Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin

Article abstract of DOI:10.1016/j.carres.2009.07.004

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and

Full text of DOI:10.1016/j.carres.2009.07.004

Carbohydrate Research 344 (2009) 2250–2254
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Carbohydrate Research
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Note
Structural characterization of an O-linked tetrasaccharide from Pseudomonas
syringae pv. tabaci flagellin
Tomoyuki Konishi a, Fumiko Taguchi b, Masako Iwaki b, Mayumi Ohnishi-Kameyama c,
Masanobu Yamamoto c, Ikuko Maeda c, Yoshihiro Nishida d, Yuki Ichinose b,
Mitsuru Yoshida c, Tadashi Ishii a,
*
a Forestry and Forest Products Research Institute, 1, Matsunosato, Tsukuba, Ibaraki 305-8687, Japan
b Graduate School of National Science and Technology, Okayama University, 1-1-1, Tsushima-naka, Okayama 700-8530, Japan
c National Food Research Institute, 2-1-12, Kannondai, Tsukuba 305-8642, Japan
d Department of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-0092, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 May 2009
Received in revised form 10 July 2009
Accepted 14 July 2009
Available online 18 July 2009
The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides
composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These
O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyr-
idine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase
high-performance liquid chromatography. These oligosaccharides are structurally characterized using
mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is
Keywords:
Bacteria
Pseudomonas syringae
Flagellin glycosylation
O-linked oligosaccharides
Structure
glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1?3)-
-Rhap-(1?2)- -Rhap-(1?2)- -Rha-(1?, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanam-
ido)-2-O-methyl-Glcp-(1?3)- -Rhap-(1?2)- -Rha-(1?, which was identified in a previous study.
Ó 2009 Elsevier Ltd. All rights reserved.
a-
L
a
-
L
a-L
a-
L
a-L
NMR
The phytopathogenic bacterium Pseudomonas syringae is classi-
fied into at least 50 pathovars on the basis of its virulence toward
different host plant species.1,2 Flagellin is a protein that is an essen-
tial component of the flagellum filament of many bacteria includ-
ing P. syringae. Flagellins of numerous plant pathogens including P.
syringae pv. tabaci 6605, P. syringae pv. glycinea race 4, P. syringae
pv. tomato DC 3000, and Acidovorax avenae are glycosylated.3,4 Re-
cently we demonstrated that the flagellin of P. syringae pv. glyci-
nea, but not P. syringae pv. tabaci, causes hypersensitive cell
death in tobacco cells,3 even though the flagellins from both path-
ovars have identical amino acid sequences. Such results when ta-
ken together with the results of biological and mutational studies
have led to the suggestion that flagellin glycosylation has a role
in P. syringae virulence and host specificity.5–8 In previous studies
we showed that six serine residues (S143, S164, S176, S183,
S193, and S201) are glycosylated in the flagellins from P. syringae
pv. tabaci and P. syringae pv. glycinea.7,9 We reported that S201
of the flagellin from P. syringae pv. tabaci and P. syringae pv.
glycinea is O-glycosylated with a trisaccharide.9 To determine if
structurally different oligosaccharides are attached to the other
serine residues, we subjected P. syringae pv. tabaci flagellin to
hydrazinolysis and labeled the released oligosaccharides with 2-
aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled
tetrasaccharide were isolated and structurally characterized by
mass spectrometry (MS) and NMR spectroscopy.
In a previous study we showed that P. syringae pv. tabaci flagel-
lin contained
We confirmed that the Rha was
L
-Rha and a modified 4-amino-4,6-dideoxyglucose.9
L
using GC and GC–MS analysis
of the trimethylsilylated (TMS) (S)-(+)-2-butyl glycosides and by
reversed-phase HPLC analysis of the S-(+)-2-tert-butyl-2-methyl-
1,3-benzodioxole-4-carboxylic acid glycose derivatives10 (data
not shown). We also confirmed the presence of the acid-labile
dideoxy sugar11,12 by treating the flagellin with anhydrous hydro-
gen fluoride (HF). This procedure cleaves the glycosidic bond of
dideoxyglycoses with limited degradation of the released gly-
cose.12 The products generated by HF treatment were reduced with
NaBD4, O-peracetylated, and then analyzed by GC–MS. The elec-
tron- impact mass spectrum (EIMS) of the acetylated alditol deriv-
ative of the 4,6-dideoxyglycose contained fragment ions at m/z 118
and 347, indicating that an O-methyl and a 3-hydroxybutanamido
group are linked to C-2 and C-4, respectively (Fig. 1). These results,
together with chemical ionization MS (CIMS) analysis, confirmed
that the glycose was 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-
* Corresponding author. Tel.: +81 29 829 8276; fax: +81 29 874 3720.
