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(Received for publication, April 4, 1996, and in revised form, May 21, 1996)
From the The biosynthetic function of the
lgtABE genetic locus of Neisseria meningitidis
was determined by structural analysis of lipopolysaccharide (LPS)
derived from mutant strains and enzymic assay for glycosyltransferase
activity. LPS was obtained from mutants generated by insertion of
antibiotic resistance cassets in each of the three genes
lgtA, lgtB, lgtE of the N. meningitidis immunotype L3 strain Diseases caused by Neisseria meningitidis remain a
significant problem worldwide (1). Both capsule and lipopolysaccharide
(LPS)1 are recognized as major virulence
determinants (2, 3) and, in the developed world, the group B capsular
type is the leading cause of meningococcal meningitis (4). LPS
comprises a heterogeneous mixture of molecules consisting of a variable
core oligosaccharide (5, 6, 7, 8, 9) and a conserved lipid A component (10). On
the basis of the structural diversity of the oligosaccharide epitopes
and their reactivity with monoclonal antibodies, the meningococci have
been classified into 12 immunotypes (11, 12, 13). Structural studies of
core oligosaccharides and the use of monoclonal antibodies have led to
the identification of both common and variable structural features
which are responsible for immunotype specificity (13). In addition,
terminal saccharide structures have been identified which are also
found on the surface of human epithelial cells or in host secretions
(14, 15). Included among these are Gal There has been increasing interest in the genetics of LPS expression in
Neisseria (18, 22, 23, 24, 25). Among the genes identified was
meningococcal galE which encodes the UDP-glucose-4-epimerase
essential for incorporation of galactose into LPS and an insertion
mutant has been constructed in the galE gene which produces
truncated LPS (22). The genetic locus responsible for expression of
lacto-N-neotetraose, its terminally GalNAc-substituted
analogue, and Gal The following strains of N. meningitidis were used in this study: immunotype L7 strain M982B
(NRCC no. 4725); lgt mutant strains N.
meningitidis strains were resuscitated from microbeads on 5%
sheep blood agar plates and incubated overnight at 37 °C, and the
proceeds of six plates were suspended in 50 mL of Difco Bacto Todd
Hewitt broth (Difco). For mutant strains, this culture was used to
inoculate 2.5 liters of the same medium containing a final
concentration of 50 µg/mL kanamycin (Sigma) and,
following incubation at 37 °C for 6-8 h, was used to inoculate 60 liters of the above medium in a New Brunswick Scientific IF-75
fermenter. Fermenter growth was overnight (approximately 17 h) at
37 °C. The culture was killed by the addition of 1% (final
concentration) phenol, chilled to 15 °C, and harvested by continuous
centrifugation. Wet weight biomass yields were in the order of 3.5 g/liter. N. meningitidis strain M982B (immunotype L7) was
grown under similar conditions as described previously (5). LPS was
isolated by the hot phenol-water extraction procedure (26) and purified
by repeated ultracentrifugation (105,000 × g, 4 °C,
2 × 5 h).
Polyacrylamide gel electrophoresis was performed using the
buffer system of Laemmli and Favre (27) as modified by Komuro and
Galanos (28) with sodium deoxycholate as the detergent.
Lipopolysaccharide bands were stained and visualized by silver staining
as described by Tsai and Frasch (29).
LPS (25 mg) was hydrolyzed in 1%
aqueous acetic acid (5 ml) for 2.5 h at 100 °C, the solution
was then cooled (4 °C), and the precipitated lipid A was removed by
low speed centrifugation. The supernatant solution was lyophilized, and
the water-soluble component was fractionated on a Sephadex G-50 gel
filtration column (2.0 × 90 cm, Pharmacia Biotech Inc.) by
elution with pyridinium acetate (0.05 M, pH 4.5) to afford
core oligosaccharide. The fractions were monitored for neutral glycoses
(30) and KDO (31).
LPS was
O-deacylated with anhydrous hydrazine under mild conditions
as described previously (32). Briefly, a sample (14 mg) was treated
with anhydrous hydrazine (1 mL) and stirred at 37 °C. After 1 h, the reaction mixture was cooled (0 °C) and hydrazine was
destroyed by addition of cold acetone (5 ml). The precipitated product
was washed with acetone (4 × 2 ml), acetone: water (4:1, 5 ml)
and then lyophilized from water.
Employing a
modification of the method of Holst et al. (33), LPS (4 mg)
was treated with hot alkali (4 M KOH, 18 h, 125 °C)
to cleave ester- and amide-linked fatty acids. The reaction mixture was
cooled to room temperature and neutralized (6 M HCI).
Completely deacylated LPS was purified by gel filtration chromatography
on a Sephadex G-10 column (1.6 × 90 cm, Pharmacia) eluted with
pyridinium acetate (0.05 M, pH 4.5).
For analysis of constituent sugars,
samples (0.5 mg each) of LPS or core oligosaccharide were hydrolyzed
with 2 M trifluoroacetic acid for 1 h at 125 °C.
The excess acid was removed by evaporation under a stream of nitrogen,
and the glycoses were determined by gas-liquid chromatography-mass
spectrometry (GLC-MS) of their derived alditol acetates as described
previously (34). GLC-MS was performed with a Varian ion trap system
fitted with a DB-17 fused silica capillary column (0.25 mm × 25 m, Quadrex Corp.) in the electron impact mode with a
temperature program starting at 180 °C for 2 min followed by an
increase of 5 °C/min to 300 °C.
