Identification and localization of glycine in the inner core
lipopolysaccharide of
Neisseria meningitidis
Andrew D. Cox, Jianjun Li and James C. Richards
Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada
The amino acid glycine is identified as a component of the
inner core oligosaccharide in meningococcal lipopolysac-
charide (LPS). Ester-linked glycine residues were consis-
tently found by mass spectrometry experiments to be located
on the distal heptose residue (HepII) in LPS from several
strains of Neisseria meningitidis. Nuclear magnetic resonance
studies confirmed and extended this observation locating the
glycine residue at the 7-position of the HepII molecule in L3
and L4 immunotype strains.
Keywords: Neisseria meningitidis; lipopolysaccharide;
glycine; NMR; mass spectrometry.
The LPS of Neisseria meningitidis contains a core
oligosaccharide unit with an inner core di-heptose-N-
acetyl-glucosamine backbone, wherein the two
L
-glycero-
D
-manno-heptose (Hep) residues can provide a point of
attachment for the outer core oligosaccharide residues [1].
Meningococcal LPS has been classified into 12 distinct LPS
immunotypes (L1-L12), originally defined by monoclonal
antibody (mAb) reactivities [2], but further defined by
structural analyses. The structures of LPS from immuno-
types L1/6 [3,4], L2 [5], L3 [6], L4/7 [7], L5 [8] and L9 [9]
have been elucidated. The structural basis of the immuno-
typing scheme is governed by the location of a phospho-

strains [3,5,7,8]. In this study we identify and structurally
characterize the presence of ester-linked glycine residues in
the core oligosaccharide of meningococcal LPS.
MATERIALS AND METHODS
Growth of organism and isolation of LPS
N. meningitidis immunotype strains L3 galE (NRCC #4720)
and L4 galE (NRCC #4719) and clinical strains BZ157 galE
B5+ (NRCC #6094) and NGH15 B5+ and B5– (NRCC
#6092 and 6093) were all grown in a 28-L fermenter as
described previously [11] yielding  100 g wet wt. of cells
from each growth. Strains BZ157 and NGH15 are from the
culture collection of E. R. Moxon. LPS was extracted by
the hot phenol/water method as described previously and
purified from the aqueous phase by ultracentrifugation
(45K, 4 °C, 5 h) [11] yielding  200 mg in each case.
O-deacylated LPS was prepared as described previously [13]
in  50% yield from the LPS. Core oligosaccharides were
prepared according to the following procedure. LPS was
hydrolysed at 100 °C for 2 h in 2% acetic acid. Insoluble
material was removed by centrifugation (6000 g, 20 min.)
and the supernatant solution was lyophilized yielding core
oligosaccharide in  50% yield.
Mass spectrometry
All ES-MS and CE-MS analyses were carried out as
described previously [12].
NMR spectroscopy
Nuclear magnetic resonance experiments were performed
on Varian INOVA 500, 400 and 200 NMR spectrometers as
Correspondence to A. D. Cox, Institute for Biological Sciences,
National Research Council, 100, Sussex Drive, Ottawa, ON K1A 0R6,

examined by ES-MS (Fig. 1). Several ions were observed
and the compositions for each glycoform are listed in
Table 1. Typical ions differing by 18 a.m.u. were observed
for each glycoform and corresponded to reducing end
anhydro and intact Kdo species due to rearrangements of
the Kdo molecule during hydrolysis. L3 galE core oligo-
saccharide consisted of two sets of ions differing by 57
a.m.u. (Fig. 1A). The ion at m/z 1110 corresponds to a
composition of Hex, 2Hep, HexNAc, PEtn, Kdo as would
be expected for the galE mutant, as further extension
beyond the first glucose (GlcI) at the proximal heptose
residue (HepI) is precluded due to the unavailability of
galactose in this genetic background. A second ion, 57
a.m.u. higher, of approximately equal intensity was
observed at m/z 1167. A mass of 57 a.m.u. corresponds to
the amino acid glycine (Gly) that has been reported
previously in the LPS of several Gram-negative bacteria
[15] but not in N. meningitidis.TheES-MSofL4galE core
oligosaccharide reflected a more complex mixture (Fig. 1B).
In addition to glycoforms corresponding to the presence or
absence of glycine, species with and without an O-acetyl
group and the PEtn residue were also observed (Table 1).
As indicated in Table 1 for L4 galE core oligosaccharide,
the ions at m/z 969 and 987 correspond to the expected core
oligosaccharides without the PEtn moiety. Ions at m/z 1011
and 1029 correspond to the same core oligosaccharides but
with an additional O-acetyl group. Ions at m/z 1092 and
1110 correspond to the expected inner core structure
without any modifications corresponding to a composition
of Hex, 2Hep, HexNAc, PEtn, Kdo. The final two sets of

