obviously wrong in the latter (1). In Table I and
Fig. 3, the proton chemical shift differences for the Glc unit between
compounds I and II are attributed to different fluorescent
aglycons.
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Site-directed Mutagenesis of MC58(L3)-Lst--
A sequence
comparison of the 126E(L1)-Lst (GenBankTM accession U60662)
and MC58(L3)-Lst (GenBankTM accession U60660)
proteins showed only 6 amino acid changes. We sequentially changed all
of the residues in MC58(L3)-Lst that differed from 126E(L1)-Lst to the
corresponding residue from 126E(L1)-Lst (E40D, R102W, S129A, G168I,
T242A, K273N). Only the G168I mutation resulted in an enzyme with
-2,6-sialyltransferase activity. We then performed additional
mutagenesis to put all possible amino acids at the position Gly-168 in
MC58(L3)-Lst to determine whether other amino acids also permit the
second enzyme activity. The results of these experiments were analyzed
by first measuring the activity of the mutants on
-N-acetyllactosamine-FCHASE, the preferred acceptor for
the MC58(L3)-Lst, to ensure the mutants were functional. Both Lst
enzymes make only a single product, the
-2,3-linked Neu5Ac, with
-N-acetyllactosamine-FCHASE. Only one of the mutations
resulted in an inactive enzyme, G168P, and this was not due to lack of
protein production (data not shown). These mutants were then analyzed
for activity on the Pk-FCHASE acceptor using equivalent
amounts of enzyme as measured on the
-N-acetyllactosamine-FCHASE acceptor. Only a few of these had significant
-2,6-sialyltransferase activity, and these data is
shown in Fig. 4. The mutants G168I and
G168L showed levels of
-2,6-sialyltransferase activity equivalent to
the 126E(L1)-Lst. The G168V mutant showed 40% of the
-2,6 activity
level of 126E(L1)-Lst, and the G168M mutant showed 25% of the level of
-2,6 activity of 126E(L1)-Lst.
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We also noted that certain mutants were less active than the wild type
on Pk-FCHASE in general but basically unaffected for
activity on -N-acetyllactosamine-FCHASE. These mutants
were G168F, G168Y, and G168W, and using the standard assay described
here, they showed a higher specificity for
-N-acetyllactosamine-FCHASE than they did for
Pk -FCHASE.
Production of the 126E(L1)-lst Knock-out-- An insertional inactivation mutant of the lst gene was made in the 126E(L1) strain, and then the LOS was analyzed by mass spectrometry. When the lst gene has been inactivated, no Neu5Ac could be detected in the LOS (data not shown). The mutation should not cause any polar effects as the next gene downstream of lst, a cytochrome c' homologue, is transcribed in the opposite direction and is not involved in sialic acid metabolism (18).
Allelic Exchange of the 126E(L1)-lst Gene with That from MC58(L3)
and with the G168I Mutant Allele--
To assess the bi-functional
nature of the lst gene from 126E(L1) in vivo, we
constructed isogenic mutants that contained either the MC58(L3)-derived
lst gene or the G168I mutation. After obtaining transformants, they were screened for sialyltransferase activity, and
then the resident lst gene was amplified by polymerase chain reaction and sequenced to ensure the correct gene had been inserted. Both gene replacement mutants produced similar levels of
sialyltransferase activity as measured with the in vitro
assay on synthetic acceptors. The activity was similar to what we had
seen in vitro; the MC58(L3)-derived enzyme did not make a
significant amount of the -2,6-linked Neu5Ac, whereas the G168I
mutant showed the same ratio of
-2,3 to
-2,6 product (data
not shown). LOS was prepared from these strains and was analyzed by
CE-electrospray mass spectrometry (Fig.
5). The level of Neu5Ac incorporation in
the 126E(L1) mutant strain with the MC58(L3)-lst gene was
very low, but certainly detectable. The level of Neu5Ac incorporation
from the G168I mutant gene was comparable with the wild-type
126E(L1) strain. Methylation analysis of these LOS samples (Table
II) showed 6-substituted Gal,
indicating that the Neu5Ac in both cases was
-2,6-linked and
that no
-2,3-linked Neu5Ac was present.
