Báo cáo khoa học: Identification of a novel inner-core oligosaccharide structure in Neisseria meningitidis lipopolysaccharide docx

Identification of a novel inner-core oligosaccharide structure
in
Neisseria meningitidis
lipopolysaccharide
Andrew D. Cox
1
, J. Claire Wright
2
, Margaret A. J. Gidney
1
, Suzanne Lacelle
1
, Joyce S. Plested
2
,
Adele Martin
1
, E. Richard Moxon
2
and James C. Richards
1
1
Institute for Biological Sciences, National Research Council, Ottawa, Canada;
2
Institute for Molecular Medicine,
John Radcliffe Hospital, University of Oxford, UK
The structure of the lipopolysaccharide (LPS) from three
Neisseria meningitidis strains was elucidated. These strains
were nonreactive with mAbs that recognize common inner-
core epitopes from meningococcal LPS. It is well established
that the inner core of meningococcal LPS consists of a

diheptosyl-N-acetylglucosamine unit, in which the distal
heptose unit (Hep II) can carry PEtn at the 3 or 6 position or
not at all, and the proximal heptose residue (Hep I) is sub-
stituted at the 4 position by a glucose residue. Additional
substitution at the 3 position of Hep II with a glucose residue
is also a common structural feature in some strains. The
structures of the O-deacylated LPSs and core oligosaccha-
rides of the three chosen strains were deduced by a combi-
nation of monosaccharide analysis, NMR spectroscopy and
MS. These analyses revealed the presence of a structure not
previously identified in meningococcal LPS, in which an
additional b-configured glucose residue was found to sub-
stitute Hep I at the 2 position. This provided the structural
basis for the nonreactivity of LPS with these mAbs. The
determination of this novel structural feature identified a
further degree of variability within the inner-core oligosac-
charide of meningococcal LPS which may contribute to the
interaction of meningococcal strains with their host.
Keywords: lipopolysaccharide; mass spectrometry; Neisse-
ria meningitidis; NMR; oligosaccharide.
The lipopolysaccharide (LPS) of Neisseria meningitidis
contains a core oligosaccharide unit with a conserved
inner-core diheptose-N-acetylglucosamine backbone, in
which 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 immunotypes (L1–L12), originally
defined by 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-
ethanolamine (PEtn) moiety on the distal heptose residue
(Hep II) at either the 3 or 6 position, or absent, or at
both positions simultaneously [10]. The length and nature
of oligosaccharide extension from the proximal heptose
residue (Hep I) and the presence or absence of a glu-
cose sugar at Hep II also dictates the immunotype. The
enzyme UDP glucose 4-epimerase (GalE) is essential for
N. meningitidis to synthesize UDP-Gal for incorporation of
galactose into its LPS and is encoded by the gene galE
[11]. The absence of galactose residues from the conserved
inner-core structure of meningococcal LPS has led to
the utilization of mutants defective in the enzyme, resulting
in truncation of the oligosaccharide chains of the LPS at
the glucose residue at Hep I, and galE mutants have been
used by our group to derive mAbs to inner-core LPS
epitopes [12] (unpublished data). Identified in this way,
mAbs with an absolute requirement for PEtn at the 3
position [12], or the 6 position (unpublished data) of Hep II
were developed. mAbs were also produced that had
specificities for PEtn at the 6 position of Hep II coupled
with the presence of a glucose residue at the 3 position of
Hep II, glucose at the 3 position of Hep II coupled with the
absence of PEtn at the 6 position of Hep II and specific for
an epitope where there was no substitution with PEtn or

