Structural Characterization of the O-antigenic Polysaccharide of
the Lipopolysaccharide from Rhizobium etli Strain CE3
A UNIQUE O-ACETYLATED GLYCAN OF DISCRETE SIZE,
CONTAINING 3-O-METHYL-6-DEOXY-L-TALOSE AND
2,3,4-TRI-O-METHYL-L-FUCOSE*
Lennart S.
Forsberg
,
U. Ramadas
Bhat§, and
Russell W.
Carlson¶
From the Complex Carbohydrate Research Center,
University of Georgia, Athens, Georgia 30602 and
§ Miles-Cutter Laboratories,
Berkeley, California 94701-1986
Received for publication, February 9, 2000, and in revised form, April 14, 2000
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ABSTRACT |
The O-antigenic polysaccharide of the
Rhizobium etli CE3 lipopolysaccharide (LPS) was
structurally characterized using chemical degradations (Smith
degradation and 
-elimination of uronosyl residues) in combination
with alkylation analysis, electrospray, and matrix-assisted laser
desorption ionization-time of flight mass spectrometry, tandem mass
spectrometry, and 1H COSY and TOCSY nuclear magnetic
resonance spectroscopy analyses of the native polysaccharide and the
derived oligosaccharides. The polysaccharide was found to be a unique,
relatively low molecular weight glycan having a fairly discrete size,
with surprisingly little variation in the number of repeating units
(degree of polymerization = 5). The polysaccharide is
O-acetylated and contains a variety of
O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major
de-O-acetylated species, including the reducing end
3-deoxy-D-manno-2-octulosonic acid (Kdo)
residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide
repeating unit having the structure
4)-
-D-GlcpA-(14)-[-3-O-Me-6-deoxy-Talp-(13)]--L-Fucp-(1. The nonreducing end of the glycan is terminated with the capping sequence
-2,3,4-tri-O-Me-Fucp-(14)--D-GlcpA-(1,
and the reducing end of the molecule consists of the
non-repeating sequence
3)--L-Fucp-(13)--D-Manp-(13)--QuiNAcp-(14)--Kdop-(2, where QuiNAc is N-acetylquinovosamine
(2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo
residue links the O-chain polysaccharide to the core region
oligosaccharide, resulting in a unique location for a Kdo residue in
LPS, removed four residues distally from the lipid A moiety. Structural
heterogeneity in the O-chain arises mainly from the
O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of
the 2,3,4-tri-O-methylfucosyl capping residues, typically
15%, are replaced with 2-O-methyl- and/or
2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the
3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl
residues are 2-O-methylated. A majority of the glucuronosyl
residues are methyl-esterified at C-6. These unique structural features
may be significant in the infection process.
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INTRODUCTION |
The Rhizobium are Gram-negative bacteria capable of
forming a nitrogen-fixing symbiosis with legumes in a host-specific
manner. The initial stages of this process have been fairly well
studied and involve an exchange of signal molecules (e.g.
flavonoids and nod factors) (1-4) between the bacterium and plant,
leading to the induction of specific bacterial and plant genes.
Relatively little is known about the molecular events occurring during
the later stages of infection, including the subsequent development of
the infection threads and bacteroids. Morphological studies have shown
that the rhizobial cell surface is in contact with the plant cell
surface throughout these stages, suggesting a prominent role for cell
surface macromolecules in these events.
Rhizobium cells cultured as free-living bacteria express a
variety of glycoconjugates on the cell surface, including the
lipopolysaccharides (LPS),1
capsular polysaccharides (K-antigens), and the extracellular polysaccharides (5-7). The LPS are major structural components of the
rhizobial outer membrane and the dominant antigens of the rhizobial
cell surface; antibodies raised to whole rhizobial cells are directed
most strongly to the LPS (5, 8-10). Studies with monoclonal antibodies
directed to the O-chain moiety of various rhizobia have revealed that
different types of LPS can be expressed by a single
Rhizobium strain and that this expression depends on the
in planta or physiological environment (11-16). The
majority of these antigenic changes appear to occur in the O-chain
portion of the LPS and are associated with the different stages of
symbiotic infection (11, 12, 14-18). In addition, recent studies have shown that certain LPS structural changes can be induced by plant flavonoids, indicating that LPS expression can be modulated by nod/nol gene products (19-21). In one example, an
Rhizobium etli LPS O-chain epitope was suppressed by adding
bean root or seed exudate to the growth medium (22), and the active
compound was identified as an anthocyanin (21). Other studies have
examined rhizobial LPS mutants and their symbiotic phenotypes and have shown that mutants that lack the O-antigen portion of their LPS or that
contain truncated O-chains are unable to form normal infection threads
(10, 23, 24) and/or are unable to invade the root nodule cells
(25-27). These studies have led to the general hypothesis that
complete LPS molecules, containing a polymeric O-chain and intact
core-lipid A, are required for normal symbiosis to occur (24, 28,
29).
The structure of the R. etli- Rhizobium
leguminosarum common core region has been elucidated (30-32) and
was found to have an entirely different structure from the typical core
regions of enterobacterial LPS, including distinct linkage and
branching patterns, the absence of both heptose and phosphate, an
abundance of galacturonic acid, and notably, the location of a Kdo
residue in the O-chain attachment region, removed four residues
distally from the lipid A moiety. The lipid A moieties of these
rhizobial LPS also differ significantly from the enterobacterial lipid
As (33). The R. etli and R. leguminosarum lipid
As are devoid of phosphate, have trisaccharide backbones containing
uronic acid, glucosamine, and 2-aminogluconic acid, and contain the
long chain fatty acid, 27-hydroxyoctacosanoic acid, not found in the
enteric lipid As. Like the enterobacterial LPS, the core-lipid A
regions of the rhizobial LPS tend to be conserved within each
Rhizobium genus, whereas structural diversity is typically
found in the O-specific polysaccharide moiety (6, 8, 29). In the case of R. etli and R. leguminosarum, the structurally
variable O-chains are strain-specific and are the dominant antigenic
determinants (8-10). In the majority of cases the structures of these
epitopes have not been defined, and it is difficult to draw precise
conclusions about their alterations or the significance of these
alterations with regard to nodulation. In this report we describe the
structure of the O-antigenic polysaccharide portion of the R. etli CE3 LPS. With the structure of this O-chain, the complete
glycosyl sequence of the R. etli LPS, including the core
region (30) and lipid A (33), is now known.
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EXPERIMENTAL PROCEDURES |
Growth of Bacteria--
R. etli CE3, the
streptomycin-resistant derivative of wild-type strain CFN42, was grown
in tryptone/yeast extract supplemented with Ca2+ as
described previously (10). Bacteria were harvested by centrifugation at
late log/early stationary phase.
Isolation of Lipopolysaccharide and the O-specific
Polysaccharide--
Lipopolysaccharides were extracted using the hot
phenol/water procedure (10) and were analyzed by polyacrylamide gel
electrophoresis (PAGE) in the presence of deoxycholate (34, 35). Water
layers containing LPS were dialyzed and treated sequentially with
ribonuclease, deoxyribonuclease, and proteinase K and then redialyzed
and subjected to size exclusion chromatography (SEC) in
triethylamine/acetic acid buffer (10). The O-polysaccharide was
isolated from the SEC-purified total LPS (both smooth and rough forms)
by mild acid hydrolysis in 1.0% acetic acid at 105 °C for 2 h.
