The terminal heptose HepIII in RM6255 is substituted at the O-3 position by a b-D-Glcp residue whereas HepIII in strains RM7290and RM6252 is substituted at O-2 by the globoside unit a-D-
Trang 1Structural analysis of lipopolysaccharides from Haemophilus
Structural diversity observed in three strains
Ha˚kan H Yildirim1, Derek W Hood2, E Richard Moxon2and Elke K H Schweda1
1 Clinical Research Centre, Karolinska Institutet and University College of South Stockholm, Sweden; 2 Molecular Infectious Diseases Group, University of Oxford Department of Pediatrics, Weatherall Institute of Molecular Medicine,
John Radcliffe Hospital, Oxford, UK
Structural elucidation of the lipopolysaccharide (LPS) from
three serotype f Haemophilus influenzae clinical isolates
RM6255, RM7290and RM6252 has been achieved using
NMR spectroscopy techniques and ESI-MS on
O-deacyl-ated LPS and core oligosaccharide material (OS) as well as
ESI-MSnon permethylated dephosphorylated OS This is
the first study to report structural details on LPS from
sero-type f strains We found that the LPSs of all strains were
highly heterogeneous mixtures of glycoforms expressing the
common H influenzae structural element L-a-D
-Hepp-(1fi2)-[PEtnfi6]-L-a-D-Hepp-(1fi3)-[b-D-Glcp-(1fi4)]-L-a-D
-Hepp-(1fi5)-[PPEtnfi4]-a-Kdo-(2fi6)-lipid A with variable
length of OS chains linked to each of the heptoses The
terminal heptose (HepIII) in RM6255 is substituted at the
O-3 position by a b-D-Glcp residue whereas HepIII in strains
RM7290and RM6252 is substituted at O-2 by the globoside unit (a-D-Galp-(1fi4)-b-D-Galp-(1fi4)-b-D-Glc) or trun-cated versions thereof The central heptose (HepII) is substituted by an a-D-Galp-(1fi4)-b-D-Galp-(1fi4)-b-D -Glcp-(1fi4)-a-D-Glcp unit in RM7290and RM6252 or truncated versions thereof Strain RM6255 does not express galactose in its LPS and only shows a cellobiose unit elon-gating from HepII (b-D-Glcp-(1fi4)-a-D-Glcp) ESI-MSnon dephosphorylated and permethylated OS provided infor-mation on the existence of additional minor isomeric glycoforms
Keywords: Haemophilus influenzae; lipopolysaccharide; NMR; ESI-MS
Haemophilus influenzae is an important cause of human
disease worldwide and exists in encapsulated (type a
through f) and unencapsulated (nontypeable) forms Type
b capsular strains are associated with invasive bacteraemic
diseases, including meningitis and epiglottis, while acapsular
or nontypeable strains of H influenzae (NTHi) are primary
pathogens in otitis media and lower respiratory tract
infections [1] Lipopolysaccharide (LPS) is an essential
surface component of these pathogens and is implicated as a major virulence factor LPS of H influenzae can mimic host glycolipids and has a propensity for reversible switching of terminal epitopes (phase variation) of the oligosaccharide portion Molecular structural studies of LPS from H influ-enzae type b, d and NTHi strains have shown that the conserved part of LPS consists of a triheptosyl inner-core moiety linked to lipid A via 3-deoxy-D -manno-oct-2-ulo-sonic acid (Kdo) Each of the heptose residues (designated HepI, HepII and HepIII) can provide a point for elongation
by oligosaccharide chains or for attachment of noncarbo-hydrate substituents (Structure 1)
Correspondence to E Schweda, University College of South
Stock-holm, 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: Ac, acetate; AnKdo-ol, reduced anhydro Kdo; CE,
capillary electrophoresis; gHSQC, gradient heteronuclear single
quantum coherence; gHMQC, gradient selected heteronuclear
mul-tiple quantum coherence; gHMBC, gradient selected heteronuclear
multiple-bond correlation; GPC, gel permeation chromatography;
Hep, heptose; L , D -Hep, L -glycero- D -manno-heptose; Hex, hexose;
Hib, H influenzae serotype b; Hif, H influenzae serotype f; Kdo,
3-deoxy- D -manno-oct-2-ulosonic acid; lipid A-OH, O-deacylated
lipid A; LPS, lipopolysaccharide; LPS-OH, O-deacylated
lipopoly-saccharide; PAD, pulsed amperometric detection; MSn, multiple step
tandem MS; Neu5Ac, N-acetyl neuraminic acid; NTHi, nontypeable
Haemophilus influenzae; OS, oligosaccharide; PCho, phosphocholine;
PEtn, phosphoethanolamine; PPEtn, pyrophosphoethanolamine.