glucose at Hep II (unpublished data). During the course of
these studies, we specifically selected LPS from clinical
isolates that were not reactive with these mAbs. Structural
analysis of meningococcal clinical isolates revealed an
additional and uniquely located glucose residue in the core
oligosaccharide that had not previously been identified in
meningococcal LPS.
Materials and methods
Growth of organism and isolation of LPS
N. meningitidis strains 1000 (NRCC No. 6156) and 1000
galE (NRCC No. 6109) were grown in a 28-L fermenter as
described previously [12], yielding  100gwetweightfor
each growth. Strains 425/93 and NM115 are from the
Correspondence to A. D. Cox, Institute for Biological Sciences,
100 Sussex Drive, National Research Council, Ottawa,
ON K1A 0R6, Canada.
Fax: + 1 613 952 9092, Tel.: + 1 613 991 6172,
E-mail:
Abbreviations: LPS, lipopolysaccharide; PEtn, phosphoethanolamine;
ESI, electrospray ionization; HSQC, heteronuclear single quantum
coherence.
(Received 21 January 2003, revised 18 February 2003,
accepted 24 February 2003)
Eur. J. Biochem. 270, 1759–1766 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03535.x
culture collection of E. R. Moxon (University of Oxford,
UK) and were grown on BHI agar plates as described [12].
Strain 1000 originates from a global collection of 34
representative serogroup B strains [13]. Strain 425/93 is a
serogroup B carriage strain from the collection of P. Kriz
(National Institute of Public Health, Prague, Czech Repub-

lic) isolated from the Czech Republic in 1993 [14]. Strain
NM115 is a serogroup B strain from R. Heyderman’s group
(Imperial College, London, UK) [15]. LPS was extracted
from the fermenter-grown strains by the hot phenol/water
method as described previously and purified from the dia-
lysed aqueous phase by ultracentrifugation (45 000 r.p.m.,
4 °C, 5 h) after treatment with DNase, RNase and pro-
teinase K [12], yielding  200 mg LPS for each strain. LPS
was extracted from the plate-grown strains by the hot
phenol/water method and ethanol precipitation of the
aqueous phase as described [12], yielding  12 mg LPS for
each strain. O-Deacylated LPS (LPS-OH) was prepared as
described previously [16], with yields of  50%. Core
oligosaccharide was prepared by the following procedure.
LPS was hydrolysed at 100 °C for 2 h in 2% acetic acid.
Insoluble material was removed by centrifugation
(8000 r.p.m., 20 min), and the supernatant was lyophilized,
resulting in  50% yield of core oligosaccharide.
Analytical methods
Sugars were determined as their alditol acetate derivatives
by GLC-MS as described previously [16].
Mass spectrometry
All electrospray ionization (ESI)-MS and capillary electro-
phoresis (CE)-ESI-MS analyses were carried out as
described previously [17].
NMR spectroscopy
NMRexperimentswereperformedonVarianINOVA500
and 400 NMR spectrometers as described previously [10].
Results
LPS was isolated from plate-grown (425/93 and NM115)

or fermenter-grown (1000 and its galE mutant) strains by
standard methods. Sugar analysis of the LPS-derived
alditol acetates from the parent strains revealed glucitol,
galactitol, glucosaminitol and
L
-glycero-
D
-manno-heptitol
in approximately equimolar ratios. Sugar analysis of the
LPS-derived alditol acetates from the galE mutant of
strain 1000 revealed glucitol, glucosaminitol and
L
-gly-
cero-
D
-manno-heptitol in approximately equimolar ratios.
O-Deacylated LPS (LPS-OH) from all strains was
prepared by hydrazinolysis, and initial analyses were
carried out by negative-ion ESI-MS and CE-ESI-MS
(Table 1). MS-MS enabled the size of the core oligosac-
charide and lipid A moiety to be deduced. A consistent
variation in the size of the lipid A moiety was again
observed; this is due to a difference in the phosphory-
lation pattern of the lipid A region (unpublished data).
MS analysis indicated that there was no PEtn in the core
oligosaccharide, and this was consistent with these strains
not reacting with mAbs requiring the presence of PEtn in
the core oligosaccharide (unpublished data). Sialylated
glycoforms were only observed in strains NM115 and
425/93. 4 Hex and sialylated 4 Hex were the major

