Structural analysis of deacylated lipopolysaccharideand F653 R3 core-type Sven Mu¨ller-Loennies, Buko Lindner and Helmut Brade Borstel Research Center, Center for Medicine and Bioscience
Trang 1Structural analysis of deacylated lipopolysaccharide
and F653 (R3 core-type)
Sven Mu¨ller-Loennies, Buko Lindner and Helmut Brade
Borstel Research Center, Center for Medicine and Biosciences, Borstel, Germany
Lipopolysaccharide (LPS) of Escherichia coli strain 2513 (R4
core-type) yielded after alkaline deacylation one major
oligosaccharide by high-performance anion-exchange
chro-matography (HPAEC) which had a molecular mass of
2486.59 Da as determined by electrospray ionization mass
spectrometry This was in accordance with the calculated
molecular mass of a tetraphosphorylated dodecasaccharide
of the composition shown below NMR-analyses identified
the chemical structure as
where L-a-D-Hep is L-glycero-a-D-manno-heptopyranose
and Kdo is 3-deoxy-a-D-manno-oct-2-ulopyranosylonic
acid and all hexoses are present as D-pyranoses
We have also isolated the complete core-oligosaccha-rides of E coli F653 LPS for which only preliminary data were available and investigated the deacylated LPS by
NMR and MS The proposed structure determined pre-viously by methylation analysis was confirmed and is shown below
In addition we have quantified the side-chain heptose substitution of the inner core with GlcpN ( 30%) and confirmed that this sugar is only present when the phosphate
at the secondL,D-Hepp residue is absent
Keywords: Escherichia coli; lipopolysaccharide; R3 core-type; R4 core-core-type; structural analysis
Correspondence to S Mu¨ller-Loennies, Borstel Research Center, Parkallee 22, 23845 Borstel, Germany.
Fax: + 49 4537 188 419, Tel.: + 49 4537 188 467, E-mail: sml@fz-borstel.de
Abbreviations: DEPT, Distortionless enhancement by polarization transfer; L , D -Hep, L -glycero-a- D -manno-heptose; HPAEC, high-performance anion exchange chromatography; Kdo, 3-deoxy-a- D -manno-oct-2-ulosonic acid; LPS, lipopolysaccharide.
(Received 4 September 2002, accepted 22 October 2002)
Trang 2Lipopolysaccharide (LPS) is the major component of the
outer leaflet of the outer membrane of Gram-negative
bacteria [1] LPS of enterobacteria consist of three
domains, namely lipid A, core-region and O-antigen [2]
Due to its exposed location, it is the major target of the
humoral immune response in mammals and the lipid A
moiety is responsible for many of the pathological effects
seen in septic shock patients Whereas the chemical
structure of the O-antigen is highly variable, the
core-region and lipid A show only a limited structural
variability within the same species This prompted many
investigators to attempt the isolation of antibodies
directed against the conserved regions of LPS, i.e lipid A
and core-region (reviewed in [3]) It was assumed that
these antibodies would be both reactive and
cross-protective against different Gram-negative pathogens
Whereas a cross-protective effect was described for a
polyclonal antiserum as early as in 1966 [4], all
subsequently isolated monoclonal antibodies failed to
show cross-reactivity in vitro and cross-protectivity in vivo
[3], except one reported by DiPadova et al (mAb
WN1 222-5) This mAbrecognized LPS from all tested
clinical isolates of E coli, Salmonella, and Shigella in
Western-blots and showed cross-protective effects in vivo
against endotoxic activities of LPS [5] The
cross-reacti-vity was attributed to a common epitope located in the
inner core-region of these LPS In order to verify this
assumption, we have determined the as yet unknown
chemical structures of those LPS that reacted with this
mAb
The chemical structures of four E coli
charides (R1, R2, R3, and K-12) and two
core-oligosac-charides of S enterica [2] are known The chemical
structure of the E coli R3 core-type was determined by
methylation analysis [6,7] Complete core-oligosaccharides
were isolated and NMR chemical shift data were
determined for core-oligosaccharides of the R1 and R2
core-types [8] whereas the chemical structure of the inner
core-region of the E coli R4 core-type was hitherto
unknown The chemical structure of the outer core region
of the latter was determined by methylation analysis [9]
We have now isolated the complete core-oligosaccharides
and investigated the chemical structure of this LPS in
detail to understand the cross-reactivity of WN1 222-5
Since these data are a prerequisite for NMR based