E-mail address: tishii@ffpri.affrc.go.jp (T. Ishii).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.07.004

T. Konishi et al. / Carbohydrate Research 344 (2009) 2250–2254
2251
CHDOAc
118
H C OCH3
118
287
100
80
60
40
20
0
−60
AcO C H
347
−42
H C NHCOCH2CH(OAc)CH3
244
305
H C OAc
CH3
347
305
315
160
287
244
250
m/z
100
150
200
300
350
Figure 1. EIMS spectrum of the alditol acetate derivative of 4,6-dideoxy 4-(3-hydroxybutanamido)-2-O-methylglucopyranose (mVio). The origins of the predominant
primary and secondary fragment ions are shown.
methylglycopyranose. This dideoxyglycose will be referred to as
modified viosamine (mVio) as it is structurally related to 4,6-dide-
oxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methylgluco-
pyranose (viosamine) that has been identified in other bacteria.13
Rhamnose and mVio were also detected when the P. syringae pv.
tabaci flagellin was treated with methanolic HCl, and the resulting
methyl glycosides were then O-peracetylated and analyzed by GC
and GC–MS (data not shown). Thus, we conclude that P. syringae
pv. tabaci flagellin glycans are composed of only -Rha and mVio.
L
The O-linked oligosaccharides of P. syringae pv. tabaci flagellin
were released by hydrazinolysis and labeled at their reducing ends
with PA. Two quantitatively major and one minor peaks were de-
tected when the labeled products were analyzed by normal-phase
high-performance liquid chromatography (HPLC) with fluores-
Table 1
NMR signal assignment of compound 1 and 2
Residue
Position
Compound 1
1H NMR
Compound 2
13C NMR
d (ppm)
43.87
1H NMR
13C NMR
d (ppm)*
43.8
d (ppm)
Multiplicity
J (Hz)
d (ppm)
Multiplicity
J (Hz)
L
-Rhamnitol R
1
1
2
3
4
5
6
3.635
3.588
3.949
3.967
3.478
3.883
1.243
dd
dd
ddd
dd
dd
dq
d
14.5, 3.1
14.5, 6.0
6.6. 6.0, 3.1
6.6, 2.2
7.4, 2.2
7.4, 6.3
6.3
3.641
3.553
3.951
3.986
3.489
3.882
1.256
nd
nd
nd
dd
dd
dq
d
nd
nd
nd
6.0, 2.3
7.4, 2.3
7.4, 6.3
6.3
78.86
70.28
75.59
68.76
20.25
79.2
70.2
75.7
68.8
20.2
a
-
-
L
-Rhap A
-Rhap E
1
2
3
4
5
6
4.911
4.129
3.786
3.543
3.733
1.063
d
1.6
101.32
72.00
81.30
72.72
71.11
18.20
4.996
3.950
3.834
3.412
3.686
1.042
br s
d
dd
dd
dq
d
100.2
80.5
71.8
73.7
71.2
18.3
dd
dd
dd
dq
d
3.1, 1.6
9.7, 3.1
9.7, 9.6
9.6, 6.3
6.3
3.3
9.8, 3.3
9.7, 9.7
9.7, 6.4
6.4
a
L
1
2
3
4
5
6
—
—
—
—
—
—
4.966
4.255
3.878
3.587
3.715
1.256
d
1.1
103.9
71.3
81.2
72.8
71.2
18.4
dd
dd
nd
nd
d
3.1, 1.1
9.7, 3.1
nd
nd
6.3
4,6-Dideoxy-2-O-Me-
glucopyranosyl T
1
2
3
4
5
6
4.655
3.118
3.523
3.609
3.529
1.204
3.605
d
8.0
105.43
84.87
74.44
58.27
72.54
18.66
61.68
4.710
3.120
3.508
3.611
3.528
1.189
3.613
d
8.1
9.1, 8.1
nd
nd
nd
105.2
85.0
74.3
58.4
72.7
18.6
61.8
dd
dd
dd
dd
d
9.5, 8.0
9.7, 9.5
10.0, 9.7
10.0, 6.2
6.2
dd
nd
nd
nd
d
6.3
2-O-Me
s
s
3-Hydroxy-butanamido
HBA
10
20
20
30
40
—
176.19
46.85
—
nd
46.9
2.432
2.409
4.185
1.224
dd
dd
ddq
d
14.1, 5.7
14.1, 7.7
7.7, 5.7, 6.3
6.3
2.399
2.423
4.174
1.217
dd
dd
ddq
d
12.8, 5.6
12.8, 7.4
7.4, 5.6, 6.3
6.3
66.71
23.75
66.7
23.8
The signals of PA were assigned as follows: H-3 (d 6.678 ppm, d, 8.2 Hz), H-4 (d 7.562 ppm, ddd, 8.2, 7.0, 1.7 Hz), H-5 (d 6.697 ppm, dd, 7.0, 5.2 Hz) and H-6 (d 7.968 ppm, dd,
5.2, 1.7 Hz), C-2 (d 160.36 ppm), C-3 (d 114.94 ppm), C-4 (d 140.45 ppm), C-5 (d 110.72 ppm), and C-6 (d 148.36 ppm).