(Received 14 April 2003, revised 21 May 2003,
accepted 27 May 2003)
Eur J Biochem 270, 3153–3167 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03693.x
Trang 2To date, HepI has been found to be substituted by
glucose (R1¼ b-D-Glcp) in all strains investigated This
glucose can provide a point for further chain extension
There is no chain extension from HepII in the type
d-derived strain Rd [2], however, in three type b strains
and a number of NTHi strains HepII is substituted by a
glucose residue (R2¼ a-D-Glcp) which can provide a
point for further chain extension HepIII is substituted
by galactose {R3¼ b-D-Galp-(1fi2) in type b strains [3];
R3¼ b-D-Galp-(1fi3), NTHi strain 176 [4]} or
oligosac-charides extending from glucose {R3¼ b-D-Glcp-(1fi2),
strain Rd, NTHi strain 1003 [5]; R3¼ b-D-Glcp-(1fi3),
NTHi strain 486 [6]} In addition, N-acetyl neuraminic acid
has been found to be a common constituent of LPS in
H influenzae[7] Prominent noncarbohydrate substituents
are phosphate (P), pyrophosphoethanolamine (PPEtn),
phosphoethanolamine (PEtn), phosphocholine (PCho),
acetate (Ac) and glycine (Gly)
The oligosaccharide portion of H influenzae LPS is
known to be subject to high-frequency phase variation of
terminal epitopes, which can lead to a very heterogeneous
population of LPS molecules within a single strain [8] Phase
variation is thought to provide an adaptive mechanism
which is advantageous for survival of bacteria confronted
by the differing microenvironments and immune responses
of the host [9] A genetic mechanism contributing to LPS
phase variation has been identified in five chromosomal loci;
lic1, lic2, lic3, lgtC and lex2 [10–12] It has been
demon-strated that expression of PCho substituents in H
influen-zaeLPS is subject to phase variation mediated by the lic1
locus [13] Genes comprising the lic2 locus have been shown
to be required for chain extension from HepII and, together
with lgtC, in the phase variable expression of the pk
epitope [a-D-Galp-(1fi4)-b-D-Galp-(1fi4)-b-D-Glcp-(1fi]
[12,14,15] The lic3 locus has been shown to encode an
a-2,3-sialyltransferase that is responsible for addition of
sialic acid (N-acetylneuraminic acid or Neu5Ac) to terminal
lactose [16] The phase-variable lex2 locus has been shown
to encode a glycosyltransferase important in further
oligo-saccharide extension from HepI in the inner core [17] A
comprehensive study of LPS biosynthetic loci has been
undertaken in the type b strain Eagan (RM153) and in
strain Rd–(RM118) [18,19] H influenzae serotype f (Hif)
has its own structurally and serologically distinct capsular
polysaccharide distinguishing it from the other five
sero-types and the nontypeable strains [20]
Historically, Hif strains have been a rare cause of disease
in children and adults [21,22] However, septic arthritis,
meningitis, sepsis, otitis media, pneumonia, cellulitis, and
bacteremia are diseases associated with Hif infections in
children who quite often have a predisposing medical
condition, including documented immunodeficiency [20,
23–25] Countries engaged in global vaccination programs
against H influenzae serotype b (Hib) experienced a rapid
decline in diseases caused by Hib strains In the UK, Hib
infection decreased 98% in children younger than 5 years of
age [22], thus resulting in a greater proportion of infections
due to serotype f strains in the Hib postvaccination era [26]
The capsular polysaccharide of type f strains is known to be
composed of b-D-GalpNAc(1fi4)-a-D-GalpNAc(1fiPO4
repeating units [27] However, no data on LPS structures
are known To gain further insight into the LPS structures
expressed by H influenzae, we report detailed structural analyses on three epidemiologically distinct type f strains In these strains we identify a novel combination of oligosac-charide extensions not seen before in H influenzae LPS
Experimental procedures
Bacterial strains, growth conditions and LPS extraction
H influenzaetype f strains RM6255, RM7290and RM6252 are epidemiologically distinct clinical isolates which are representative of the limited genetic diversity observed within type f isolates RM6252 was isolated in 1966 from the nasal pus of a 59-year-old male from Newcastle, England, who suffered from subacute sinusitis RM6255 was isolated
in 1967 from the sputum of a 72-year-old male from Newcastle, England, who suffered from a postoperative chest infection RM7290was isolated in 1974 from the sputum of a 30-year-old female from Kuala Lumpur, Malaysia, who suffered from respiratory infection and malnutrition Bacteria were grown in brain–heart infusion broth supplemented with haemin (10 lgÆmL)1) and NAD (2 lgÆmL)1) LPS was extracted from lyophilized bacteria using phenol/chloroform/light petroleum as described pre-viously but with the modification that the LPS was precipitated with 6 vols diethyl ether/acetone (1 : 5, v/v) [28] LPS was purified by ultracentrifugation (82 000 g,
4C, 12 h) 11, 49 and 56 mg LPS was extracted from 1.6, 5.3, 3.4 g lyophilized bacteria from RM6255, RM7290and RM6252, respectively
Chromatography Gel permeation chromatography (GPC) was performed using a Bio-Gel P4 column (2.5· 80cm) with pyridinium acetate (0.1M, pH 5.3) as eluent and a differential refrac-tometer as detector GLC was carried out using a Hewlett-Packard 5890instrument with a DB-5 fused silica capillary column (25 m· 0.25 mm; 0.25 lm internal diameter) and
a temperature gradient of 160C (1min) fi 250 C at
3C min)1 HPAEC was performed on a Dionex Series 4500i chromatography system using a CarboPac PA1 column (4· 250mm) and pulsed amperometric detection Samples were eluted using a linear gradient of 0–500 mMNaOAc in 0.1MNaOH over 20min and a flow rate of 1 mLÆmin)1 Preparation of oligosaccharides
O-Deacylation of LPS with hydrazine O-Deacylation of LPS was achieved as previously described [29] Briefly, LPS (1 mg) was mixed with anhydrous hydrazine (0.1 mL) and stirred at 37C for 1 h The reaction mixture was cooled and cold acetone (1 mL) was added to destroy excess hydrazine The precipitated O-deacylated LPS (LPS-OH) was centrifuged (48 200 g, 20 min), the pellet was washed twice with cold acetone, once with diethyl ether, and then dissolved in water followed by lyophilization