conformational analysis of the inner core region of
enterobacterial LPS and epitope mapping of WN1 222-5
we have in addition isolated the complete
core-oligosac-charides of E coli F653 (R3-core) and determined NMR
chemical shift values
M A T E R I A L S A N D M E T H O D S
Bacteria and bacterial LPS
E colistrains 2513 and F653 were cultivated and used for
the isolation of LPS by
phenol/chloroform/petrolether-extraction as reported [10]
Analytical methods
Neutral sugars, GlcN, Kdo and bound organic phosphate
were determined as described [11]
Preparation of deacylated LPS ofE coli 2513 LPS (5 g) was de-O-acylated by mild hydrazinolysis [7] (yield 3.84 g) and 400 mg of the latter were subjected to alkaline de-N-acylation as described [12] After neutraliza-tion by addineutraliza-tion of ion exchanger Amberlite IRA120 H+ (Serva), 160 mg of the deacylated oligosaccharide fraction (yield 217 mg) was subjected to high-performance anion-exchange chromatography (HPAEC; eight runs of 20 mg each) using a semipreparative CarboPak PA100 column (9· 250 mm) and a DX300 chromatography system (Dio-nex, Germany) The main (fraction 2; oligosaccharide 1, yield 31.44 mg) and the minor oligosaccharide (fraction 1; oligosaccharide 2, yield 10.96 mg) were collected, neutral-ized and desalted as described above by addition of ion-exchanger followed by lyophilization Conditions for semipreparative and analytical HPAEC were as described previously [13]
Preparation of deacylated LPS ofE coli F653 LPS (2.11 g) was de-O-acylated by mild hydrazinolysis (yield 1.425 g) and 902.5 mg were further subjected to alkaline de-N-acylation as above The solution was neut-ralized by addition of 8MHCl and extracted three times with dichloromethane Subsequent desalting was achieved
by gelfiltration on Sephadex G10 (2.5· 65 cm) in 10 mM ammonium carbonate (yield 420 mg) A portion (417 mg)
of the desalted oligosaccharide mixture was subjected to semipreparative HPAEC as described above The sample was redissolved in water at a concentration of 90 mgÆmL)1 and 450 lL per run loaded onto the HPAEC column Elution and separation was achieved by a linear gradient of 2–600 mMNaOAc over a time of 70 min Fractions were analyzed by analytical HPAEC and appropriately com-bined Desalting was achieved by gelfiltration as described above Two pure oligosaccharides were obtained (fraction 1; oligosaccharide 3, 145.22 mg; fraction 2; oligosaccharide 4, 70.7 mg)
NMR-spectroscopy NMR-spectra were recorded of samples of deacylated LPS (11 mg each of R4 oligosaccharides 1 and 2 and 10 mg each of R3 oligosaccharides 3 and 4) in 0.5 mL solutions in
D2O using a Bruker DRX 600 Avance spectrometer equipped with a multinuclear probehead with z-gradient Acetone served as a reference 2.225 p.p.m (1H) and 31.5 p.p.m (13C) All spectra were run at a temperature
of 300 K
NMR of oligosaccharide 1 (R4 core) Two-dimensional homonuclear1H,1H-COSY was performed with a double quantum filter and time-proportional phase incrementation (TPPI) (DQF-COSY) The BrukerCOSYDFTPpulseprogram was modified to allow water suppression with 10 Gaussian shaped pulses of 100 ms defined by 1024 points during the relaxation delay Five-hundred and twelve experiments of
4096 data points each were recorded over a spectral width of 6.5 p.p.m in each dimension Prior to Fourier transforma-tion F1 was zero-filled to 1024 data points
TOCSY was performed at a spinlock field strength of
8 kHz for 75.15 ms using the Bruker pulse
Trang 3program and the same experimental parameters that were
used for TOCSY-ROESY (TORO)
A TORO-spectrum [14–17] was recorded as a
two-dimensional experiment using a fixed delay as the second
mixing time (ROESY-step) The spectrum was recorded
phase-sensitive by applying TPPI Four-thousand and
ninety-six data points were recorded over 512 experiments
consisting of 40 scans each over a spectral width of 8 p.p.m
in each dimension Water suppression was achieved by
presaturation on resonance during the relaxation delay
Prior to FT the data were multiplied by a shifted sine bell
function and zero-filled in F1, 1024 data points
NOESY was recorded phase-sensitive using the Bruker
NOESYPRTP pulse program Four-thousand and ninety-six
data points in F2 and 512 experiments in F1 were recorded
over a spectral width of 10 p.p.m in both dimensions Prior
to Fourier transformation, the FID was multiplied with a
shifted sine bell window function and zero-filling was
