All sugars were present as pyranose rings, as indicated by 1H- and 13C-NMR chemical shifts and by the HMBC spectrum that showed for all residues intraresidual scalar connectivity between
Trang 1A novel type of highly negatively charged lipooligosaccharide
Serena Leone1, Viviana Izzo2, Alba Silipo1, Luisa Sturiale3, Domenico Garozzo3, Rosa Lanzetta1,
Michelangelo Parrilli1, Antonio Molinaro1and Alberto Di Donato2
1
Dipartimento di Chimica Organica e Biochimica and2Dipartimento di Chimica Biologica, Universita` degli Studi di Napoli Federico II, Napoli, Italy;3Istituto per la Chimica e la Tecnologia dei Materiali Polimerici – ICTMP – CNR, Catania, Italy
Pseudomonas stutzeriOXI is a Gram-negative
microorgan-ism able to grow in media containing aromatic
hydrocar-bons A novel lipo-oligosaccharide from P stutzeri OX1
was isolated and characterized For the first time, the
pres-ence of two moieties of 4,6-O-(1-carboxy)-ethylidene
resi-dues (pyruvic acid) was identified in a core region; these two
residues were found to possess different absolute
configur-ation The structure of the oligosaccharide backbone was
determined using either alkaline or acid hydrolysis Alkaline
treatment, aimed at recovering the complete carbohydrate
backbone, was carried out by mild hydrazinolysis
(de-O-acylation) followed by de-N-acylation using hot KOH The
lipo-oligosaccharide was also analyzed after acid treatment,
attained by mild hydrolysis with acetic acid, to obtain
information on the nature of the phosphate and acyl groups
The two resulting oligosaccharides were isolated by gel permeation chromatography, and investigated by composi-tional and methylation analyses, by MALDI mass spectro-metry, and by1H-,31P- and13C-NMR spectroscopy These experiments led to the identification of the major oligosac-charide structure representative of core region-lipid A All sugars areD-pyranoses and a-linked, if not stated otherwise Based on the structure found, the hypothesis can be ad-vanced that pyruvate residues are used to block elongation
of the oligosaccharide chain This would lead to a less hydrophilic cellular surface, indicating an adaptive response
of P sutzeri OX1 to a hydrocarbon-containing environment Keywords: Pseudomonas stutzeri OXI; lipopolysaccharide; NMR spectroscopy; mass spectrometry; pyruvic acid
Environmental pollution is recognized worldwide as an
emergency for its negative effects on the biosphere and on
human health Bioremediation strategies have recently been
devised, based on microbial biotransformations, given the
metabolic potential of selected microorganisms, in
partic-ular by Gram-negative bacteria, and their adaptability to
many different pollutants [1]
Pseudomonas stutzeriOX1 is a Gram-negative bacterium
isolated from the activated sludge of a wastewater treatment
plant, and endowed with unusual metabolic capabilities for
the degradation of aromatic hydrocarbons [2] In fact, in
contrast with other Pseudomonas strains, this microrganism
is able to grow on a large spectrum of aromatic compounds
including phenol, cresol and dimethylphenol, and on
nonhydroxylated molecules such as toluene and o-xylene,
the most recalcitrant isomer of xylene Moreover, it is able to metabolize tetrachloroethylene (PCE), one of the ground-water pollutants commonly resistant to degradation [3] Degradation of aromatic hydrocarbons by aerobic bac-teria comprises an upper pathway, which produces dihy-droxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates to molecules that enter the citric acid cycle [4] We have recently cloned, expressed and charac-terized three different enzymatic systems from P.stutzeri OX1: (a) toluene-o-xylene monooxygenase (ToMO) [5], endowed with a broad substrate specificity [6] and (b) phenol hydroxylase (PH) [7], both belonging to the upper pathway; and (c) catechol 2,3 dioxygenase (C2,3O) (A di Donato, unpublished observations), the gateway enzyme
to the lower pathway However, chemical toxicity of wastes can hamper the use of this and other microorganisms in bioremediation strategies, especially when organic solvents are present at high concentrations
Several mechanisms have been described that contribute
to solvent resistance in Gram-negative bacteria, all based on structural changes in outer and inner membranes [8] Different short- and long-term responses have been observed including modifications of the fatty acid and phospholipid composition of the membrane, extrusion mechanisms using vesicles, and energy-dependent active efflux pumps that export toxic organic solvents outside the cytoplasm [9]
Correspondence to A Molinaro, Dipartimento di Chimica Organica e
Biochimica, Universita` di Napoli Federico II, Complesso
Universitario Monte S Angelo, via Cintia 4, 80126 Napoli, Italy.