Mild acid hydrolysis of LPS Core oligosaccharide frac-tions were obtained from LPS (10mg RM6255, 22 mg RM7290, 30 mg RM6252) following mild acid hydrolysis
Trang 3(1% acetic acid, pH 3.1, 100C, 2 h) The insoluble lipid A
was separated from the hydrolysis mixtures by
centrifuga-tion The reducing agent, borane-N-methylmorpholine
complex (1, 2 and 3 mg, respectively, for RM6255,
RM7290and RM6252), was included in the hydrolysis
mixture Following purification by GPC on the Biogel P4
column oligosaccharide (OS) fractions OS6255 (2.5 mg),
OS7290(4.0mg) and OS6252 (9.7 mg) were obtained and
investigated in this study
Dephosphorylation OS6255, OS7290and OS6252 (1 mg
each) were incubated with 48% hydrogen fluoride (0.1 mL)
for 48 h at 4C Then, the samples were placed into an ice
bath and HF was evaporated under a stream of nitrogen
gas The samples, OS6255-P, OS7290-P and OS6252-P,
were dissolved in water and lyophilized
Mass spectrometry GLC-MS was carried out with a
Hewlett-Packard 5890chromatograph connected to a
NERMAG R10-10H quadrupole mass spectrometer using
the same conditions for GLC as described above ESI-MS
on LPS-OH and OS samples was recorded on a VG Quattro
triple quadrupole mass spectrometer (Micromass,
Man-chester, UK) in the negative ion mode The samples were
dissolved in a mixture of water/acetonitrile (1 : 1, v/v)
Sample solutions were injected via a syringe pump into a
running solvent of H2O/CH3CN (1 : 1) at a flow rate of
10 lLÆmin)1 Multiple step tandem ESI-MS (ESI-MSn)
experiments were performed on a Finnigan LCQ iontrap
mass spectrometer (Finnigan-MAT, San Jose, CA, USA)
Samples were dissolved in 1 mM sodium acetate in 70%
MeOH/30% H2O The applied flow rate was 10 lLÆmin)1
All experiments were in the positive mode Capillary
electrophoresis (CE)-ESI-MSn was carried out with a
Crystal model 310CE instrument (ATI Unicam, Boston,
MA, USA) coupled to an API 3000 mass spectrometer
(Perkin-Elmer/Sciex, Concord, Canada) via a
MicroIon-spray interface as described previously [30]
Analytical methods
Sugars were identified by GLC-MS as their alditol acetates
as previously described [31] Methylation was performed
with methyl iodide in dimethylsulfoxide in the presence of
lithium methylsulfinylmethanide [32] The methylated
com-pounds were recovered using a SepPak C18 cartridge and
subjected to sugar analysis or ESI-MSn The relative
proportions of the various alditol acetates and partially
methylated alditol acetates obtained in sugar- and methyl-ation analyses, discussed below, correspond to the detector response of the GLC-MS The absolute configuration of glycoses was determined as previously described [33] The presence of glycine was determined by HPAEC following treatment of LPS with 0.1MNaOH at 20–22C for 30min N-acetylneuraminic acid was determined by treating
LPS-OH (0.2 mg) with 20 mU
10mM NaOAc, pH 5.0, at 37C for 4 h One unit will liberate 1.0 lmol of Neu5Ac per min at pH 5.0at 37C The reaction mixture was analyzed by HPAEC as previ-ously described without further work-up [30] The enzyme cleaves terminal Neu5Ac residues linked a-2,3, a-2,6 or a-2,8 to oligosaccharides Fatty acids were identified as described previously [34]
NMR spectroscopy NMR spectra were recorded for OS samples in deuterium oxide (D2O) at 30C Spectra were acquired on JEOL
500 MHz and Varian UNITY 600 MHz spectrometers using standard pulse sequences The LPS-OH samples were solubilized by adding perdeutero-EDTA (2 mM) and per-deutero-SDS (10mgÆmL)1) to the D2O solution Chemical shifts were reported in p.p.m., and externally referenced
to sodium 3-trimethylsilylpropanoate-d4 (d 0.00,1H) and acetone (d 29.8,13C) COSY, TOCSY with a mixing time of 180ms, gradient selected heteronuclear single quantum coherence (gHSQC), gradient selected heteronuclear mul-tiple quantum coherence (gHMQC), and gradient selected heteronuclear multiple-bond correlation (gHMBC) experi-ments were performed according to standard pulse sequences For interresidue correlation, two-dimensional NOESY experiments with a mixing time of 250ms were used
Results
Characterization of LPS
H influenzaetype f strains RM6255, RM7290and RM6252 were grown in liquid culture and the LPS was isolated by phenol/chloroform/light petroleum extraction [6]
Compositional analysis of the LPS samples for strains RM7290and RM6252 identified D-glucose (Glc),
D-galactose (Gal), 2-amino-2-deoxyglucose (GlcN) and
L-glycero-D-manno-heptose (Hep) as the constituent sugars
by GLC-MS of the derived alditol acetates and 2-butyl
Table 1 Sugar analysis data for LPS-OH and OS samples derived from H influenzae strains RM6255, RM7290 and RM6252.
Relative detector response percentage
Sugar residuea
a Sugars were identified by GLC-MS as their alditol acetates.
FEBS 2003 Structural diversity of LPS in H influenzae (Eur J Biochem 270) 3155
Trang 4glycoside derivatives on oligosaccharide material (Table 1).
Strain RM6255 showed the same sugars except for Gal
In addition, glycine was detected as a substituent of each
LPS by HPAEC with pulsed amperometric detection
(HPAEC-PAD) as described earlier [30] Neu5Ac can be
detected in H influenzae LPS by a method involving
analysis by HPAEC-PAD of terminal N-acetyl neuraminic
2
acid residues released by neuraminidase treatment [7]
However, by applying this method in our study, N-acetyl
neuraminic acid was not detected in any strain
O-Deacyla-tion of LPS by treatment with anhydrous hydrazine
under mild conditions afforded water-soluble material
(LPS-OH), which was subjected to ESI-MS The ESI-MS
spectra of the samples (negative mode) revealed abundant
molecular peaks corresponding to triply and quadruply
deprotonated ions The MS data (Table 2), pointed to
the presence of glycoforms in which each molecular
species contains the conserved PEtn-substituted triheptosyl
inner-core moiety attached via a phosphorylated Kdo
linked to the O-deacylated lipid A (structure 1) Two
populations of glycoforms were observed which differed by
123 Da (i.e the mass of a PEtn group) As observed for other
H influenzae strains, this would be consistent with either
phosphate or pyrophosphoethanolamine (PPEtn)
substitu-tion at the O-4 posisubstitu-tion of the Kdo residue [2,3,35]
Thus, for strain RM6255, abundant quadruply/triply
charged ions were observed at m/z 649.4/866.1 and 680.1/
907.0 indicating Hex4 glycoforms with the respective
compositions Hex4ÆHep3ÆPEtn1,2ÆP1ÆKdo1ÆLipidA-OH