Table 1. Negative ion ES-MS data and proposed compositions of core oligosaccharide from N. meningitidis strains. L3 galE,L4galE,NGH15B5
–
and B5
+
and BZ157 galE B5
+
. Average mass units were used for calculation of molecular mass based on proposed composition as follows: Glc,
162.15; Hep, 192.17; GlcNAc, 203.19; Kdo, 220.18; PEtn, 123.05. Gly, 57.05; OAc, 42.00.
Strain
Observed Ions (m/z) Molecular Mass (Da)
Relative
intensity
Proposed composition
(M-H)
–
(M-2H)
2–
Observed Calculated
L3 galE 1091.4 – 1092.3 1092.9 0.3 Glc, GlcNAc, 2Hep, PEtn, aKdo
1109.3 554.2 1110.5 1110.9 1.0 Glc, GlcNAc, 2Hep, PEtn, Kdo
1148.5 – 1149.5 1149.9 0.5 Gly, Glc, GlcNAc, 2Hep, PEtn, aKdo
1166.4 582.5 1167.4 1167.9 0.9 Gly, Glc, GlcNAc, 2Hep, PEtn, Kdo
L3 galE O-deac 1091.5 – 1092.6 1092.9 0.2 Glc, GlcNAc, 2Hep, PEtn, aKdo
1109.4 554.2 1110.5 1110.9 1.0 Glc, GlcNAc, 2Hep, PEtn, Kdo
L4 galE 968.3 – 969.3 969.8 0.2 Glc, GlcNAc, 2Hep, aKdo
986.4 – 987.3 987.8 0.2 Glc, GlcNAc, 2Hep, Kdo
1010.2 – 1011.4 1011.8 0.4 OAc, Glc, GlcNAc, 2Hep, aKdo
1028.1 – 1029.1 1029.8 0.6 OAc, Glc, GlcNAc, 2Hep, Kdo
1091.5 – 1092.4 1092.9 0.3 Glc, GlcNAc, 2Hep, PEtn, aKdo
1109.3 – 1110.4 1110.9 0.3 Glc, GlcNAc, 2Hep, PEtn, Kdo

Ó FEBS 2002 Glycine in meningococcal LPS (Eur. J. Biochem. 269) 4171
also performed on two other meningococcal strains of
clinical origin and the compositions of the glycoforms
observed are listed in Table 1. In each case where glycine
was identified, MS-MS studies located this residue to the
HepII molecule (data not shown).
The glycine residue was assumed to be ester-linked
because it had not been observed in O-deacylated material
examined from these samples [6,7]. Base-labile ester-linked
residues are readily removed under the alkaline conditions
for O-deacylation. To confirm this, O-deacylated LPS
from L3 galE was hydrolysed with 2% acetic acid in
order to afford the O-deacylated core oligosaccharide.
ES-MS analysis for this sample revealed a simplified
spectrum without ions corresponding to glycine con-
taining glycoforms (Table 1), thus confirming that the
glycine residue is attached to the core oligosaccharide via
an ester linkage.
In order to confirm and further characterize the presence
of the glycine residue, NMR studies were performed. Initial
experiments on commercial glycine suggested the
1
Hand
13
C resonances of the -CH
2
- group were at 3.56 and
41.5 p.p.m., respectively. The core oligosaccharide from L3
galE was chosen for NMR studies as the MS data had
indicated a less complex mixture than the core oligosaccha-