The hydrolysates were subjected to freeze/thaw (three repetitions), and
the precipitated lipid A was removed by ultracentrifugation at
160,000 × g for 1 h. The supernatants, containing
the solubilized O-polysaccharide and the core region oligosaccharides,
were passed through a column of polymyxin-agarose to remove remaining
traces of free lipid A and intact LPS (30). The flow-through eluants
were then chromatographed on Bio-Gel P-2 (45-90-µm fine, 1.5 × 90 cm) equilibrated in 50 mM ammonium formate, pH 6.5, to
separate the O-chain from the core oligosaccharides.
Analysis of Glycosyl Residues--
Carbohydrate compositions of
the O-polysaccharide and derived fractions were determined by
preparation of the TMS methyl glycosides with GLC-MS (electron impact)
analysis (36) using a 30-m DB-5 fused silica capillary column (J & W
Scientific). Carbohydrate identities and the location of endogenous
O-methyl groups were also determined by analysis of the
alditol acetate derivatives, using a 30-m SP-2330 capillary column
(Supelco). Alditol acetates were prepared by hydrolysis in 2 M trifluoroacetic acid (121 °C, 2 h), followed by
reduction (NaBD4) and acetylation (acetic
anhydride/trifluoroacetic acid) (37). Acidic glycosyl residues were
analyzed as the alditol acetates by converting the carboxyl groups to
methyl esters (1 M methanolic HCl, 80 °C for 2 h),
followed by carboxyl reduction with NaBD4 in water,
trifluoroacetic acid hydrolysis, and conversion to the alditol acetates
(33). Authentic N-acetylquinovosamine (QuiNAc) was prepared
by chemical synthesis by Dr. R. Hollingsworth and provided as a gift.
An authentic standard of 6-deoxy-L-talose was obtained from
a streptococcal cell wall glycan (38).
Linkage analysis of neutral sugars was performed by permethylation
(Hakomori method), conversion to the partially methylated alditol
acetates (PMAAs) (36), and GLC-MS analysis. The Kdo and uronic acid
linkages were identified by sequential permethylation, reduction of the
carboxymethyl groups with lithium triethylborodeuteride (Aldrich), mild
hydrolysis (0.1 M trifluoroacetic acid, 100 °C, 30 min)
to cleave ketosidic linkages, reduction (NaBD4), normal hydrolysis (2 M trifluoroacetic acid, 121 °C, 2 h),
and conversion to the PMAAs (30). Methylations using trideuteriomethyl
iodide were also performed to confirm sugar identities and the
locations of endogenous O-Me ether groups. -Elimination
in conjunction with linkage analysis was performed by treating
permethylated samples with 2 M dimethyl sulfoxide anion
(potassium salt) in Me2SO for 8 h; the elimination
products were then ethylated using ethyl iodide, and the products were
converted to the corresponding partially methylated, ethylated alditol
acetates and analyzed by GLC-MS (33). Where necessary, the identities
of TMS methyl glycoside and PMAA derivatives were confirmed by chemical
ionization-MS, using a 30-m DB-1 column and ammonia as the reactant gas.
Chemical Modifications and Smith
Degradation--
De-O-acetylation of the O-specific
polysaccharide was performed in 10 mM NaOH at 4 °C for
10 h. The progress of the reaction was followed by 1H
NMR and MALDI-TOF mass spectrometry. Total -elimination
(depolymerization) was performed on portions of the native or
de-O-acetylated polysaccharide using the following
procedure. The polysaccharide (23 mg) was reduced with
NaBD4 (10 mg/3 ml 0.3 M NH4OH)
overnight to stabilize the reducing end Kdo residue. The polysaccharide
was then methyl-esterified by treating 3 times with methanol containing
1 to 2 drops of aqueous 1 M HCl and evaporating to dryness.
The fully esterified polysaccharide was then treated with aqueous NaOH
(0.25 M, 38 °C, 18 h), and the products were
chromatographed by (SEC) using Bio-Gel P-10 (1.5 × 92 cm) in
water. Further fractionation was performed on Bio-Gel P-4 (<45 mm,
1 × 120 cm) or P-2 (<40 mm, 1 × 120 cm). Column eluants
were monitored for neutral carbohydrate using the phenol sulfuric acid
assay (36) and for the unsaturated 
4,5-hexuronosyl
residues by measuring absorbance at 232 nm. Appropriate fractions
containing oligosaccharides were combined and analyzed by ESI-MS,
MALDI-TOF MS, and NMR as described below.
The Smith degradation (39) was performed by dissolving a sample of the
de-O-acetylated O-chain polysaccharide in 50 mM
sodium acetate buffer, pH 4.5, mixing with an equal volume of 0.2 M NaIO4, and maintaining at 4 °C for 72 h in the dark. Excess periodate was destroyed (ethylene glycol), and
the sample was reduced with NaBH4 with the solution
adjusted to pH 7.5. The resulting oxidized-reduced glycan
("polyol") was purified by chromatography on Bio-Gel P-10 and
lyophilized. The polyol was subjected to mild hydrolysis (0.5 M trifluoroacetic acid, 23 °C, 48 h); the acid was
removed by evaporation at room temperature, and the products were
separated on Bio-Gel P-2. The resulting oligosaccharides were analyzed
for structural information.
Mass Spectrometry--
Electrospray ionization mass spectrometry
(ESI-MS) was performed on a SCIEX API-III triple-quadrupole mass
analyzer (PE/SCIEX Thornhill, Ontario, Canada) operated in the positive
ion mode with an orifice potential of 35-50 V (15). Spectra are the
accumulation of 10-60 scans collected over the m/z range
200-2400 with a mass step of 0.2-1.0 atomic mass units at 1 ms/step.
All water-soluble samples were desodiated by dialysis, followed by
treatment with a strong cation exchange resin (H+ form),
and then filtered using 0.45-µm nylon spin filters and stored in
polypropylene tubes. Underivatized samples were analyzed at a
concentration of 2 µg/µl with a flow rate of 3-5 µl/min using a
solution of 15% v/v methanol in deionized water containing 0.5% v/v
acetic acid. Permethylated polysaccharide samples were analyzed in
100% methanol containing 1% ammonium formate. Tandem mass
spectrometry (MS/MS) was performed on the SCIEX instrument by selecting
a parent ion for collision-induced dissociation using argon as
collision gas. The predicted molecular weights of the various
de-O-acylated LPS were calculated using the following
average incremental mass values, based on the atomic weights of the
elements: hexose, 162.1424; hexuronic acid, 176.1259;
4-deoxyhex-4-enopyranosiduronic acid, 158.1106; 3-deoxy-2-octulosonic
acid (Kdo), 220.1791; anhydro Kdo, 202.1638; 6-deoxyhexose, 146.1430;
mono-O-methyl-6-deoxyhexose, 160.1699;
di-O-methyl-6-deoxyhexose 174.1968;
tri-O-methyl-6-deoxyhexose, 188.2236;
2-N-acetamido-2,6-dideoxyhexose, 187.1955; free reducing end, 18.0153.
Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry
was performed on an LDI 1700XP time-of-flight (TOF) spectrometer
(Linear Scientific, Reno, NV) in the positive and negative modes, using
a matrix of 100 mM 2,5-dihydroxybenzoic acid in 90%
methanol. The instrument was operated at an accelerating voltage of 30 kV and an extractor voltage of 9 kV. The sample was ionized with a
nitrogen laser (
= 337 nm) with a pulse width of 3 ns and
4-7.5-µJ pulse. The following settings were used: detector
sensitivity, 1275 mV full scale; voltage, 4.75 kV. Mass spectra were
recorded over a m/z range of 1-50,000; spectra are the
summation of 50-250 acquisitions. Polygalacturonic acid or malto-oligosaccharides were used as calibration standards.
High Pressure Liquid Chromatography Fractionation of
Oligosaccharides--
Oligosaccharides derived from the O-chain by
-elimination were separated by SEC as described above and then
fractionated using a Vydac 238TP510 5-µm monomeric C18
semi-preparative reverse phase column (10 × 250 mm) using a
linear gradient (0-30%) of methanol in water, containing 0.05%
trifluoroacetic acid with a flow rate of 3 ml/min. Elution was
monitored using an evaporative light scattering detector (Richard
Scientific), using a 9:1 split in which 90% of the eluant was diverted
from the detector and collected into fractions for structural analysis.
NMR Spectroscopy--
1H spectra were recorded with
a Varian 300 or 600 spectrometer in D2O at 35 °C using
acetone as internal standard, 
2.22. Two-dimensional NMR spectra
(COSY and TOCSY) were performed using the Varian software.
Glycosyl Configuration--
The enantiomeric configuration of
the glycosyl residues was determined by GLC-MS analysis of the TMS
(
)-2-butyl glycosides of the polysaccharide in comparison with the
authentic sugars (40). The Kdo was assumed to be in the
D-configuration.
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RESULTS |
Isolation, Molecular Size, and Composition
Analysis--
Phenol/water extraction of R. etli strain CE3
typically allows recovery of both the smooth and rough LPSs in the
water layer (~800 mg of LPS/50 g of lyophilized cells), as shown in
the deoxycholate-PAGE profile in Fig. 1,
inset. MALDI-TOF-MS analysis of these LPSs is also shown in
Fig. 1. It can be seen that the major MALDI component, corresponding to
the LPS-I band, has an apparent molecular mass centered at around 6600 atomic mass units, using neutral maltooligosaccharides as the TOF
calibrant. The molecular mass of this smooth LPS is consistent with the
previously characterized masses for the R. etli lipid A
moiety (major species2 1987 atomic mass units (33)), and the core region oligosaccharide (major
species3 1531 atomic mass
units) (30). These results suggested an approximate size for the
O-chain of around 3100 atomic mass units.
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Fig. 1.
Positive ion MALDI-TOF mass spectrum and
deoxycholate-PAGE analysis (inset) of the total
R. etli LPS, isolated from the water layer of
phenol/water extracts by Sepharose 4B chromatography. The strain
CE3 LPSs consists of distinct smooth and rough forms (10, 29). The
LPS-I species contains the intact O-chain; LPS II is a rough LPS that
lacks N-acetylquinovosamine and the O-chain repeating unit
residues. LPS III and IV appear to carry truncated O-chains (one
repeating unit) based on immunoblot results (10, 29). In the mass
spectrum, the apparent size heterogeneity of these LPS is due, in part,
to fatty acid heterogeneity in the lipid A, to glycosyl heterogeneity
in both the core region and the O-chain, and presumably to sodium
adduct formation.
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The LPS was purified and subjected to mild acid hydrolysis, which
selectively cleaves the Kdo ketosidic linkages yielding the O-chain
polysaccharide, the core oligosaccharides, and the free lipid A. The
released O-chain and core region oligosaccharides each carry a Kdo
residue at the reducing end that can undergo a variety of degradative
reactions as discussed below. Following hydrolysis and lipid A removal,
the soluble products were separated on Bio-Gel P-2, yielding the
O-specific polysaccharide fraction (PS) at the void volume, and the
lower molecular weight core region oligosaccharide fraction (OS) (data
not shown). As shown in Fig. 2A, rechromatography of the PS
fraction on Bio-Gel P-10 yielded a single symmetrical peak having a
Kav = 0.18. MALDI-TOF-MS analysis of this PS
revealed three major components, with an approximate molecular mass
around 3000 atomic mass units (Fig. 2B). Inspection of these
components showed each to be broad, with small incremental differences
of 42 ± 14 mass units, suggesting the presence of O-acetyl and O-methyl groups (Fig. 2B,
inset). The 1H NMR spectrum of PS confirmed this,
showing signals for both O-acetyl and N-acetyl
methyl group protons at 2.12 and 2.03, respectively (Fig.
3A). The ratio of
O-acetyl to N-acetyl protons was approximately
5:1. Other signals in the proton spectrum indicated the presence of
R-CH3 protons, ~ 1.2, typical of
6-deoxyhexoses, and intense signals from 3.4 to 3.5, typical of
O-CH3 protons. The anomeric resonances were
broad, presumably a result of the O-acetyl and
O-methyl substitution. A set of doublets at 2.60 are
consistent with H-3 methylene protons of an anhydro Kdof
residue (41, 42)
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Fig. 2.
Chromatographic properties and molecular size
estimation of the O-chain polysaccharide on Bio-Gel P-10 and by
MALDI-TOF mass spectrometry. A, the Bio-Gel P-10
profile compares the native O-chain (Kav = 0.18)
and the de-O-acetylated O-chain (Kav = 0.39). B, the negative ion MALDI spectrum is of the native
glycan and shows three major components as described in the text.
B, weak ions marked with arrows differ by
approximately 480 mass units and appear to arise from small amounts of
truncated or incompletely synthesized O-chains, which contain fewer
repeating units. Inset, O-acetyl and
O-methyl heterogeneity.
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Fig. 3.
The 1H NMR spectra of the native
(A) and de-O-acetylated O-chain
(B). The native glycan is O-acetylated
and also contains N-acetyl group(s). Inset,
doublets at 2.60 are indicative of H-3 methylene protons of an
anhydro Kdof residue (41, 42). Other features of the
spectrum are described in the text. The sample in B was
analyzed after 10 h of "mild" de-O-acetylation, at
which point O-acetyl removal appeared complete.