Signals corresponding to minor glycoforms with
composi-tions Hex2ÆHep3ÆPEtn1,2ÆP1ÆKdo1ÆLipidA-OH and Hex3Æ
Hep3ÆPEtn1,2ÆP1ÆKdo1ÆLipidA-OH were observed at m/z
568.2/758.2, 599.2/799.1, 608.5/811.7, and 639.5/853.5,
respectively The ESI-MS spectrum of LPS-OH from
RM7290revealed major quadruply/triply charged ions at
m/z649.4/866.2, 730.5/974.3, and 761.3/1015.3 which
indi-cated the occurrence of glycoforms with compositions
Hex4ÆHep3ÆPEtn1ÆP1ÆKdo1ÆLipidA-OH, and Hex6ÆHep3Æ
PEtn1,2ÆP1ÆKdo1ÆLipidA-OH, respectively Less abundant
quadruply charged ions at m/z 608.8, 639.6, 680.2, 690.0,
811.8 and 842.4 together with their triply charged
counter-parts corresponded to glycoforms with the following
compositions Hex3ÆHep3ÆPEtn1,2ÆP1ÆKdo1ÆLipidA-OH,
Hex4ÆHep3ÆPEtn2ÆP1ÆKdo1ÆLipidA-OH, Hex5ÆHep3ÆPEtn1Æ
P1ÆKdo1ÆLipidA-OH, and Hex8ÆHep3ÆPEtn1,2ÆP1ÆKdo1Æ
LipidA-OH, respectively
The ESI-MS spectrum (negative mode) of LPS-OH from
strain RM6252 showed ions corresponding to the same
glycoforms as observed for strain RM7290 However,
additional ions corresponding to Hex7 glycoforms with
compositions Hex7ÆHep3ÆPEtn1,2ÆP1ÆKdo1ÆLipidA-OH were
observed at m/z 1028.2 and 1068.8 In general, the ions
corresponding to the higher glycoforms (Hex6 to Hex8)
were of greater abundance in the spectrum of RM6252 than
in the one of RM7290 From ESI-MS data it was also
evident that PCho was not a major substituent in the LPS
of the type f strains
Characterization of the Kdo-lipid A-OH element
The structure of the lipid A-OH region in all H influenzae
strains investigated so far has been found to consist of
a b-1,6-linked D-glucosamine disaccharide substituted by N-linked 3-hydroxytetradecanoic acid at C-2 and C-2¢ and phosphomonoester groups at C-1 and C-4¢ [2,3,5,6,34,36]
In this investigation, ESI-MS data (Table 2) and fatty acid compositional analysis yielding 3-hydroxytetradeca-noic acid indicated the presence of the same lipid A-OH structure in Hif In addition, in a 1H–31P coupled gHMQC experiment, a 31P signal at d )0.5 correlated
to1H d 5.48, which was assigned to the anomeric1H shift
of the a-linked GlcN (GlcNI) in the lipid A In the same spectrum, a31P signal at d 3.4 correlated to a1H signal at
d 4.05 which was identified as the H-4 signal of the b-GlcN residue (GlcNII)
Characterization of oligosaccharides Mild acid hydrolysis of LPS with dilute aqueous acetic acid afforded insoluble lipid A and core oligosaccharide, which after purification by gel filtration resulted in oligosaccharide samples OS6255, OS7290and OS6252
Sugar analysis of the OS samples shown in Table 1 revealed D-glucose (Glc), L-glycero-D-manno-heptose in all three strains as well as D-galactose (Gal) in RM7290 and RM6252 The absence of 2-amino-2-deoxyglucose (GlcN) in the OS samples confirmed this sugar to be part
of lipid A After dephosphorylation of OS samples with 48% HF, methylation analysis of the resulting material (OS6255-P) showed terminal Glc, 4-substituted Glc, 3-substituted-Hep, 2,3-disubstituted Hep and 3,4-disubsti-tuted Hep as the major sugar components; linkage analysis
of OS7290-P and OS6252-P revealed terminal Glc, terminal Gal, 4-substituted Glc, 4-substituted Gal, 3,4-disubstituted Hep, 2,3-disubstituted Hep and 2-substituted Hep in the relative proportions as shown in Table 3 The data was consistent with triantennary structures, containing the common inner-core element, L-a-D-Hepp-(1fi2)-L-a-D -Hepp-(1fi3)-[b-D-Glcp-(1fi4)]-L-a-D-Hepp-(1fi5)-a-Kdop
of H influenzae LPS The presence of 3-substituted Hep in OS6255-P indicated substitution at the O-3 position of HepIII whereas in strains RM7290and RM6252 substitu-tion at O-2 of HepIII was indicated (structure 1) ESI-MS
on OS samples (Table 2) indicated OS6255 to be acetylated and OS7290and OS6252 to be both acetylated and glycylilated Thus in the ESI-MS spectrum of OS6255 (negative mode) major doubly charged ions were observed
at m/z 783.8 and 804.7 corresponding to glycoforms with respective compositions Hex4ÆHep3ÆPEtn1ÆAnKdo-ol and
Ac1ÆHex4ÆHep3ÆPEtn1ÆAnKdo-ol Minor doubly charged ions at m/z 621.6, 642.8, 702.8, and 723.8 corresponded
to Hex2ÆHep3ÆPEtn1ÆAnKdo-ol, Ac1ÆHex2ÆHep3ÆPEtn1Æ AnKdo-ol, Hex3ÆHep3ÆPEtn1ÆAnKdo-ol and Ac1ÆHex3Æ Hep3ÆPEtn1ÆAnKdo-ol, respectively The ESI-MS spectra
of OS7290and OS6252 indicated 15 and 22 glycoforms, respectively The Hex6 glycoforms were the most predom-inant ones in OS7290, comprising 64% of the OS molecules They were detected as doubly charged ions at m/z 946.1, 966.9, and 995.3 corresponding to the respective composi-tions Hex6ÆHep3ÆPEtn1ÆP1ÆKdo1ÆAnKdo-ol, Ac1ÆHex6ÆHep3Æ PEtn1ÆP1ÆKdo1ÆAnKdo-ol, and Ac1ÆGly1ÆHex6ÆHep3ÆPEtn1Æ
P1ÆKdo1ÆAnKdo-ol The most predominant doubly charged ions in the ESI-MS spectrum of OS6252 at m/z 804.9 and 1129.2 corresponded to Hex4 and Hex8 glycoforms
Trang 5with compositions Ac1ÆHex4ÆHep3ÆPEtn1ÆAnKdo-ol and
Ac1ÆHex8ÆHep3ÆPEtn1ÆAnKdo-ol, respectively
ESI-MS/MS
Some information on the location of the acylation sites in
OS7290and OS6252 was provided by ESI MS/MS
following on-line separation by CE in the positive mode
[30,36] Thus, product ion spectra obtained from the
doubly charged ions at m/z 998 in OS7290(composition:
AcÆGlyÆHexÆHepÆPEtnÆAnKdo-ol) and m/z 1160in
OS6252 (composition: Ac1ÆGly1ÆHex8ÆHep3ÆPEtn1Æ AnKdo-ol) showed ions at m/z 204 and 235 corresponding
to AcÆHex and AcÆHep, respectively, indicating mixed acetylation at either a hexose residue or a heptose No ions indicating glycylilation could be observed in the spectrum
of OS6252 However, the spectrum of OS7290showed ions at m/z 280and 292 corresponding to GlyÆAnKdo-ol and AcÆGlyÆHep, respectively, indicating glycylilation at either Kdo or a heptose residue No more detailed information could be obtained from the spectra which showed very random fragmentation patterns
Table 2 Negative ion ESI-MS data and proposed compositions for LPS-OH and OS samples of H influenzae f-type strains RM6255, RM7290 and RM6252 Average mass units were used for calculation of molecular mass values based on proposed composition as follows: Hex, 162.14; Hep, 192.17; Kdo, 220.18; AnKdo-ol, 222.18; P, 79.98; PEtn, 123.05; Ac, 42.04; Gly, 57.05; and LipidA-OH 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 5% of the base peak intensity are not included in the table.