Fax: + 39 081 674393, Tel.: + 39 081 674123,
E-mail: molinaro@unina.it
Abbreviations: DEPT, distorsionless enhancement by polarization
transfer; GlcN, 2-amino-2-deoxy-glucose; Hep, L -glycero- D
-manno-heptose; Kdo, 3-deoxy- D -manno-oct-2-ulosonic acid;
LOS, lipooligosaccharide; LPS, lipopolysaccharide.
(Received 1 March 2004, revised 23 April 2004,
accepted 30 April 2004)
Trang 2Even though lipopolysaccharides (LPSs) are major
components of the outer membrane of Gram-negative
bacteria, little is known about their role and their chemical
modifications under environmental stress [1,9] It is certain,
however, that LPSs are unique and vital components of
these microorganisms and that they play an important role
in their survival and their interaction with the environment
[10,11] Smooth-form lipopolysaccharides (S-LPSs) include
three regions, the O-specific polysaccharide (or O-antigen),
the oligosaccharide region (core region) and the lipid part
(lipid A) Conversely, rough (R) form LPSs do not possess
an O-specific polysaccharide and are frequently named
lipo-oligosaccharides (LOSs) LOSs have been found either in
wild-type strains and in mutant strains harboring mutations
in the genes encoding enzymes of the biosynthesis and/or
the transfer of the O-specific polysaccharide [12,13]
The core region from both smooth and rough forms of
enteric bacteria generally includes oligosaccharides built of
up to 11 units [12,13], and consists of two distinct domains:
an inner core, characterized by the presence of 3-deoxy-D
-manno-oct-2-ulosonic acid (Kdo) and L-glycero-D
-manno-heptose (Hep), and an outer core, which contains common
sugars It is worth noting that the core oligosaccharide of
LOSs has been reported to play a role in the interaction of
the microorganism with the environment [12,13]
In this paper, the structural characterization of the
carbohydrate backbone of the rough form LPS of P stutzeri
OX1 is reported, obtained by chemical analyses,
MALDI-TOFmass spectrometry and two-dimensional NMR
spectroscopy This novel oligosaccharide chain was found
to possess unusual structural features, which might be
biologically relevant Among these is a GalN residue
substituted by two gluco-configured residues, which are
blocked at position O-4 and O-6 by a pyruvate ketal linkage,
a structure peculiar and new to lipopolysaccharide core
regions
Based on this finding, the hypothesis can be advanced
that the insertion of pyruvate residues at the end of the
oligosaccharide chain blocks its elongation, thereby leading
to a shorter LOS and hence to a less hydrophilic cellular
surface Moreover, as it has already been proposed [1,9],
these residues may also contribute to the rigidity and
stability of the Gram-negative cell wall by binding cations
Experimental procedures
Bacterial growth and LPS extraction
Cells were routinely grown on M9-agar plates supplemented
with 10 mMmalic acid as the sole carbon source, at 27C
For growth in liquid medium, 1 mL was inoculated with a
single colony from a fresh plate, and grown for 18 h at 27C
with constant shaking This saturated culture was used to
inoculate 100 mL of the same medium and grown at 27C
until D600 1 Final growth was started by inoculating the
appropriate volume of the latter culture into 1 L of fresh
medium, to D600¼ 0.02 Cells were grown at 27 C, until
D600¼ 1 was reached and then recovered by centrifugation
at 3000 g for 15 min at 4C, washed with an isotonic buffer
and lyophilized Growth was carried out in M9 salt medium
supplemented with 4 mM phenol as the sole carbon and
energy source Dried cell yield was 0.13 gÆL)1
Dried cells were extracted three times with a mixture of aqueous 90% phenol/chloroform/petroleum ether (50 mL,
2 : 5 : 8 v/v/v) as described previously [14] After removal of the organic solvents under vacuum, the LOS fraction was precipitated from phenol with water, washed first with aqueous 80% (v/v) phenol, and then three times with cold acetone, each time centrifuged as above, and lyophilized (the yield was 90 mg of LOS, about 4.3% of the dry mass) Sodium dodecyl sulfate polyacrylamide gel electrophor-esis (SDS/PAGE) was performed as described previously [15] For detection of LPS and LOS, gels were stained with silver nitrate [15]
Isolation of oligosaccharides