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The glycosyl composition of the PS fraction is reported in Table
I. Analysis of the TMS methyl glycosides
revealed that glucuronic acid, fucose, and an unidentified
3-O-methyl-6-deoxyhexose were the major components, along
with lesser amounts of mannose, N-acetylquinovosamine, Kdo,
and several partially O-methylated sugars. The PS fraction did not contain any fatty acids, N-acetylglucosamine,
galacturonic acid, or other previously identified lipid A or core
oligosaccharide components (30, 33), indicating that the PS consisted
entirely of the O-chain polysaccharide. Analysis of the alditol
acetates indicated that the 3-O-methyl sugar had a retention
time identical to that of 3-O-methyl-6-deoxytalose. In an
earlier study, it was reported that the total LPS from these cells
contained 3-O-methylrhamnose, presumably the O-chain
component (43). However, in the course of our present study it became
clear that this component has a slightly different retention time from
that of 3-O-methylrhamnose, when derivatized by either the
TMS methyl glycoside, alditol acetate, or partially methylated alditol
acetate procedures. The derivatives from this R. etli
O-chain component had retention times identical to those from an
authentic standard of 6-deoxytalose (38) but differed from the
corresponding derivatives prepared from rhamnose, fucose, or quinovose.
The O-polysaccharide contained a small amount of a dideoxy amino sugar,
a 2-N-acetamido-2,6-dideoxyhexose, identified by the
electron impact and chemical ionization mass spectra of its
corresponding TMS (m/z 173, 204, 217, 247, 259, 316 inter alia), alditol acetate (m/z 85, 103, 145, 159, 201, 231, 242, 260, 302), and PMAA derivatives (shown below). This
residue was determined to have the gluco configuration,
since the retention times of these derivatives were identical to those
obtained from authentic N-acetylquinovosamine. In addition,
the 1H ring proton connectivities observed during TOCSY
analysis of oligosaccharides derived from the O-chain support the
assignment of the gluco configuration for this sugar, as
discussed below. GLC-MS analysis of the TMS ()-2-butyl glycosides
derived from the polysaccharide indicated that all fucose residues were
of the L-configuration, and the glucuronic acid and mannose
residues were of the D- configuration. The configurations
for N-acetylquinovosamine, 3-O-Me-6-deoxytalose,
2,3,4-tri-O-Me-fucose, and the other endogenously methylated
glycosyl residues could not be clearly assigned. Another interesting
feature of the O-chain is that although it contains substantial
glucuronic acid (Table I), most of the native polysaccharide (~70%)
fails to bind to DEAE-Sephadex, and the remaining 30% binds only
weakly, due to the single Kdo residue that survives the mild acid
hydrolysis (data not shown). This indicates that the GlcA carboxyl
groups are blocked by some moiety.
A portion of the O-chain was subjected to de-O-acylation to
allow accurate molecular mass assignment and facilitate chemical degradations, e.g. periodate oxidation. It was found,
however, that the typical conditions used to effect
de-O-acylation (e.g. 0.1 M NaOH,
1 h at room temperature) resulted in extensive depolymerization of
the polysaccharide, due to base-catalyzed elimination. This was
confirmed by the appearance of a UV maximum at 232 nm (not shown) and
subsequently by MS and NMR analyses discussed below. It thus became
necessary to find suitable conditions for complete O-acetyl
removal while allowing the recovery of an intact polysaccharide. Graded
treatments with alkali under a variety of conditions were tried on a
small scale, and the reactions were monitored by 1H NMR
(Fig. 3B) and MALDI-TOF MS (Fig.
4). It was found that 10 mM
NaOH at 4 °C for 10 h was sufficient to result in complete de-O-acetylation without depolymerization. It can be seen
that this resulted in a slight overall reduction in molecular mass accompanied by a sharpening of the molecular ions, due to the loss of
O-acetyl heterogeneity (Figs. 4 and 2A). Removal
of the O-acetyl groups also allowed accurate molecular mass
analysis by ESI-MS as shown in Fig.
5A. In the ESI-MS spectrum,
the three major molecular species previously identified by MALDI-TOF
are clearly resolved and are shown to differ by 202 and 187 atomic mass
units, corresponding to the incremental masses of anhydro Kdo and
QuiNAc residues, respectively. Hence, the O-polysaccharide consists of
three major species that differ by loss of a Kdo and a QuiNAc residue.
One possible explanation for this is a sequential loss of these labile
residues from the reducing end of the molecule, during either the mild
acid treatment or the de-O-acylation reaction, conclusions
that were further substantiated by experiments described below.
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Fig. 4.
Sequential MALDI-TOF-MS analysis of the
de-O-acetylation reaction of the O-chain
polysaccharide. The polysaccharide was subjected to mild
de-O-acetylation at 4 °C, and the reaction was followed
by MALDI-TOF MS (negative ion mode). Mild de-O-acetylation
resulted in a slight reduction in the mass of the three major
components and a tightening of the pseudo molecular ions, due to
removal of O-acetyl heterogeneity. Arrows mark
the locations of lower molecular weight products that differ by
approximately 480 mass units and that arise from the loss of repeating
units due to -elimination. In the bottom tracing, these
depolymerization products become the dominant species when the
polysaccharide is treated with strong alkali (0.25 M NaOH,
35 °C, 4 h). RT, room temperature.
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Fig. 5.
Positive ion ESI-MS spectra of
de-O-acylated O-chain (A) and
permethylated O-chain (B).
De-O-acetylation reduces the complexity of the spectra and
allows accurate mass assignment for the various molecular species. The
inset shows the total spectrum of the de-O-acyl
O-chain. There are three major molecular species represented by three
ion clusters as follows: the intact polysaccharide contains the
anhydro-Kdo and QuiNAc residues (m/z 1657 ion family). A
portion of the Kdo residues is lost during mild hydrolysis and
de-O-acetylation, resulting in the m/z 1555 family. The 3-linked QuiNAc residue is also somewhat labile and
undergoes -elimination from C-3 to some extent, accounting for the
m/z 1462 family. All ions are (M + 2H)2+;
differences of 7 m/z correspond to increments of 14 mass
units, reflecting the endogenous O-methyl heterogeneity in
the O-chain. The calculated and observed mass of a major intact
species, having an anhydro-Kdo residue, is 3312.1 (ion m/z
1657.0). amu, atomic mass units. B, the
permethylated O-chain yielded both doubly and triply charged
protonated, ammoniated molecular ions for the major species
(m/z 1179, experimental mass 3500). The mass differences,
for both the doubly charged and triply charged species, correspond to
what would be expected for loss of a di-O-methylated QuiNAc
residue and a di-O-Me-anhydro-Kdo residue from the
polysaccharide and confirm the assignments proposed for the
non-methylated sample. For example, among the triply charged
pseudomolecular ions, 1 m/z = 72 corresponds to an incremental mass of 216, that of a
di-O-Me-QuiNAc residue, and 2
m/z = 77 corresponds to the incremental mass of a
di-O-Me-anhydro-Kdo residue, 231.