applied in F1, 1024 points The mixing time was 200 ms
For heteronuclear1H,13C-NMR correlation spectroscopy
the Bruker standard pulse programs INV4PRST (HMQC),
INV4NDTP(HMQC without decoupling during acquisition),
INV4MLPRTP (HMQC-TOCSY),INDECOBIMLTPPR
(DEPT-HMQC-TOCSY), and INV4LRNDPR (HMBC) were used
These spectra were recorded with 4096 data points in F2
and 512 experiments in F1 over spectral widths of 10 and
120 p.p.m, respectively Zero-filling was applied to 1024
data points in F1 For TOCSY a spinlock period of 81 ms
was applied at a field strength of 8.3 kHz For
DEPT-HMQC-TOCSY the sweep width was reduced to 15 p.p.m
in F1 and 3.5 p.p.m in F2 Two-hundred and fifty-six
experiments were recorded at 2048 data points per
incre-ment and a TOCSY mixing time of 67 ms For HMBC, F1
was enlarged to 180 p.p.m and the delay for the evolution
of long-range couplings was set to 50 ms
31P spectroscopy was performed after addition of NaOD
(Sigma) until all signals appeared as sharp singuletts The
pD was then approximated using pH paper and found to be
pD 9
31P,1H-HMQC was performed using a modified Bruker
pulse program (INVIPRTP) which was using continuous wave
instead of composite pulse decoupling during acquisition
The spectrum consisted of 128 experiments of 2048 data
points covering 10 p.p.m in both dimensions The delay for
evolution of couplings was adjusted for a3JP,Hof 10 Hz
(d4¼ 25 ms)
NMR of oligosaccharides 3 and 4 (R3 core)
DQF-COSY and NOESY were recorded as described above In
COSY the spectral width was reduced to 5.5 p.p.m in each
dimension TOCSY was performed using the DIPSI-2
composite pulse for spin-lock at a field strength of 7.8 kHz
and a spectral width of 5.5 p.p.m in each dimension
Five-hundred and twelve experiments were recorded consisting of
4096 data points each Water presaturation was achieved by
a shaped pulse as described for DQF-COSY
ROESY-TOCSY (ROTO) was performed as TORO (see
above) as a two-dimensional experiment but using a fixed
delay as the TOCSY mixing time The spectrum was
recorded phase-sensitive by applying TPPI Two-thousand
and forty-eight data points were recorded of 256
experi-ments consisting of 32 scans each over a spectral width of
10 p.p.m in each dimension Water suppression was
achieved by presaturation on resonance during the relaxa-tion delay Prior to Fourier transformarelaxa-tion, the data were multiplied by a shifted sine bell function and zero-filled in F1 and F2, 4096 and 512 data points, respectively The spin-lock for ROE of 5.6 kHz was applied for 150 ms and TOCSY was performed with a mixing time of 77 ms The field strength of the TOCSY spin-lock in this experiment was 9.4 kHz
HMQC, HMBC and HMQC-TOCSY were recorded as z-gradient experiments using standard Bruker software The experimental setup was otherwise identical to the same spectra recorded of oligosaccharide 1 DEPT-HMQC-TOCSY was run as described above but the spectral width was reduced to 30 p.p.m in F1 and 5 p.p.m in F2 One-hundred and twenty-eight experiments of 64 scans with 2048 data points were recorded The TOCSY spinlock was applied for 90 ms
Mass spectrometry Mass spectra were recorded in the negative ion mode of the mixture of oligosaccharides prior to HPAEC, of the isolated main oligosaccharide of deacylated LPS and of acylated purified LPS from E coli F2513 (R4 core-type)
In addition, the deacylated minor core-oligosaccharide of
E coli F653 (R3 core-type) was analyzed Negative ion electrospray ionization mass spectra were recorded on a Fourier Transform Ion Cyclotron Resonance FT-ICR mass spectrometer (APEX II, Bruker Daltonics, Billerica, USA) equipped with a 7 Tesla actively shielded magnet and an Apollo ion source Samples were dissolved at a concentration of 10 ngÆlL)1in a 50 : 50 : 0.001 (v/v/v) mixture of 2-propanol, water, and triethylamine and sprayed at a flow rate of 2 lLÆmin)1 For straightforward interpretation the spectra were charge-deconvoluted
R E S U L T S
Structural analysis ofE coli 2513 core-oligosaccharide (R4 core)
Compositional analysis of LPS identified Gal, Glc, GlcN, Kdo,L,D-Hep, and -P in a molar ratio given in Table 1 In addition 3OH-C14:0, C12, and C14 were found in
Table 1 Composition of E coli R4 LPS Kdo AcP , Kdo determination after hydrolysis in acetate buffer pH 4.5; Kdo HCl , Kdo determination after hydrolysis in 0.1 M HCl.
Component nmol component per mg Molar ratio a
3OH-C14 : 0 636 3.3
a Relative to GlcN ¼ 2.0.