glycoforms observed for all three strains but not for the
galE mutant strain, and indeed were the only glycoforms
observed for strain NM115, whereas 3 Hex glycoforms
were also observed for strains 425/93 and 1000 (Table 1).
Core oligosaccharide from the 1000 galE mutant strain
was also prepared and examined by MS. A range of
molecular masses was found for the core oligosaccharide
consistent with a composition of Kdo, 2Hep, GlcNAc,
2Glc with nonstoichiometric substitution with glycine and
acetyl groups as observed previously for meningococcal
LPS (Table 1) [18].
To elucidate the exact locations and linkage patterns of
the oligosaccharide chains in the LPS, NMR studies were
performed on the three LPS-OHs from the parent strains.
LPS-OH from strain 425/93 (Fig. 1) and strain NM115
gave good
1
H-NMR spectra simply by dissolving in D
2
O.
However, LPS-OH from strain 1000 initially gave a poor
1
H-NMR spectrum, which was better resolved on the
addition of deuterated SDS (5 mg) and EDTA (0.5 mg).
The
1
H-NMR spectra of the three LPS-OH samples were
very similar. However, the anomeric signals of the lipid A
amino sugars were only weakly visible for the LPS-OH
from strain 1000, presumably because of extensive aggre-

gation of this region of the molecule to suppress their
signals.
1
H resonances of the LPS-OH from the three
strains were assigned by COSY and TOCSY experiments.
Figure 2 shows a region of the TOCSY spectrum for the
O-deacylated LPS from strain 425/93. Assignments were
made by comparison with reported data for other
meningococcal oligosaccharides [5–8,10], and are summar-
ized in Table 2. In addition to the assignments tabulated,
peaks corresponding to the axial ( 1.80 p.p.m.) and
equatorial ( 2.75 p.p.m.) H-3 protons of the sialic acid
residue were observed in the
1
H-NMR spectra of strains
425/93 and NM115. Peaks corresponding to the acetyl
groups of the N-acetylglucosamine residues and the
equatorial and the axial H-3 protons of the Kdo residues
were unresolved because of overlap with the N-linked
fatty acid residues and the axial H-3 resonance of the
sialic acid, respectively.
In the representative spectrum of the LPS-OH from
strain 425/93, spin systems arising from heptose residues
(Hep I and Hep II) were readily identified from their
anomeric
1
H resonances at 5.45 and 5.42 p.p.m. (Hep I)
and 5.54 p.p.m. (Hep II) and from the appearance of
their spin systems, which pointed to manno-pyranosyl
ring systems. The heterogeneity observed for the ano-

meric proton of Hep I was thought to be due to
variation in phosphate substitution in the lipid A region
of the molecule (unpublished data). The a-configurations
were evident for the heptosyl residues from the occur-
rence of intraresidue NOEs between the H-1 and H-2
resonances only. The remaining resolved residues in the
a-anomeric region at 5.07 and 5.39 p.p.m. and a minor
signal at 5.50 p.p.m. were determined to be gluco-
pyranose amino sugars, from the appearance of their
spin systems and the fact that the H-2 resonances of 3.89
and 3.92 p.p.m. correlated in a
13
C-
1
H heteronuclear
1760 A. D. Cox et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Table 1. CE-ESI-MS data and proposed compositions of O-deacylated LPS and core oligosaccharide (OS) from N. meningitidis strains 1000, 425/93 and NM115. Average mass units were used for calculation
of molecular mass based on proposed composition as follows: Sial, 291.00; Hex, 162.15; Hep, 192.17; HexNAc, 203.19; Kdo, 220.18; OAc, 42.00; Gly, 57.00. The average molecular mass of the O-deacylated
lipid A (Lipid A-OH) is as indicated. Lipid A-OH consists of two glucosamine residues each bearing an N-linked 3-OH C
14:0
fatty acid and a phosphate group. Variation in lipid A-OH sizes observed are due
to the presence of an additional phosphate group (1032), and additional PEtn moiety (1075), an additional phosphate group and an additional PEtn moiety (1155) and one additional phosphate group and
two additional PEtn moieties (1278). Relative intensity of each glycoform/phosphoform is expressed relative to the largest peak.
Strain
Observed ions (m/z) Molecular mass (Da)
Relative intensity Lipid A
a
Core OS Proposed composition(M)3H)
3–
(M)2H)