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The ESI-MS spectrum of the de-O-acylated PS also revealed
the multiplicity of species arising from O-methyl
heterogeneity (Fig. 5A). The doubly charged protonated
molecular ions (M + 2H)2+ differ by increments of 7 m/z units, hence corresponding to 14 mass units, as would be
expected for the replacement of a hydroxyl group with
-O-CH3. The locations of these methyl group
variations presumably involve one or more of the endogenously
O-methylated glycosyl residues. Another interesting feature
of the R. etli O-chain revealed by the MALDI and ESI-MS
analysis is the remarkably uniform molecular size of this
polysaccharide. As shown in Figs. 2B and 4, several low
intensity ions occurring at regular intervals of approximately 480 mass
units correspond to repeating unit increments, and presumably these
weak ions reflect the endogenous repeating unit variability in the
glycan. The intensity of these ions relative to the three major
molecular species increases during strong base treatment, a result of
-elimination at each repeating unit (discussed below).
In order to collapse the O-Me heterogeneity, the
de-O-acetylated O-chain was permethylated and analyzed by
ESI-MS (Fig. 5B). As was the case for the non-methylated
material, pseudomolecular ions for three main components were observed,
yielding one set of doubly charged and one set of triply charged ions.
Within each set, the m/z values are consistent with the
loss of a permethylated anhydro-Kdo residue (e.g.
1-methoxy-6,8-di-O-methyl-2,7-anhydro-Kdof, increment mass 230.2) and a permethylated QuiNAc residue
(e.g. 1, 4-di-O-methyl-N-methylacetamido-quinovosamine,
increment mass 215.2). Both of these residues are monosubstituted
(shown below). These results confirm the following. 1) The mass
decrease attributed to loss of a QuiNAc residue (incremental mass
187.2) from the non-methylated polysaccharide was indeed due to loss of
QuiNAc and not loss of a 2,3,4-tri-Me-Fuc residue (incremental mass
188.2), since the incremental mass for the 2,3,4-tri-Me-Fuc residue
would have remained at 188.2 even after permethylation. 2) The Kdo
residue was most likely in an anhydro form, rather than a lactone,
since methylation of the latter would open the lactone yielding
additional sites of alkylation. 3) The results support the likelihood
that a QuiNAc residue is penultimate to the reducing end Kdo, as
previously reported for the R. etli mutant CE359 LPS V (30).
These results also argue against the possibility that strain CE3
synthesizes any O-chains that lack a QuiNAc residue, since a molecular
species lacking only a QuiNAc residue was not observed during ESI-MS analysis.
Methylation Analysis--
The O-chain was reduced with sodium
borodeuteride to stabilize the reducing end and then subjected to
sequential methylation, carboxyl reduction, remethylation, and
conversion to the PMAA derivatives (Table
II). The major linkages and their
corresponding derivatives were 3,4-linked Fuc
(1,3,4,5-tetra-O-acetyl-2-O-methylfucitol, m/z 118 and 275); 3-linked Fuc
(1,3,5-tri-O-acetyl-2,4-di-O-methylfucitol, m/z 118, 131, 234, and 247); 4-linked GlcA
(1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-6,6-2H-glucitol,
m/z 47, 118, 235, and 279); and
terminal-3-O-methyl-6-deoxy-Tal (1,5-di-O-acetyl-6-deoxy-2,3,4-tri-O-methyltalitol,
m/z 118, 131, 162, and 175). These results suggested a
trisaccharide or tetrasaccharide repeating unit comprised of
terminal-3-O-Me-6-deoxy-Tal, 4-linked GlcA, and 3,4-linked
Fuc branch points, possibly also containing 3-linked Fuc residues.
Minor components in the linkage analysis included 3-linked Man,
3-QuiNAc, 4-Kdo, and T-Fuc. The 4-linked Kdo residue yielded two
derivatives having identical mass spectra
(4-O-acetyl-3-deoxy-1,2,5,6,7,8-hexa-O-methyl-1,1,2-2H-octitol,
primary fragments m/z 45, 89, 92, 133, 222, 266, and 308),
present as a diastereomeric pair due to nonstereospecific reduction at
C-2 with borodeuteride. The presence of this derivative, containing
deuterium at C-2 and O-methyl groups at C-2 and C-6, could
only arise from a Kdo residue susceptible to borodeuteride reduction
prior to methylation, i.e. a reducing end Kdo. This derivative results from that portion of the reducing end Kdo residues that did not assume an anhydro form during the mild acid hydrolysis, since a 4-linked 2,7-anhydro Kdo residue, an "internal glycoside," would not be reduced by borodeuteride. The "terminal" fucosyl derivative, i.e.
1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol
(m/z 118, 131, 162, and 175), arises at least in part from
the endogenous 2,3,4-Me-Fuc residue present in the glycan (Table I).
The other endogenously methylated glycosyl components listed in Table I (2, 3-Me-Fuc and 2-Me-Fuc) could also be linked either terminally or 4- or 3,4-linked, and the present results cannot distinguish between these
possibilities. The PMAA derivative arising from the 3-linked QuiNAc
residue
(1,3,5-tri-O-acetyl-4-O-methyl-2- (N-methylacetamido)glucitol,
primary fragments m/z 131, 159, and 275 and secondary
fragments 117 and 243) was further confirmed by CI-MS (M + H)+ = m/z 363.
To determine unequivocally the linkages of the endogenously methylated
sugars, methylation analysis of the native polysaccharide was
performed with trideuteriomethyl iodide. The
3-O-methyl-6-deoxytalose residue yielded exclusively
1,5-di- O-acetyl-2,4-di-O-trideuteriomethyl-3-O-methyl-6-deoxytalitol acetate (m/z 121, 134, 165, and 178), indicating that this
residue is indeed terminal. Furthermore, no deuterated derivatives were observed that would arise from a terminal 2-methyl or
2,3-di-O-methyl 6-deoxytalose residue, indicating that
methyl group heterogeneity does not occur at this location.
In contrast, methyl group heterogeneity was observed at the fucosyl
residues. Examination of the trimethyl derivative arising from the
fucosyl residue(s) indicated that approximately 85% of this derivative
consisted of
1,5-di-O-acetyl-2,3,4-tri-O-methylfucitol acetate
(m/z 118, 131, 162, and 175), whereas 15% consisted of the
deuteriomethylated derivatives
1, 5-di-O-acetyl-4-O-trideuteriomethyl-2,3-di-O-methylfucitol acetate (m/z 118, 134, 162, and 178) and
1,5-di-O-acetyl-3,4-di-O-trideuteriomethyl-2-O-methylfucitol acetate (m/z 118, 134, 165, and 181). The incorporation of
deuteriomethyl groups into these derivatives, their locations, and
fragment ion ratios indicates that this derivative is composed of
~85% terminally linked (T)-2,3,4-Me-Fuc, and ~15% T-2,3-Me-Fuc
and T-2-Me-Fuc.