Samples
were analyzed in the negative or positive ion mode on a VG Quattro
triple quadrupole mass spectrometer (Fisons Instruments) fitted with an
electrospray ion source. Oligosaccharide samples were dissolved in
water, this was diluted by 50% with
acetonitrile:H2O:MeOH:1% ammonia (4:4:1:1), and then the
mixture was introduced by direct infusion at 4 µl/min with a Harvard
syringe pump 22. The electrospray tip voltage was 3.5 kV, and the mass
spectrometer was scanned from m/z 50-2500 with a scan time
of 10 s. Data were collected in multichannel analysis mode, and
data processing was handled by the VG data system (Masslynx). For MS-MS
experiments, precursor ions were selected using the first quadrupole
mass analyzer, and fragment ions, formed by collisional activation with
argon in the rf-only quadrupole collision cell, were mass analyzed by
scanning the third quadrupole. Collision energies were typically 60 eV.
NMR spectra
were obtained on a Bruker AMX 500 spectrometer using standard Bruker
software. Measurements were made at 37 °C on solutions in 0.5 ml of
D2O subsequent to several lyophilizations with
D2O.
Proton NMR spectra were measured at 500 MHz using a spectral width of
6.0 KHz and a 90° pulse. Proton and 13C chemical shifts
were referenced to that of the methyl resonances of internal acetone
( Heteronuclear two-dimensional 1H-13C chemical
shifts correlations were measured in the 1H-detected mode
via multiple quantum coherence (HMQC) with proton decoupling in the
13C domain (37), using data sets of 1024 × 256 points
and spectral widths of 4.5 and 13.8 KHz for 1H and
13C domains, respectively; 128 scans were acquired for each
t1 value.
Phosphorous-31 spectra were measured at 202 MHz with a spectral width
of 13 KHz and phosphoric acid (85%) was used as the external standard
( Basic DNA manipulations were performed as
described previously (38). A polymerase chain reaction was performed
with Pwo polymerase as described by the manufacturer
(Boehringer Mannheim). DNA sequencing was performed with an Applied
Biosystems (ABI) model 370A automated DNA sequencer using the cycle
sequencing kit from ABI. The expression vector used for the
lgtB gene has been described previously (38).
p-Aminophenylglycoside (10 mg)
(Sigma) was dissolved in 0.5 ml of 0.2 M
triethylamine acetate buffer, pH 8.2. 5-(Fluorescein-carboxamido)-hexanoic acid succimidyl ester (10 mg,
single iosmer) (FCHASE, Molecular Probes) was dissolved in 0.5 ml of
methanol and added to the aminophenylglycoside solution. The mixture
was stirred in the dark for 3 h at room temperature and then dried
in a Savant Speedvac. The dry mixture was resuspended in 200 µl of
50% acetonitrile and spotted on a 1-mm thick 20 cm × 20-cm
Silica 60 TLC plate (E. Merck). The TLC plate was developed with the
following solvent system: ethyl acetate/methanol/water/acetic acid
7:2:1:0.1. After air drying in a fume hood, the bright yellow product
was scraped off the plate and eluted with five 10-ml washes of
distilled water. The water eluates were pooled, and the product was
concentrated and desalted by binding to a Sep-Pak C18
reverse phase cartridge. After washing the cartridge with 20 ml of
water, the product was eluted in 1-3 ml of 50% acetonitrile. The
product was quantitated by spectrophotometry with
E494 = 68,000 M Capillary electrophoresis (CE)
was performed with a Beckman P/ACE 5510 equipped with an Argon ion
laser-induced fluorescence detector, Cell extracts were prepared using an Avestin
B3 Emulsiflex cell disrupter (Avestin Ottawa, Ontario). The clarified
cell extracts were centrifuged at 100,000 × g for
1 h to pellet the cell membrane. Glycosyltransferase reactions
were performed at 37 °C in 20-µl volumes and contained MES buffer
50 mM, pH 6.7, 10 mM MnCl2, 5 mM dithiothreitol, 1.0 mM labeled acceptor, 1 mM UDP-Gal or 1 mM UDP-GlcNAc as donor, and
various amounts of enzyme, either from crude bacterial extracts or
extracts of recombinant Escherichia coli with the cloned
lgtB gene. The reactions were terminated by the addition of
an equal volume of 2% SDS and heated to 75 °C, for 3 min. These
samples were then diluted appropriately in water prior to analysis by
CE.
After the reaction the FCHASE-aminophenylglycosides were bound to a
Sep-Pak C18 reverse phase cartridge, desalted by washing
with water and then eluted in 50% acetonitrile. After drying under
vacuum, the samples were dissolved in water, and glycosidase assays
were performed as described by the enzyme manufacturer (Oxford
Glycosystems). These samples were then diluted with water and again
analyzed by CE.
Lacto-N-neotetraose-deficient mutant strains of
N. meningitidis were constructed by insertion of kanamycin
resistance cassettes into the lgtABE locus (mutants
lgt A, lgt B, and lgt E) and the
galE gene (mutant gal E) of the L3 immunotype
strain ( Deoxycholate-PAGE analysis of the LPS from the immunotype L3-derived
mutants revealed single-band patterns that were faster migrating than
meningococcal LPS containing the complete
lacto-N-neotetraose epitope (Fig. 1). The
observed electrophoretic mobilities of the bands correspond to low
molecular weight LPS composed of a lipid A and a core oligosaccharide.
LPS from lgt A, lgt B, and lgt E
showed consecutively faster relative mobilities than that of immunotype
L7 LPS consistent with successive sugar deletions in the core
oligosaccharide regions (Fig. 1). gal E LPS gave a more
diffuse band that exhibited similar electrophoretic mobility to
lgt E LPS. LPS from the N. meningitidis
immunotype L7 strain M982B has been shown (9) to contain the
immunotype L3 basal structure with complete expression of the
lacto-N-neotetraose epitope.
