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Structural characterization of a novel branching pattern in the
lipopolysaccharide from nontypeable
Haemophilus influenzae
Martin Ma
˚
nsson
1
, Derek W. Hood
2
, E. Richard Moxon
2
and Elke K. H. Schweda
1
1
Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Novum, Huddinge, Sweden;
2
Molecular Infectious Diseases Group and Department of Paediatrics, Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford, UK
Structural analysis of the lipopolysaccharide (LPS) from
nontypeable Haemophilus influenzae strain 981 has been
achieved using NMR spectroscopy and ESI-MS on
O-deacylated LPS and core oligosaccharide (OS) material as
well as by ESI-MS
n
on permethylated dephosphorylated
OS. A heterogeneous glycoform population was identified,
resulting from the variable length of the OS branches
attached to the glucose residue in the common structural
element of H. influenzae LPS,
L
-a-
D
-Hepp-(1fi2)-
[PEtnfi6]-
L
-a-
D
-Hepp-(1fi3)-[b-
D
-Glcp-(1fi4)]-
L
-a-
D
-Hepp-
(1fi5)-[PPEtnfi4]-a-Kdop-(2fi6)-Lipid A. Notably, the
O-6 position of the b-
D
-Glcp residue was either substituted
by PCho or the disaccharide branch b-
D
-Galp-(1fi4)-
D
-a-
D
-Hepp, while the O-4 position was substituted by the
globotetraose unit, b-
D
-GalpNAc-(1fi3)-a-
D
-Galp-(1fi4)-
b-
D
-Galp-(1fi4)-b-
D
-Glcp, or sequentially truncated ver-
sions thereof. This is the first time a branching sugar residue
has been reported in the outer-core region of H. influenzae
LPS. Additionally, a PEtn group was identified at O-3 of the
distal heptose residue in the inner-core.
Keywords: Haemophilus; lipopolysaccharide; phosphocho-
line; structural analysis; ESI-MS
n
.
Haemophilus influenzae is a Gram-negative pathogen that
routinely colonizes the human upper respiratory tract and
which can be found both in encapsulated (types a–f) and
unencapsulated (nontypeable) forms. While the incidence
of disease caused by H. influenzae type b (invasive diseases,
including meningitis and pneumonia) has been greatly
reduced in recent years as a result of the development of
conjugate vaccines, there exists no vaccine against nontype-
able H. influenzae (NTHi). NTHi strains are a common
cause of otitis media and respiratory tract infections [1] and
its lipopolysaccharide (LPS) molecule has been shown to be
important for colonization and bacterial persistence during
infection. H. influenzae LPS is composed of a membrane-
anchoring lipid A moiety linked by a single phosphorylated
3-deoxy-
D
-manno-oct-2-ulosonic acid (Kdo) residue to a
variable core oligosaccharide (OS) portion. The carbo-
hydrate regions provide targets for recognition by host
immune responses and some of these OS epitopes mimic
human antigens, suggesting that the bacteria may use these
structures to evade the host immune system [2,3]. Moreover,
the OS portion of H. influenzae LPS has a propensity for
reversible switching of terminal epitopes (phase variation),
which is a major factor in the generation of the vast
intrastrain LPS heterogeneity usually found [4]. This
heterogeneity is thought to be an advantage to the bacteria,
allowing them to better confront different host compart-
ments and microenvironments and to survive the host
immune response [5]. The availability of the complete
genome sequence of H. influenzae strain Rd [6] has facili-
tated a comprehensive study of LPS biosynthetic loci in the
homologous strain RM118 [7] and in the type b strains
Eagan (RM153) and RM7004 [8]. Gene functions have been
identified that are responsible for most of the steps in the
biosynthesis of the OS portion of their LPS molecules.
Molecular structural studies of LPS from H. influenzae
strains [9–23] have resulted in a structural model consisting
of a conserved phosphoethanolamine (PEtn)-substituted
triheptosyl inner-core moiety (labelled HepI–HepIII) in
which each of the heptose residues can provide a point for
attachment of OS chains or noncarbohydrate substituents
(Fig. 1). Notably, HepIII and the b-
D
-Glcp residue that is
linked to HepI (labelled GlcI) have an especially wide range
of alternatives in the substitution pattern. HepIII has been
found to be substituted by a b-
D
-Glcp residue (either at O-2
[13] or O-3 [16]) or a b-
D
-Galp residue (either at O-2 [10] or
O-3 [19]). Analysis of LPS from lpsA mutants established in
a number of strain backgrounds supports a role for LpsA in
each of the alternative glycose substitutions of HepIII.
HepIII has also been found to be substituted by the
Correspondence to E. Schweda, University College of South
Stockholm, Clinical Research Centre, NOVUM, S-141 86 Huddinge,
Sweden. Fax: + 46 8585 838 20, Tel.: + 46 8585 838 23,
E-mail: elke.schweda@kfc.ki.se
Abbreviations: CE, capillary electrophoresis; PCho, phosphocholine;
PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine;
Hep, heptose;
D
,
D
-Hep,
D
-glycero-
D
-manno-heptose;
L
,
D
-Hep,
L
-glycero-
D
-manno-heptose; Hex, hexose; HexNAc, N-acetylhexos-
amine; HMBC, heteronuclear multiple-bond correlation; lipid A-OH,
O-deacylated lipid A; Kdo, 3-deoxy-
D
-manno-oct-2-ulosonic acid;
LPS, lipopolysaccharide; LPS-OH, O-deacylated LPS; MS
n
, multiple
step tandem mass spectrometry; Neu5Ac, N-acetylneuraminic acid;
NTHi, nontypeable Haemophilus influenzae; OS, oligosaccharide;
AnKdo-ol, reduced anhydro Kdo.
(Received 27 February 2003, revised 13 May 2003,
accepted 16 May 2003)
Eur. J. Biochem. 270, 2979–2991 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03675.x
noncarbohydrate substituents Ac (either at O-2 [16] or O-3
[15]), Gly [16,24], P (atO-4[11])andPEtn[9].ForGlcI,the
O-4 position has been found to be substituted by a b-
D
-Galp
residue [9] or a b-
D
-Glcp residue [10], while in other strains,
O-6 was substituted by phosphocholine (PCho) [13],
D
-glycero-
D
-manno-heptose (
D
,
D
-Hep) [14] or
L
-glycero-
D
-
manno-heptose (
L
,
D
-Hep) [21]. In several strains, a disubsti-
tution-pattern of GlcI has been observed, including b-
D
-
Glcp (at O-4)/PCho (at O-6) [17], b-
D
-Galp (at O-4)/PCho
(at O-6) [20], Ac (at O-4)/PCho (at O-6) [18] and Ac (at
O-4)/
L
,
D
-Hep (at O-6) [21]. In each strain analysed, PCho
addition has been shown to be directed by the products of
the lic1 locus. Our recent studies have focussed on the
structural diversity of LPS expression, and the genetic basis
for that diversity, in a representative set consisting of 25
NTHi clinical isolates obtained from otitis media patients
[25]. In the present investigation we report on the structural
analysis of LPS from one of these isolates (NTHi strain
981), which provides evidence for a novel branching pattern
at GlcI.
