Tài liệu Báo cáo khoa học: The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGLB pdf

The structure and biological characteristics of the
Spirochaeta aurantia
outer membrane glycolipid LGL
B
Evgeny Vinogradov
1
, Catherine J. Paul
2
, Jianjun Li
1
, Yuchen Zhou
2
, Elizabeth A. Lyle
3
, Richard I. Tapping
3
,
Andrew M. Kropinski
2
and Malcolm B. Perry
1
1
Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada;
2
Queen’s University, Kingston, ON, Canada;
3
University of Illinois, Urbana, IL, USA
In an attempt t o i solate lipopolysaccharide from Spirocha-
eta aurantia, Darveau-Hancock extraction of the cell mass
was performed. While no lipopolysaccharide was found, two
carbohydrate-containing compounds were detected. They

were resolved by size-exclusion chromatography into high
molecularmass(LGL
A
) and low molecular mass (LGL
B
)
fractions. H ere w e present the results of the a nalysis of t he
glycolipid LGL
B
. Deacylation of LGL
B
with hydrazine a nd
separation of the products by using anion-exchange chro-
matography gave two major p roducts. Their structure w as
determined by using chemical methods, NMR and mass
spectrometry. All monosaccharides had the
D
-configuration,
andasparticacidhadthe
L
-configuration. Intact LGL
B
contained two fatty groups at O-2 a nd O-3 of the glycerol
residue. Nonhydroxylated C14 to C18 fatty a cids were
identified, which were p redominantly unsaturated or bran-
ched. LGL
B
was able t o gel Limulus amebocyte lysate, albeit
at a lower level t han that observed f or Escherichia coli O113
lipopolysaccharide. However, even large amounts of LGL

B
were unable to stimulate any Toll-like receptor (TLR)
examined, including TLR4 a nd TLR2, previously shown
to be sensitive t o lipopolysaccharide and glycolipids from
diverse bacterial origins, including other spirochetes.
Keywords: glycolipid; Spirochaeta aurantia;structure.
Spirochetes are a group of bacteria unified by spiral or
flattened-waveform cell morphology and periplasmic endo-
flagella; Spirochaeta is one of the six genera within this
phylum [1]. This b acterium is a f ree-living nonpathogenic
spirochete, originally isolated from pond mud and able to
fix atmospheric nitrogen [2–4]. Other members of this
phylum include the human pathogens Borrelia burgdorferi
(Lyme disease), the Leptospira (leptospiroses), Treponema
pallidum (syphilis), and T. denticola, T. brennaborense,and
T. maltophilum, which are i mplicated in periodontal disease
[5–7]. Although classified as Gram-negative, controversy
exists over the existence of lipopolysaccharide (LPS) in the
outer membranes o f spirochetes. Clear genetic a nd bio-
chemical evidence exists for the presence of LPS in
Leptospira [8] and for i ts absence i n T. pallidum and Borrelia
[9,10]. Limited structural analysis suggests that several o ral
treponemes (T. brennaborense and T. maltophilium [6],
T. medium [11], and T. denticola [12]) pos sess a surface
glycolipid similar to the lipotechoic acid of Gram-positive
bacteria. Recently, several small surface glycolipids were
identified in B. burgdorferi [13,14].
Toll-like r eceptors (TLR) a re an important component of
the host response to invading bacteria, with TLR4 required
for signal transduction and the inflammatory response

following exposure of cells to LPS derived from Gram-
negative enteric b acteria [15–17]. A lthough LPS der ived
from enteric bacteria is a potent agonist for TLR4, other
nonenteric bacterial L PS, such as that derived from
Legionella pneumophila, Leptospira interrogans and at least
one strain of Porphyromonas gingivalis can act as agonists
for TLR2 [8,18,19].
The glycolipids isolated from T. denticola, T. brennabo-
rense,andT. maltophilum appear to have functional
similarity to LPS in that t hey possess some ability to gel
Limulus amebocyte lysate (LAL) [12,20], a standard assay
for endotoxin activity. In addition, while glycolipid derived
from T. brennaborense stimulates immune cells through
TLR4, the glycolipids from T. denticola and T. maltophilum
stimulate cells through TLR2 [ 5,6,20]. T he strict correlation
between t he structure of the LPS molecule wit h that of TLR
specificity remains undefined but it is clear that TLR2 is
capable of r ecognizing a w ider range of potential lipid A
structures than TLR4 [21].
S. aurant ia has simple growth r e quirements t hat f acilitate
studies otherwise limited by the amount of cell mass, a
problem often limiting studies on other spirochetes [2]. We
describe here the structural characterization o f the carbo-
hydrate s keleton and fatty acids of one of its glycolipids,
LGL
B
. In addition we present evidence which suggests that
Correspondence to E. Vinogradov, Institute for Biological Sciences,
National Research Counci l, 1 00 Sussex Dr., Ottawa, ON, Canada
K1A 0R6. Fax: +1 613 952 9092, Tel.: +1 613 990 0832,