2–
(M + H)
+
Observed Calculated
1000wt 943.0 1415.3 – 2832.6 2831.7 1.0 952 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
O-deac – 1334.0 – 2669.8 2669.5 0.2 952 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1000 galE – 1151.3 – 2304.5 2304.5 1.0 952 1352 2Hex, HexNAc, 2Hep, 2Kdo, Lipid A-OH
O-deac
1000 galE
core – – 1133.0 1132.0 1132.0 0.2 – – 2Hex, HexNAc, 2Hep, aKdo
OS – – 1151.0 1150.0 1150.0 0.3 – – 2Hex, HexNAc, 2Hep, Kdo
– – 1175.0 1174.0 1174.0 0.5 – – 2Hex, HexNAc, 2Hep, aKdo, OAc
– – 1190.0 1189.0 1189.0 0.1 – – 2Hex, HexNAc, 2Hep, aKdo, Gly
– – 1193.0 1192.0 1192.0 1.0 – – 2Hex, HexNAc, 2Hep, Kdo, OAc
– – 1208.0 1207.0 1207.0 0.1 – – 2Hex, HexNAc, 2Hep, Kdo, Gly
– – 1232.0 1231.0 1231.0 0.6 – – 2Hex, HexNAc, 2Hep, aKdo, OAc, Gly
– – 1250.0 1249.0 1249.0 0.6 – – 2Hex, HexNAc, 2Hep, Kdo, OAc, Gly
425/93 889 – – 2670 2669.5 0.2 952 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
O-deac 916 – – 2750 2759.5 0.1 1032 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
930 – – 2793 2792.5 0.2 1075 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
957 – – 2873 2872.5 0.1 1155 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
998 – – 2996 2995.5 0.1 1278 1718 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
943 – – 2832 2831.7 0.2 952 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
970 – – 2912 2911.7 0.2 1032 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
984 – – 2955 2954.7 0.6 1075 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1011 – – 3035 3034.7 0.6 1155 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1052 – – 3158 3157.7 0.2 1278 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
986 – – 2961 2960.5 0.1 952 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1013 – – 3041 3040.5 0.1 1032 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1027 – – 3084 3083.5 0.2 1075 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH

1054 – – 3164 3163.5 0.2 1155 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1095 – – 3287 3286.5 0.1 1278 2009 Sial, 3Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1040 – – 3123 3122.7 0.4 952 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1067 – – 3203 3202.7 0.5 1032 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1081 – – 3246 3245.7 0.8 1075 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1108 – – 3326 3325.7 1.0 1155 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1149 – – 3449 3448.7 0.3 1278 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
Ó FEBS 2003 Novel inner-core structure in meningococcal LPS (Eur. J. Biochem. 270) 1761
single quantum coherence (HSQC) experiment with
13
C
resonances of  56.0 p.p.m., this
13
C chemical shift being
diagnostic of amino-substituted carbons. The signals at
5.39 and 5.50 p.p.m. were attributable to the a-glucos-
amine residue of lipid A. The heterogeneity of this
residue was probably due to variation in phosphate
substitution patterns in the lipid A region of the molecule
(unpublished data). There were no other residues in the
a-anomeric region, and of particular interest was the
absence of an a-glucose residue that is often found
substituting the 3 position of the Hep II residue in mAb
B5 nonreactive strains such as immunotype strains L2 [5]
and L5 [8] and clinical strain BZ157 [10]. The remainder
of the anomeric resonances in the low-field region (4.45–
6.00 p.p.m.) of the spectrum were all attributable to
b-linked residues by virtue of their chemical shifts and, in
the case of resolved residues, their high J
1,2