Experimental procedures
Bacterial culture and preparation of LPS
NTHi strain 981 was obtained from the Finnish Otitis
Media Study Group and is an isolate obtained from the
middle ear [25]. Bacteria were grown in brain-heart
infusion broth supplemented with haemin (10 lgÆmL
)1
),
NAD (2 lgÆmL
)1
)andN-acetylneuraminic acid (Neu5Ac)
(25 lgÆmL
)1
). LPS was extracted by the phenol/chloro-
form/light petroleum method, as described previously
[16].
Chromatography
Gel filtration chromatography and GLC were carried out as
described previously [16].
Preparation of OS material
O-Deacylation of LPS. O-Deacylation of LPS was
achieved with anhydrous hydrazine, as described previously
[16,26].
Mild acid hydrolysis of LPS. Reduced core OS material
was obtained after mild acid hydrolysis (1% aqueous acetic
acid, pH 3.1, 100 °C, 2 h) and simultaneous reduction
(borane-N-methylmorpholine complex, 20 mg) of LPS
(120 mg). The insoluble lipid A (62 mg) was separated
by centrifugation and the water-soluble part was repeatedly
chromatographed on a P-4 column, giving a major (OS-1,
8.5 mg) and a minor (OS-2, 3.7 mg) OS-containing
fraction.
Dephosphorylation of OS. Dephosphorylation of OS
material was performed with 48% aqueous HF, as
described previously [18].
Mass spectrometry
GLC-MS and ESI-MS were performed as described
previously [16,21]. ESI-MS
n
on permethylated dephos-
phorylated OS was performed using a Finnigan-MAT
LCQ ion trap mass spectrometer (Finnigan-MAT; San
Jose, CA, USA) in the positive ion mode. The samples
were dissolved in methanol/water (7 : 3) containing 1 m
M
NaOAc to a concentration of about 1 mgÆmL
)1
,andwere
injected into a running solvent of identical composition at
10 lLÆmin
)1
.
NMR spectroscopy
NMR spectra were obtained at 25 °C(OS)or20°C
[O-deacylated LPS (LPS-OH)] either on a Varian UNITY
600 MHz spectrometer or on a JEOL JNM-ECP500
spectrometer, using the previously described experiments
[16,18,21].
Analytical methods
Sugars were identified as their alditol acetates, as previously
described [27]. Methylation analysis was performed as
described previously [16]. The relative proportions of the
various alditol acetates and partially methylated alditol
acetates obtained in sugar- and methylation analyses
correspond to the detector response of the GLC-MS.
Permethylation of dephosphorylated OS was performed in
the same way as in the methylation analyses [16], but
without the prior acetylation step. The absolute configura-
tions of the hexoses were determined by the method devised
by Gerwig et al. [28]. The total content of fatty acids was
analysed as described previously [29].
Results
Characterization of LPS
NTHi strain 981 was cultured in liquid media and the
LPS was extracted using the phenol/chloroform/light pet-
roleum method. Compositional sugar analysis of the LPS
Fig. 1.
5
Structural model of the lipopolysaccharide from Haemophilus
influenzae. R
1
¼ H, phosphocholine (PCho),
D
-glycero-
D
-manno-
heptose (
D
,
D
-Hep) or
L
-glycero-
D
-manno-heptose (
L
,
D
-Hep); R
2
,R
4
,
R
5
¼ H, glucose (Glc), galactose (Gal) or acetate (Ac)
6
;R
3
¼ HorGlc,
Y ¼ Gly, P or phosphoethanolamine (PEtn). Note that substitution
with Gly has also been reported at HepI, HepII and Kdo [24].
2980 M. Ma
˚
nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003
sample indicated
D
-glucose (Glc),
D
-galactose (Gal),
2-amino-2-deoxy-
D
-glucose (GlcN), 2-amino-2-deoxy-
D
-galactose (GalN),
D
-glycero-
D
-manno-heptose (
D
,
D
-Hep)
and
L
-glycero-
D
-manno-heptose (
L
,
D
-Hep) in the ratio
25:26:18:1:5:25,asidentifiedbyGLC-MSoftheir
corresponding alditol acetate and 2-butyl glycoside deriva-
tives [28]. In previous investigations the LPS was found
to contain ester-linked glycine [24] and a low level of
Neu5Ac [30].
On treatment of the LPS with anhydrous hydrazine
under mild conditions, water-soluble O-deacylated LPS
(LPS-OH) was obtained. ESI-MS data (Table 1) indicated a
heterogeneous mixture of glycoforms consisting of two
subpopulations: a major subpopulation in which the
glycoform compositions comprised three heptoses and, to
agreatextent,PCho (Hep3-glycoforms); and a minor
subpopulation with compositions comprising four heptoses
but lacking PCho (Hep4-glycoforms). Quadruply charged
ions were observed at m/z 640.5/671.3 (minor), 680.8/
711.5 (major), 721.4/751.7 and 772.3/802.9, corresponding
to Hep3-glycoforms with the compositions PChoÆHex
2
Æ
Hep
3
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH, PChoÆHex
3
ÆHep
3
Æ
PEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH, PChoÆHex
4
ÆHep
3
ÆPEtn
2)3
ÆP
1
Æ
KdoÆLipid A-OH and PChoÆHexNAc
1
ÆHex
4
ÆHep
3
ÆPEtn
2)3
Æ
P
1
ÆKdoÆLipid A-OH, respectively. Minor quadruply charged
ions were also found at m/z 639.8/670.3, consistent with
Hep3-glycoforms that were lacking PCho with the compo-
sitions Hex
3
ÆHep
3
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH. Quadruply
charged ions at m/z 646.9/677.8, 687.7/718.4 (minor) and
728.1/758.8 (major) corresponded to Hep4-glycoforms
with the compositions Hex
2
ÆHep
4
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid
A-OH, Hex
3
ÆHep
4
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH and Hex
4
Æ
Hep
4
ÆPEtn
2)3
ÆP
1
ÆKdoÆLipid A-OH, respectively.
Characterization of OS
Core OS material was obtained after partial acid hydrolysis
of LPS with dilute aqueous acetic acid. The OS material was
repeatedly chromatographed on a P-4 column with inter-
mediate selection for the Hep3- and Hep4-glycoforms (by
ESI-MS). This procedure resulted in a major fraction (OS-1,
8.5 mg) in which the Hep3-glycoforms accounted for about
95%, and a minor fraction (OS-2, 3.7 mg) in which the
Hep4-glycoforms accounted for about 80% (Table 2). In
the ESI-MS spectrum of OS-1 (Fig. 2A), doubly charged
ions at m/z 684.9 (minor), 766.0 (minor), 847.2 (major) and
928.3, corresponded to the respective compositions PChoÆ
Hex
1)4
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol, while ions at m/z 949.1 (very
minor) and 1029.9 were consistent with the composi-
tions PChoÆHexNAc
1
ÆHex
3)4
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol, res-
pectively. In the ESI-MS spectrum of OS-2 (Fig. 2B),
doubly charged ions at m/z 698.4 (minor), 779.6 (major),
860.6, 941.8 (major) and 1022.5 (very minor) were consistent
with the respective compositions Hex
1)5
ÆHep
4
ÆPEtn
2
ÆAnK-
do-ol. In both OS fractions, minor peaks were observed
corresponding to the above-mentioned compositions but
additionally containing glycine or a phosphate group.