*
13C data were obtained from HSQC analysis. nd, not determined; br s, broad singlet; s, singlet.

2252
T. Konishi et al. / Carbohydrate Research 344 (2009) 2250–2254
cence detection. These peaks were collected and then analyzed by
positive-ion mode electrospray ionization mass spectrometry
(ESIMS) and by NMR spectroscopy. The first peak co-eluted with
PA and was not further analyzed. The ESIMS spectrum of the
second peak (compound 1), together with 1H and 13C NMR spectro-
scopic data (see Table 1 and Figs. 2A and 3A), is consistent with the
sequence N-[4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-
residue derived from the former reducing end of the oligosaccha-
ride released by hydrazinolysis were assigned in the HSQC spec-
trum (Fig. 3B, Table 1). The second
to be linked to O-2 of the PA-labeled
relation of H-1–C-2 and the low-field shift of C-2 (d 79.2) (Fig. 3B,
Table 1). The third -Rhap residue E was shown to be linked to O-2
of the second -Rhap residue A by HMBC correlations of H-1–C-2
L
-Rhap residue A was deduced
L
-rhamnitol, R by a HMBC cor-
L
L
Glcp-(1?3)-
a
-
L
-Rhap-(1?2)-
L
-rhamnitol]-2-aminopyridine
and the low-field shift of C-2 (d 80.5) (Fig. 3B, Table 1). The struc-
ture of the non-reducing terminal glycosyl residue T was identified
as 4-amino-4,6-dideoxyglucose by comparison of the 1H and 13C
NMR data (Table 1) with those of 4,6-dideoxy-4-(3-hydroxy-3-
methylbutanamido)-2-O-methylglucopyranose.13 The mVio was
shown to be linked to O-3 of Rhap residue, E by HMBC correlations
(Fig. 2A). A trisaccharide with this sequence has previously shown
to be attached to serine 201 of the flagellins from P. syringae pv.
tabaci 6605 and P. syringae pv. glycinea race 4.9
The ESIMS spectrum of the third peak (compound 2) contained
an ion at m/z 780 that corresponds to the [M+H]+ ion of an oligosac-
charide that is composed of one mVio, two rhamnosyl, one rhamn-
itol, and one PA unit. The product ion spectrum of the protonated
ion at m/z 780 contained Yn type ions14 at m/z 535, 389, and 243.
Such results are consistent with the sequence mVio-Rha-Rha-
rhamnitol-PA. The primary structure of compound 2 was deter-
mined by 1H NMR spectroscopy. The complete assignments of
chemical shifts are given in Table 1. Three signals were detected
in anomeric region (Fig. 2B). The broad signals at d 4.996 and
4.966 were assigned to H-1 of the internal Rhap residues A and
E, respectively. These chemical shifts and their small coupling con-
of mVio H-1 to L-rhamnosyl residue E C-3 and the low-field shift of
C-3 (d 81.2, Fig. 3B, Table 1). Modification of the viosamine residue
by O-methylation on C-2 was confirmed by a HMBC experiment.
Attachment of the 3-hydroxybutyl group to viosamine through
amide linkage was deduced from the high similarity of 1H and
13C NMR chemical shifts to those of compound 1 although HMBC
correlation of the carbonyl to H-4 of mVio was not observed for
compound 2. We conclude that compound 2 is N-[4,6-dideoxy-
4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1?3)-
2)- -Rhap-(1?2)- -rhamnitol]-2-aminopyridine (Fig. 2B).
Our present results demonstrate that P. syringae pv. tabaci fla-
a-L-Rhap-(1?
a-
L
L
stants were consistent with a
-linkages.15 The H-1 resonance of the
non-reducing terminal mVio, T was at d 4.710, and its large cou-
pling (J 8.1 Hz) is consistent with b-linkage13,15 (Fig. 2B, Table 1).