An aliquot of LOS (40 mg) was dissolved in anhydrous hydrazine (2 mL), stirred at 37C for 90 min, cooled, poured into ice-cold acetone (20 mL), and allowed to precipitate The precipitate was then centrifuged (3000 g,
30 min, 4C), washed twice with ice-cold acetone, dried, dissolved in water and lyophilized (32 mg, 80% of the LOS) This material was de-N-acylated with 4MKOH as described [16] Salts were removed using a Sephadex G-10 (Pharmacia) column (50· 1.5 cm) The resulting oligosac-charide 1 constitutes the complete carbohydrate backbone
of the lipid A-core region (16 mg, 40% of the LOS) Another aliquot of LOS (40 mg) was hydrolyzed in 1% (v/v) acetic acid (100C, 2 h) and the precipitate (lipid A) was removed by centrifugation (8000 g, 30 min) The supernatant was separated by gel-permeation chromatog-raphy on a P-2 column (85· 1.5 cm) Two fractions were obtained, the first contained oligosaccharide 2 (28 mg, 70%
of the LOS), whereas the second fraction contained a mixture of reducing pyranose, furanose, anhydro and lactone forms of 3-deoxy-D-manno-oct-2-ulosonic acid (3 mg, 7.5% of the LPSs)
General and analytical methods Determination of Kdo, neutral sugars, carbamoyl analysis, including the determination of the absolute configuration of the heptose residues, organic bound phosphate, absolute configuration of the hexoses, fatty acids and their absolute configuration, GLC and GLC-MS were all carried out as described previously [17–21] For methylation analysis of Kdo region, LOS was carboxy-methylated with methanolic HCl (0.1M, 5 min) and then with diazomethane to improve its solubility in dimethyl sulfoxide Methylation was carried out as described [22,23] LOS was hydrolyzed with 2M
trifluoroacetic acid (100C, 1 h), carbonyl-reduced with NaBD4, carboxy-methylated as described above, carboxyl-reduced with NaBD4(4C, 18 h), acetylated and analyzed
by GLC-MS
Methylation of the complete core region was carried out
as described previously [22–24] The sample was hydrolyzed with 4Mtrifluoroacetic acid (100C, 4 h), carbonyl-reduced with NaBD4, acetylated and analyzed by GLC-MS NMR spectroscopy
For structural assignments of oligosaccharides 1 and 2, 1D and 2D1H-NMR spectra were recorded on a solution of
Trang 35 mg in 0.6 mL of D2O, at 55C or at 30 C, at pD 14 and
7 (uncorrected values), respectively 1H- and 13C-NMR
experiments were carried out using a Varian Inova 500 or a
Varian Inova 600 instrument, whereas for31P-NMR spectra
a Bruker DRX-400 spectrometer was used Spectra were
calibrated with internal acetone [dH 2.225, dC 31.45]
Aqueous 85% phosphoric acid was used as external
reference (0.00 p.p.m.) for31P-NMR spectroscopy
Nuclear Overhauser enhancement spectroscopy
(NOESY) and rotating frame Overhauser enhancement
spectroscopy (ROESY) were measured using data sets
(t1· t2) of 4096· 1024 points, and 16 scans were acquired
A mixing time of 200 ms was used Double quantum-filtered
phase-sensitive COSY experiments were performed with
0.258 s acquisition time, using data sets of 4096· 1024
points, and 64 scans were acquired Total correlation
spectroscopy experiments (TOCSY) were performed with a
spinlock time of 80 ms, using data sets (t1· t2) of
4096· 1024 points, and 16 scans were acquired In all
homonuclear experiments the data matrix was zero-filled in
the F1 dimension to give a matrix of 4096· 2048 points and
was resolution enhanced in both dimensions by a shifted
sine-bell function before Fourier transformation Coupling
constants were determined on a first-order basis from 2D
phase sensitive double quantum filtered correlation
spectro-scopy (DQF-COSY) [25,26] Intensities of NOE signals were
classified as strong, medium and weak using cross-peaks
from intraring proton-proton contacts for calibration
Heteronuclear single quantum coherence (HSQC) and
heteronuclear multiple bond correlation (HMBC)
experi-ments were measured in the1H-detected mode via single
quantum coherence with proton decoupling in the 13C
domain, using data sets of 2048· 512 points, and 64 scans
were acquired for each t1value Experiments were carried
out in the phase-sensitive mode according to the method of
States et al [27] A 60 ms delay was used for the evolution
of long-range connectivities in the HMBC experiment In all
heteronuclear experiments the data matrix was extended to
2048· 1024 points using forward linear prediction