Treatment of the LPS samples with anhydrous hydrazine under mild
conditions afforded water-soluble O-deacylated samples that
were suitable for mass spectral analysis by ESI. In the last few years,
ESI-MS has proved to be a valuable tool for structural analysis and for
probing structural heterogeneity of low molecular weight LPS (39, 40, 41, 42).
ESI-MS, recorded in the negative ion mode, for the lgt
mutant O-deacylated LPS (LPS-OH) are shown in Fig.
2. For each sample, the mass spectrum is dominated by
molecular peaks corresponding to doubly and triply deprotonated ions
arising from a single molecular species and this is in agreement with
the deoxycholate-PAGE results. For the lgt B LPS-OH sample
(Fig. 2A), triply [M
Negative ion ESI-MS data and proposed compositions for O-deacylated LPS
of N. meningitidis MC58 lgt and gal E mutant strains
Removal of ester and amido linked fatty acid groups of the lgt
B LPS effected by treating it with strong alkali according to
established procedures (33) afforded a backbone oligosaccharide sample.
ESI-MS of the LPS backbone oligosaccharide sample, so obtained, in the
negative ion mode revealed abundant triply [M The sequence of the glycose residues within the oligosaccharide
component of the lgt B LPS was confirmed by NMR spectroscopy
(45). This was achieved by measurement of nuclear Overhauser effects
(NOEs) between protons on contiguous residues in the backbone
oligosaccharide sample and required initial complete assignment of ring
1H resonances.
As would be expected, the 1H detected 13C NMR
spectrum of the backbone oligosaccharide derived from the lgt
B LPS sample showed resonances in the low field region (90-105
ppm), corresponding to the anomeric carbons from eight aldose residues.
In addition, diagnostic signals from the methylene carbons of two KDO
residues were observed at 35.1 and 35.7 ppm. The 1H NMR
spectrum showed characteristic resonances (34, 46) in the high field
region from the H-3 methylene protons from the two
Proton chemical shifts and coupling constants for the deacylated
backbone oligosaccharides derived from N. meningitidis lgt B LPS
Proton NOE data for the deacylated backbone oligosaccharides derived
from N. meningitidis lgt B LPS
Volume 271, Number 32,
Issue of August 9, 1996
pp. 19166-19173
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,

Institute for Biological Sciences, National
Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada and the
§ Molecular Infectious Diseases Group and Department of
Paediatrics, Institute for Molecular Medicine, John Radcliffe Hospital,
Headington, Oxford, OX3 3DU, United Kingdom
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
3 MC58. LPS from the parent
strain expresses the terminal lacto-N-neotetraose
structure, Gal
1
4GlcNAc
1
3Gal
1
4Glc. Mild
hydrazine treatment of the LPS afforded O-deacylated
samples that were analyzed directly by electrospray ionization mass
spectrometry (ESI-MS) in the negative ion mode. In conjunction with
results from sugar analysis, ESI-MS revealed successive loss of the
sugars Gal, GlcNAc, and Gal in lgt B, lgt A,
and lgt E LPS, respectively. The structure of a sample of
O- and N-deacylated LPS derived by aqueous KOH
treatment of lgt B LPS was determined in detail by
two-dimensional homo- and heteronuclear NMR methods. Using a synthetic
-GlcNAc acceptor and a
-lactose acceptor, the glycosyltransferase
activities encoded by the lgtB and lgtA genes
were unambiguously established. These data provide the first definitive
evidence that the three genes encode the respective
glycosyltransferases required for biosynthesis of the terminal
trisaccharide moiety of the lacto-N-neotetraose structure
in Neisseria LPS. From ESI-MS data, it was also determined
that the Gal-deficient LPS expressed by the lgt E mutant is
identical to that of the major component expressed by immunotype L3
galE-deficient strains. The galE gene which
encodes for UDP-glucose-4-epimerase plays an essential role in the
incorporation of Gal into meningococcal LPS.
1
4Gal (immunotype L1),
lacto-N-neotetraose (e.g. immunotypes L3 and L7),
and lactose (e.g. immunotype L8) (16). Endogenous
sialylation of the terminal galactosyl residue of the
lacto-N-neotetraose epitope has also been demonstrated (9,
17). It is believed that the presence of these epitopes may facilitate
the evasion of the host immune system (14). Phase variation in these
terminal structures has been demonstrated (18), and recent virulence
studies implicate differential expression of these terminal LPS
epitopes in the pathogenesis of N. meningitidis (2,
19, 20, 21).
1
4Gal in Neisseria gonorrhoeae LPS
has been identified and characterized (25). In N. meningitidis, a gene locus named lgtABE, having
homology to the gonococcal locus has been recently shown to be
required for the biosynthesis of the terminal trisaccharide,
Gal
1
4GlcNAc
1
3Gal, of the lacto-N-neotetraose
epitope and to provide the mechanism by which phase variable expression
of the epitope is controlled (18). Mutants generated in each of three
genes in the lgtABE locus were implicated by immunological
and PAGE analysis of derived LPS to have a role in the synthesis of
glycosyltransferase enzymes (18). In this study, the molecular
structures for LPS produced by the three lgt mutant strains
are determined providing, for the first time, definitive evidence that
this genetic locus encodes the glycosyltransferases required for
sequential addition of glycoses to the growing
lacto-N-neotetraose end group. A relationship between the
structures of the LPS elaborated by lgt E and gal
E mutants is established. Moreover, the
-1,4-galactosyltransferase
activity encoded by the lgtB gene and the
-1,3-N-acetylglucosaminyltransferase activity of the
lgtA gene were unequivocally demonstrated by enzymic assay
using synthetic acceptors.