Minor peaks could also be observed corresponding to
glycoforms containing only one PEtn group or containing
one PEtn group and a phosphate group.
In order to obtain sequence and branching information,
the OS fractions were dephosphorylated and permethylated
and subjected to ESI-MS
n
[21,31]. Sodiated adduct ions
were observed in the MS spectra (positive mode) corres-
ponding to the compositions Hex
1)4
ÆHep
3
ÆAnKdo-ol, Hex-
NAc
1
ÆHex
3)4
ÆHep
3
ÆAnKdo-ol, Hex
1)5
ÆHep
4
ÆAnKdo-ol and
HexNAc
1
ÆHex
5
ÆHep
4
ÆAnKdo-ol (Tables 3 and 4). The
Table 1. Negative ion ESI-MS data and proposed compositions for O-deacylated lipopolysaccharide (LPS-OH) of nontypeable Haemophilus influ-
enzae (NTHi) strain 981. Average mass units were used for calculation of molecular mass values based on proposed compositions, as follows: Hex
(hexose), 162.14; HexNAc (N-acetylhexosamine), 203.19; Hep (heptose), 192.17; Kdo (3-deoxy-
D
-manno-oct-2-ulosonic acid), 220.18; P (phos-
phate), 79.98; PEtn (phosphoethanolamine), 123.05; PCho (phosphocholine), 165.13 and lipid A-OH (O-deacylated lipid A), 953.02. Relative
abundance was estimated from the area of molecular ion peak relative to the total area (expressed as percentage). Peaks representing less than 3% of
the base peak were not included in the table.
Observed ions (m/z) Molecular mass (Da)
Relative abundance (%) Proposed composition
(M-4H)
4–
(M-3H)
3–
Observed Calculated
639.8 853.4 2563.2 2562.2 1 Hex
3
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
670.3 893.7 2684.6 2685.3 2 Hex
3
ÆHep
3
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
640.5 854.1 2565.6 2565.2 1 PChoÆHex
2
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
671.3 894.7 2688.2 2688.2 2 PChoÆHex
2
ÆHep
3
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
680.8 907.8 2726.8 2727.3 17 PChoÆHex
3
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
711.5 948.9 2849.8 2850.4 26 PChoÆHex
3
ÆHep
3
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
721.4 962.0 2889.3 2889.5 3 PChoÆHex
4
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
751.7 1003.2 3011.7 3012.5 4 PChoÆHex
4
ÆHep
3
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
772.3 1029.7 3092.6 3092.7 3 PChoÆHexNAc
1
ÆHex
4
ÆHep
3
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
802.9 1070.8 3215.5 3215.7 5 PChoÆHexNAc
1
ÆHex
4
ÆHep
3
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
646.9 862.8 2591.5 2592.2 4 Hex
2
ÆHep
4
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
677.8 903.9 2715.0 2715.3 4 Hex
2
ÆHep
4
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
687.7 917.0 2754.4 2754.4 1 Hex
3
ÆHep
4
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
718.4 958.6 2878.2 2877.4 1 Hex
3
ÆHep
4
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
728.1 971.0 2916.2 2916.5 9 Hex
4
ÆHep
4
ÆPEtn
2
ÆP
1
ÆKdoÆLipid A-OH
758.8 1012.2 3039.4 3039.6 17 Hex
4
ÆHep
4
ÆPEtn
3
ÆP
1
ÆKdoÆLipid A-OH
Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2981
monosaccharide sequence and branching for the different
glycoforms were obtained following collision-induced dis-
sociation (CID) of the glycosidic bonds [21,31]. Through the
ion mass distinction between reducing, nonreducing and
internal fragments resulting from the bond ruptures [21,31],
the topology could be determined for nearly all composi-
tions found in the MS profiling spectra (Tables 3 and 4). In
those cases, where the same glycoform was found in both
OS-1 and OS-2 (Table 2), experiments showed that identical
structures were present in both fractions.
For the major Hep3-glycoform with the composition
Hex
3
ÆHep
3
ÆAnKdo-ol ([M + Na]
+
1671.8 Da), ion selec-
tion and collisional activation of the precursor ion at m/z
1671.8 provided the MS
2
spectrum shown in Fig. 3. The
fragmentation pattern was consistent with two nonreducing
terminal branches attached to a disubstituted heptose
(HepI)whichislinkedtotheterminalAnKdo-ol residue.
The ions at m/z 1409.7 and 1161.6 corresponded to losses of
terminal-Hep (t-Hep) and t-Hep–Hep, respectively, which
indicated the presence of a HepIII–HepII-disaccharide
branch. Furthermore, ions at m/z 1453.7, 1249.6 and
1045.5, corresponding to the respective loss of t-Hex,
t-Hex–Hex and t-Hex–Hex–Hex, indicated the presence of
a Hex–Hex–Hex-trisaccharide branch. Loss of the terminal
AnKdo-ol residue was also observed from the ion at m/z
1393.6. The assigned structure was confirmed by MS
3
experiments on selected product ions (data not shown). The
structures of the other Hep3-glycoforms were obtained in an
analogous manner (data not shown) and for all glycoforms
the hexoses were found to be members of a linear chain
attached to HepI (Table 3).
For the major Hep4-glycoform with the composition
Hex
4
ÆHep
4
ÆAnKdo-ol ([M + Na]
+
2124.0 Da), ions in the
MS
2
spectrum (precursor ion [M + 2Na]
2+
1073.5 Da)
(Fig. 4A) at m/z 1861.8 (loss of t-Hep) and 1613.8 (loss of
t-Hep–Hep) indicated the presence of a HepIII–HepII-
branch attached to HepI, as was also found in the Hep3-
glycoforms (Tables 3 and 4). An OS extension consisting
of four hexose residues and one heptose residue was
indicated to be linked to HepI from the occurrence of an
ion at m/z 1045.6 (counterpart at m/z 1101.6) correspond-
ing to loss of the entire Hex4Hep moiety. The fragmen-
tation pattern clearly indicated this OS extension not to be
arranged in a linear chain as fragment ions were found at
m/z 1905.8 (loss of t-Hex), 1701.7 (loss of t-Hex–Hex) and
1657.7 (either loss of t-Hex–Hep or t-Hep–Hex), consis-
tent with the presence of a Hex–Hex-disaccharide branch
and a second disaccharide branch that either could be
Hex–Hep- or Hep–Hex- (the former alternative was
shown, see below). Ions resulting from double glycosidic
bond cleavage could also be observed at m/z 1395.7 (e.g.
loss of t-Hep–Hep/t-Hex) and at m/z 1191.5 (loss of
t-Hep–Hep/t-Hex–Hex). An MS
3
experiment on m/z
1613.8 (Fig. 4B) gave fragment ions at m/z 1101.6 (coun-
terpart at m/z 535.3), as a result of glycosidic cleavage
between the Hex4Hep moiety and HepI. Ions at m/z
1395.7, 1191.6 and 1147.4, corresponding to the respective
loss of t-Hex, t-Hex–Hex and t-Hex–Hep, indicated that
the Hex4Hep moiety consisted of two nonreducing ter-
minal disaccharide branches (Hex–Hex- and Hex–Hep-)
attached to a disubstituted hexose. To confirm the assigned
structure, an MS
4
experiment on m/z 1191.6 was per-
formed (Fig. 4C), which showed the expected ions at m/z
973.5 (loss of t-Hex) and 725.3 (loss of t-Hex–Hep). The
topology of the other Hep4-glycoforms were determined in
a similar manner (data not shown). For the higher
molecular mass Hep4-glycoforms (Hex3–Hex5 and Hex-
NAc1Hex5), the hexose residue attached to HepI was
found to be disubstituted with a Hex–Hep-branch and a
second branch containing hexoses (Table 4).