Trang 4accordance with the common acylation pattern of E coli
lipid A [18]
Analytical HPAEC revealed that the deacylated LPS
fraction contained one major oligosaccharide isolated by
semipreparative HPAEC The charge deconvoluted
negat-ive ion-mode ESI-FT-ICR mass spectrum of the major
oligosaccharide fraction (Fig 1) obtained by deacylation of
LPS revealed a prominent ion with a mass of 2486.59 m/z
This was indicative of a composition of two HexN-, two
Kdo-, three heptose-, five hexose-residues, and four
phos-phates (theoretical mass [M-H]– 2485.577) Thus, this
oligosaccharide was a dodecasaccharide carrying four
phosphate substituents, in agreement with the compositional
analysis Further sodium and potassium ion-clusters were
observed that indicated the loss of one phosphate group
(m/z 80 lower mass) A 31P,1H-COSY NMR-spectrum
revealed that the phosphate group at the 4¢ position of
lipid A was missing (data not shown)
In addition ESI-FT-ICR contained the signals of a
molecular ion with m/z 2566.56, which was 80 Da higher
than the main fraction and was indicative of an additional
phosphate residue Due to its low abundance this fraction
could not be isolated and subjected to a more detailed
analysis Further molecular ions with a mass of 18 m/z
lower were observed which were not present in the same
spectrum of purified LPS prior to deacylation (not shown)
NMR-spectroscopy of the main oligosaccharide
con-firmed the compositional and mass spectrometrical
analy-ses The1H-NMR spectrum (Fig 2) contained 10 signals of
anomeric protons (Table 2) In addition, two pairs of
signals originating from 3-deoxy protons of Kdo-residues
were present Full assignment of proton and carbon
chemical shifts and determination of 3JH,H-coupling
con-stant values identified two pyranosidic Kdo-residues Their
a-configuration was evident from the resonance frequencies
of the deoxy-protons (equatorial H3 > 2.4 p.p.m for
b-Kdop) and the chemical shift values of the H-5 protons
[19] All sugars were present as pyranoses which was
deduced from their C-4 carbon chemical shifts (above
80 p.p.m for furanoses, Table 3) Correlation signals from
anomeric protons to intraresidue C-5 in HMBC
corrobor-ated the pyranose-configuration of sugar residues All other
sugars except two were also a-configurated which was determined by the analysis of JC-1,H-1-coupling constants (> 172 Hz) from a HMQC spectrum recorded without decoupling during acquisition Signals of anomeric carbons
at 99.75 p.p.m and 103.20 p.p.m were assigned to a b-GlcpN (164 Hz, residue B) and b-Galp (164 Hz, residue M), respectively Their b-configuration was confirmed by their
3JH,H-coupling constants ( 8 Hz) and their intraresidual NOE connectivities between H-1, H-3 and H-5 Three signals of anomeric protons showed 3JH,H-coupling con-stant values of less than 1 Hz and thus the H-2 in these residues was in equatorial position as in manno-configurated sugars (residues E, F, H) The determination of the spin system and coupling constants revealed that they belonged
toL,D-Hepp-residues The analysis of a CH2-edited DEPT-HMQC spectrum and a DEPT-DEPT-HMQC-TOCSY spectrum allowed the assignment of H-7a/band C-7 of L,D-Hepp residues F and H The chemical shift of H-6 of residue F could also be assigned in the latter spectrum The chemical shift of the H-6 proton of residue H, however, could not be identified in this spectrum Analysis of the1H,13C correla-tion spectrum indicated its chemical shift at 4.025 p.p.m., in agreement with the chemical shift of the same proton in previously analyzed oligosaccharides from E coli J-5 [13] Further residues were identified as a-Glcp (residues G and I), a-Galp (residues K and L), and a-GlcpN (A)
The analysis of an HMBC spectrum showing intraresid-ual cross-correlation signals from anomeric protons to carbons C-3 and C-5 was important for the assignment of spin-systems and chemical shifts of carbon Additionally, long-range correlations between protons of adjacent sugar residues across the glycosidic bond established their sequence; this was confirmed by the analysis of a NOESY spectrum and a 2D-TOCSY-ROESY (TORO) [14–17] spectrum (Fig 3) This latter experiment facilitated the assignment because all protons connected by scalar cou-plings and part of a spin system detectable by TOCSY show connectivities to protons close in space to any of these protons Therefore, more correlation signals are observed in the region of anomeric protons that resolves signal overlap, the identification of ROE signals is simplified and corro-borated by further correlation signals within the adjacent residue Furthermore, due to the asymmetry of the experi-ment with respect to the magnetization transfer mechanism the pulse sequence generates signals in the vertical plane
Fig 1 Charge deconvoluted ESI-FT-ICR mass spectrum of the
deac-ylated LPS of E coli strain 2513 (R4 core) Shown are the spectra of
the mixture prior to separation (A) and of the isolated oligosaccharide
1 (B).
Fig 2 1 H-NMR spectrum of E coli 2513 deacylated LPS (R4 core, oligosaccharide 1).