Bacterial Strains
3 lgt B,
3 lgt A, and
3 lgt E; and mutant strains
MC58 gal E and H44/76 gal E. The insertional
mutants containing a kanamycin-resistance (kanr) cassette
in each of the genes of the lgtABE locus were constructed
from N. meningitidis immunotype L3 strain
3 (MC58) as
described previously (18). UDP galactose-deficient strains were
constructed by insertion of kanr cassette in the capsular
polysaccharide biosynthetic locus copy of the galE gene of
N. meningitidis MC58 and H44/76 (22).
H, 2.225 ppm;
C, 31.07 ppm).
Two-dimensional homonuclear proton correlation experiments (COSY) (35)
were measured over a spectral width of 2.3 or 1.3 KHz, using data sets
(t1 × t2) of 256 × 2048 or 512 × 2048 points; 32 or 64 scans were acquired,
respectively. Spectra were processed in magnitude mode with
symmetrization about the diagonal. Two-dimensional nuclear Overhauser
effect experiments (NOESY) (36) were performed using a data set of
256 × 2048 points, a spectral width of 2.3 KHz, a 400 ms mixing
time and 128 scans.
P, 0.0 ppm). 1H-31P
correlations (HMQC) were made in the 1H-detected mode by
using a data matrix of 16 × 1024 points, sweep widths of 10 KHz for 31P and 1.3 KHz for 1H, and a mixing
time of 60 ms.
1cm
1.
= 488 nm. The capillary was a
standard 75 µm × 50-cm bare silica, with the detector at 47 cm.
The capillary was conditioned before each run by washing with 0.2 M NaOH for 2 min, water for 2 min, then 20 mM
sodium dodecyl sulfate/25 mM sodium tetraborate, pH 9.4, for 2 min. Samples were introduced by pressure injection for 2-5 s,
and the separation was performed at 12 kV, 53 µA. Peak integration
was performed with the Beckman System Gold (version 8) software.
3) MC58 (18, 22). LPS was obtained from fermenter-grown
cells by extraction with aqueous phenol (26) followed by
ultracentrifugation of the dialyzed and concentrated aqueous phase.
Meningococcal LPS, representative of the parent strain, has been
reported (5) to be comprised of D-galactose (Gal),
D-glucose (Glc), 2-amino-2-deoxy-D-glucose
(GlcN),
L-glycero-D-manno-heptose
(Hep), and 3-deoxy-D-manno-octulosonic acid
(KDO) in the molar ratio of 2:1:4:2:2. Compositional analysis of
lgt B and lgt A LPS revealed the presence of
these constituent sugars, but significantly less Gal was detected in
both LPS samples as well as a diminished amount of GlcN in
lgt A LPS. Quantitative analysis of the respective core
oligosaccharides indicated the presence of equal molar amounts of Glc
and Gal. Gal was not detected among the complete acid hydrolysis
products of lgt E and gal E LPS indicating the
absence of Gal in these LPS samples.
Fig. 1.
Silver-stained deoxycholate-PAGE of LPS (0.6 µg) from N. meningitidis M982B (immunotype L7)
(lane 1) and MC58 (immunotype L3 strain
3)-derived
mutant strains, lgt B (lane 2), lgt
A (lane 3), lgt E (lane 4), and
gal E (lane 5).
3H]3- and doubly
[M
2H]2- charged ions were observed at m/z
876.1 and m/z 1314.3, respectively. These data indicate
that the composition of the major O-deacylated LPS species
(Mr 2631) contains one less hexose residue than
that predicted for the complete lacto-N-neotetraose epitope
of immunotype L7 LPS-OH (i.e. Mr 2793). The
MS-MS spectrum of lgt B LPS-OH produced by low energy
collisional activation of either the triply or doubly deprotonated ion
afforded a major fragment ion at m/z 951 arising from
cleavage of the KDO-
-glucosamine bond in which the ketosidic oxygen
is retained by the O-deacylated lipid A forming a Y-type
fragment ion (43) (data not shown). Cleavage of this linkage in LPS-OH
to yield the O-deacylated lipid A singly charged ion appears
to be the dominant fragmentation pathway in the negative ion mode (40,
42). The mass of this fragment is in accord with the proposed structure
of N. meningitidis lipid A (10). The ESI-MS and MS-MS data
are consistent with the lgt B LPS containing a truncated
lacto-N-neotetraose chain (GlcN-Gal-Glc) attached to the
basal inner core region of the molecule and this was confirmed by a
detailed NMR analysis of the LPS backbone oligosaccharide (see below).
ESI-MS analysis of lgt A and lgt E LPS-OH gave
triply and doubly charged ions of lower mass (Fig. 2, B and
C) corresponding to consecutive loss of hexosamine (GlcN)
and a hexose (Gal) in the respective LPS samples. As expected (18, 22),
the gal E LPS-OH sample gave an ESI-MS that was similar
to that of lgt E (data not shown). In addition to this major
LPS-OH species (Mr 2265.9), the ESI-MS of the
gal E sample revealed a minor component (5-10% of the
total) containing one extra hexose residue (Mr
2427.6) which was indicated to be Glc from sugar analysis. The ESI-MS
data and the proposed compositions are summarized in Table
I and the structural relationships are presented in Fig.
3.
Fig. 2.