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DISCUSSION |
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The proteins involved in the addition of the sialic acid to the outer core oligosaccharide portion of the LOS from C. jejuni, H. influenzae, N. meningitidis, and N. gonorrhoeae have been identified (7, 17, 18). These proteins have a variety of acceptor specificities, and only the C. jejuni and H. influenzae enzymes share some sequence identity with each other. No structure/function analysis for any of these enzymes has so far been reported. Since there is no conserved sequence in the group of enzymes as a whole, it has been difficult to predict which residues might be involved in donor/acceptor recognition or catalysis.
Sialic acid transfer to the LOS of N. meningitidis species
has been shown to be performed by the product of the lst
gene (18). The terminal oligosaccharide produced in most strains is a
mimic of the human glycolipid oligosaccharide
sialyllacto-N-neotetraose. An alternate sialylated LOS
structure with the terminal oligosaccharide -2,6-Neu5Ac-Pk has been shown to be produced in the
126E(L1) strain (1, 20), but the enzyme for producing this novel
-2,6-sialylated structure was not reported. We have now determined
the enzyme responsible for the addition of the
-2,6-linked Neu5Ac in
the 126E(L1) strain and have shown it to be a bi-functional version of
the Lst enzyme that previously had been shown to transfer the
-2,3-linked Neu5Ac in N. meningitidis MC58(L3) strain.
The first reported multifunctional enzyme involved in
lipopolysaccharide biosynthesis was the
3-deoxy--D-mannooctulosonic acid (KDO) from
Chlamydia pneumoniae, which adds KDO to the lipid A core
through a
-2,6 linkage to GlcNAc and then in an
-2,4 linkage to
the first KDO residue, and then in an
-2,8 linkage to the second KDO
(27). Recently a bi-functional sialyltransferase from C. jejuni was described where the enzyme could also use two very
different acceptors, namely a
-linked Gal or a
-2,3-linked Neu5Ac
(17). The sialyltransferase enzyme from the 126E(L1) strain of N. meningitidis described in this paper is also bi-functional but
uses the same terminal sugar acceptor for the reaction and makes an
alternate linkage (
-2, 6) in addition to the more usual
-2,3-linkage. This activity is also found only with terminal Gal
residues that are in an
-linkage with the previous galactose residue. We have never seen any second (
-2,6) product formed during
in vitro reactions on
-linked galactose residues. We
detected the
-2,6 product produced during in vitro enzyme
reactions, but only about 10% of the synthetic Pk-FCHASE
acceptor is converted to the
-2,6 linkage product, whereas the
majority of the product formed in vitro is the
-2,3-sialyl-Pk structure. The MC58(L3)-Lst enzyme does
produce detectable
-2,6-linked Neu5Ac, but the amount of
-2,6-linked Neu5Ac was about 45-fold less than was made by
126E(L1)-Lst, and this could only be seen using the ultra-sensitive
laser-induced fluorescence detector on the CE. The isomeric
-2,3-sialyl-Pk structure has not been detected in
vivo in the N. meningitidis 126E(L1) strain.