extra-polation [28,29]
MALDI-TOF analysis
MALDI mass spectra were carried out in the negative
polarity in linear or in reflector mode on a Voyager STR
instrument (Applied Biosystems, Framingham, MA, USA)
equipped with a nitrogen laser (k¼ 337 nm) and provided
with delayed extraction technology Ions formed by the
pulsed laser beam were accelerated through 24 kV Each
spectrum is the result of approximately 200 laser shots A
saturated solution of 2,4,6-trihydroxyacetophenone was
used as the matrix
Results
Isolation and characterization of the LOS fraction
The LOS fraction was isolated from dried cells by extraction
with phenol/chloroform/petroleum ether, and further
puri-fied with gel permeation chromatography SDS/PAGE
showed, after silver nitrate gel staining, the presence of fast
migrating species in agreement with their oligosaccharide
nature Compositional monosaccharide analysis of the LOS fraction led to the identification of L,D-Hep, D-GalN,
D-GlcN, D-Glc, Kdo (2 : 1.0 : 3.2 : 1.1 : 1.8) and trace amounts ofL-Rha 7-O-Carbamoyl-L,D-Hep was present in
a stoichiometric ratio withL,D-Hep Methylation analysis showed the presence of terminal Kdo, 6-substituted-HexN, 3-substituted-Hep, 4,5-disubstituted-Kdo, 3,4-disubstituted-HexN, 4,6-disubstituted-Glc, 4,6-disubstituted-HexN and,
in small amounts, terminal-Rha and 6-substituted-Glc
In addition, the disaccharide 7-O-carbamoyl-Hep-(1fi3)-Hep was found
Fatty acid analysis revealed the presence of typical fatty acids of pseudomonads LPS [30], i.e (R)-3-hydroxydodec-anoic acid [C12:0 (3-OH)], present exclusively in amide linkage and (R)-3-hydroxydecanoic [C10:0 (3-OH)] (S)-2-hydroxydecanoic [C12:0 (2-OH)] and dodecanoic acid (C12:0), present in ester linkage Moreover, phosphate colorimetric assays gave positive results
The LOS fraction was then subjected to both alkaline and acid degradations and complete structural characterization
NMR spectroscopy and MALDI-TOF MS spectrometry
of oligosaccharide 1 Oligosaccharide 1 was isolated by gel permeation chroma-tography after complete deacylation of the LOS of
P stutzeriOX1 The complete structure of fully deacylated oligosaccharide 1 (Fig 1) was determined by1H-,31P- and
13C-NMR spectroscopy Chemical shifts were assigned using DQF-COSY, TOCSY, NOESY, ROESY, 1H,13 C-DEPT-HSQC,1H,31P-HSQC,1H,13C-HMBC and1H,13 C-HSQC-TOCSY experiments Anomeric configurations were assigned on the basis of 1H and13C chemical shifts,
of3JH1,H2values determined from the DQF-COSY experi-ment (Table 1), and of1JC1,H1values derived by 1H,13 C-HSQC spectrum recorded without decoupling during acquisition
All sugars were present as pyranose rings, as indicated by
1H- and 13C-NMR chemical shifts and by the HMBC spectrum that showed for all residues intraresidual scalar connectivity between H-1/C-1 and C-5/H-5 atoms (for Kdo units, between C-2 and H-6) The anomeric region of the
1H-NMR spectrum (Fig 2) showed seven major signals in the region between 5.46 and 4.47 p.p.m relative to seven different spin systems (A–G, in order of decreasing chemical shift), and in addition two AB methylene resonances at high fields, typical of Kdo residues (I–L) Each spin system was completely assigned by COSY and TOCSY starting from anomeric resonances For Kdo residues I and L the starting point was the H-3 diastereotopic methylene resonance Both spin systems A and D (5.46 and 5.27 p.p.m.) were characterized by low3JH1, H2and3JH2, H3values, indicative
of two a-manno-configured residues Moreover, all other cross peaks within each spin system were assigned in the TOCSY spectrum from H-2 proton signals, leading to their identification as two heptoses Residue B was identified as a-gluco-configured hexosamine on the basis of chemical shifts and 3JH,Hvalues Moreover, based on its anomeric signal at 5.42 p.p.m present as a double doublet (3JH1,H2¼ 2.9 Hz and3JH1,P¼ 8.3 Hz), with one of the couplings due
to a phosphate signal as shown below, it was identified as GlcN I of the lipid A skeleton The spin system at
Trang 4Table 1 1 H, 13 C and 31 P NMR chemical shifts (p.p.m) of deacylated core-lipid A backbone (oligosaccharide 1) of LOS from P stutzeri OX1 Chemical shifts are relative to acetone and external aq 85% (v/v) phosphoric acid (1H, 2.225 p.p.m.;13C, 31.45 p.p.m.;31P, 0.00 p.p.m at 55 C).