Sample
Observed ions (m/z) Molecular mass (Da) Relative abundance (%)
(M-4H) 4– (M-3H) 3– (M-2H) 2– Observed Calculated 6255 72906252 Proposed composition
LPS-OH –a 758.2b 2277.6 2277.02 Hex 2 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH
599.2 b 799.1 b 2400.6 2400.1 3 Hex 2 ÆHep 3 ÆPetn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 608.9 811.5 2438.6 2439.2 6 6 6 Hex 3 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 639.7 853.5 2563.2 2562.2 3 2 4 Hex 3 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 649.4 866.1 2601.5 2601.4 37 16 16 Hex 4 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 680.3 906.7 2724.2 2724.4 49 4 6 Hex 4 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 689.9 920.1 2763.5 2763.4 5 11 Hex 5 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 720.7 960.8 2886.1 2886.5 3 Hex 5 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 730.4 973.5 2924.6 2925.6 37 8 Hex 6 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 761.3 1015.1 3048.8 3048.6 23 7 Hex 6 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 771.0 1028.3 3088.0 3087.7 10 Hex 7 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 801.9 1068.8 3210.5 3210.8 6 Hex 7 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH 811.5 1082.2 3249.6 3249.9 12 Hex 8 ÆHep 3 ÆPEtn 1 ÆP 1 ÆKdo 1 ÆLipidA-OH 842.3 1123.1 3372.8 3372.9 2 13 Hex 8 ÆHep 3 ÆPEtn 2 ÆP 1 ÆKdo 1 ÆLipidA-OH
OS 621.6 b 1245.2 1246.06 Hex 2 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol
642.8 b 1287.6 1288.03 Ac 1 ÆHex 2 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 702.8b 1407.6 1408.2 5 Hex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 723.5 1449.01450 2 5 2 3 Ac 1 ÆHex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 745.01492.01492.2 1 Ac 2 ÆHex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 752.7 1507.0 1507.2 2 7 Ac 1 ÆGly 1 ÆHex 3 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 783.9 1569.8 1570.3 40 1 2 Hex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 805.1 1611.8 1612.3 42 8 11 Ac 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 826.01653.01654.3 7 6 Ac 2 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 833.6 1669.2 1669.4 2 1 Ac 1 ÆGly 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 854.1 1710.2 1711.4 6 2 Ac 2 ÆGly 1 ÆHex 4 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 865.2 1732.8 1732.4 1 1 Hex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 886.01774.01774.4 3 7 Ac 1 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 907.0 1815.8 1816.4 6 Ac 2 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 913.6 1829.2 1831.5 1 2 Ac 1 ÆGly 1 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 936.01873.6 1873.5 2 Ac 2 ÆGly 1 ÆHex 5 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 945.7 1893.8 1894.6 4 1 Hex 6 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 967.01936.8 1936.6 36 6 Ac 1 ÆHex 6 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 995.9 1993.8 1993.6 24 1 Ac 1 ÆGly 1 ÆHex 6 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1027.0 2055.8 2056.7 2 Hex 7 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1048.1 2098.0 2098.7 9 Ac 1 ÆHex 7 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1076.8 2156.8 2155.8 4 Ac 1 ÆGly 1 ÆHex 7 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1108.0 2218.2 2218.9 2 Hex 8 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1129.1 2260.4 2260.9 2 14 Ac 1 ÆHex 8 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol 1157.6 2318.1 2317.9 1 9 Ac 1 ÆGly 1 ÆHex 8 ÆHep 3 ÆPEtn 1 ÆAnKdo-ol
a
Trace amounts of a signal at m/z 568.2 were detected.bThe observed m/z-values correspond to spectra from RM6255 All other m/z-values correspond to spectra from RM6252 Observed values in RM7290and RM6255 varied ± 0.8 Da.