( 8Hz)
coupling constants. Three of these resonances at 4.72
(GlcNAc), 4.57 (Glc I) and 4.50 (Glc II) p.p.m. were
assigned to the gluco-configuration from the appearance
of their spin systems. The resonance at 4.72 p.p.m. was
attributed to an amino sugar because its H-2 resonance
correlatedina
13
C-
1
H HSQC experiment with a
13
C
resonance of  55 p.p.m. The remaining two resonances
in the low-field region at 4.55 (Gal II) and 4.45
(Gal I) p.p.m. were assigned to galacto-pyranosyl residues
from the appearance of their characteristic spin systems
to the H-4 resonance in a TOCSY experiment.
The sequence of glycosyl residues of the LPS-OH from
strain 425/93 was determined from interresidue
1
H-
1
H
NOE measurements between anomeric and aglyconic
protons on adjacent glycosyl residues (Table 2). Thus a
lacto-N-neotetraose oligosaccharide unit attached to the
proximal heptose residue was readily identified, as was
the inner-core diheptosyl moiety substituted with
N-acetylglucosamine. These are common structural motifs

which have been identified in most N. meningitidis
immunotypes. Intriguingly, a novel NOE contact was
observed between the anomeric resonance of the Glc II
residue at 4.50 p.p.m. and the H-2 resonance of the
Hep I residue at 4.20 p.p.m. (Fig. 3A). Similarly there
was a NOE connectivity between the H-1 resonance of
the Hep I residue at 5.45 p.p.m. and the H-1 resonance
of the Glc II resonance at 4.50 p.p.m. (Fig. 3B). Taken
together these NOE data suggested that the Hep I
residue was also substituted at the 2 position by the
Glc II residue. This behaviour has been observed previ-
ously for a b-Glc residue replacing a heptose residue at
the 2 position [19], because of the proximity of the
heptose H-1 proton enabling a NOE effect between the
anomeric protons. This structural arrangement has not
been previously observed in meningococcal LPS. Con-
firmatory data for this novel linkage were obtained from
a2D
13
C-
1
H HSQC experiment in which the
1
H
resonance for the H-2 proton of Hep I at 4.20 p.p.m.
gave a
13
C cross-peak at 78.0 p.p.m. consistent with
substitution at the 2 position of this Hep I residue
(Fig. 4), when compared with a

13
C chemical shift of
 69–71 p.p.m. for the H-2 resonance of the Hep I
residue from the meningococcal immunotype strains LPS
[6,7]. Additional evidence for a novel substitution pattern
at Hep I was provided by an alteration in the inter-NOE
Table 1. (Continued).
Strain
Observed ions (m/z) Molecular mass (Da)
Relative intensity Lipid A
a
Core OS Proposed composition
(M)3H)
3–
(M)2H)
2–
(M + H)
+
Observed Calculated
NM115 943 – – 2832 2831.7 0.4 952 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
O-deac 970 – – 2912 2911.7 0.3 1032 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
984 – – 2955 2954.7 0.8 1075 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1011 – – 3035 3034.7 0.3 1155 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1052 – – 3158 3157.7 0.2 1278 1880 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1040 – – 3123 3122.7 0.7 952 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1067 – – 3203 3202.7 0.4 1032 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1081 – – 3246 3245.7 1.0 1075 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1108 – – 3326 3325.7 0.4 1155 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
1149 – – 3449 3448.7 0.1 1278 2171 Sial, 4Hex, 2HexNAc, 2Hep, 2Kdo, Lipid A-OH
a

As determined by MS-MS analysis.
1762 A. D. Cox et al.(Eur. J. Biochem. 270) Ó FEBS 2003
contacts observed from the H-1 resonance of the Glc I
residue. When Glc I substitutes Hep I at the 4 position,
NOE contacts are usually observed between the anomeric
proton resonance of the Glc I residue and the H-4 and
H-6 proton resonances of the Hep I residue [10]. In the
present structure, a NOE contact was only observed to
the H-4 resonance, suggesting that a change in confor-
mation has occurred at Hep I caused by the presence of
the Glc II residue at the 2 position. It was also possible
to discriminate between the 4 position of Hep I and the 2
position of Hep II, as the location of the Glc I residue,
because of the absence of characteristic H-1 to H-1 NOE
contacts normally observed for substitution of a heptose
residue at the 2 position [19]. Almost identical results
were obtained from the LPS-OH of strain 1000 and
strain NM115. Chemical shifts for the Gal II residue of
strain 1000 were different because in this strain, Gal II is
a terminal residue whereas in strain 425/93 and NM115
it is substituted by sialic acid at the 3 position, as
evidenced by the differences in the chemical shifts for the
H-3 proton resonances (Table 2). The
1
H-NMR data for
strain 1000 and NM115 are summarized in Table 2,
confirming that each strain had the same LPS structure,
the major 4 Hex glycoform of which is depicted in
Fig. 5.
To confirm the novel linkage pattern at Hep I, methy-