31
31
13
Fig 1 The structure of oligosaccharide 1 obtained by alkaline hydrolysis of the core region of the LPS of P stutzeri OX1.
Trang 55.32 p.p.m (C;3JH1, H2¼ 3.6 Hz) was identified as a-GalN
by its JH,Hvalues for H-3/H-4 and H-4/H-5, diagnostic of a
galactoconfiguration (3.4 Hz and less than 1 Hz,
respect-ively) Three spin systems E, F and G (doublets;3JH1, H2¼
8.6, 7.8 and 7.7 Hz, respectively) were identified as
b-gluco-configured monosaccharides given their large3JH,H-values
A further indication of their b configuration was the
observation of NOE contacts in the ROESY spectrum
among H-1, H-3 and H-5, for all E, F, G residues
The H-3 methylene signals of two a-Kdo residues were
present at 1.82 p.p.m (H-3ax) and 2.07 p.p.m (H-3eq)
(residue I), and 2.00 p.p.m (H-3ax) and 2.34 p.p.m
(H-3eq) (residue L), respectively Their a configuration
was established on the basis of the chemical shift of their
H-3eq protons and by measurement of the 3JH7,H8aand
3JH7,H8b coupling constants [31,32] Two methyl singlet
signals were present at higher fields, at 1.48 and 1.62 p.p.m.,
respectively Each methyl signal was in a 3 : 1 ratio with the
anomeric signals, i.e in a stoichiometric ratio
The13C-NMR chemical shifts could be assigned by a
DEPT-HSQC experiment, using the assigned 1H-NMR
spectrum Seven anomeric carbon resonances were
identi-fied (Table 1), numerous carbon ring signals and four
nitrogen-bearing carbon signals assigned to C-2 of B, C, F
and G spin systems Considering the13C chemical shifts of
nonsubstituted residues [33], several low-field shifted signals
indicated substitutions at O-3 of residue A, O-6 of residue B,
O-3 and O-4 of residue C, O-3 of residue D, O-4 and O-6 of
residues E and F, O-6 of residue G, O-5 and O-4 of residue
L, whereas I was a terminal residue In the high field region
of the spectrum two cross peaks at 1.48/25.2 and 1.62/ 17.2 p.p.m were present
Phosphate substitution was established on the basis of
31P-NMR spectroscopy The31P-NMR spectrum showed the presence of five monophosphate monoester signals (Table 1) The site of substitution was inferred by a
1H,31P-HSQC spectrum that showed correlations of 31P signals with H-1 B (GlcN), H-4 A and H-2 A (Hep I), H-4 G (GlcN) and H-6 D (Hep II)
The sequence of the monosaccharide residues was determined using NOE effects of the ROESY (Fig 3) and NOESY spectra, and by1H,13C-HMBC correlations The typical lipid A carbohydrate backbone was eventually assigned on the basis of the NOE signal between H-1 G and H-6a,b B In the case of Kdo units, which lack the anomeric proton, the sequence was inferred by NOE contacts between the methylene-proton H-3eq of Kdo L and H-6 of Kdo I, whereas Kdo L was substituted by heptose A as indicated by the NOE effect found between H-1 A and H-5 L, and, in addition, between H-5 A and H-3axL All of these NOE contacts were characteristic of the sequence a-L-glycero-D -manno-heptose-(1fi5)-[a-D-Kdo-(2fi4)]-a-D-Kdo [34,35]
Heptose A was, in turn, substituted at the O-3 position
by heptose D, as demonstrated by the NOE cross peak between H-1 D and H-3 A A disaccharide
7-O-carbamoyl-Fig 2.1H-NMR spectrum of oligosaccharide 1 The spectrum was recorded under the following conditions: 5 mg of oligosaccharide 1 in 0.6 mL
D 2 O, pD 14 at 30 C.