FEBS 2003 Structural diversity of LPS in H influenzae (Eur J Biochem 270) 3157
Trang 6Sequence analysis of OS6255-P, OS7290-P
and OS6522-P by MSn
OS6255-P, OS7290-P and OS6252-P were permethylated
and analyzed by ESI-MSn in order to determine the
sequence and branching details of the various glycoforms
This method was introduced by Reinhold et al and has
been applied by us recently [36–38] In the ESI-MS spectrum
of OS6255-P (positive mode), shown in Fig 1A, four major
sodiated parent ions were observed at m/z 1264.3, 1468.1,
1672.4 and 1876.6 corresponding to the dephosphorylated
and permethylated Hex1 to Hex4 glycoforms (Hex1)4ÆHep3Æ
AnKdo-ol) These ions were further fragmented in MS2
experiments, and some of the resulting product ions
underwent further fragmentation (MS3) to confirm the
existence of the proposed glycoforms shown in Table 4
Two isomeric Hex1 glycoforms were identified by
performing MS2 on parent ion m/z 1264.3 Two product
ions at m/z 797.4 and 737.3 corresponding to the neutral
losses of tHexÆHepIII and HepIÆAnKdo-ol, respectively,
confirmed the presence of a Hex1 glycoform in which
HepIII is substituted The glycoform with a hexose
elonga-ting from HepI was confirmed by product ions m/z 1001.4
and 753.3, due to losses of t-HepIII and tHepIIIÆHepII,
respectively
Performing MS2 on ion m/z 1468.3, and subsequent
MS3 on the resulting product ions determined four
isomeric Hex2 structures The ion corresponding to the
loss of t-HepIII at m/z 1205.5 was further fragmented to
give the ion at m/z 957.5 corresponding to the loss of an
unsubstituted HepII This confirmed the structure of the
glycoform in which a disaccharide moiety substitutes
HepI Furthermore, in the same MS3 spectrum an ion
was detected at m/z 753.5 due to the loss of a tHex–
HepII unit thus evidencing a structure with one hexose
residue substituted to both HepI and HepII The ion at
m/z 548.9 corresponded to the loss of tHex–Hex–HepII
Finally, a structure with one hexose residue each linked
to HepI and HepIII could be determined when the
product ion, m/z 1001.5, corresponding to the loss of a
terminal hexose linked to HepIII, was further fragmented
to give an ion at m/z 753.3 due to the loss of HepII
Five isomeric Hex3 glycoforms were determined by
performing MS2 on the parent ion m/z 1672.4 and
subsequent MS3 on resulting product ions at m/z 1409.6
and 1205.6 due to losses of a terminal heptose and a terminal Hex–Hep residue, respectively When the ion at m/z1409.6 was further fragmented in a MS3experiment it gave, inter alia, product ions at m/z 883.4 and 753.4 due to
Table 3 Methylation analysis data of the dephosphorylated OS samples derived from H Influenzae type f strains RM6255, RM7290, and RM6252.
Methylated sugar a T gmb Linkage assignment
Relative abundance OS6255-P OS6252-P OS7290-P
2,3,6-Me 3 -Glc 1.18 )4)- D -Glcp-(1- 18 31 36 3,4,6,7-Me 4 -Hep 1.43 )2)- L , D -Hepp(1- 5 12 2,4,6,7-Me 4 -Hep 1.45 )3)- L , D -Hepp-(1- 10
2,6,7-Me 3 -Hep 1.50 )3,4)- L , D -Hepp-(1- 3 13 14 4,6,7-Me 3 -Hep 1.55 )2,3)- L , D -Hepp-(1- 13 13 8
a 2,3,4,6-Me 4 -Glc represents 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl- D -glucitol-1-d 1 , etc b Retention times (T gm ) are reported relative to 2,3,4,6-Me 4 -Glc.
Fig 1 ESI-MS spectra (positive mode) of the permethylated (A) OS6255-P (B) OS7290-P and (C) OS6252-P The peaks marked by asterisks
5 indicate glycoforms with mono-methylated phosphate groups due to glycoforms resulting from incomplete dephosphorylation.
Trang 7losses of HepIÆAnKdo-ol and HexÆHexÆHepII elements,
respectively Thus two structures with terminal HepIII were
identified: the first with a trisaccharide group linked to
HepII and the second with a disaccharide group linked to
HepII and a hexose linked to HepI When the ion at m/z
1205.6 was further fragmented in a MS3experiment product ions at m/z 957.6, 753.4, and 549.3 were observed defining the losses of single substituted HepII, t-HexÆHepII, and HexÆHexÆHepII A single Hex4 glycoform was identified in RM6255 comprising terminal hexose residues substituted to
Table 4 Structures of glycoforms from OS6255-P, OS7290-P and OS6252-P elucidated by ESI-MSn Subscripts denoted by the letters a, b and c indicated the number of hexose residues in the following structure:
Relative abundancea(%) Structure 6255 Structure 7290Structure 6252 Relative abundanceb
6255 72906252 a b c a b c a b c 6255 7290 6252
Hex2 17.5 1.2 3.3 2 002 002 00Trace Trace Trace
Hex3 23.3 2.4 12.4 1 2 01 2 01 2 0Medium Low Low
2 01 1 1 1 1 1 1 Trace Medium Low
Hex4 52.9 25.5 27.7 1 2 1 1 2 1 1 2 1 High Low Medium
1 4 2 1 4 2 Medium Medium
a
Relative abundance for each glycoform Calculated from the intensity of the molecular ion peak relative to the total intensity of all molecular ion peaks in the MS spectrum expressed as percentage b Relative abundance for structural isomers of each glycoform Calculated from the intensity of the product ions in the MS 2 spectrum and indicated as follows: high (over 80%), medium (30–80%), low (2–30%), trace (below 2%).
FEBS 2003 Structural diversity of LPS in H influenzae (Eur J Biochem 270) 3159
Trang 8both HepI and HepIII, and a HexÆHex unit substituted to
HepII This structure is defined by a MS2experiment on
molecular ion m/z 1876.9 resulting in fragment ions m/z
1409.7 and 1145.5 due to the loss of tHexÆHepIII and
tHexÆHepÆAnKdo-ol, respectively This structure is further
supported by the MS3experiment on m/z 1409.7 in which
the resulting fragment ions m/z 753.4 and 679.4 are due to
losses of tHexÆHexÆHepII and tHexÆHepIÆAnKdo-ol,
respectively
In the ESI-MS spectra of permethylated OS7290-P and
OS6252-P (Figs 1B and C) seven sodiated parent ions
were observed corresponding to Hex2 to Hex8
glyco-forms (Hex1)8ÆHep3ÆAnKdo-ol) In order to obtain
sequence and branching information, these molecular
ions were further fragmented in MS2 experiments, and if
necessary MS3 experiments were used The fragment ions
produced in MS2and MS3 experiments were analyzed in
analogy to OS6255-P described above and provided
evidence for 22 structures present in OS7290-P and 33
structures present in OS6252-P as shown in Table 4 It
was obvious that OS6252-P is a slightly more
hetero-geneous mixture of glycoforms than OS7290-P The Hex4
and Hex6 glycoforms are the most abundant in
OS7290-P, while the abundance is almost equally distributed
between Hex3 and Hex8 glycoforms in OS6252-P The
Hex6 glycoform is the most abundant in OS7290-P and
comprises only one structure while the same glycoform
has five isomers in OS6252-P Thus, when the ion at m/z
2283.6 in OS7290-P was further fragmented in a MS2
experiment (see Fig 2A) it showed, inter alia, an ion at
m/z 1613.8 due to the loss of a t-Hex–Hex–HepIII unit
A MS3 experiment on m/z 1613.8 revealed, inter alia,
the ion at m/z 884.3 due to the loss of a tHex–HepI–
AnKdo-ol unit, which gave evidence for the Hex6
glycoform in OS7290-P being substituted by a
disacchar-ide element at HepIII and a trisacchardisacchar-ide unit at HepII
(see Fig 2B) Ions corresponding to this glycoform were
identified in the same manner in OS6252-P However,
when the parent ion at m/z 2283.5 in OS6252-P was fragmented further in a MS2 experiment it revealed additional ions at m/z 1817.8 and 1410.3 due to losses of tHex–HepIII and tHex–Hex–Hex-HepIII units, respect-ively (see Fig 3A) The MS3 experiment on m/z 1817.8
Fig 2 The ESI-MS n analysis of permethyl-ated OS7290-P (A) Product ion spectrum of m/z 2283.6 corresponding to the sodium adduct of the Hex6 glycoform (B) The MS3 spectrum of the fragment ion m/z 1613.8 resulting from the MS 2 of m/z 2283.6 The relevant fragmentation pathways are shown to the right of each spectrum.