Other signals of these residues were assigned on the basis of the
correlations in the DQF-COSY and TOCSY spectra (Table 1). Some
gellin is O-glycosylated with a trisaccharide (compound 1) and a
tetrasaccharide (compound 2). Six serine residues (S143, S164,
S176, S183, S193, and S201) are known to be glycosylated in the
flagellin protein from P. syringae pv. tabaci and P. syringae pv. glyci-
nea.7,9 At least one of these residues (S201) is glycosylated with the
trisaccharide (compound 1).9 The ratios of a trisaccharide (com-
pound 1) and a tetrasaccharide (compound 2) on LC by fluores-
cence detection were about 5:1, indicating that about five serine
residues were glycosylated with a trisaccharide (compound 1).
of the 1H signals of
L-rhamnitol residue R could not be unambigu-
ously assigned because of contaminating material. Due to the small
amount of compound 2 available, a 13C NMR spectrum was not ob-
tained; rather the 13C chemical shifts were deduced from an HSQC
experiment (Fig. 3B, Table 1). The two H-1 protons of L-rhamnitol
Figure 2. 1H NMR and chemical structure of compounds 1 and 2. (A) compound 1; (B) compound 2. Arrows show HMBC correlations indicating the linkage pattern between
sugars. Letters A, E, R, T, and HBA on the spectra show sugars and a modification group on the oligosaccharides as indicated in Table 1. Numbers after the letters show
positions on each sugar. Asterisks show peaks due to contaminating material.

T. Konishi et al. / Carbohydrate Research 344 (2009) 2250–2254
2253
A
B
Figure 3. Partial overlap of HSQC (black) and HMBC (red) spectra of compounds 1 and 2. (A) Compound 1; (B) compound 2. Letters A, E, R, T, and HBA on the spectra show
sugars and a modification group on the oligosaccharides as indicated in Table 1. Numbers after the letters show positions on each sugar. Asterisks show peaks due to
contaminating material.
Elucidating the glycosylation patterns of these pathovars will re-
quire the complete structural characterization of the glycopeptides
generated from the flagellin proteins. The glycans of the flagellin of
GC–MS was performed with a JEOL JMS-DX303HF mass spec-
trometer interfaced with an Agilent Technologies 6890N gas chro-
matograph. GC-chemical ionization MS (GC–CIMS) was performed
with ammonia as the reagent gas and a source temperature at
180 °C. GC–electron impact ionization MS (GC–EIMS) was per-
formed with an ionization current of 70 eV and an ion source tem-
perature of 180 °C. ESIMS spectra were recorded with a LCQ DUO
mass spectrometer (Thermo Quest, Tokyo, Japan) operated in the
positive-ion mode with a spray voltage of 4.55 kV, a capillary volt-
age of 3.1 V, and a capillary temperature of 180 °C. Solutions of PA
oligosaccharides in 50% aq (v/v) MeOH containing 0.5% (v/v)
P. syringae pv. tabaci contain
glycinea flagellin contains
L
-Rha, whereas the P. syringae pv.
D
and
L
Rha.9 Thus, the possibility
cannot be discounted that the chirality of the Rha residues together
with the glycosylation pattern and the degree of polymerization of
the flagellin oligosaccharide side chains are factors that determine
host specificity and virulence.
1. Experimental
HCOOH were infused into the electrospray source at 5 l
L minꢀ1
with a syringe pump. Spectra were obtained between m/z 150
and 2000 with a step size of 0.1 amu and a dwell time of 25 ms.
HF treatment was performed at the Peptide Institute (Osaka,
Japan). Isolated flagellin (10 mg) was dried in vacuo over P2O5 for
16 h. The dry residue was dissolved in anhyd HF (1 mL) in a HF
apparatus and kept for 3 h at room temperature.12 The HF was re-
moved under diminished pressure, and the last traces of HF were
removed by co-distillation with Et2O.12 The resulting monosaccha-
rides were reduced with NaBD4 and converted into their alditol
acetate derivatives.16
(S)-(+)-2-butanol was purchased from Sigma–Aldrich Japan
(Tokyo, Japan). TMSI-H (trimethylsilyling reagent) was obtained
from GL Science (Tokyo, Japan). D2O was obtained from Kanto Ka-
gaku (Tokyo, Japan). (R)-(ꢀ)-2-butanol, 5% methanolic hydrogen
chloride solution, and other chemicals were purchased from Wako
Pure Chemicals (Osaka, Japan).