Trang 5only for protons within the preceeding residue and in the
horizontal plane only for protons within an attached residue
(most importantly the anomeric proton, if present) The
same result is obtained for a ROESY-TOCSY (ROTO)
experiment where a mirror image of the TORO spectrum is
obtained For example, the well resolved anomeric proton
E1 shows cross-correlation signals not only to E2 but also
interresidual connectivities to protons C5, C6, C7, C8a and
C8b The latter cross-correlation signals appeared in
opposite phase in the spectrum The correlation signal
between I1 and G1 is only seen in the vertical plane, proving
that residue I is attached to residue G Due to the opposite
phases, cancellation or diminished signal intensity may
occur and signals from direct ROE effects are still present
For some overlapping signals arising from the two different
pathways, mixed phase signals instead of complete
cancel-lation was observed However, even if some signals may
have cancelled out, this possible disadvantage is more than
compensated for by the additional information provided
The results of these experiments are summarized in Table 5
Residue B was thus connected to residue A in b1fi 6
linkage and these represented the lipid A backbone Char-acteristic NOEs between protons H-3a (weak) and H-3e (strong) of a-Kdop (residue C) and H-6 of a-Kdop (residue D) confirmed their 2fi 4-linkage [20] The heptose-region was composed of three heptose residues of the sequenceL
-a-D-Hepp-(1fi 7)-L-a-D-Hepp-(1fi 3)-L-a-D-Hepp which was connected to the inner Kdo (residue C) in position 5 The residues G, I, K, L, and M were those of the outer core and the NMR analyses confirmed the results obtained by methylation analysis [9] Long-range NOEs were observed between H-3a (strong) and H-3e (very weak) of a-Kdop (residue C) and H-1 ofL,D-Hepp (residue H) and between the anomeric proton of the innerL,D-Hepp residue (E) and the equatorial H-3 of the side-chain a-Kdop (residue D)
Four phosphate residues were identified (Fig 4A, Table 4) that were shown by HMQC to be linked to protons A1 (a-GlcpN), B4 (b-GlcpN), E4 (L,D-Hepp I) and F4 (L,D -Hepp II) The substitution with phosphate led to significant downfield shifts of protons and carbons at these positions The additional scalar coupling led to splitting of the corresponding signals in1H-, and13C-NMR spectroscopy
Table 2 1 H-NMR chemical shifts (p.p.m.) of deacylated LPS of E coli strain 2513 (R4 core-type, 1) and F653 (R3 core-type, 3 major and 4 minor) Compound Residue H-1 H-2 H-3ax H-3eq H-4 H-5 H-6a H-6bH-7a H-7b H-8a H-8b
1 A fi 6-aGlcN 1P 5.664 3.417 3.902 3.611 4.127 3.765 4.293
3 5.652 3.411 3.913 3.608 4.144 3.770 4.296
4 5.664 3.418 3.921 3.602 4.154 3.770 4.306
1 B fi 6-bGlcN 4P 4.860 3.076 3.859 3.796 3.740 3.443 3.666
3 4.870 3.062 3.850 3.767 3.725 3.453 3.668
4 4.861 3.081 3.859 3.773 3.740 3.458 3.690
1 C fi 4,5-aKdo 1.932 2.137 4.126 4.277 3.706 3.841 3.619 3.915
3 1.948 2.139 4.136 4.270 3.728 3.852 3.634 3.916
4 1.952 2.130 4.148 4.276 3.721 3.838 3.655 3.919
1 D aKdo 1.775 2.147 4.105 4.045 3.653 3.995 3.745 3.945
3 1.780 2.156 4.108 4.051 3.663 3.999 3.751 3.957
4 1.787 2.163 4.133 4.044 3.649 3.998 3.742 3.985
1 E fi 3-aHep 4P 5.289 4.061 4.128 4.415 4.218 4.097 3.917 3.766
3 5.287 4.067 4.136 4.412 4.223 4.102 3.903 3.784
4 5.293 4.084 4.168 4.430 4.205 4.129 3.934 3.795
1 F fi 3,7-aHep 4P 5.089 4.402 4.115 4.405 3.849 4.245 3.698 3.698
3 5.092 4.385 4.093 4.354 3.826 4.260 3.696 3.696
4 5.176 4.380 4.059 4.012 3.732 4.146 3.674 3.752
1 G fi 3-aGlc 5.195 3.638 4.073 3.732 3.885 3.891 3.785
3 5.198 3.595 4.185 3.776 3.878 3.863 3.729
4 5.289 3.705 4.116 3.787 3.880 3.926 3.749
1 H aHep 4.934 3.964 3.857 3.846 3.638 4.042 3.739 3.739
3 4.943 3.968 3.866 3.848 3.648 4.003 3.673 3.758
4 fi 7-aHep 4.944 3.993 3.874 3.849 3.621 4.233 3.909 3.730
1 I fi 2,4-aGlc 5.754 3.743 4.034 3.727 4.189 4.014 3.816
3 fi 2,3-aGal 5.938 4.189 4.342 4.319 4.313 3.770 3.770
4 5.903 4.225 4.321 4.358 4.299 3.800 3.769
1 K fi 2-aGal 5.542 3.990 4.097 3.857 4.072 3.779 3.734
3 fi 2-aGlc 5.512 3.735 3.885 3.470 3.765 3.922 3.746
4 5.550 3.746 3.910 3.495 3.765 3.931 3.771
1 L aGal 5.247 3.840 3.943 3.983 4.138 3.700–3.777 3.700–3.777
3 aGlc 5.222 3.552 3.752 3.453 3.907 3.929 3.771
4 5.208 3.566 3.756 3.450 3.935 3.933 3.840
1 M bGal 4.451 3.538 3.652 3.913 3.708 3.522 3.618
3 aGlcN 5.415 3.382 3.902 3.556 4.057 3.950 3.814