Fig 3 The ESI-MSnanalysis of permethylated OS6252-P (A) Prod-uct ion spectrum of m/z 2283.6 corresponding to the sodium addProd-uct of the Hex6 glycoform The proposed fragmentations are shown beside the spectrum The number of hexoses, Hex x , linked to HepI and HepII are determined by MS 3 (B) MS 3 of the ions m/z 1410.3, 1613.9 and 1817.8 resulting from MS 2 of m/z 2283.5 The fragmentation pathways are shown at the bottom of the spectra.
Trang 9showed ions at m/z 1088.6 and 957.9 due to losses of
tHex–HepI–AnKdo-ol and tHex–Hex–Hex–HepII,
respect-ively This defined two Hex6 glycoforms having either a
trisaccharide or a tetrasaccharide unit linked to HepII
(see Fig 3B) When the ion at m/z 1410.3 was further
fragmented in a MS3experiment it gave ions at m/z 884.4
and 679.9 due to losses of a HepI–AnKdo-ol and tHex–
HepI–AnKdo-ol units, respectively This gave evidence
for two Hex6 glycoforms having either a disaccharide or
a trisaccharide unit linked to HepII (see Fig 3B)
NMR spectroscopy on LPS-OH from RM6255
and O-deacylated OS7290 and OS6252
The1H NMR spectra were assigned using chemical shift
correlation techniques (COSY, TOCSY, HMQC, HMBC
and HSQC experiments) The chemical shift data
corres-ponding to Hif 6255 is given in Table 5 NMR data
corresponding to Hif 7290and 6252 were found to be
identical and are combined in Table 6 Subspectra
corres-ponding to the individual glycosyl residues were identified
on the basis of spin-connectivity pathways delineated in
the1H chemical shift correlation maps, the chemical shift
values, and the vicinal coupling constants The chemical
shift data are consistent with eachD-sugar residue being
present in the pyranosyl ring form Further evidence for this
conclusion was obtained from NOE data [Table 7 and
Fig 4 (RM6255) and Fig 5 (RM6252)], which also served
to confirm the anomeric configurations of the linkages and
the monosaccharide sequence The Hep ring systems were
identified on the basis of the small J1,2-values and their
a-configurations were confirmed by the occurrence of single intraresidue NOE between the respective H-1 and H-2 resonances
The structure of the Hex4 glycoform in strain RM6255 was determined by examining the1H-NMR spectrum of its LPS-OH in detail Anomeric1H/13C NMR resonances of HepI, HepII and HepIII were identified at d 5.17/99.22, 5.78/97.6 and 5.08/100.1, respectively Four anomeric signals corresponding to Glc residues I to IV were observed
at d 4.57/101.7, 5.26/100.2, 4.54/102.4 and 4.54/99.3, respectively In addition, the anomeric signals of the a- and b-linked GlcN residues of the lipid A part were observed at d 5.48/93.3 and 4.62/101.6, respectively Chem-ical shift data are consistent with GlcI, III and IV being terminal residues and GlcII being substituted in O-4 in agreement with methylation analyses
The occurrence of interresidue NOESY connectivities between the proton pairs HepIII H-1/HepII H-2, HepII H-1/HepI H-3, and HepI H-1/Kdo H-5 confirmed the sequence of the heptose-containing trisaccharide unit in the inner-core region and the point of attachment to Kdo (structure 1) The occurrence of interresidue NOE between H-1 of GlcI and H-4/H-6 of HepI confirmed the 1,4 linkage between GlcI and HepI Interresidue NOE connectivities between proton pairs GlcIII H-1/GlcII H-4/H-6 and GlcII H-1/HepII H-2/H-3 established the sequence of a disaccharide unit and its attachment point
to HepII as b-D-Glcp-(1fi4)-a-D-Glcp-(1fi3)-L-a-D -Hepp-(1fi Interresidue NOE between H-1 of GlcIV and H-3/ H-2 of HepIII gave evidence for the a b-D-Glcp-(1fi3)-L -a-D-Hepp-(1fi unit
Table 5.1H and13C NMR chemical shifts for O-deacylated LPS of H influenzae type f strain RM6255 Data was recorded in D 2 O containing 2 m M
perdeutero-EDTA and 10mgÆmL)1perdeutero-SDS at 30 C 3 J H,H -values for anomeric 1 H resonances are given in parenthesis ND, Not determined; NR, not resolved (small coupling).