The derivative arising from 3-linked fucosyl residues consisted
primarily (~90%) of
1,3,5-tri-O-acetyl-2,4-di-O-trideuteriomethylfucitol (m/z 121, 134, 240, and 253), which would arise from a
3-linked fucose. Approximately 10% of this derivative consisted of
1,3,5-tri-O-acetyl-4-O-trideuteriomethyl-2-O-methylfucitiol (m/z 118, 134, 237, and 250), as estimated from the
m/z 121/118, 240/237, and 253/250 ratios. Therefore,
approximately 10% of the 3-linked fucosyl residues in the glycan are
actually 3-linked 2-O-methylfucosyl residues. The derivative
arising from the 3,4-linked fucosyl residues contained primarily
(~75%) 2-O-trideuteriomethylfucitol acetate
(m/z 121 and 278), indicating that most of this derivative arises from 3,4-linked fucose. However, approximately 25% of this derivative consisted of
1,3,4,5-tetra-O-acetyl-2-O-methylfucitol (m/z 118 and 275), as estimated from the m/z
121/118 and 278/275 ratios. This indicates that a substantial portion
(~1/4) of the 3,4-linked branching fucosyl residues in the glycan are
endogenously methylated at O-2. Thus, most of the 2-Me-Fuc residues
present in the glycan and listed in Table I arise from the branch point fucosyl residues.
Glycosyl Sequence Determination of the Repeating
Oligosaccharide--
The arrangement of substituents at the branching
3,4-linked fucosyl residue was investigated by -elimination of the
uronosyl residues in combination with methylation and ethylation
analysis. The polysaccharide was methylated and then treated with the
potassium dimethylsulfinyl anion. The -elimination products were
ethylated and then hydrolyzed and converted to the corresponding
partially methylated, ethylated alditol acetates (Table II). The only
ethylated derivative observed was
1,3,5-tri-O-acetyl-4-O-ethyl-2-O-methylfucitol (m/z 118, 145, 248, and 261), and the presence of the ethyl
group at O-4 indicates that a glucuronosyl residue was originally
linked to this position. Thus, the branching fucosyl residues are
substituted at O-4 by glucuronosyl residues and at O-3 by some other
repeating residue, most likely either the terminal
3-O-Me-6-deoxytalose residues or possibly 3-linked fucosyl
residues. The following trisaccharide (Structure 1) or tetrasaccharide
(Structure 2) structures can thus be proposed for the repeating
unit.
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Other alternatives are possible for the tetrasaccharide unit.
3-Linked Man was also detected in the total mixture of -elimination products (Table II); however, glycosyl composition and linkage analysis
of the native O-chain indicated that this residue occurs to only a
small extent and therefore is probably not part of a repeating
structure. Rather, the 3-Man is part of a non-repeating sequence that
occurs at the reducing end of the polysaccharide (described below).
To distinguish between the structural alternatives, the Smith
degradation was applied to the de-O-acetylated glycan. A
sample was treated with periodate and reduced with borohydride, and the reaction was chromatographed on Bio-Gel P-10. The oxidized-reduced product (polyol) eluted as a broad, tailing peak having a
Kav = 0.47 (not shown). A sample of the polyol
was isolated and subjected to composition and linkage analysis (Tables
I and II, respectively). As expected, only the 4-linked glucuronosyl
residues were susceptible to the periodate oxidation. A substantial
portion of the Kdo also survived the periodate treatment, suggesting
that this terminal Kdo residue was present as a 2,7-anhydro
Kdof, rather than a 2,5-anhydro pyranose, which would be
susceptible to oxidation. The remainder of the polyol was subjected to
mild hydrolysis, constituting the Smith degradation, and the
oligosaccharide products were isolated on Bio-Gel P-4. The major
oligosaccharide fraction was analyzed by ESI-MS/MS (Fig.
6A) which revealed a major
oligosaccharide (parent ion m/z 457.5; calculated M + H+ 457.4) consistent with Structure 3.
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In Structure 3, the tetronic acid derives from carbons 6, 5, 4, and 3 of the oxidized-reduced 4-linked glucuronosyl residues. Following
total hydrolysis of this oligosaccharide in trifluoroacetic acid, the
presence of a tetronic acid was confirmed by GLC-MS analysis of the
carboxyl-reduced alditol acetate (m/z 145, 147, 217, and
219). The tetronic acid in this case would be erythronic acid; however,
the configuration was not examined. Methylation analysis of the polyol
with carboxyl reduction confirmed the presence of terminally linked
6-deoxytalose, 3-linked fucose, and a 3-substituted tetronic acid
derivative,
1,2,3-tri-O-acetyl-4-O-methyl-1,1-2H-tetritol,
primary fragments m/z 45, 117, 147, 189, and 219 (data not
shown).
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Fig. 6.
A, positive ion ESI-MS/MS spectrum of
the major Smith degradation product (parent ion m/z 457.5)
derived from the repeating unit of the de-O-acylated
O-chain. The products obtained from Smith degradation were fractionated
on Bio-Gel P-4 and analyzed by CID-MS/MS. The ion at m/z 425 arises by loss of methanol from the parent ion. Loss of water from the
B1, B2, and parent ion gives rise to ions
m/z 143, m/z 289, and m/z 439.5 (B3), respectively. Ions m/z 279 and
m/z 265 are assigned to double cleavage products resulting
from the loss of the 3-O-Me-6-deoxyhexose residue (increment
mass 160.2) from the m/z 439.5 and m/z 425 ions,
respectively. B, positive ion ESI-MS/MS spectrum of the
minor Smith degradation product (parent ion m/z 471.5) also
derived from repeating units of the de-O-acylated O-chain.
Ions indicative for the presence of the
O-methyl-6-deoxyhexose residue (2-O-MeFuc) are
B2 (m/z 321), and the double cleavage product is
m/z 279.
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A second Smith derived oligosaccharide (M + H+ = 471.5),
which co-eluted with the major oligosaccharide on P-4, was identified by ESI-MS/MS as containing two contiguous O-methyl
6-deoxyhexosyl residues linked directly to a terminal tetronic acid
aglycon (Fig. 6B). These results are consistent with an
oligosaccharide having the following Structure 4.
This glycosyl sequence is also predicted from the linkage analysis
using trideuteriomethyl iodide. Both of these oligosaccharides are
consistent with a trisaccharide repeating unit, as shown in Structure 1 above. The occurrence of a methyl ester in each of these
oligosaccharides presumably indicates the C-6 substituent of the
glucuronosyl residues. As discussed previously, methylation analysis of
the native polysaccharide revealed substantial 3-linked Fuc (Table II),
suggesting the possibility of a tetrasaccharide repeating unit.
However, as shown below, the intact polysaccharide contains only one
3-linked Fuc residue, which originates from the non-repeating glycosyl
sequence at the reducing end of the molecule. The additional 3-linked
Fuc reported in Table II appears to arise from -elimination and
subsequent degradation of some of the esterified GlcA residues during
the methylation with dimsyl anion. This also results in a slight
decrease in the amount of 3,4-linked Fuc, with respect to the terminal
6-deoxy-Tal and 4-linked GlcA derivatives (Table II). Clearly, the
results of methylation analysis of intact polysaccharides can only
approximate the structures deduced by other means, such as MS/MS
analysis of the Smith-derived fragments.
Oligosaccharides derived from the non-repeating sequences at both the
reducing and nonreducing ends of the O-chain were also detected during
the Smith procedure; however, their yields were relatively low,
presumably because they are not repeated sequences and because some
were lost during the work up of the polyol. Several repetitions of the
oxidation-reduction procedure, including chromatography steps, were
required to preclude inter-residue hemiacetal formation and thus ensure
complete oxidation. A more direct procedure for the isolation of the
reducing and nonreducing end oligosaccharides was employed using
-elimination, as described below.