4 5.427 3.404 3.926 3.571 4.079 3.957 3.810
4 N aGlcN 5.224 3.352 3.944 3.494 3.763 3.778 3.778
Trang 6In accordance with all experimental data the chemical
structure of deacylated E coli 2513 LPS (R4 core-type) is as
depicted in Fig 5
Structural analysis ofE coli F653 core-oligosaccharides
(R3 core)
We have subjected purified LPS from E coli F653 to
alkaline deacylation by hot alkali and separated the
oligosaccharide mixture obtained by HPAEC Two main
fractions were obtained The 1H-NMR spectrum of the major oligosaccharide (Fig 6A) contained 10 signals of anomeric protons and two pairs of signals originating from 3-deoxy protons characteristic of aKdop The assignment of
1H and13C-resonances (Tables 2 and 3) and determination
of vicinal coupling constants (3JHn, Hn+1) revealed that it was composed of three GlcpN (residues A, B, M, Fig 8), two Kdop (C, D), threeL,D-Hepp (E, F, H), three Glcp (G,
K, L) residues and one Galp (I) residue All sugars except one were present as a-pyranoses as evident from3JH-1,H-2 and JC-1,H-1coupling constants and13C chemical shifts The chemical shift and the 3JH-1,H-2 coupling constant of the anomeric proton (8.5 Hz) of one GlcpN (residue B) as well
as NOE correlation signals between H-1, -3 and -5 confirmed its b-configuration In addition, four phosphate residues were present (Fig 4B, Table 4) which were located
at positions 1 and 4¢ of the lipid A backbone (GlcpN-disaccharide at the reducing end, residues A and B) and at positions 4 of both L,D-Hepp-residues E and F as deter-mined by31P,1H-COSY, as well as1H- and13C-chemical shift analysis The sequence of residues was determined by NOESY, ROTO, and HMBC In NOESY the anomeric protons of residues B to G showed NOEs in agreement with the chemical structure present in the R4-core (see above)
Fig 4 31 P-NMR spectra of deacylated LPS from E coli 2513 [R4
core, oligosaccharide 1 (A)], and from E coli F653 [R3 core,
oligosac-charide 3 (B), and oligosacoligosac-charide 4 (C)].
Fig 5 Chemical structure of E coli strain 2513 deacylated LPS (R4 core, oligosaccharide 1).
Fig 3.1H-TORO-NMR spectrum of E coli 2513 deacylated LPS (R4 core, oligosaccharide 1) In the lower right corner the boxed area of the spectrum is shown expanded.
Trang 7and in the corresponding partial structure isolated
previ-ously from the Rc-mutant strain E coli J-5 [13] In addition,
interresidual NOEs were observed from I1 to G3, M1 to I3,
K1 to I1 and I2, and L1 to K1 and K2 establishing the
sequence of these residues as a-D-Glcp1fi 2-a-D
-Glcp1fi 2-[a-D-GlcpN1fi 3]-a-D-Galp1fi 3-a-D-Glcp
These residues thus represented the outer core and
con-firmed the results previously obtained by methylation
analyses [6,7,9,21] The sequence deduced from observed
NOEs was corroborated by cross-correlation signals in
HMBC across the glycosidic bonds
In comparison to the major oligosaccharide 2,
1H- (Fig 6B) and 13C-NMR-spectra of the minor core
oligosaccharide 3 contained an additional set of signals
originating from an a-D-GlcpN-residue which was
there-fore a tridecasaccharide NOE NMR-spectra, ROTO and
HMBC confirmed that this residue (N, Fig 7) was located
at position 7 of the side chain L,D-Hepp (residue H)
leading to downfield shifts of protons F7a and F7bas well
as F6 NOE correlations were also observed between
proton H1 of this GlcpN-residue (residue N) to protons
H-6, H-7a and H-7bof residue H Simultaneous upfield
shifts of proton and carbon 4 of residue F (secondL,D
-Hepp) indicated that this position was not substituted with
phosphate in contrast to the major oligosaccharide This
was corroborated by only three phosphorus resonances in
31P-spectra (Fig 4C, Table 4) A1H,31P-NMR correlation
spectrum confirmed that apart from two phosphates in the
lipid A (positions 1 and 4¢) only position 4 of residue E
was phosphorylated
ESI-FT-MS of this oligosaccharide showed prominent
ions with a mass of 2566.70 m/z (Fig 8) and was consistent
with the composition deduced from NMR
D I S C U S S I O N
LPS of E coli strain 2513 (R4 core-type) contains the major oligosaccharide shown in Fig 5 ESI-FT-MS also revealed the presence of a minor fraction devoid of one phosphate residue and NMR-analysis showed that the 4¢-phosphate group was missing A molecular ion with a mass difference