Table 2. Negative ion ESI-MS data and proposed compositions for oligosaccharide (OS)-1 and (OS)-2 derived from the lipopolysaccharide (LPS) of
nontypeable Haemophilus influenzae (NTHi) strain 981. Average mass units were used for calculation of molecular mass values based on proposed
compositions as follows: Hex (hexose), 162.14; HexNAc (N-acetylhexosamine), 203.19; Hep (heptose), 192.17; AnKdo-ol (reduced anhydro Kdo),
222.20; PEtn (phosphoethanolamine), 123.05; and PCho (phosphocholine), 165.13. Relative abundance was estimated from the area of molecular
ion peak relative to the total area (expressed as a percentage). Minor peaks were observed corresponding to the proposed compositions but
additionally containing glycine or a phosphate group. Minor peaks could also be observed corresponding to glycoforms containing only one PEtn
group or containing one PEtn group and a phosphate group. ND, not detected.
Observed ions
(m/z) (M-2H)
2)
Molecular mass (Da) Relative abundance (%)
Proposed compositionObserved Calculated OS-1 OS-2
764.8 1531.6 1531.2 ND 9 Hex
3
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
684.9 1371.8 1372.1 3 3 PChoÆHex
1
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
766.0 1534.0 1534.2 5 5 PChoÆHex
2
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
847.2 1696.4 1696.4 70 3 PChoÆHex
3
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
928.3 1858.6 1858.5 11 ND PChoÆHex
4
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
949.1 1900.2 1899.6 Trace
a
ND PChoÆHexNAc
1
ÆHex
3
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
1029.9 2061.8 2061.7 8 ND PChoÆHexNAc
1
ÆHex
4
ÆHep
3
ÆPEtn
2
ÆAnKdo-ol
698.4 1398.8 1399.1 ND 3 Hex
1
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
779.6 1561.2 1561.3 ND 36 Hex
2
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
860.6 1723.2 1723.4 ND 7 Hex
3
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
941.8 1885.6 1885.5 3 34 Hex
4
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
1022.5 2047.0 2047.7 Trace Trace Hex
5
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
1124.4 2250.8 2250.9 Trace ND HexNAc
1
ÆHex
5
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol
a
Trace amounts, defined as peaks representing less than 3% of the base peak.
2982 M. Ma
˚
nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Methylation analysis of dephosphorylated OS-1 indi-
cated terminal-Gal (t-Gal), 4-substituted-Gal (4-Gal),
4-Glc, 3-Gal, t-
L
,
D
-Hep, 4,6-disubstituted-Glc (4,6-Glc),
4-
D
,
D
-Hep, 2-
L
,
D
-Hep and 3,4-
L
,
D
-Hep in the relative
proportions 24 : 5 : 31 : 2 : 9 : 3 : 3 : 10 : 13 together with
trace amounts of t-Glc and t-GalN. The methylation
analysis of intact OS-1 showed significantly decreasing
amounts of 2-
L
,
D
-Hep and t-
L
,
D
-Hep, which could be
derived from the presence of PEtn substituents at HepII and
HepIII, respectively (see below). Methylation analysis of
dephosphorylated OS-2 showed t-Gal, 4-Glc, 6-Glc, t-
L
,
D
-
Hep, 4,6-Glc, 4-
D
,
D
-Hep, 2-
L
,
D
-Hep and 3,4-
L
,
D
-Hep in the
ratio 32 : 15 : 2 : 10 : 5 : 15 : 10 : 11 together with trace
amounts of t-Glc, 4-Gal, 3-Gal and t-
D
,
D
-Hep. The increas-
ing amounts (compared to dephosphorylated OS-1) of
t-Gal, 4-
D
,
D
-Hep, 6-Glc and 4,6-Glc indicated that the
structural element Hex–Hep–Hex–(–) in the Hep4-glyco-
forms was probably Gal-(1fi4)-
D
,
D
-Hep-(1fi4,6)-Glc, and
this was confirmed and detailed by NMR spectroscopy (see
below).
Characterization of OS fractions and LPS-OH by NMR
The
1
H NMR resonances of OS fractions and LPS-OH
were assigned by
1
H–
1
H chemical shift correlation experi-
ments (DQF-COSY and TOCSY). Subspectra correspond-
ing to the individual glycosyl residues were identified on the
basis of spin-connectivity pathways delineated in the
1
H
chemical shift correlation maps, the chemical shift values,
and the vicinal coupling constants. From the glycoform
compositions of the different OS fractions indicated by
ESI-MS (Table 2), the spin-systems could more easily be
identified as originating from either the Hep3- or the Hep4-
glycoform population. The
13
C NMR resonances of OS
material and LPS-OH were assigned by heteronuclear
1
H–
13
C chemical shift correlation in the
1
H detected mode
(HSQC). The chemical shift data obtained for the Hep3-
and Hep4-glycoforms are presented in Tables 5 and 6,
respectively, and are consistent with each
D
-sugar residue
being present in the pyranosyl ring form. Further evidence
for this conclusion was obtained from NOE data, which
also served to confirm the anomeric configurations of the
linkages and, together with a heteronuclear multiple-bond
correlation (HMBC) experiment on OS-2, determined the
monosaccharide sequence and branching pattern.
Characterization of the Kdo-lipid A-OH region. ESI-MS
data (Table 1), fatty acid compositional analysis (yielding
3-hydroxytetradecanoic acid) and NMR experiments on
LPS-OH (data not shown, giving similar results as for
NTHi strains 486 [16] and 1003 [18]), indicated the presence
of the common Kdo-(2fi6)-lipid A-OH element in NTHi
strain 981. As observed previously [16,18], two spin-systems
Table 3. Structures of the Hep3-glycoforms of nontypeable Haemophilus influenzae (NTHi) strain 981, as indicated by ESI-MS
n
on permethylated
dephosphorylated oligosaccharide (OS). ND, not determined.