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
GlcNI fi6)-a- D -GlcpN-(1fi 5.48 3.84 ND ND ND ND ND
93.3 53.40 GlcNII fi6)-b- D -GlcpN(1fi 4.62 3.88 ND 4.03 ND ND ND
101.6 55.6 72.0 HepI fi3,4)- L -a- D -Hepp(1fi I 5.17 (NR) 4.19 4.13 4.35 – 4.04 3.68 3.68
99.22 69.7 72.4 72.3 67.7 63.1 HepII fi2,3)- L -a- D -Hepp(1fi II 5.78 (NR) 4.28 4.08 ND 3.88 4.59 ND ND
97.6 78.1 78.3 ND 73.3 HepIII fi3)- L -a- D -Hepp(1fi III 5.08 (NR) 4.04 4.21 ND ND ND 3.73 3.86
GlcI b- D -Glcp (1fi IV 4.57 (8.0) 3.39 3.47 3.31 3.47 3.98 3.77
101.7 73.3 75.8 70.0 75.8 60.3 GlcII fi4)-a- D -Glcp(1fi V 5.26 (4.0) 3.59 3.86 3.70 3.89 3.97 3.89
100.0 71.2 71.3 78.6 70.8 59.4 GlcIII b- D -Glcp (1fi VI 4.54 (8.1) 3.41 3.52 3.44 3.52 3.95 3.78
102.4 72.5 75.069.2 75.5 60 3 GlcIV b- D -Glcp (1fi VII 4.54 (7.9) 3.42 3.44 3.41 3.44 3.93 3.76
99.3 72.5 75.069.3 75.060 3 Kdo fi4,5)-a-Kdop-(2fi 2.03/2.37 ND 4.29 ND 3.80
61.4 39.4
60.6 39.5
FEBS 2003 Structural diversity of LPS in H influenzae (Eur J Biochem 270) 3161
Trang 10The ESI-MS data of OS6255 (Table 2) indicated that the
most abundant glycoform in this strain was
monoacetyl-ated The acetylation site was identified by NMR on
OS6255 (data not shown) It was observed that chemical
shift values for several HepIII resonances in OS6255
differed considerably from the corresponding values in
LPS-OH Thus resonances for H-1/C-1, H-2/C-2 and
H-3/C-3 were observed at d 5.13/97.6, 5.16/68.9 and 4.47/
73.8, respectively The chemical shift values were consistent
with acetylation at O-2 as the H-1 (+ 0.05 p.p.m), H-2
(+ 1.2), H-3 (+ 0.26 p.p.m) and C-2 (+ 1.3 p.p.m) signals
were shifted downfield, while C-1 () 2.5 p.p.m) and C-3
() 2.7 p.p.m) were shifted upfield [39] From the combined
data it could thus be concluded that the Hex4 glycoform of
RM6255 has the structure 2
Structural details of the oligosaccharide epitopes in
RM7290and RM6252 were provided by 1H-NMR and
13C-NMR data of OS7290and OS6252 (Table 6) Several
signals for methylene protons of AnKdo-ol were observed
in the COSY and TOCSY spectra in the region 2.25–
1.65 p.p.m This is due to the fact that several
anhydro-forms of Kdo are formed during the hydrolysis by
elimination of phosphate or pyrophosphoethanolamine
from the C-4 position [40] Anomeric resonances of HepI,
HepII and HepIII of OS7290were identified at d 5.06–5.16, 5.68–5.72 and 5.08, respectively As observed earlier, several anomeric signals for HepI and HepII appear due to the microheterogeneity caused by the anhydro-forms of Kdo [40] Subspectra corresponding to GlcI to IV, GalI and GalII were identified in the two-dimensional COSY and TOCSY spectra at d 4.54, 5.33, 4.58, 4.46, 4.47, and 4.44, respectively In addition, a minor spin system corresponding
to a-D-Galp was identified at d 4.96 Chemical shift data are consistent with GlcI and GalI being terminal residues in
Table 6. 1H and13C NMR chemical shifts for OS7290 and OS6252 Data was recorded in D 2 O at 30 C 3
J H,H -values for anomeric1H resonances are given in parenthesis Signals corresponding to PCho methyl protons and carbons occurred at 3.20and 53.6 p.p.m., respectively Pairs of deoxy protons of reduced, AnKdo were identified in the DQF-COSY at d 1.91–2.30 3 J H,H -values for anomeric 1 H resonances are given in parenthesis.
NR, Not resolved (small coupling); ND, not determined.
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
Hex6 glycoform
HepI fi3,4)- L -a- D -Hepp(1fi I 5.06–5.16 (NR) 4.01 4.06 4.15 ND 4.31 ND
96.2 69.7 76.8 74.2 72.8 HepII fi2,3)- L -a- D -Hepp(1fi II 5.68–5.72 (NR) 4.29 4.12 ND 3.73 4.56 3.79 3.90
97.9 77.8 77.4 71.2 73.9 61.7 HepIII fi2)- L -a- D -Hepp(1fi III 5.08 (NR) 4.01 4.08 ND ND ND ND
98.7 77.8 69.1 GlcI b- D -Glcp (1fi IV 4.54 (8.5) 3.35 3.47 3.41 3.47 3.77 3.96
10 2.2 73.3 76.072.076.060 6 GlcII fi4)-a- D -Glcp(1fi V 5.33 (4.8) 3.62 3.87 3.71 3.89 3.97 3.90
99.6 71.1 71.078.071.059.6 GlcIII fi4)-b- D -Glcp (1fi VI 4.58 (8.0) 3.39 3.67 3.71 3.71 3.84 4.00
10 2.0 72.074.4 78.071.059.6 GalI b- D -Galp(1fi VII 4.47 (8.4) 3.55 3.68 3.94 3.70ND
102.5 76.5 72.0 68.0 74.9 GlcIV fi4)-b- D -Glcp (1fi VIII 4.46 (8.1) 3.42 3.703.69 3.69 3.85 4.0 0
101.5 69.6 74.3 78.0 70.9 59.6 GalII b- D -Galp(1fi IX 4.44 (8.4) 3.57 3.703.95 3.70ND
102.5 76.5 72.0 68.0 74.9
62.3 40.0 Hex8 glycoform
GalI fi4)-b- D -Galp(1fi VII 4.54 (7.8) 3.59 3.76 4.07 3.81 ND
102.3 70.5 72.3 76.8 75.5 GalIII a- D -Galp(1fi X 4.98 (3.9) 3.84 3.904.0 5 4.37 3.72 3.72
99.8 68.0 68.7 72.2 70.5 60.0 ND GalII fi4)-b- D -Galp(1fi IX 4.52 (7.8) 3.61 3.76 4.07 3.81 ND
102.3 70.5 72.3 76.8 75.5 GalV a- D -Galp(1fi XI 4.96 (3.9) 3.86 3.92 4.05 4.37 3.72 3.72
99.8 68.0 68.8 72.2 70.6 60.0 ND