Trang 6Hep-(1fi3)-Hep was also identified by methylation analysis
of the intact LOS, thus, the carbamoyl group should be
attached to O-7 of the heptose moiety D The GalN C was
attached to the O-3 position of this last heptose as shown by
the NOE effect between the anomeric proton of GalN and
H-3 of D
GalN is the branching point of the chain and,
conse-quently, it should carry two sugar residues at O-3 and O-4
Indeed, the anomeric proton of b-glucose E gave a NOE
effect with H-3 of GalN, whereas the anomeric proton of
b-glucosamine F gave a NOE effect with H-4 of GalN
In determining the L Kdo location, its linkage to unit G
was deduced by exclusion In particular, the linkage to O-6
of G was inferred by taking into account the downfield shift
of the carbon signal C-6 (63.7 p.p.m., Table 1) indicating its
involvement in a glycosydic linkage
The HMBC spectrum confirmed the sequence proposed
for oligosaccharide 1, as it contained the significant
long-range correlations required for the determination of the
sequence and of the attachment points In fact, together
with intraresidual long-range cross-peaks, interresidual
long-range connectivity was found between H-5/C-5 L
and C-1/H-1 A, H-3/C-3 A and C-1/H-1 D, H3/C-3 D and
C-1/H-1 C, H-1/C-1 E and C-3/H-3 C, H-1/C-1 F and C-4/
H-4 C, H-1/C-1 G and C-6/H-6 B
The HMBC experiment was also crucial for the
identification and localization of the two methyl
groups belonging to noncarbohydrate constituents Plain
long-range correlations (Fig 4A) were found in the spectrum for each methyl signal The signal at 1.48 p.p.m correlated to two different carbon signals at 101.9 and 175.5 p.p.m., whereas the signal at 1.62 p.p.m correlated to two other signals at 99.5 and 175.8 p.p.m None of these four carbon signals was present in the HSQC spectrum These data pointed to two cyclic ketals
of pyruvic acid present on two distinct residues, namely E and F, whose C-4 and C-6 signals experienced a downfield displacement This was confirmed by the HMBC spec-trum where each ketal carbon signal of pyruvate residues correlated to H-4 and H-6 of E and F residues (b-D-Glc and b-D-GlcN, respectively) It should be noted that the signal discrepancy in the1H and13C chemical shifts of the two pyruvate moieties is due to the different absolute configuration at C-2 In fact, the methyl signal occurring
at 1.48 and 25.2 p.p.m is assigned to the S-pyruvate group, whereas the one occurring at 1.62 and 17.2 p.p.m
is assigned to an R-pyruvate group, as already described [36] Moreover, the ROESY spectrum (Fig 4B) was in complete agreement with the assignment above In fact, the methyl signal of the R-pyruvate residue at 1.62 p.p.m gave a strong NOE effect with H-4 and H-6aof residue F This is in agreement with an axial orientation of the methyl group on a 1,3-dioxane ring in a chair-like conformation in which H-4 and H-6aare sin diaxial with respect to it The methyl signal of S-pyruvate, being in equatorial orientation, only gave NOE effect with the
Fig 3 Section of the ROESY spectrum of oligosaccharide 1 Monosaccharide labels are as indicated in Fig 1 NOE cross-peaks are in black, in antiphase with diagonal (grey lines) Spectrum was recorded at pD 14, 55 C.