Negative ion ESI-MS of
O-deacylated LPS from N. meningitidis lgt B
(A), lgt A (B), and lgt
E (C). The calculated molecular masses and proposed
structures are given in Table I. Low abundance peaks observed at
[M
3H]3- + 26 and/or [M
3H]3- + 13 corresponding to M + n (39), where
n = 1 or 2, may indicate adducts containing one or two
potassium ions. Minor ions at [M
3H]3-
6, observed particularly in spectrum C, represents M
H2O as previously observed for LPS-OH (39).
Mutant strain
Observed
ion
Molecular mass
Proposed compositionb
(M
3H)3
(M
2H)2
Observed
Calculateda
m/z
Da
lgt
B
876.1
1314.3
2631.0
2631.4
HexNAc2·Hex2·Hep2·PEA1·KDO2·LipidA-OH
lgt
A
808.2
1212.6
2427.4
2428.2
HexNAc1·Hex2·Hep2·PEA1·KDO2·LipidA-OH
lgt
E
754.0
1131.5
2265.0
2266.1
HexNAc1·Hex1·Hep2·PEA1·KDO2·LipidA-OH
gal
E
754.3
1132.0
2265.9
2266.1
HexNAc1·Hex1·Hep2·PEA1·KDO2·LipidA-OH
a
Average mass units were used for calculation of
molecular weight values based on proposed compositions as follows (39):
HexNAc, 203.20; Hex, 162.14; Hep, 192.17; PEA, 123.05; KDO, 220.18;
Lipid A-OH, 953.01.
b
Sugar analysis indicates HexNAc corresponds to
D-GlcNAc; Hex corresponds to one D-Glc and one
D-Gal in Igt B and Igt A LPS, and to
D-Glc only in lgt E and gal E LPS;
and, Hep corresponds to L-glycero-D-manno
heptose.
Fig. 3.
Structure of the
lacto-N-neotetraose epitope in N. meningitidis
O-deacylated LPS represented by immunotype L3 (IT L3)
or L7 (9, 10). See Fig. 6 for the position and configurations of
the linkages of the lacto-N-neotetraose epitope. The
positions at which this epitope is truncated in LPS of lgt
B, lgt A, lgt E, and gal E mutants are
indicated below the structure and the observed
Mr are indicated above. Incremental mass values
for consecutive sugar deletions is also indicated. The sequence of the
sugar residues was confirmed for the fully deacylated lgt B
LPS (present study).
3H]3- and doubly [M
2H]2- charged
ions at m/z 682.7 and 1024.5 corresponding to a
decasaccharide trisphosphate
(Hex2·Hep2·HexN4·KDO2·(H2PO3)3)
as the major molecular species (Mr 2051.2).
Correspondingly, the sample gave ions at m/z 513.7, 684.7, and 1026.4 from [M + 4H]4+, [M + 3H]3+ and
[M + 2H]2+ multiply charged protonated ion species
(Mr 2050.9) in the positive ion spectrum. MS-MS
of the doubly deprotonated ion (m/z 1024.5) afforded a major
fragment ion at m/z 499.1 from the glucosamine disaccharide
bisphosphate derived from the lipid A portion of the molecule pointing
to the presence of a single phosphate substituent in the core
oligosaccharide region. A comparison of these results with the ESI-MS
data obtained for lgt B LPS-OH (Table I) clearly indicates
loss of ethanolamine from the PEA moiety, which is known to be
substituted at O-3 of the penultimate heptose in immunotype L3 LPS (9).
This most likely occurred under the alkaline KOH conditions used in the
deacylation procedure. Elimination of ethanolamine accompanied by
phosphate migration in Haemophilus influenzae LPS under
these reaction conditions has been recently
observed.2
-linked-KDO
residues at 1.84 ppm (t, 1H, H-3ax), 2.00 ppm (t,
1H, H-3
ax) and 2.15 ppm (m, 2H, H-3eq/H-3
eq). The low field region of
the 1H spectrum (5.8-4.4 ppm) was complex indicating the
sample to be a mixture of two related decasaccharides. The anomeric
region of the two-dimensional 1H-13C
correlation map was especially revealing since a doubling of anomeric
1H signals from several residues in the inner core
oligosaccharide region was readily discernable (Fig. 4).
An approximate 60/40 ratio of two decasaccharides is indicated from
estimation of the area of related anomeric 1H signals,
e.g.
-GlcN anomeric protons at 5.42/5.40 ppm. The proton
resonances were assigned by two-dimensional homonuclear correlation
(COSY) (45) and the component monosaccharide units were identified from
the 1H chemical shift (9, 47) and coupling constant values
(48). The chemical shift data (Table II) is consistent
with each D-sugar residue being present in the pyranosyl
ring form. Further evidence for this was obtained from intraresidue NOE
data (Table III) which also served to confirm the
anomeric configurations of the linkages.
Fig. 4.
Heteronuclear two-dimensional
1H-13C chemical shift correlation map of the
anomeric region of the lgt B deacylated LPS oligosacharide
sample. Assignments of the glycose residues are indicated.