lation analysis was carried out on the core oligosaccharide
from the galE mutant of strain 1000. As expected, 1,2,3,4,5-
O-acetyl-6,7-di-O-methylheptitol was identified (data not
shown), consistent with the presence of the 2,3,4-trisubsti-
tuted Hep I residue.
Discussion
Strains 425/93 and NM115 were initially identified in a
collection of meningococcal serogroup B isolates by virtue
of their lack of reactivity with mAbs specific for defined
meningococcal LPS inner-core epitopes (unpublished data).
Strain 425/93 came from a collection of carriage isolates [14]
whereas the NM115 strain was isolated from patients with
meningococcal sepsis [15]. Strain 1000 was initially identified
in a collection of meningococcal clinical isolates by virtue of
its lack of reactivity with mAbs identified to require PEtn at
the distal heptose residue (Hep II) in the inner core ([12];
unpublished data). All previous structural studies on
meningococcal LPS had revealed the same substitution
pattern at the proximal heptose residue (Hep I), wherein
Hep II substituted Hep I at the 3 position, and the first
glucose residue (Glc I) of the oligosaccharide chain
substituted Hep I at the 4 position. Structural analysis of
LPS from meningococcal strains 1000, 425/93 and NM115
revealed the same arrangement. However, an additional
glucose residue (Glc II) was also identified and found to
substitute Hep I at the 2 position. This organization at
Fig. 2. b-Anomeric region of the TOCSY spectrum from the O-deac-
ylated LPS of N. meningitidis strain 425/93. The spectrum was recor-
dedinD
2

Oat25°C.
Fig. 1. Anomeric region of the 1D
1
H-NMR
spectrum of the O-deacylated LPS from
N. meningitidis strain 425/93. The spectrum
was recorded in D
2
Oat25°C.
Ó FEBS 2003 Novel inner-core structure in meningococcal LPS (Eur. J. Biochem. 270) 1763
Hep I has not been observed previously in meningococcal
LPS. The observed lack of reactivity with several inner-core
mAbs would suggest that this novel substitution pattern
either alters the inner-core conformation or masks inner-
core epitopes. Molecular modelling studies are underway to
determine if the 2,3,4-trisubstituted Hep I residue adopts a
unique conformation because of the constraints of substi-
tution at the three ring positions. A unique conformation
would also be consistent with the observed differences in the
NOE contacts for the Glc I to Hep I linkage. In all three
strains examined, in which the Hep I residue of the LPS
bears the additional Glc II residue at the 2 position, only an
interresidue NOE to the H-4 proton of Hep I is observed
from Glc I. The well-established NOE contacts between the
anomeric proton resonance of the Glc I residue and the H4
and H6 proton resonances of the Hep I residue [10],
observed in previous studies of meningococcal LPS, were
not observed, consistent with an altered pattern of substi-
tution at Hep I, suggesting that a change in conformation
has occurred caused by the presence of the Glc II residue at

the 2 position of Hep I. Experiments will be initiated to
attempt to identify the gene encoding the glucosyltrans-
ferase responsible for the addition of this b-glucose residue
to the 2 position of Hep I. Trisubstitution of the Hep I
residue of LPS has been observed in other bacterial species.
An additional glucose residue has been identified previously
at the 6 position of the Hep I residue of LPS from Vibrio
cholerae O22 [20] and Mannheimia haemolytica [21]. Other
residues have been identified as substituents at the 2 position
of Hep I with a b-configured glucuronic acid residue in
the LPS of Vibrio parahaemolyticus O12 [22], and an
a-configured galactose residue in the LPS from Campylo-
bacter lari [23,24]. The arrangement of residues at Hep I in
the LPS of V. parahaemolyticus O12 is identical with that
identified here except that in the meningococcal strains
investigated here it is a glucose residue at the 2 position. It is
intriguing that only a small number of the meningococcal
strains so far examined elaborate this LPS structure, and it
will be interesting to see how common this substitution
pattern is and, perhaps more crucially, how many men-
ingococcal strains possess the genetic machinery required to
elaborate this novel structure. These analyses have therefore
revealed further potential for variation in the inner-core
LPS of meningococcal strains. The potential to vary the
degree of substitution at the Hep I residue provides
N. meningitidis with additional mechanisms to alter the
conformation of its LPS epitopes and possibly affect its
interaction with the host.
Table 2.
1