Trang 7adjacent H-6 of residue E Thus, all main spin systems
were assigned in the NMR spectra, and all chemical data
found a rational explanation
The presence of a minor spin system (10%) belonging to
rhamnose (anomeric signal at 4.89 p.p.m) and 6-substituted
glucose (overlapped with terminal glucose) might be
explained by the presence of a second outer core glycoform
in which rhamnose is attached at O-6 of the glucose residue,
which obviously lacks the pyruvate group
The MALDI mass spectrum confirmed the proposed
structure In fact, an ion peak at m/z 2188.4 (Fig 5A) was
present, corresponding to the complete carbohydrate
back-bone bearing five phosphate goups and two pyruvic acid
acetal residues Moreover, at higher laser intensity (Fig 5B)
various ion peaks related to fragments were found, all fitting
with the structure shown in Fig 1
In conclusion, the data above allowed the identification
of the carbohydrate backbone from alkaline degradation of
the rough form LPS from P stutzeri OX1
Isolation, NMR and MS analyses of oligosaccharide 2
from acetic acid hydrolysis
Further information on alkaline labile groups that could
be present in the core region (i.e acyl groups) was obtained
by treating the LOS with acetic acid to split the Kdo
linkage An oligosaccharide mixture was isolated after gel
permeation chromatography, which was purified further and the resulting oligosaccharide 2 (Fig 6) analyzed by compositional/methylation analyses, 2D NMR and mass spectrometry
Compositional and methylation analyses led to the identification of 3-substituted-L,D-Hep, 7-O-carbamoyl-3-substituted-L,D-Hep 3,4-disubstituted-D-GalN, terminal
D-glucose and terminal D-GlcN Traces of 5-substituted Kdo, 6-substituted-D-Glc and terminal L-rhamnose were also found
The1H-NMR spectrum revealed the absence of anomeric signals from GlcN I and GlcN II of Lipid A, the lack of pyruvate methyl groups, as a consequence of the cleavage of the ketal group under acid treatment, and the presence
of singlet signals at 2.00 p.p.m Methylene signals of Kdo were spread because of its presence as reducing end unit, i.e pyranose, furanose, anhydro and lactone forms present at same time The anomeric region of the spectrum consisted
of six main signals (Fig 6), five of which belonging to the main oligosaccharide backbone, named U–Z All
resonanc-es of the monosaccharidresonanc-es (Table 2) were obtained from 2D NMR spectroscopy (DQF-COSY, TOCSY, NOESY, ROESY, 1H,13C-DEPT-HSQC 1H,31P-HSQC, 1H,13 C-HMBC and 1H,13C-HSQC-TOCSY) Evaluation of chemical shifts and of 3JH,H coupling constants led to identification of residues Hep (U), 7-O-carbamoyl-Hep (V), GalN (X), GlcN (W), Glc (Z)
Fig 4 Sections of the high field region of the (A) ROESY and (B) HMBC spectra Correlations of pyruvate methyl groups are shown (A) The 4,6 Pyr-GlcN residue is drawn in the middle of the figure with arrows indicating the relevant NOE contacts between methyl protons of the R-pyruvate group and H-4 and H-6 of GlcN residue Spectra were recorded at pD 14, 55 C.
Trang 8Low-field shifted signals were present in the HSQC
spectrum indicating substitutions at O-3 (U, V and X) and
O-4 (X), whereas residues W and Z were not substituted
Cross-peaks were also detected only for two nitrogen atoms
bearing carbon signals, at 4.26/49.8 p.p.m (H-2/C-2 X) and
3.67/56.1 p.p.m (H-2/C-2 W), in agreement with the
absence of Lipid A disaccharide and with the presence of
a a-galacto and a b-gluco configured 2-amino-2-deoxy
hexoses Moreover, given the downfield H-2 chemical shifts
of the X and W residues, the amino groups should have
been present as acylamido
The high field proton region of the HMBC gave clues for
the identification of the nature of acyl groups Three
different singlet signals were present in this region (Fig 6),
two with a smaller area that probably account for the same
methyl group that experienced oligosaccharide
heterogen-eity All signals in the region of 2.0 p.p.m showed
long-range correlations with a carbonyl signal at 174.6 p.p.m
which in turn correlated to protons at 3.67 p.p.m (H-2 W)
and 4.26 p.p.m (H-2 X) These correlations indicated the
presence of two acetamido groups at the C-2 of GlcN W and GalN X Thus, the two smaller methyl signals are both due to GalN X and are consequences of oligosaccharide heterogeneity, possibly due either to the adjacent heptose V bearing heterogeneous phosphate substitution (see below)
or to a reducing Kdo residue
In addition, all diagnostic interresidue NOE effects were found in the ROESY spectrum This confirmed the oligosaccharide sequence as determined in the previous paragraph
Other information on noncarbohydrate substituents (phosphate and carbamoyl groups) was gained by the observation of the downfield displaced heptose signals, namely H-2/C-2 and H-4/C-4 of heptose U and H-6/C-6 and H-7a,bof heptose V
The H-7a,bdownfield shift was clearly due to the presence
of a carbamoyl group that did not undergo hydrolysis in mild acid conditions, and that has already been located at position O-7 of the second heptose residue on the basis of methylation analysis In agreement with this assignment, a
Fig 5 Negative ion MALDI-TOF mass spectra of oligosaccharide 1 obtained in linear mode at normal (A) and higher laser intensity (B) Assignments
of main ion peaks are shown P, phosphate; Pyr, pyruvic acid.