charide to give the resulting fragment ions.
Diagnostic ions that localized the glycine
residue and the O-acetylation status of the
GlcNAc residue are indicated in the inset
figures.
4172 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002
with relay between the carbonyl carbon and the CH
2
protons of the glycine moiety, thus confirming the identi-
fication of glycine.
NMR provided evidence for the exact location of the
glycine moiety on the HepII residue of the inner core
oligosaccharide. There are only a few positions available for
attachment on this substituted residue in the meningococcal
LPS inner core. Linkage to HepI occurs from the anomeric
position of HepII, the N-acetyl-glucosamine residue substi-
tutes HepII at the 2-position and PEtn substitutes HepII at
the 3-position in immunotype L3 and at the 6-position in
immunotype L4. Ring formation occurs at position 5 in this
pyranose sugar. There are therefore only two locations
available for the glycine moiety namely the 4-position or the
7-position of the HepII residue common to both immuno-
types. If the glycine residue was linked to the 4-position of
HepII one might expect the CH
2
protons of the glycine
molecule to be split by the plane of symmetry from the
carbohydrate ring. However, as the CH
2
protons appear as

1
HHMQCand
31
P-
1
H HMQC-TOCSY
experiments (Fig. 4) were performed on O-deacylated L4
galE oligosaccharide. Oligosaccharide derived from the L4
immunotype LPS was chosen for this analysis because of
the inherent difficulties in accessing the exocyclic protons in
a heptosyl spin-system from the anomeric proton resonance.
The HepII residue of immunotype L4 LPS is substituted at
the 6-position by a PEtn residue and therefore this
configuration was taken advantage of in
31
P-
1
HNMR
experiments [7]. The
31
P-
1
H HMQC experiment revealed
cross-peaks from the
31
P-resonance of the PEtn molecule at
the 6-position of HepII to
1
H-resonances at 4.58 p.p.m.
which is characteristic for substitution of the 6-position of

5.51 4.37 4.40 4.13 3.70
a
Data for the O-deacylated core oligosaccharide.
Fig. 4. Region of the 2D-
31
P-
1
H-HMQC-TOCSY
NMR spectrum of the core oligosaccharide from
N. meningitidis strain L4 galE.
Ó FEBS 2002 Glycine in meningococcal LPS (Eur. J. Biochem. 269) 4173
HepII with PEtn and resonances at 3.32 and 4.18 p.p.m.
diagnostic for the ethanolamine protons distal and proximal
to the phosphorus atom, respectively [7]. The
31
P-
1
H
HMQC-TOCSY experiment revealed additional cross
peaks at 3.85 and 3.75 p.p.m. consistent with nonsubstituted
H-7
1
H-resonances [7]. When these experiments were
performed on L4 galE oligosaccharide the
31
P-
1
H cross-
peaks observed were identical, apart from an addi-
tional

elaboration of the amino acid glycine, and it is interesting
to note that in strain BZ157 galE a glycine residue is
elaborated at this HepII residue even in glycoforms that
contain two PEtn moieties (data not shown). It is important
to note that incorporation of glycine is not simply a
consequence of the galE mutation. The LPS from the parent
strain of immunotype L3 and its lgtB and lgtA mutants also
elaborated a glycine residue (data not shown). Glycine has
been identified in the LPS of several Gram-negative bacteria
including Escherichia, Salmonella, Hafnia, Citrobacter and
Shigella species [16], but in each case the relevance of this
finding is unclear. Recently glycine was identified as a
common component in the LPS of Haemophilus influenzae
[17]. However in H. influenzae the glycine residue is not
found consistently in one location in the inner core
oligosaccharide as appears to be the case for N. meningitidis.
Depending on the strain of H. influenzae glycine could be
found at each of the heptose residues of the inner core tri-
heptosyl group or on the Kdo residue. Glycine has also been
localized in the core oligosaccharide of Proteus mirabilis
serotype O28 [18]. The glycine moiety was found to be
amide-linked to the amino group of a glucosamine residue.
Interestingly in this arrangement the -CH
2
- protons of the
glycine moiety are split and are found at 3.80 and
4.00 p.p.m., which when compared to the sharp singlet at
3.56 p.p.m. observed for the -CH
2
- protons of the glycine

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