H-NMR chemical shifts (recorded at 25 °C, in D
2
O relative to HOD at 4.78 p.p.m) and NOE data for the LPS-OH from strains (i) 1000,
(ii) 425/93, and (iii) NM115. ND, not determined.
Residue Strain H-1 H-2 H-3 H-4 H-5 NOEs
Hep I i 5.42 4.21 4.12 4.15 ND Kdo H-5, Glc II H-1
ii 5.45 4.20 4.10 4.14 ND Kdo H-5, Glc II H-1
5.42 4.20 ND ND ND Kdo H-5, Glc II H-1
iii 5.48 4.18 4.11 4.13 ND Kdo H-5, Glc II H-1
Hep II i 5.51 4.14 ND ND ND Hep-I H-3
ii 5.50 4.15 ND ND ND Hep-I H-3
iii 5.50 4.15 ND ND ND Hep-I H-3
a-GlcNAc i 5.06 3.89 3.80 3.55 ND Hep-II H-2, Hep-II H-1
ii 5.07 3.89 3.80 3.55 ND Hep-II H-2, Hep-II H-1
iii 5.06 3.89 3.80 3.55 ND Hep-II H-2, Hep-II H-1
a-GlcN (lipid A) i ND ND ND ND ND –
ii 5.39 3.92 3.80 ND ND –
5.50 3.97 3.84 ND ND –
iii 5.42 3.80 ND ND ND –
b-Glc (Glc I) i 4.55 3.44 3.64 3.64 3.64 Hep-I H-4
ii 4.57 3.44 3.64 3.64 3.64 Hep-I H-4
iii 4.57 3.44 3.64 3.64 3.64 Hep-I H-4
b-Gal (Gal I) i 4.45 3.60 3.77 4.16 ND Glc-I H-4
ii 4.45 3.57 3.76 4.16 ND Glc-I H-4
iii 4.45 3.60 3.76 4.16 ND Glc-I H-4
b-GlcNAc i 4.72 3.81 3.75 3.75 3.59 Gal-I H-3, Gal-I H-4
ii 4.72 3.82 3.75 3.75 3.58 Gal-I H-3, Gal-I H-4
iii 4.72 3.82 3.75 3.75 3.58 Gal-I H-3, Gal-I H-4
b-Gal (Gal II) i 4.48 3.55 3.68 3.93 ND GlcNAc H-4
ii 4.55 3.60 4.12 3.96 ND GlcNAc H-4

iii 4.55 3.50 4.11 3.96 ND GlcNAc H-4
b-Glc (Glc II) i 4.50 3.39 3.49 ND ND Hep-I H-2, Hep-I H-1
ii 4.50 3.38 3.42 3.68 3.55 Hep-I H-2, Hep-I H-1
iii 4.50 3.36 3.42 3.68 3.55 Hep-I H-2, Hep-I H-1
b-GlcN (lipid A) i ND ND ND ND ND ND
ii 4.65 3.80 ND ND ND ND
iii 4.62 3.80 ND ND ND ND
1764 A. D. Cox et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Acknowledgements
We are grateful to P. Kriz (National Institute of Public Health, Prague)
and M. Maiden (Oxford) who kindly provided strain 425/93 from a
collection of carriage strains from the Czech Republic. We gratefully
acknowledge the contribution of O. Harrison, C. Ison and R. Heyder-
man (Imperial College, London) who provided strain NM115. We
thank J. Li for CE-MS-MS studies and D. W. Hood for valuable
discussions.
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