Glycosyl Sequence of the Nonreducing and Reducing
Ends--
Portions of the O-chain polysaccharide were pre-reduced with
borodeuteride and subjected to base-catalyzed -elimination as described under "Experimental Procedures." The oligosaccharide products were chromatographed on Bio-Gel P-10 (Fig.
7). The P-10 profile shows clearly that
these depolymerization products are of lower molecular weight than that
of either the native or de-O-acetylated polysaccharides
(compare Fig. 2A) and have absorbance at 232 nm, characteristic of a 4-O-substituted hexuronate
-elimination product, i.e.
4-deoxyhex-4-enopyranosiduronate residues. The major fraction, P-102
(Kav = 0.59), was isolated and chromatographed
on Bio-Gel P-4; however, the material failed to undergo further
separation and yielded a poorly resolved, peak/shoulder that migrated
near the void volume of the P-4 column (not shown). Samples were taken from both the leading and tailing edges and were analyzed for glycosyl
composition (Table I) and by ESI-MS. Five molecular ions were observed,
indicating that this fraction consisted of a mixture of five
oligosaccharides, designated OS-1 to OS-5. The major molecular species
and their proposed formulas are summarized in Table
III. The relative proportion of these
molecular ions varied between the leading and tailing edge of the P-4
peak: the leading edge (higher molecular weight) was enriched in
OS-2-5, and the tailing edge (lower molecular weight) was enriched in
OS-1. As will be shown below, OS-1 originates from the nonreducing end of the O-chain, and OS-2-5 originate from the reducing end of the
molecule. The ESI-MS spectrum showing the molecular ions for OS-1-5 is
shown in Fig. 8.
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Fig. 7.
Bio-Gel P-10 fractionation of the
-elimination products derived from the
O-chain. Oligosaccharides derived from both the nonreducing and
reducing ends of the O-chain co-eluted in P-102
(Kav = 0.59, compare Fig. 2). The
oligosaccharides in this fraction, designated OS-1 to OS-5, were
further separated by Bio-Gel P-4 chromatography and reverse phase HPLC
and analyzed for glycosyl composition and linkage by GLC-MS, ESI-MS/MS,
and 1H NMR. Fraction P10-1 contained partially degraded
O-chain fragments, characterized by some intact repeating unit
sequences, in which some of the glucuronosyl residues did not undergo
-elimination. Fraction P10-3 consisted of
3-O-Me-6-deoxytalose, fucose, and unidentified degradation
products arising from these residues due to -elimination of the
interior glucuronosyl residues.
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Table III
Ions derived from electrospray mass spectrometry of the
oligosaccharides produced from -elimination of the R. etli CE3
O-chain polysaccharide
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Fig. 8.
Positive ion ESI-MS spectra of
oligosaccharides OS-1 (inset) and OS-2-5 released by
base-catalyzed -elimination of the O-chain
polysaccharide. All molecular ions are of the formula M + H+. Oligosaccharides OS-2-5 represent different forms of
the reducing end of the O-chain, where OS-4 and OS-5 terminate with an
unsaturated residue, a 4-deoxyhex-4-enopyranosyluronic acid, resulting
from elimination of the 4-O-substituent from the
glucuronosyl residues. OS-1 derives from the nonreducing end of the
O-chain. The structures are summarized in Table III.
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To establish the glycosyl sequence of these oligosaccharides, the major
molecular ions were selected for CID-MS/MS. For most of the spectra,
the fragment ions consisted of Y-type ions resulting from glycosidic
cleavage with charge retention on the reducing end, and B-type ions,
arising from oxonium ion formation with charge retention on the
nonreducing end (44). Second generation fragment ions resulting from
double cleavages were also observed in several of the spectra. The
ESI-MS/MS spectrum of OS-1 is shown in Fig.
9. The results show that OS-1 contains
the 2,3,4-tri-Me-Fuc residue, and OS-1 thus represents the nonreducing
end of the glycan. Glycosyl composition and linkage results previously
indicated that this residue occurs only once in the glycan, as a
terminally linked or "capping" residue. One of the reasons for the
low recovery of this oligosaccharide during the Smith degradation is
that most of the glucuronic acid was oxidized by periodate, leaving
only the single 2,3,4-tri-Me-fucosyl residue as a representative of this nonreducing end "sequence." During the -elimination, a
portion of the glucuronosyl residues survived the base treatment,
allowing detection of this intact sequence (OS-1).
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Fig. 9.
Positive ion ESI-MS/MS spectrum and proposed
glycosyl sequence of the nonreducing-end oligosaccharide OS-1 (parent
ion m/z 689; calculated M + H+ 689.7)
generated by base-catalyzed -elimination of
the O-chain. The oligosaccharide was isolated by Bio-Gel
chromatography. Consecutive loss of water from m/z 511 gives
rise to m/z 493 and 475; loss of methanol from the
trimethyl-Fuc oxonium ion (m/z 189) yields the
m/z 157 ion.
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Examination of oligosaccharides OS-2-5 (Figs. 8 and 10 and Table III)
revealed them to be a series of related structures that differ in the
nature of their reducing and nonreducing end residues. OS-4 and OS-5
terminate at the nonreducing end with a
4-deoxyhex-4-enopyranosiduronate residue, resulting from the
-elimination. Since the glycosidic linkage of these unsaturated
residues is labile, a portion of these residues is lost during the
-elimination and isolation, yielding OS-2 and OS-3. Hence, OS-4 and
OS-5 are each 158 mass units greater than OS-2 and OS-3, respectively,
corresponding to the interval mass of the 4,5-hexuronate
residue. In addition, OS-3 and OS-5 terminate at the reducing end with
a Kdo alditol, resulting from borodeuteride reduction, and OS-2 and
OS-4 terminate with a 2,7-anhydro Kdo residue, which is resistant to
reduction. The mass difference between OS-2 and OS-3 is 21 atomic mass
units, which is due to the replacement of an anhydro Kdo residue with a
reducing Kdop (+18 atomic mass units) reduced at C-2 with
borodeuteride (i.e. C=O D-C-OH = +3
atomic mass units). An identical difference exists for the OS-4/OS-5
pair. Furthermore, prereduction of the polysaccharide with borohydride,
rather than borodeuteride, resulted in a difference of 20 atomic mass
units for these molecular species (i.e. OS-2 and OS-4
remained at m/z 1022.5 and 1180.5, respectively, whereas
OS-3 and OS-5 were shifted to m/z 1042.5 and 1200.5, respectively) (data not shown). Further confirmation of the charge
state and type of cationization (i.e. protonation) was
obtained by the controlled addition of sodium, which resulted in an
increase of 22 m/z units for each of these species. These
results support the conclusions drawn from linkage and ESI-MS analysis
of the intact polysaccharide, namely that both types of Kdo residues
(anhydro and reducing) are present at the reducing end of the glycan.