of an additional 80 Da indicated the presence of a fifth phosphate residue This compound, however, was present only in minute amounts and was not seen in HPAEC It is known that in enterobacterial LPS the first heptose (residue E) is substituted in position 4 with 2-aminoethanol diphos-phate instead of a monophosdiphos-phate [2] The origin of this oligosaccharide can thus be explained by the possibility that the strong alkaline treatment did not hydrolyze the 2-aminoethanol diphosphodiester between phosphate groups but to a small extent between the 2-aminoethanol and phosphate leading to a diphosphate at this position A mass spectrum of purified LPS (not shown) recorded to explain the origin of this molecular ion and of additional molecular ions with a lower mass of m/z 18, which were present in the spectra of alkali treated LPS (Fig 1), only contained molecular ions with a mass difference of m/z 123 (2-aminoethanol monophosphate) but not with additional m/z
80 (phosphate) Therefore an additional monophosphate substitution at a different location may be excluded Furthermore, the spectrum did not contain molecular ions with a lower mass of Dm/z 18 which thus were artefacts Similar artefacts have been described previously by Olstho-orn et al [22]
There was no heterogeneity with respect to the substitution by additional sugar residues attached to the side chain-heptose as observed in other LPS-core struc-tures (E coli F470, E coli F653, Shigella sonnei, Shigella flexneri, Erwinia carotovora FERM P-7576, Proteus mirabilis R110/1959, Citrobacter freundii O23) [2] Notably, the phosphate substitution at position 4 of the second heptose (residue F) is quantitative and no smaller molecule is present which lacks the side chain heptose (residue H) as found in an Rc-mutant strain of E coli F653 (E coli J-5, R3 core-type) [13]
The NMR analyses of the oligosaccharides derived from
E coliF653 (R3 core) confirmed the results of methylation analyses published earlier [6,7] The relative amounts of GlcpN in the preparation were approximately 30% Important from a biosynthetic point of view, we observed
a correlation of side-chain heptose substitution with GlcpN and the lack of phosphate substitution at the second heptose
of the inner core This has been observed the first time in the rough mutant strain E coli J-5 [13] We have therefore reinvestigated the minor core-oligosaccharide of E coli F470 [8] which also contains this substitution by1H,1 H-DQF-COSY The correlation signals of H-4 of the second heptose, which in case of phosphorylation are shifted downfield ( 4.5 p.p.m), were not present at this frequency but had moved upfield Therefore, we concluded that also in this case the phosphate group at this position is missing and this correlation seems to be true in general, at least for all
E coli LPS Obviously, this phosphate residue must be removed either prior, during or after the transfer of this GlcpN The enzymatic activity and the corresponding gene locus have not been identified so far It is tempting to speculate that this GlcpN-transferase may be able to act as a
Fig 6.1H-NMR spectra of E coli F653 major (oligosaccharide 3, A)
and minor (oligosaccharide 4, B) oligosaccharide.
Trang 8phosphorylase and that both reactions occur simultaneously
since no molecules are detected which contain both, GlcpN
and phosphate, or no GlcpN and no phosphate at this
position The presence of phosphate at this position in
oligosaccharides, which possess a side-chain heptose residue
is explained by the fact that the activity of
heptosytrans-ferase III (WaaQ) is dependent on the phosphate at this
position [23] Thus, it may be that the reaction of this
GlcpN-transferase is energetically driven by the removal of the phosphate It may also be that two different enzymatic activities are active and the lack of the side-chain GlcpN substitution in the R4 core oligosaccharide can be either due
to the lack of the responsible GlcpN-transferase or by the lack of a phosphatase if the phosphate has to be removed prior to glycosylation
The reactivity of E coli 2513 LPS with mAbWN1 222-5 can be explained by an inner-core structure identical to those found in LPS from all core-types of E coli and Salmonella Investigation of the inter-residual NOE con-nectivities revealed that no long range NOEs indicative of a backfolding of the outer core were found Therefore, it is apparent that the outer core and the inner core form two structural confined domains, which may be a prerequisite for the accessibility of the inner core sugars for the binding