Proton resonance
Core oligosaccharide residue
assignmentsa
Deacylated lipid A residue
assignments
-GlcNI
-Gal
-Glc
-HepI
-HepII
-GlcNKDOI
KDOII
-GlcNII
-GlcN-P
H-1
5.00
4.54 4.50
4.58
4.59
5.29 5.28
5.41 5.32
5.42 5.40
4.84
5.75
(J1,2)
(8.1)
(8.9)
(8.9)
(8.9)
(<3) (<3)
(<3)
(<3)
(3.8)
(3.8)
(8.2)
(
)b
H-2
3.12
3.71 3.72
3.48 4.43
4.12 4.15
4.53
4.38
3.38 3.38
3.16
3.48
(J2,3)
(9.9)
(10.0)
(9.3)
(9.5)
(
) (
)(~3)
(~3)
(9.6) (9.6)
(9.6)
(10.0)
H-3
3.70
3.90
~3.64 3.69
4.09 4.11
4.41
4.25
3.92
3.99
2.00/2.15c
1.84/2.15c
3.91
3.92
(J3,4)
(10.5)
(3.4)
(
)
(9.8)(
)
(
)(10.5,10.0d)(10.1)
(9.9)(10.0)

(
)(10.2)
(10.1)
H-4
3.50
4.19
~3.64 3.59
4.29 4.25
4.08
4.45
3.59 3.52
4.22

3.95
3.62
(J4,5)
(
)(~1)
(
)
(
)(
) (
)(9.0) (9.8, 10.0d)
(~10)
(10.0)
(
)
(
)(9.9)
H-5
3.51
3.71
3.53 3.60
4.18 4.28
3.81 3.82
3.89
3.90
4.28

3.63
4.15
H-6
e~3.80f
4.08
4.10






4.12
3.81/4.29f
H-7




3.85
H-8


a
Data recorded on the left are for the
decasaccharide corresponding to the LPS backbone oligosaccharide (9).
Data on the right are for proton resonances shifted (
0.01
ppm) in the decasaccharide which carries a phosphate substitute at the
O-4 position in
-HepII.
b
(
), coupling constant value not determined.
c
H-3 (ax) and H-3 (eq) value, respectively.
d
Value for 3JH,P.
e
, chemical shift unresolved.
f
H-6 and H-6
value.
-HepII.
Anomeric
proton
Observed proton
Partial sequence
Intraresidue NOE
Transglycosidic NOE
ppm
ppm
5.00
(
-GlcNI)3.70
(H-3), 3.51 (H-5)
3.90
(H-3 of
-Gal)GlcNI
1
3Gal
4.54 (
-Gal)3.90 (H-3), 3.71 (H-5)
3.64 (H-4 of
-Glc)Gal
1
4Glc
4.50 (
-Gal*)3.90 (H-3), 3.71 (H-5)
3.59 (H-4 of
-Glc*)
4.58 (
-Glc)3.64 (H-3), 3.53 (H-5)
4.29, 4.08 (H-4, H-6 of
-HepI)Glc
1
4HepI
4.59
(
-Glc*)3.69 (H-3), 3.60 (H-5)
4.25, 4.10 (H-4, H-6 of
-HepI*)
5.29 (
-HepI)4.12 (H-2)
4.27 (H-5 of
-KDOI)HepI
1
5KDOI
5.28 (
-HepI*)4.15
(H-2)
4.28 (H-5 of
-KDOI*)
5.42 (
-GlcN)3.38
(H-2)
4.53 (H-2 of
-HepII)GlcN
1
2HepII
5.40
(
-GlcN*)3.38 (H-2)
4.38 (H-2 of
-HepII*)
5.41
(
-HepII)4.53 (H-2)
4.09, 4.12 (H-3, H-2 of
-HepI)HepII
1
3HepI
5.32 (
-HepII*)4.38
(H-2)
4.11, 4.15 (H-3, H-2 of
-HepI*)
4.84
(
-GlcNII)3.91 (H-3), 3.63 (H-5)
4.29, 3.81 (H-6
, H-6 of
-GlcN-P)GlcNII
1
6GlcN-P
5.75 (
-GlcN-P)3.48
(H-2)
Proton spin-systems corresponding to the monomeric components from an
-glucosamine (
-GlcN), two heptoses (
-HepI,
-HepII), a
-glucose (
-Glc), and a
-galactose (
-Gal) in each of the two
decasaccharides were identified (Table II). In addition to the two KDO
residues, unique single spin-systems were identified for the two
glucosamine residues in the deacylated lipid A region (
-GlcN-P,
-GlcNII) and for the terminal
-linked glucosamine (
-GlcNI).
Correlation of the H-2 resonances with the directly attached
13C resonances (HMQC experiment), which occurred in the
13C-chemical shift region (50-60 ppm) diagnostic of amino
substituted carbons, confirmed the identity of the GlcN residues.
A comparison of the 1H NMR data for the residues listed in
Table II revealed significant differences in chemical shifts and
coupling patterns for the 1H resonances associated with the
residues assigned to
-HepII. This was readily apparent from the
downfield shifted values for
-HepII H-3 (4.41 ppm,
3JH,p ~ 10 Hz) and
-HepII* H-4
(4.45 ppm, 3JH,p ~ 10 Hz), and is
attributed to phosphate substitution at different sites in the two
oligosaccharides. In the 31P NMR spectrum, three signals
were observed at 1.37, 0.55 and
1.84 ppm, of which the latter two
showed strong correlations in the 1H-31P
correlation experiment to H-4 of
-GlcNII and H-1 of
-GlcN-P,
respectively, confirming the presence of monophosphate groups at the
corresponding positions in the GlcN
1
6GlcN moiety. As expected for
the deacylated oligosaccharide derived from the parent strain LPS (9),
a strong correlation was observed between
-HepII H-3 (4.41 ppm) and
one of the 31P resonances (0.55 ppm) in the
1H-31P correlation map indicating substitution
by phosphate of the C-3 position. In the related oligosaccharide, the
phosphate substituent is located at the C-4 position of the heptose as
indicated by the occurrence of a strong correlation between
-HepII*
H-4 (4.45 ppm) and the 31P signal at 1.37 ppm.