8
Hex1 Hex2 Hex3 Hex4 HexNAc1Hex3 HexNAc1Hex4
Hex-Hep-AnKdo-ol Hex
2
-Hep-AnKdo-ol Hex
3
-Hep-AnKdo-ol Hex
4
-Hep-AnKdo-ol ND HexNAc-Hex
4
-Hep-AnKdo-ol
|| | | |
Hep Hep Hep Hep Hep
|| | | |
Hep Hep Hep Hep Hep
Fig. 2. Negative ion ESI-MS spectra of oligosaccharide (OS)-1 (A) and
OS-2 (B) derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981 showing doubly charged ions.
(A) The peak at m/z 684.9 corresponds to a glycoform with the com-
position, phosphocholine (PCho)ÆHex
1
ÆHep
3
Æphosphoethanolamine
(PEtn)
2
ÆAnKdo-ol. (B) The peak at m/z 698.4 corresponds to a gly-
coform with the composition Hex
1
ÆHep
4
ÆPEtn
2
ÆAnKdo-ol. Minor
peaks were observed corresponding to the compositions proposed in
Table 2 but additionally containing glycine (indicated by · )ora
phosphate group (indicated by °). Minor peaks could also be observed
corresponding to glycoforms containing only one PEtn group (indi-
cated by e) or containing one PEtn group and a phosphate group
(indicated by +).
Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2983
could be traced for the single a-linked Kdo residue,
probably owing to the partial occurrence of PEtn attached
to the phosphate group at O-4 of Kdo [11,13,16].
Structure of the core region of the Hep3-glycoforms. In
the
1
H NMR spectrum of OS-1 (Fig. 5A), anomeric
resonances of HepI–HepIII were observed at d 5.89–5.82
(1H, not resolved), 5.24–5.22 (1H, not resolved) and 5.15–
5.01 (1H, not resolved). The Hep ring systems were
identified on the basis of the observed small J
1,2
values,
and their a-configurations were confirmed by the occurrence
of single intraresidue NOE between the respective H-1 and
H-2 resonances, as observed previously [16,18]. An intense
signal from methyl protons of an N-acetyl group was obser-
ved at d 2.04, which correlated to a
13
Csignalatd 22.7 in the
HSQC spectrum. A crosspeak from the ester-linked glycine
substituent was observed at d 4.00/40.7 (in the HSQC
spectrum) as a result of correlation between the methylene
proton and its carbon. Several signals for methylene protons
of AnKdo-ol were observed in the DQF-COSY and
TOCSY spectra of OS-1 in the region d 2.26–1.67, as
observed and explained previously [10,16,21]. The mono-
saccharide sequence within the inner-core region, as
indicated by ESI-MS
n
(described above), was confirmed
and detailed from transglycosidic NOE connectivities
between the proton pairs HepIII H-1/HepII H-2, HepII
H-1/HepI H-3 (LPS-OH and OS-1) and HepI H-1/Kdo H-5
and H-7 (LPS-OH), evidencing the sequence as
L
-a-
D
-
Hepp-(1fi2)-
L
-a-
D
-Hepp-(1fi3)-
L
-a-
D
-Hepp-(1fi5)-a-Kdop.
Relatively large J
1,2
values ( 7.8 Hz) of the anomeric
resonances observed at d 4.64, 4.62, 4.55, 4.52 and 4.46,
indicated that each of the corresponding residues had the
b-anomeric configuration. The residues with anomeric
signals at d 4.94 (J 4.0 Hz) and 4.91 (J 4.0 Hz) were
identified as having the a-anomeric configuration. The
assignments of the anomeric configurations were also
supported by the occurrence of intraresidue NOE between
the respective H-1, H-3 and H-5 resonances (b-configur-
ation) or between H-1 and H-2 (a-configuration). On the
basis of the chemical shift data and the large J
2,3
, J
3,4
and
Table 4. Structures of the Hep4-glycoforms of nontypeable Haemophilus influenzae (NTHi) strain 981, as indicated by ESI-MS
n
on permethylated dephosphorylated oligosaccharide (OS).
9
Hex1 Hex2 Hex3 Hex4 Hex5 HexNAc1Hex5
Hex Hex
a
Hex Hex Hex
|| | | |
Hep Hep Hep Hep Hep Hep
|| | | | |
Hex-Hep-AnKdo-ol Hex-Hep-AnKdo-ol Hex-Hex-Hep-AnKdo-ol Hex
2
-Hex-Hep-AnKdo-ol Hex
3
-Hex-Hep-AnKdo-ol HexNAc-Hex
3
-Hex-Hep-AnKdo-ol
|| | | | |
Hep Hep Hep Hep Hep Hep
|| | | | |
Hep Hep Hep Hep Hep Hep
Hep
b
|
Hex
2
-Hex-Hep-AnKdo-ol
|
Hep
|
Hep
a
Major isomer of the Hex3 glycoform (estimated from the intensity of the fragments in MS
2
experiments).
b
Minor isomer of the Hex3 glycoform (estimated from the intensity of the fragments in
MS
2
experiments).
Fig. 3. ESI-MS
2
analysis of permethylated dephosphorylated oligosac-
charide (OS) derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981. The product ion spectrum
is shown of [M + Na]
+
m/z 1671.8, corresponding to a glycoform
with the composition Hex
3
ÆHep
3
ÆAnKdo-ol. The proposed structure is
shown in the inset.
2984 M. Ma
˚
nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003
J
4,5
values ( 9 Hz), the residues with anomeric shifts of d
4.55 and 4.64 could be attributed to the 4-Glc (GlcI and
GlcII) identified by methylation analysis. On the basis of
low J
3,4
and J
4,5
values (<4 Hz) and chemical shift data, the
residues with anomeric resonances at d 4.46, 4.94, 4.52, 4.91
and 4.62 were attributed to the t-Gal (GalI and GalII),
4-Gal (GalI*), 3-Gal (GalII*) and t-GalNAc (GalNAc)
identified by linkage analysis.
Signals for the methyl protons of PChowereobservedat
d 3.23 and characteristic spin-systems for ethylene protons
from this residue and from the two PEtn residues were
found at d 4.41/3.69 (PCho), 4.15/3.29 (PEtnI) and 4.17/
3.29 (PEtnII).
1
H–
31
P NMR correlation studies (Fig. 6)
demonstrated PChotobelocatedatGlcIandthetwoPEtn
residues to be situated at HepII and HepIII, respectively.
Intense
31
P resonances from phosphodiesters were observed
at d 0.33, )0.50 and )0.62. Correlations between the former
signal and the signals from H-6 of HepII (d 4.55) and the
methylene proton pair of PEtnI (d 4.15) in the
1
H–
31
P
HMQC experiment evidenced substitution by PEtn at O-6
of HepII. The second PEtnresiduewasdemonstratedtobe
linked to O-3 of HepIII as the
31
P resonance at d )0.50
correlated to the signals from H-3 of HepIII (d 4.33) and the
methylene proton pair of PEtnII (d 4.17). Correlations
between the signal at d )0.62 and the signals from the H-6
protons of GlcI (d 4.30) and the methylene protons of PCho
(d 4.41) established the PCho substituent to be located at
O-6 of this residue.