b y mAb WN1 222-5
Previously, an NOE between the anomeric proton of the side-chain heptose (residue H) and the axial H-3 of the first aKdop (residue C) has been observed in the analysis
of the R1 and R2 core oligosaccharides [8] and a
Table 3 13 C-NMR chemical shifts (p.p.m.) of deacylated LPS of E coli strain 2513 (R4 core, 1) and F653 (R3 core, 3 major and 4 minor) Compound Residue C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8
1 A fi 6-aGlcN 1P 92.15 55.57 70.37 70.83 73.67 70.85
1 B fi 6-bGlcN 4P 100.61 56.74 73.09 75.48 74.18 63.55
3 100.60 56.76 73.17 73.17 75.15 63.21
4 100.73 56.81 73.37 73.52 75.22 63.71
1 C fi 4,5-aKdo ND ND 35.54 71.56 70.84 73.41 70.81 64.96
3 175.7 ND 35.55 71.60 71.14 73.45 70.81 64.98
1 D aKdo ND ND 36.06 67.08 67.78 73.44 71.61 64.25
3 176.1 ND 36.05 67.16 67.79 73.44 71.21 64.25
1 E fi 3-aHep 4P 100.21 72.12 79.00 71.14 73.77 70.28 64.41
3 100.35 72.09 79.15 71.14 73.86 70.29 64.38
4 100.40 72.27 78.26 71.22 73.88 71.50 64.41
1 F fi 3,7-aHep 4P 103.76 71.22 80.25 69.88 73.48 68.60 69.68
3 103.85 71.22 80.36 69.66 73.45 68.65 69.68
4 103.52 70.66 79.03 67.19 73.31 69.05 70.50
1 G fi 3-aGlc 102.49 71.51 77.63 71.59 73.73 61.14
3 102.68 71.59 75.81 71.97 73.72 61.05
4 101.12 71.60 76.85 71.84 73.54 61.24
1 H aHep 101.31 70.44 71.65 67.54 72.74 70.28 64.19
3 101.40 71.19 71.89 67.53 72.79 70.40 64.20
4 fi 7-aHep 101.60 71.15 71.71 67.36 73.04 69.04 71.46
1 I fi 2,4-aGlc 95.86 74.96 70.18 79.17 71.33 60.30
3 fi 2,3-aGal 95.14 69.37 72.31 65.55 71.09 61.78
1 K fi 2-aGal 93.50 73.43 69.18 71.06 72.24 62.30
3 fi 2-aGlc 92.57 76.27 72.81 70.93 75.43 61.89
1 L aGal 96.20 68.54 69.72 70.79 71.55 61.59
3 aGlc 97.10 72.74 74.36 70.56 73.34 62.06
1 M bGal 103.20 71.26 72.84 68.99 75.68 63.76
3 aGlcN 91.87 55.03 71.08 70.87 73.55 61.59
4 N aGlcN 97.42 55.33 71.08 70.84 73.50 61.67
Table 4.31P-NMR chemical shifts (p.p.m.) of deacylated LPS of E coli
strain 2513 (R4 core-type, 1) and F653 (R3 core-type, 3 major and 4
minor).
Residue
Compound
A fi 6-aGlcN 1P 3.16 3.20 3.19
B fi 6-bGlcN 4P 4.38 4.40 4.41
E fi 3-aHep 4P 5.06 5.10 4.96
F fi 3,7-aHep 4P 5.00 5.05
Trang 9computational calculation of a partial oligosaccharide
excluding phosphate substitution performed in this study
revealed a distance of 5–6 A˚ of these protons, inconsistent
with reported results [8] and interpreted as spin diffusion
Also these NOEs were not observed in NOESY spectra of
smaller oligosaccharides from the Rc-mutant E coli strain
J-5 [13] The same long-range interactions between the
side-chainL,D-Hepp (residue H) and the inner a-Kdop (C),
and between the adjacent L,D-Hepp (residue F) and the
side-chain a-Kdop (residue D) were observed in the
analysis of the R3 and R4 core oligosaccharides To
provide further evidence we have therefore conducted a
series of NOESY experiments with varying mixing times
that showed that these were present also at mixing times as
short as 50 ms They may therefore indicate a
conform-ational change of the inner core with respect to molecules
that do not have an outer core rather than the result of
spin diffusion In order to arrive at meaningful
calcula-tions, extensive NMR measurements will have to be
performed and pH and the presence of divalent cations
should be taken into account
With respect to the recognition of these structures by
antibodies and the structural characterization of epitopes
leading to cross-reactivity, the full assignment of carbon
and, in particular, proton resonances now provides the
basis for detailed NMR-based conformational analysis of the inner core region of enterobacterial LPS Furthermore,
it will allow the interpretation of saturation transfer difference measurements [24] aiming at further character-ization of the epitope recognized by the cross-reactive mAbWN1 222-5
A C K N O W L E D G E M E N T S
We greatfully acknowledge the technical assistance of V Susott, and Helga Lu¨thje as well as the kind gift of the minor core oligosaccharide from E coli rough mutant F470 by Drs O Holst and E Vinogradov.
This research was financially supported by the Deutsche Forschungsg-emeinschaft Grants DFG L1-448 (BL).
R E F E R E N C E S
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Fig 7 Chemical structure of the deacylated E coli R3 LPS The major
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Intraresidual contact
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