The two decasaccharides were shown to have identical sugar residue
sequences from transglycosidic NOE measurements. NOE connectivities
were observed between anomeric and aglyconic protons on contiguous
residues (Table III). Thus, the occurrence of NOEs between the proton
pairs
-GlcNI H-1/
-Gal H-3,
-Gal H-1/
-Glc H-4,
-Glc
H-1/
-HepI H-4, and
-HepI/KDOI H-5 established the partial
sequence of the main chain:
GlcNI
1
3Gal
1
4Glc
1
4HepI
1
5KDOI.
In N. meningitidis LPS (5), HepI forms a branch point to
which the disaccharide GlcN
1
2HepII is attached. This was
confirmed for lgt B LPS from the observed NOEs between
-GlcN H-1/
-HepII H-2 and
-HepII H-1/
-HepI H-3 protons. KDO
in the main chain (KDOI) is known (49, 50) to be substituted at O-4 by
a second unit (KDOII) and to link the core oligosaccharide to the
putative O-deacylated lipid A. The 1H NMR and
ESI-MS data is in accord with this inference and, as expected, a
transglycosidic NOE is observed between H-1of
-GlcNII and the
H-6/H-6
proton pair of
-GlcN-P in the deacylated lipid A region.
The structures of the two decasaccharides are depicted in Table II.
Glycosyltransferase activity of the lgtB gene was
established using a fluorescence-labeled synthetic acceptor in a
capillary electrophoresis-based assay. Cell extracts of MC58 (L3
immunotype strain
3) and the lgt B mutant were used in
glycosyltransferase assays with FCHASE-aminophenyl-
-GlcNAc as an
acceptor molecule. Capillary electrophoresis analysis of the reaction
mixture from MC58 is shown in Fig. 5. Three major peaks
were observed in the reaction mixture from MC58. The fastest migrating
peak (peak 1) was identified as
FCHASE-aminophenyl-
-LacNAc as its migration time (12.9 min.) is
identical to authentic FCHASE-
-LacNAc (data not shown). In addition,
this peak is sensitive to
-galactosidase as shown in Fig.
5B. The second peak having a migration time of 13.1 min
corresponded to FCHASE-aminophenyl-
-GlcNAc. The third peak at 14.6 min results from the action of an endogenous hexosaminidase activity
present in the extracts. The appearance of peak 2 was
dependent on the addition of UDP-Gal and MnCl2 to the
reaction mixture and the presence of
-linked GlcNAc as an acceptor.
This same enzyme activity could be expressed in E. coli
carrying the lgt B gene in an expression vector (data not
shown). The cell extracts of the lgt B mutant strain failed
to catalyze addition of
-Gal to the GlcNAc acceptor (data not
shown).
3) after 3 h incubation with
FCHASE-aminophenyl-
-GlcNAc (A) and following subsequent
treatment of the reaction mixture with
-galactosidase
(B). Peak 1, FCHASE-aminophenyl
-LacNAc;
peak 2, FCHASE-aminophenyl-
-GlcNAc; peak 3,
FCHASE-aminophenol.
A similar analysis was performed for the function of the
lgtA gene. Cell extracts of MC58 had
-N-acetylglucosaminyltransferase activity when
FCHASE-aminophenyl-
-lactose was used as an acceptor and UDP-GlcNAc
was used as a donor. The product peak from this reaction was also shown
to be sensitive to
-N-acetylhexosamidase (data not
shown). Cell extracts of the lgt A mutant were unable to
transfer
-GlcNAc to the lactose acceptor.
The lgtABE genetic locus is required for the
biosynthesis of the lacto-N-neotetraose terminal LPS
structure in N. meningitidis (18). Insertion mutants have
been constructed in each of the three genes in the lgtABE
locus of the immunotype L3 strain (
3) of MC58. In previous work,
immunological and PAGE analysis of LPS from mutants lgt B,
lgt A, and lgt E suggested structural alterations
in lacto-N-neotetraose epitope, but the determination of the
chemical structures of the LPS core oligosaccharide regions had not
been reported. To unequivocally assign functions to lgtABE
locus, determination of the detailed structure of the LPS core
oligosaccharide regions was necessary. In this study, the complete
molecular structure of deacylated lgt B LPS was determined
by electrospray mass spectrometry and detailed NMR analytical methods.
A structural model from this LPS is shown in Fig. 6.
The structure of the N. meningitidis immunotype L3 LPS has
been determined in detail (9). In complete expression of the L3
immunotype, the terminal
-galactose of the
lacto-N-neotetraose epitope is capped by
-2,3-linked
sialic acid residues. LPS of other N. meningitidis
immunotypes, notably L7, also express the
lacto-N-neotetraose epitope, but not the sialylated variant.
LPS from lgt B only differs from that of the L7 immunotype
in that it lacks the terminal
-galactose residue of the
lacto-N-neotetraose epitope (9) and this is consistent with
the observed inability of this mutant strain to bind the type-specific
L3 monoclonal antibody Mn4A8-B2 (18). A comparison of
O-deacylated LPS derived from lgt B with those
from lgt A and lgt E by ESI-MS revealed further
sugar truncations in the lacto-N-neotetraose epitope arising
from respective loss of
-GlcNAc and
-GlcNAc-
-Gal residues. It
was further established that the lgt E LPS is identical to
the major LPS component expressed by the gal E mutant, a
galactose deficient LPS resulting from inactivation of
UDP-Glc-4-epimerase which is required for synthesis of UDP-Gal
(22).
The structural data for the mutant LPS clearly indicate that the
lgtABE locus encodes the glycosyltransferases for the
biosynthesis of lacto-N-neotetraose terminal epitope. A
mutation in the lgtE gene affords the truncated LPS
containing a 1,4-linked
-Glc terminal group attached to
-HepI.