Trang 9signal at 160.0 p.p.m in the HMBC spectrum correlated
with both protons H-7 of V
The degree of phosphorylation and localization of
phosphate substituents was established by 1D and 2D
31P-NMR spectroscopy (Fig 7, Table 2) Several signals
were found in the 31P-NMR spectrum, whose chemical
shift clearly indicated that they derived from phosphate
groups present in different magnetic/chemical
environ-ments In fact, in addition to a number of phosphate
monoester signals in the region of 1.4–3.2 p.p.m., two
peaks of lower intensity were present at )5.5 and
)9.7 p.p.m These last two signals derived from a
diphosphate monoester bond, i.e a pyrophosphate group
In particular, the signal at)5.5 p.p.m could be identified
as the distal phosphate group, while the phosphate at
)9.7 p.p.m was identified as the proximal phosphate
group The 1H,31P-HSQC spectrum showed correlation
between H-2 and H-4 of heptose U, with typical signals of
a phosphate monoester group The H-6 V resonance,
present as two different signals, showed two different
cross-peaks, one with a phosphate monoester group at
4.46/3.2 p.p.m and the other at 4.63/)9.7 p.p.m., with the
proximal phosphate of a diphosphate monoester residue
Thus, heptose U is substituted at O-2 and O-4 by a
phosphate group, whereas heptose V carries at O-6 a phosphate group or alternatively, a pyrophosphate group The MALDI-TOFmass spectrum (Fig 8) of oligosac-charide 2 confirmed all the assignments, as all ion peaks corresponding to the structures above were present In fact, ion peaks characteristic of an oligosaccharide were found, composed of two HexNAc, one Hex, two Hep, one Kdo, one carbamoyl group and two, three, four and five phosphate groups Moreover, additional peaks at Dm/z
146 accounted for the presence of a second core glycoform, which differs from the most abundant one by an additional rhamnose residue that must be linked at O-6 of glucose Ion peaks derived from the loss of water from molecular ions, probably Kdo lactone or anhydro-Kdo forms, were also present Furthermore, the MALDI-TOF mass spectrum also accounted for the presence of a very small amount of pentaphosphorylated species, which was not detected by NMR Because no different phosphate substitution was visible in 1D and 2D31P-NMR, we propose that the fifth phosphate group is present as pyrophosphate on heptose U
In conclusion, information derived from both acid and alkaline hydrolysis leads to the proposal of the following structure of the major oligosaccharide from the LOS of
P stutzeriOX1
Fig 6.1H-NMR spectrum of oligosaccharide 2 obtained by acetic acid hydrolysis The spectrum was recorded under the following conditions: 5 mg
of oligosaccharide 2 in 0.6 mL D 2 O, pD 7 Monosaccharides are as shown; rhamnose residue anomeric signal is not labeled as it belongs to the minor oligosaccharide Dotted bonds indicate a nonstoichiometric linkage Chemical shifts are shown in Table 2.
Trang 10Table 2. 1H,13C and31P NMR chemical shifts (p.p.m.) of the oligosaccharide product deriving from acetic acid treatment of the LOS from P stutzeri OX1 O-6 V resonances are given in parentheses when this position is monophosphorylated P/P refers to both resonances of pyrophosphate Because Kdo signals are spread due to its multiple forms, resonances are not given Resonances of the minor fragment Rha-C1fi6)-Glc are also shown at the bottom Chemical shifts are relative to acetone and external aq 85% (v/v) phosphoric acid (1H, 2.225 p.p.m.;13C, 31.45 p.p.m.;
31 P, 0.00 p.p.m at 30 C).
1
H/13C/31P
)9.7/)5.5 (3.2)
62.4
Fig 7 Section of the 1 H, 31 P-HSQC spectrum of oligosaccharide 2 The spectrum shows cross peaks relevant for the localization of the phosphate groups.