Pseudomonas syringae pv. tabaci 6605 was maintained in King’s
B medium at 27 °C.3 It was grown in LB medium containing 10 mM
MgCl2 for 48 h at 25 °C.3 The cells were harvested by centrifuga-
tion, resuspended in one-third volume of minimal medium
[50 mM potassium phosphate buffer, 7.6 mM (NH4)2SO4, 1.7 mM
MgCl2, and 1.7 mM NaCl (pH 5.7)] supplemented with mannitol
and fructose (10 mM each), and then kept for 24 h at 23 °C. Flagel-
lin was purified by the method of Taguchi et al.3
The monosaccharide compositions of flagellin glycans were
determined by GC analysis of the acetate derivatives of the methyl
glycosides. Samples were dried by stream of dry air, suspended in
250 lL of 5% methanolic HCl, and heated at 80 °C overnight.

2254
T. Konishi et al. / Carbohydrate Research 344 (2009) 2250–2254
tert-Butanol (200
l
L) was added to the tube, and the methanolic
three-channel inverse (1H/13C [15N]) CryoProbe (Bruker Biospin,
Karlsruhe, Germany) at a temperature of 303 K. 1D 13C NMR spec-
tra were recorded at 125.77 MHz on a Bruker Avance 500 spec-
trometer with a dual 13C [1H] CryoProbe (Bruker Biospin) at
303 K. The methyl signals of 2-methyl-2-propanol, H d 1.230 and
C d 31.30 were used as references for 1H and 13C chemical shifts.
HCl was removed by stream of dry air. The methyl glycosides were
acetylated with Ac2O and pyridine (1:1 v/v) for 20 min at 121 °C.
The acetylated glycosides were separated by GC–MS on a DB-1 col-
umn (15 m ꢁ 0.25 mm) as described.16 The absolute configuration
of rhamnose was determined by GC and GC–MS analyses of the
trimethylsilylated (S)-(+)-2-butyl glycosides of
scribed.17 The retention time of
-rhamnose, for which a standard
was not available, was determined by chromatography of the trim-
-rhamnose. Absolute
configuration of rhamnose was also determined by fluorescent
labeling with (S)-(+)-2-tert-butyl-2-methyl-1,3-benzodioxole-4-
carboxylic acid (TBMB), followed by separation of the fluores-
cent-labeled rhamnose by reversed-phase HPLC.10
L-rhamnose as de-
D
Acknowledgments
ethylsilylated (R)-(ꢀ)-2-butyl glycosides of
L
We thank M. A. O’Neill (Complex Carbohydrate Research Cen-
ter, The University of Georgia, Athens, GA, USA) for critical reading
of the manuscript. This work was supported by the Program for
Promotion of Basic Research Activities for Innovative Biosciences
(PROBRAIN).
Selective hydrazinolysis was used to release O-linked oligosac-
charides from flagellin of P. syringae pv. tabaci 6605. Hydrazinoly-
sis was performed at Masuda Chemicals Inc. Ltd (Kagawa, Japan).
Briefly, solutions of flagellin (10–15 mg) were concentrated to dry-
ness using a vacuum centrifuge and then stored for 16 h under vac-
uum over P2O5. The material was then subjected to hydrazinolysis
for 5 h at 60 °C according to the method of Patel et al.18 The re-
leased oligosaccharides were labeled with PA according to the
method of Hase et al.19
References
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PA-labeled oligosaccharides from flagellin were analyzed by
normal-phase HPLC using fluorescence (kex = 320 nm, kem
=
400 nm) detection. Normal-phase HPLC was performed using a
TOSOH Amide-80 column (4.6 ꢁ 250 mm, TOSOH, Tokyo, Japan)
eluted at 0.5 mL minꢀ1 at 40 °C. The column was eluted as follows:
eluent A, 90% (v/v) of 50 mM ammonium formate (pH 4.5) and 10%
CH3CN; eluent B, 10% of 50 mM ammonium formate (pH 4.5) and
90% CH3CN, and a linear gradient of eluent B from 80% (v/v) to
70% (v/v) in 20 min. The fluorescent-positive fractions (retention
time 12.5 min and 15.0 min for compounds 1 and 2, respectively)
were collected manually, concentrated by rotary evaporation, and
then freeze-dried. The purified oligosaccharides were analyzed by
ESIMS, 1H and 13C NMR spectroscopies, and their glycosyl residue
compositions were determined by GC analysis.
The purified PA-labeled oligosaccharides were dissolved in
99.96% isotopically enriched D2O and then freeze-dried. The sam-
ples were dissolved in 99.96% enriched D2O. 1D 1H NMR and 2D-
DQFCOSY, 2D-TOCSY, HSQC, and HMBC spectra were recorded at
800.13 MHz with a Bruker Avance 800 spectrometer with a
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