The lgt A mutant which contains a functional lgtE
gene is capable of adding
-Gal in a 1,4-linkage to this
-Glc to
form the terminal lactose structure. Thus, the lgtE gene
encodes for a
-galactosyltransferase. It is worthy to note that the
structure of the lgt A mutant LPS is identical to that
elaborated by immunotype L8 (5). Correspondingly, the lgt B
mutant containing a functional lgtA gene is capable of
adding
-GlcNAc in a 1,3-linkage to the terminal
-Gal of the
lactose epitope. It follows that the lgtA gene encodes the
specific
-N-acetylglucosamine transferase for synthesis
of the GlcNAc
1
3Gal terminal unit. Finally, the parent immunotype
L3 strain (MC58), which contains a functional lgtB gene, is
capable of elaborating the complete lacto-N-neotetraose unit
which indicates that this gene encodes the
-galactosyltransferase
for catalyzing addition of the 1,4-linked
-Gal to the terminal
-GlcN. The function of this gene was firmly established by
demonstrating
-galactosyltransferase enzyme activity with a
synthetic
-GlcNAc acceptor. Correspondingly, the
-N-acetylglucosaminyltransferase activity encoded by
lgtA was confirmed with a synthetic
-lactose acceptor,
whereas experiments to assay transferase activity of the
lgtE gene using a synthetic
-Glc acceptor were
unsuccessful. It is likely that the latter enzyme has a more stringent
acceptor specificity and requires
-Glc linked to heptose, precluding
recognition of our synthetic acceptor.
Identification and characterization of the lgt genetic locus was first reported (25) in N. gonorrhoea and it was postulated that the genes within the locus encoded for glycosyltransferases involved in the biosynthesis of lacto-N-neotetraose and its GalNAc containing analogue in gonococcal LPS. In meningococci, the lgtABE locus contains three genes which are homologous to the gonococcal lgtA, lgtB, and lgtE genes (18). The role of these genes in meningococcal LPS phase variation has been demonstrated (18) and it was recently shown (51) that LPS phase variation in N. gonorrhoea occurs by a similar genetic mechanism. The evidence presented in the present study unequivocally demonstrates the glycosyltransferase functions of this Neisserial gene locus. The site where the specific transferases function in the biosynthesis of the lacto-N-neotetraose epitope is shown in Fig. 6.
Recently Lee et al. (52) reported that a meningococcal
gal E mutant (strain NMB-SS3) expresses
glycosyltransferase activity capable of adding one or two additional
glucose residues to the immunotype L2 inner core LPS structure, and it
was suggested that this provided an alternative biosynthetic pathway to
the normal wild-type lacto-N-neotetraose structure. The
structure of the LPS of immunotype L2 is similar to that of immunotype
L3 in that it contains the lacto-N-neotetraose
oligosaccharide epitope and the sialylated analogue, but differs in the
inner core region where it contains an additional
-Glc moiety that
is 1,3-linked to HepII (8). In the present study, the gal E
mutant of immunotype L3 (strains MC58 and H44/76) produced, in addition
to the expected LPS,
(GlcNAc1·Glc1·Hep2·PEA1·KDO2·LipidA),
a minor amount of a second LPS species containing one additional
glucose (<10% by ESI-MS).3 The extra
glucose containing species,
GlcNAc1·Glc2·Hep2·PEA1·KDO2·LipidA,
was not detectable by ESI-MS in the LPS-OH sample obtained from the
lgt E mutant, a strain known to contain a mutation in a
single gene (22); only the
GlcNAc1·Glc1·Hep2·PEA1·KDO2·LipidA-OH
species was observed (Figs. 2C and 3). These data are
consistent with either the galactosyltransferase encoded by the
lgtE gene possessing the capability to mediate the addition
of
-Glc to the Glc
1
4HepI acceptor at low levels, or the
presence of a meningococcal glycosyltransferase which manifests itself
when there is a build up of this acceptor.
Mutational analysis of genes involved in LPS biosynthesis has involved
examination of changes in the LPS reactivity with monoclonal
antibodies, their migration patterns on PAGE and, in some cases,
measurement of radioactive sugar incorporation into endogenous LPS
glycosyltransferase acceptors (22, 25, 44). Very little data exist on
the measurement of glycosyltransferase activity involved in LPS
biosynthesis. Our enzymatic assay of
-1,4-galactosyltransferase
activity with the parent MC58
3, the lgt B mutant, and
the recombinant E. coli expressing lgtB
unequivocally demonstrates this gene encodes a
-1,4-galactosyltransferase which requires a
-linked GlcNAc as an
acceptor. To the best of our knowledge, this represents the first
correlation of a proposed bacterial glycosyltransferase gene with that
enzymatic activity using a synthetic glycosyltransferase acceptor. We
are now in the process of characterizing this enzyme.
To whom correspondence should be addressed: Institute for
Biological Sciences, National Research Council Canada, 100 Sussex Dr.,
Ottawa, Ontario, K1A 0R6. Tel.: 613-990-0854; Fax: 613-941-1327;
E-mail: RICHARDS{at}biologysx.lan.nrc.ca.
We thank D. W. Griffith for large-scale production of cells, N. Eichler for isolation of LPS, F. Cooper for GLC-MS analyses, D. Krajcarski for ESI-MS, and A. Cunningham for CE analyses. We also thank Dr. H.J. Jennings for providing us with an authentic sample of N. meningitidis immunotype L7, and Dr. H. Masoud for helpful discussions.