The ability of the enzyme to make these two products in
vitro may not be too surprising since side reactions are possible with a synthetic acceptor molecule, but what is most important to bear
in mind is that in vivo, only the -2,6-Neu5Ac linkage is
formed in the 126E(L1) strain. In our examination of the enzyme, we
were able to pinpoint a single residue in the MC58(L3)-Lst, Gly-168,
which when mutated to Ile (the residue at position 168 in 126E(L1),
permits the enzyme to form the same level of
-2,6-sialylated product
either in vitro on the synthetic Pk acceptor or
in vivo on the LOS. What was surprising, however, was the
fact that in vivo the MC58(L3)-Lst could form a very small amount of sialylated LOS in the 126E(L1) strain and that this material
was also
-2,6-linked. The amount of Neu5Ac in this 126E(L1) mutant
(MC58(L3)-Lst) was too small to analyze by NMR, but methylation analysis showed around 6% of the wild type level of the 6-substituted Gal. This level of Neu5Ac incorporation is much higher than what was
expected, given the 45-fold lower
-2,6-sialytransferase activity of
the MC58(L3)-Lst enzyme measured in vitro. These data
suggest that some factor in vivo completely blocks the O3
position of the terminal
-galactose and that something also must
promote the formation of the
-2,6-linked product since this is the
linkage formed exclusively in L1. At present we do not know if this
factor is another protein that may bind either Lst or the LOS, the
conformation of the LOS during biosynthesis, or another molecule
present at the site of biosynthesis. Molecular modeling of the 126E(L1)
LOS suggests the conformation of the terminal trisaccharide could produce a sterically hindered product where the O3 of the terminal
-Gal is effectively blocked by the phosphoethanolamine substituent on Hep-2 of the inner
core.2
Consequently the Lst variants with very low -2,6 activity would be
inactive in L1 strains when LgtC is functional. Since a relatively
subtle change (Gly to Ile/Leu) results in the
-2,6-sialyltransferase activity, we speculate that the 126E(L1) strain maintained this version
of Lst because it was advantageous for its survival in the human host,
and as this oligosaccharide structure has not been described in man, it
may or may not be molecular mimicry. We do not yet know if the
specificity of the 126E(L1)-Lst is in all L1 immunotype strains. Data
by Griffiss et al. (20) would suggest the structure we
described in strain 126E(L1) and the lst gene variant occurs
in other L1 immunotype strains.
Our mutagenesis of position 168 in the MC58(L3)-Lst revealed that all
amino acid side chains are tolerated there except for proline.
Surprisingly, the activity level of all of the mutants was similar on
the acceptor -N-acetyllactosamine-FCHASE, except G168P,
which was not active at all likely due to distortion of the enzyme
active site. The mutants G168Y, G168F, and G168W maintained activity on
-N-acetyllactosamine-FCHASE but have very reduced activity on the Pk-FCHASE compared with the MC58(L3)-Lst or
126E(L1)-Lst. When this residue is Ile or Leu, it then directs the
specificity of the reaction to allow the formation of the
-2,6
linkage as well as the 126E(L1)-Lst. The presence of the aromatic amino
acid side chains reduced the activity of the enzyme toward the
Pk-FCHASE acceptor significantly. With the CE-based assay,
the overall activity of the G168W/G168F/G168Y mutants were 4-9-fold
less active on this acceptor (data not shown), whereas their activity
on
-N-acetyllactosamine-FCHASE appeared very similar to
the wild type MC58(L3)-Lst protein. This suggests that the steric
hindrance from the large inflexible ring containing side chains
specifically block the enzyme from utilizing the
-linked terminal
Gal residue.
We postulate that the position 168 in this enzyme must be in a cavity
that has enough space to accommodate both the and
anomers of
the terminal Gal. It will be essential to obtain structural information
about this enzyme that will show us what the active site looks like to
understand how these two different acceptor conformations are
accommodated such that the product of the 126E(L1)-Lst reaction is
-2,6-linked Neu5Ac, whereas the MC58(L3)-Lst produces
-2,3-linked
Neu5Ac. Other factors in the synthesis of the 126E(L1)-LOS influence
the Neu5Ac linkage formation so it will also be important to identify
what these are to fully understand the formation of this novel
glycolipid structure.
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ACKNOWLEDGEMENTS |
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We thank Melissa J. Schur for expert technical assistance in the capillary electrophoresis analysis of the sialyltransferase assays.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Immunochemistry
Program, Institute for Biological Sciences, National Research Council
of Canada, 100 Sussex Dr., Ottawa, Ontario, Canada K1A 0R6. Fax:
613-941-1327; E-mail: warren.wakarchuk@nrc.ca.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M011293200
2 J. R. Brisson, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: LOS, lipooligosaccharide; CE, capillary electrophoresis; Neu5Ac, N-acetylneuraminic acid; FCHASE, 6-(5-fluoresceincarboxamido)hexanoic acid succimidyl ester.
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