The MS/MS spectrum of OS-2 is shown in Fig.
10. Together with composition and
linkage analyses, these results indicate that the reducing end of
the polysaccharide contains the non-repeating sequence
3)-Fucp-(13)-Manp-(13)-QuiNAcp-(14)-Kdo-(2, where C-3 of the fucosyl residue is substituted by the first repeating unit in the polysaccharide.
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Fig. 10.
Positive ion ESI-MS/MS spectrum and proposed
glycosyl sequence of oligosaccharide OS-2 (parent ion m/z
1022.5), derived from the reducing end of the O-chain by
-elimination. The spectrum is characterized by
consecutive Y-type fragment ions and a single B ion (B5),
all derived from the parent ion. During the electrospray MS/MS, much of
the fragmentation appears to be directed by the amino sugar residue.
Thus, the Y1 ion (m/z 408), containing the amino
sugar, is particularly stable. The B5 ion, which terminates
in the amino sugar, does not undergo further B-type fragmentation and
instead gives rise to a series of second generation fragment ions
(m/z 642, 496, 350, and 188) due to consecutive
-cleavages from the nonreducing end.
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The oligosaccharide mixture (OS-1
OS-5) was fractionated by reverse
phase HPLC, yielding a major fraction that contained OS-2 and OS-4 and
a second fraction containing OS-1, OS-3, and OS-5, as determined by
ESI-MS (data not shown). Further separation was not attempted, and the
structurally related OS-2/OS-4 mixture was analyzed by 1H
COSY and TOCSY experiments (Fig. 11).
The distinctive signal at 5.98 arises from H-4 of the
4-deoxyhex-4-enopyranosiduronate residue, and complete connectivities
are shown for this ring system. The chemical shifts for the ring
protons of this residue are H-1 ( 5.36), H-2 ( 3.96), H-3 ( 4.27), and H4 ( 5.98). A comparison of the H-1 shift and coupling
constant with literature values (45) suggests an -linkage for this
unsaturated residue, indicating that the glucuronosyl residues have the
-anomeric configuration in the intact polysaccharide. Five other
ring systems were also identified (Fig. 11), and the chemical shifts of
the anomeric protons for all of these glycosyl residues also suggests
they are -linked, with the exception of two resonances, at 4.55 and 4.62. The signal at 4.55 is attributed to a -mannosyl
residue, due to weak H-1/H-2/H-3 coupling, suggesting the
manno configuration for this system. The signal at 4.63 is attributed to a -N-acetylquinovosamine residue, since
cross-peaks can be detected for C-6 methyl protons as well as all ring
protons in this system. The chemical shifts for the QuiNAc ring protons
are H-1 ( 4.62), H-2 ( 3.72), H-3 ( 3.36), H-4 ( 3.75), H-5
( 3.52), and H-6 ( 1.31). Thus, the QuiNAc residue
(6-deoxy-gluco configuration) shows coupling throughout all
ring protons, whereas the other three 6-deoxyhexose methyl protons show
strong coupling only from H-6 to H-5, characteristic of fuco
and talo configurations.
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Fig. 11.
Partial 1H TOCSY spectrum of the
OS-2/OS-4 mixture. A, the solid tracing
delineates the four-ring protons of the
4-deoxyhex-4-enopyranosiduronate ring system; the dotted
lines locate the anomeric and other protons from the remaining
glycosyl residues. B, the H-6 protons from the four
6-deoxyhexose residues are shown. The N-acetylquinovosamine
residue (6-deoxy-gluco configuration) shows strong coupling
throughout all ring protons, whereas the other three 6-deoxyhexose
residues show strong cross-peaks only for H-6/H-5, characteristic of
fuco and talo configurations. The anhydro Kdo H-3
methylene protons were at 2.72 (not shown).
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The anomeric configuration of the Kdo residue was investigated
indirectly, by examining the Kdo residues in a rough LPS synthesized by
the R. etli mutant CE358. The LPS from this mutant contains a core oligosaccharide that terminates at the nonreducing end with the
distal Kdo residue, which normally serves as the site of O-chain
attachment in those LPS that contain an O-chain (30). The core region
of this mutant, as with the core region of the parent strain, contains
a total of three Kdo residues, including two in the interior portion of
the core. 1H COSY analysis of this de-O-acylated
rough LPS showed intense signals for all Kdo methylene protons at 2.02 (H3a) and 1.78 (H3e), and the chemical shift difference
( = 0.24 ppm) is indicative of -linked Kdop
residues (31, 46).
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DISCUSSION |
The glycosyl sequence of the O-antigenic polysaccharide from the
R. etli CE3 LPS is shown in Fig.
12. The polysaccharide is characterized
by a number of rather novel structural features for an O-chain,
including a relatively low molecular weight (3330 Da), uniform size
(five repeating units with little variation in the number of repeats),
numerous endogenously O-methylated components
(3-O-methyl-6-deoxytalose,
2,3-di-O-methylfucose, 2-O-methylfucose, and 2,3,4-tri-O-methylfucose), and
O-acetylation. The latter features appear to confer a fairly
high level of hydrophobicity to the glycan, a property that may be
important during infection thread development and the establishment of
symbiosis4 (6, 47). With the
elucidation of this structure, the complete glycosyl sequence of the
R. etli CE3 LPS, including the unique core region (30) and
lipid A moiety (33), is now known for the laboratory-cultured
bacterium. The results presented here also show that
3-O-methylrhamnose is not a glycosyl component of the
O-chain repeating unit (rather, what was thought to be 3-O-methylrhamnose is actually
3-O-methyl-6-deoxytalose), as was suggested in earlier
reports (6, 43) that were based on incomplete preliminary data.
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Fig. 12.
Complete glycosyl sequence of the O-specific
polysaccharide from the R. etli CE3 LPS.
R1 = CH3 (80%) or H (20%); R2 = CH3 (25%) or H (75%); and R3 = CH3 (10%) or H (90%). Most of the 2-MeFuc in the
polysaccharide originates from the branching fucosyl residues
(R2). The formula of a major species (lactone form)
terminating in an anhydro-Kdo residue is
Tri-MeFuc1GlcA5Fuc53-Me6dTal52-MeFuc1Man1QuiNAc1aKdo1,
calculated mass 3312.1, (M+2H)+2 ion m/z 1657 as
shown in Fig. 5.
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Studies with rhizobial LPS mutants and their symbiotic phenotypes have
helped define our current understanding of the role of these molecules
in symbiotic infection. These studies have led to the general
observation that complete LPS molecules, synthesized in normal amounts
and containing a polymeric O-chain, core oligosaccharide, and lipid A
moiety, are required for effective symbiosis (24, 28). Of recent
interest from a structural and biosynthetic standpoint is the R. etli mutant CE166, (Ndv+, Fix
) which
produces reduced amounts of a smooth LPS (LPS-I) containing a
structurally modified O-chain. The symbiotic defect of this mutant can
be partially suppressed by the incorporation of a multicopy plasmid
carrying R. etli CE3 DNA, a modification th
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ABSTRACT
INTRODUCTION