The occurrence of interresidue NOE connectivities
between the proton pairs GalI H-1/GlcII H-4, GlcII H-1/
GlcI H-4 and GlcI H-1/HepI H-4 and H-6 established
the presence of the tetrasaccharide unit b-
D
-Galp-(1fi4)-
b-
D
-Glcp-(1fi4)-[PChofi6]-b-
D
-Glcp-(1fi4)-
L
-a-
D
-Hepp-
(1fi). This element was shown to be further chain elongated
by an a-
D
-Galp residue because transglycosidic NOE
between GalII H-1/GalI* H-4 and GalI* H-1/GlcII H-4
were observed. Further chain extension by a b-
D
-GalpNAc
residue was evidenced from the occurrence of interresidue
NOE between GalNAc H-1/GalII* H-3 and GalII* H-1/
GalI* H-4. From the combined data, the structure in Fig. 7
is proposed for the fully extended globotetraose-containing
Hep3-glycoform (HexNAc1Hex4) of NTHi strain 981. It
was thus established that the Hex4 and Hex3 glycoforms are
sequential truncations of this structure. It is also likely that
the Hex2 and Hex1 glycoforms are further sequential
truncations, which is consistent with the occurrence of small
amounts of t-Glc in the methylation analysis. Indeed, two
weak crosspeaks are observed in the COSY spectrum at d
4.57/3.50 and 4.56/3.38, which possibly arise from the two
b-
D
-Glcp residues appearing as terminal residues; however,
this could not be confirmed because of a significant overlap
of signals.
Structure of the core region of the Hep4-glycoforms. In
the
1
H NMR spectrum of OS-2 (Fig. 5B), anomeric
resonances of HepI–HepIII were observed at d 6.01–5.87
(HepII, not resolved), 5.17–5.03 (HepI, not resolved) and
5.14/5.10 (HepIII, not resolved). The chemical shift values
for the other resonances of HepI–HepIII (data not shown)
were similar to the corresponding values in the Hep3-
glycoforms. Transglycosidic NOE connectivities (data simi-
lar to that of the Hep3-glycoforms) confirmed the sequence
of the characteristic triheptosyl unit. Anomeric signals of the
fourth heptose were identified at d 5.06/4.94 (HepIV, not
resolved) and single intraresidue NOE between H-1 and H-2
confirmed its a-configuration. This residue could be
attributed to the 4-
D
,
D
-Hep identified in the methylation
analysis on the basis of chemical shift data (Table 6).
1
H–
31
P NMR correlation studies (data similar to that of the
Hep3-glycoforms) demonstrated the PEtn substituents to be
located at O-6 of HepII and at O-3 of HepIII, as previously
observed (see above).
Relatively large J
1,2
values ( 7.8 Hz) of the anomeric
resonances observed at d 4.57, 4.54, 4.53 (very weak), 4.51,
4.50, 4.49 and 4.48, indicated each of the corresponding
residues to have the b-anomeric configuration. The residue
with an anomeric signal at d 4.95 (very weak, J 4.0 Hz)
was identified as having the a-anomeric configuration.
The anomeric configurations were also confirmed from
Fig. 4. ESI-MS
n
analysis of permethylated dephosphorylated oligosac-
charide (OS) derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981. (A) MS
2
spectrum of
[M + 2Na]
2+
m/z 1073.5, corresponding to a glycoform with the
composition Hex
4
ÆHep
4
ÆAnKdo-ol. The proposed structure is shown
in the inset in B. (B) MS
3
spectrum of the fragment ion at m/z 1613.8.
(C) MS
4
spectrum of the fragment ion at m/z 1191.6
7
.
Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2985
intraresidue NOE, as described earlier (see above). The
residues with anomeric shifts of d 4.50, 4.54 and 4.57 could
be attributed to the 6-Glc (GlcI), 4,6-Glc (GlcI*) and 4-Glc
(GlcII) identified by methylation analysis, on the basis of the
chemical shift data and the large J
2,3
, J
3,4
and J
4,5
values
( 9 Hz). On the basis of low J
3,4
and J
4,5
values (<4 Hz)
and chemical shift data, the residues with anomeric
resonances at d 4.48, 4.95, 4.51/4.49 and 4.53 were attributed
to the t-Gal (GalI, GalII and GalIII) and 4-Gal (GalI*)
identified by linkage analysis.
Interresidue NOE were observed between the proton
pairs GalIII H-1 (d 4.51)/HepIV H-4 (d 3.92), HepIV H-1
(d 4.94)/GlcI H-6A, H-6B and GlcI H-1/HepI H-4 and H-6,
which established the tetrasaccharide unit b-
D
-Galp-(1fi4)-
D
-a-
D
-Hepp-(1fi6)-b-
D
-Glcp-(1fi4)-
L
-a-
D
-Hepp-(1fi of the
Hex2 glycoform. The structure of the Hex4 glycoform was
concluded from interresidue NOE between the proton pairs
GalIII H-1 (d 4.49)/HepIV H-4 (d 3.90), HepIV H-1 (d
5.06)/GlcI* H-6A, H-6B, GlcI* H-1/HepI H-4 and H-6 and
between the proton pairs GalI H-1/GlcII H-4, GlcII H-1/
GlcI* H-4, which evidenced the hexasaccharide unit b-
D
-
Galp-(1fi4)-b-
D
-Glcp-(1fi4)-[b-
D
-Galp-(1fi4)-
D
-a-
D
-Hep
p-(1fi6)]-b-
D
-Glcp-(1fi4)-
L
-a-
D
-Hepp-(1fi. This monosac-
charide connectivity was also confirmed by transglycosidic
correlations in an HMBC experiment, where correlations
were seen between GalIII H-1/HepIV C-4, HepIV
Table 5.
1
Hand
13
C NMR chemical shifts for Hep3-glycoforms of oligosaccharide (OS)-1 derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981. Data were recorded in D
2
Oat25 °C. Signals corresponding to N-acetyl-
D
-galactosamine (GalNAc) and
phosphocholine (PCho) methyl protons and carbons occurred at 2.04/22.7 and 3.23/54.5 p.p.m., respectively. Pairs of deoxyprotons of AnKdo-ol
(reduced anhydro 3-deoxy-
D
-manno-oct-2-ulosonic acid) were identified in the DQF-COSY spectrum at 2.26–1.67 p.p.m.
Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6
A
/C-6 H-6
B
H-7
A
/C-7 H-7
B
HepI fi3,4)-
L
-a-
D
-Hepp-(1fi 5.01–5.15
a
3.96–4.04
a
3.96–4.01
a
4.28
b
–
c
4.09–4.11
a,b
––
97.1–98.6
a
70.8–70.9
a
72.3–72.4
a
74.1 – 68.2 –
HepII fi2)-
L
-a-
D
-Hepp-(1fi 5.82–5.89
a
4.23–4.25
a
3.89–3.90
a
3.93 3.77 4.55 3.70 3.89
6
›
PEtn
98.4–98.7
a
79.6 69.8 66.7 71.8 75.0 63.0
HepIII
L
-a-
D
-Hepp-(1fi 5.22–5.24
a
4.24–4.26
a
4.33 3.87 – – – –
3
›
PEtn
101.6 70.2 76.6 65.8 – – –
GlcI fi4)-b-
D
-Glcp-(1fi 4.55 3.51 3.60 3.70 3.68 4.30 4.30
6
›
PCho
103.4 73.9 75.0 78.5 74.1 64.1
GlcII fi4)-b-
D
-Glcp-(1fi 4.64 3.30 3.64 3.72 3.61 3.84 3.93
102.7 73.6 74.9 78.5 75.0 60.2
GalI b-
D
-Galp-(1fi 4.46 3.54 3.66 3.92 3.72
d
––
103.4 71.4 73.0 69.0 75.8 –
GalI*
e
fi4)-b-
D
-Galp-(1fi 4.52 3.58 3.74 4.03 3.78
d
––
103.7 71.3 72.6 77.8 75.9 –
GalII a-
D
-Galp-(1fi 4.94 3.83 3.90 4.02 4.35 3.70 –
100.8 69.0 69.8 69.4 71.3 –
GalII*
e
fi3)-a-
D
-Galp-(1fi 4.91 3.89 3.95 4.25 4.38 3.68 –
100.9 68.1 79.2 69.5 70.8 –
GalNAc b-
D
-GalpNAc-(1fi 4.62 3.94 3.75 3.94 3.67
d
––
103.8 53.0 – 68.2 75.4 –
PEtnI 4.15 3.29
62.5 40.5
PEtnII 4.17 3.29
62.5 40.5
PCho 4.41 3.69
60.1 66.5
Gly 4.00
– 40.7
a
Several signals were observed for HepI, HepII and HepIII owing to heterogeneity in the AnKdo moiety.
b
H-4/H-6 of HepI were identified
at d 4.28/4.09–4.11 by NOE from GlcI.
c
–, not obtained owing to the complexity of the spectrum.
d
Tentative assignment from NOE data.
e
Residue marked with * denotes a further substituted analogue of the corresponding residue without *.
2986 M. Ma
˚
nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003
H-1/GlcI* C-6, GalI H-1/GlcII C-4 and GlcII H-1/GlcI* C-
4. This element was shown to be further chain extended by
an a-
D
-Galp residue, providing the Hex5 glycoform, as
transglycosidic NOE between GalII H-1/GalI* H-4 and
GalI* H-1/GlcII H-4 were observed. From the combined
data, the structure in Fig. 8 is proposed for the Hex5
glycoform in the Hep4-glycoform population of NTHi
strain 981. The Hex3 and Hex1 glycoforms are probably
sequential truncations of the established structures, which is
consistent with the low amounts of t-Glc and t-
D
,
D
-Hep
found in the methylation analysis. A weak crosspeak in the
COSY spectrum at d 4.55/3.36 may arise from the terminal
Table 6.
1
Hand
13
C NMR chemical shifts for Hep4-glycoforms of oligosaccharide (OS)-2 derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981. Data were recorded in D
2
Oat25°C. Chemical shift values for resonances of HepI-HepIII, PEtn and
Gly were similar to the corresponding values in the Hep3-glycoforms (see Table 5).
Residue Glycose unit H-1/C-1 H-2/C-2 H-3/C-3 H-4/C-4 H-5/C-5 H-6
A
/C-6 H-6
B
H-7
A
/C-7 H-7
B
GlcI fi6)-b-
D
-Glcp-(1fi 4.50 3.51 3.42 3.57 3.56 3.81 4.09
103.6 74.0 77.4 70.2 74.4 65.3
HepIV
a
fi4)-
D
-a-
D
-Hepp-(1fi 4.94 4.07 3.88 3.92 4.17 –
b
––
99.3 69.9 70.4 78.8 71.2 – –
5.06 4.05 3.80 3.90 4.28 – – –
100.6 69.2 70.4 80.2 71.4 – –
GalIII
a
b-
D
-Galp-(1fi 4.51 3.57 3.68 3.93 3.76
c
––
103.6 71.5 73.1 69.1 76.1 –
4.49 3.78 3.67 3.94 3.77
c
––
104.0 71.4 73.1 69.1 76.1 –
GlcI*
d
fi4,6)-b-
D
-Glcp-(1fi 4.54 3.58 – 3.65 3.65 3.90 4.16
103.6 74.0 – 77.7 73.8 66.1
GlcII fi4)-b-
D
-Glcp-(1fi 4.57 3.40 3.52 3.79 3.85 3.87 3.91
102.5 73.5 75.2 78.1 74.1 60.1
GalI b-
D
-Galp-(1fi 4.48 3.74 3.66 3.93 3.74
c
––
103.7 71.2 73.1 69.1 75.9 –
GalI*
d
fi4)-b-
D
-Galp-(1fi 4.53 3.81 3.78 4.05 – – –
103.8 – 72.8 77.9 – –
GalII a-
D
-Galp-(1fi 4.95 3.84 3.91 4.03 – – –
– 69.0 – 69.3 – –
a
Two spin-systems were found for the residue probably as a result of differences in the chemical environments of the various glycoforms.
The first spin-system is attributed to the Hex2 glycoform, while the second spin-system is attributed to the Hex4 and Hex5 glycoforms.
b
–,
not obtained owing to the complexity of the spectrum.
c
Tentative assignment from NOE data.
d
Residue marked with * denotes a further
substituted analogue of the corresponding residue without *.
Fig. 5. The 600-MHz
1
H NMR spectra of oligosaccharide (OS)-1 (A)
and OS-2 (B) derived from the lipopolysaccharide (LPS) of nontypeable
Haemophilus influenzae (NTHi) strain 981 showing the anomeric
regions. (A) Anomeric resonances that are characteristic for the Hep3-
glycoforms are labelled. Also indicated is an ethylene proton signal
from phosphocholine (PCho) at d 4.41. (B) Anomeric resonances that
are characteristic for the Hep4-glycoforms are labelled.
Fig. 6. Part of the 500-MHz heteronuclear
1
H–
31
PHMQCspectrumof
oligosaccharide (OS)-1 derived from the lipopolysaccharide (LPS) of
nontypeable Haemophilus influenzae (NTHi) strain 981. Assignments
are labelled.
Ó FEBS 2003 Structural analysis of LPS from NTHi strain 981 (Eur. J. Biochem. 270) 2987
b-
D
-Glcp residue in the major isomer of the Hex3 glycoform
(Table 4), but, as a result of overlapping signals, this could
not be confirmed. Given the structure of the HexNAc1Hex5
glycoform by ESI-MS
n
(Table 4), a terminal b-
D
-GalpNAc
residue is assumed in analogy of the Hep3-glycoforms.
Discussion
This investigation has shown that LPS from NTHi strain
981 contain either three heptoses (Hep3-glycoforms) or four
heptoses (Hep4-glycoforms). In both of these glycoform
subpopulations, either the fully assembled globotetraose
unit [b-
D
-GalpNAc-(1fi3)-a-
D
-Galp-(1fi4)-b-
D
-Galp-
(1fi4)-b-
D
-Glcp] or sequentially truncated versions thereof
were found to be linked to O-4 of the b-
D
-Glcp residue
(labelled GlcI) that is attached to HepI of the conserved
triheptosyl inner-core moiety. This is the first example of an
H. influenzae strain expressing globotetraose in this mole-
cular environment. However, recently the H. influenzae
type b strain, RM7004, was reported to express a trun-
cated epitope, the globoside trisaccharide (globotriose)
[a-
D
-Galp-(1fi4)-b-
D
-Galp-(1fi4)-b-
D
-Glcp]inthesame
molecular environment [22]. Additionally, in that strain,
the globotriose epitope was expressed as a terminal unit of a
tetrasaccharide extension from HepII, the same tetrasac-
charide that previously had been found in the type b strain,
RM153, at that location [11]. A third molecular environ-
ment for the globotriose epitope has been reported for
H. influenzae strain RM118 (Rd) [13] and several NTHi
isolates [21], where globotriose/globotetraose are present as
terminal OS extensions at HepIII. The globotriose epitope is
found on many human cells (p
k
blood group antigen), and
in H. influenzae is thought to be important for virulence by
acting as a mimic of these host structures, thus allowing the
organism to evade some element of the host immune
response [5,32]. For strain RM118, the glycosyltransferases
involved in the assembly of its globotetraose side-chain
were recently identified [7]. The lpsA gene was shown to be
involved in the addition of a b-
D
-Glcp residue in a
1,2-linkage to initiate chain extension from HepIII.
Furthermore, the lic2A, lgtC and lgtD genes were shown
to encode glycosyltransferases involved in sequential addi-
tion of b-1,4-linked Galp (Lic2A), a-1,4-linked Galp (LgtC)
and b-1,3-linked GalpNAc (LgtD), resulting in the fully
assembled globotetraose structure. Both lic2A and lgtC are
phase-variable genes [32,33] as is lex2 [34], which is the gene
responsible for the addition of a b-
D
-Glcp residue in a
1,4-linkage to GlcI in strain RM7004 (R. Aubrey, A. D.
Cox, K. Makepeace, J. C. Richards, E. R. Moxon & D. W.
Hood, unpublished results). Genetic analysis has indicated
the presence of lex2, lic2A, lgtC and lgtD gene homologues
in NTHi strain 981 (data not shown), and although it would
Fig. 7. Structure proposed for the fully extended Hep3-glycoform (HexNAc1Hex4) of nontypeable Haemophilus influenzae (NTHi) strain 981.
Fig. 8. Structure proposed for a Hep4-glycoform (Hex5) of nontypeable Haemophilus influenzae (NTHi) strain 981.
2988 M. Ma
˚
nsson et al. (Eur. J. Biochem. 270) Ó FEBS 2003
[...]... Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus in uenzae strain 176 Carbohydr Res 337, 409–420 Cox, A. D., Hood, D.W., Martin, A. , Makepeace, K.M., Deadman, M.E., Li, J., Brisson, J.-R., Moxon, E.R & Richards, J.C (2002) Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus. .. (1992) Structural characterization of the cell surface lipooligosaccharides from a nontypable strain of Haemophilus in uenzae Biochemistry 31, 4515–4526 10 Schweda, E.K.H., Hegedus, O.E., Borrelli, S., Lindberg, A. A., Weiser, J.N., Maskell, D.J & Moxon, E.R (1993) Structural studies of the saccharide part of the cell envelope lipopolysaccharide from Haemophilus in uenzae strain AH1-3 (lic3+) Carbohydr... [16,18,40,41] The lic 3A gene, which has been shown to encode a sialyltransferase that adds Neu5Ac in an a- 2,3linkage to lactose in other H in uenzae strains, is present in strain 981 (data not shown) Sialyllactose is known to contribute to the resistance of H in uenzae strains to the killing effect of normal human serum [41] Ó FEBS 2003 ˚ 2990 M Mansson et al (Eur J Biochem 270) Acknowledgements The authors... Zahringer, U (1988) Chemical structure of ¨ the lipopolysaccharide of Haemophilus in uenzae strain I-69 Rd–/ b+ Eur J Biochem 177, 483–492 ˚ 30 Bauer, S.H.J., Mansson, M., Hood, D.W., Richards, J.C., Moxon, E.R & Schweda, E.K.H (2001) A rapid and sensitive procedure for determination of 5-N-acetyl neuraminic acid in lipopolysaccharides of Haemophilus in uenzae: a survey of 24 nontypeable H in uenzae... located at HepIII; however, for the majority of strains, the molecular environment has not yet been established [12,14,40] The present investigation locates the additional PEtn group to O-3 of HepIII In the type b strain, RM153, a phosphate group was identified at O-4 of HepIII [11] MS analysis has indicated the presence of a phosphate group in several other strains [12–14], but, owing to the low abundance... residues in the outer-core regions of its LPS Structural studies have shown the LPS to have much in common with H in uenzae LPS, including an identical inner-core region (apart from an absence of PEtn at HepII) and lipid A [36] The D,D-Hep residue has been shown to be 1,6-linked to a b-D-Glcp residue attached to HepI (as in H in uenzae LPS, see above) and interrupts what otherwise would have been sialyl-lacto-N-neotetraose... & Jeanes, A (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography Anal Chem 37, 1602–1604 28 Gerwig, G.J., Kamerling, J.P & Vliegenthart, J.F (1979) Determination of the absolute configuration of monosaccharides in complex carbohydrates by capillary G.L.C Carbohydr Res 77, 1–7 29 Helander, I.M., Lindner, B., Brade, H., Altmann, K., Lindberg, A. A.,... & Hansen, E.J (1986) Antigenic and phenotypic variations of Haemophilus in uenzae type b lipopolysaccharide and their relationship to virulence Infect Immun 51, 69–79 5 Weiser, J.N & Pan, N (1998) Adaption of Haemophilus in uenzae to acquired and innate humoral immunity based on phase variation of lipopolysaccharide Mol Microbiol 30, 767–775 6 Fleischmann, R.D., Adams, M.D., White, O., Clayton, R .A. ,... Richards, J.C (2001) Structural analysis of the lipopolysaccharide from the nontypable Haemophilus in uenzae strain SB 33 Eur J Biochem 268, 5278–5286 ˚ Mansson, M., Hood, D.W., Li, J., Richards, J.C., Moxon, E.R & Schweda, E.K.H (2002) Structural analysis of the lipopolysaccharide from nontypeable Haemophilus in uenzae strain 1003 Eur J Biochem 269, 808–818 Schweda, E.K.H., Li, J., Moxon, E.R & Richards,... Structural analysis of LPS from NTHi strain 981 (Eur J Biochem 270) 2989 seem reasonable that these genes are responsible for the expression of the globotetraose epitope, and truncated versions thereof, in this strain, this remains to be determined Genetic analysis has also shown that the lpsA gene may be nonfunctional, which is consistent with the lack of OS extension at HepIII (M Deadman, personal . Structural characterization of a novel branching pattern in the
lipopolysaccharide from nontypeable
Haemophilus in uenzae
Martin Ma
˚
nsson
1
,. of
conjugate vaccines, there exists no vaccine against nontype-
able H. in uenzae (NTHi). NTHi strains are a common
cause of otitis media and respiratory tract
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