E-mail:
Abbreviations: EU, endotoxin units; FAME, fatty acid methyl esters;
GalNAcA, N-acetylgalactosaminuronic acid; GSL, glycosphinogo-
lipids; Fuc3N, 3-ami no-3,6-dideoxygalactose; Kd o, 2-keto-3-deoxy-
D
-manno-oct-2-ulosonic acid; LA L, Limulus amebocyte lysate; LBP ,
LPS-binding protein; LPS, lipopolysaccharide; SGM, spirochaete
growth medium; TLR, Toll-like receptor; TNF-a, tumour necrosis
factor-a.
(Received 9 August 2004, revised 30 September 2004,
accepted 13 October 2004)
Eur. J. Biochem. 271, 4685–4695 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04433.x
while superfic ially resembling other spirochetal glycolipids,
LGL
B
is a multisaccharide glycolipid and is unable to
stimulate any TLR examined.
Experimental procedures
Bacterial strain and growth conditions
The S. aurantia strain, M 1, us ed in this study, was obtained
originally from E. P. Greenberg (Ohio State University,
Columbus, OH, USA). It was propagated in spirochete
growth medium (SGM) containing 0.4% (w/v) maltose
(Sigma-Aldrich, St. Louis, MO, USA), 0.2% (w/v) tryptone
and 0.2% (w/v) yeast extract (Difco), at pH 7.5. Cells were
grown at 30 °C with gentle aeration ( 30 r.p.m.; orbital
shaker; Forma Scientific, Marietta, O H, USA) for 24–48 h.
Cell stocks were maintained in SGM in liquid nitrogen.
Glycolipid isolation
Isolation of LGL from S. aurantia. Bacteria were harves-

ted f rom a total of 55 L of SGM and the c ombined cell
pellet was extracted fo llowing the method of Darveau &
Hancock [ 22]. The final product w as extracted once with
cold 95 % (v/v) ethanol and twice with chloroform/meth-
anol (2 : 1, v/v) to remove phospholipids and carotenoids.
The r esidue was resuspended in distilled water, and
contaminating protein was removed by treatment with
pronase (25 lgÆmL
)1
)for18hat37°C. A final extraction
by chloroform/methanol (2 : 1, v/v) was followed by
dialysis against distilled water using Slide-a-lyzerÒ 10K
cassettes (Pierce Chemical Company, Rockford, IL, USA)
and lyophilization. The overall yield was determined by
comparing the mass of a white powdery material left after
dialysis and lyophilization ( 547 mg), to the original d ry
weight of lyophilized whole cells of S. aurantia from which
that glycolipid material had been isolated (3.51 g).
Column chromatography. Crude LGL (1.5 g) was dis-
solved in sample buffer [20 m
M
Tris/HCl, pH 8; 50 m
M
EDTA; 10% w/v) SDS] and fractionated on a 5.5 · 40 cm
column of Sephacryl S-300 HR (Sigma-Aldrich) at 25 °C
[column buffer: 10 m
M
Tris/HCl, pH 8; 1 0 m
M
EDTA;

0.2
M
NaCl; 0.3% (w/v) SDS]. Fractions of  2.1 mL were
collected at an average fl ow rate of 1.5 mLÆmin
)1
.The
fractions containing the l ow m olecular mass m aterial
(LGL
B
), as determined by standard SDS/PAGE with silver
stain [23], were pooled, precipitated with cold 0.375
M
MgCl
2
in 95% (w/v) ethanol, suspended in distilled water
and subjected to a second chromatography to ensure
homogeneity. Material was then reprecipitated, suspended
in distilled water, dialyzed, lyophilized and weighed in
preparation for further analysis.
Tricine–SDS/PAGE. Tricine–SDS/PAGE [15% (w/v)
resolving gel; 1 0% (w/v) s pacer g el; 4.5% ( w/v) stacking
gel) was u sed to e xamine the low molecular mass portions of
LPS and LGL [24]. LPS from Salmonella enterica sv.
typhimurium wild type , Sal. enterica sv. t yphimurium TV 119
(Ra mutant) a nd Sal. enterica sv. m innesota R5 (Rc mutant)
were p urchased f rom S igma-Aldrich. Products in acryl-
amide gels we re visualized by silve r staining [23].
NMR spectroscopy and general methods
NMR spectra were recorded at 25 °CinD
2

OonaVarian
UNITY INOVA 600 instrument using acetone as the
external reference (
1
H, 2.225 p.p.m.,
13
C, 31.45 p.p.m.).
Varian standard programs COSY, N OESY (mixing time
of 300 ms), TOCSY (spinlock time 120 ms), HSQC,
HMQCTOCSY, and gHMBC (evolution delay of
100 ms), were used.
Capillary electrophoresis-electrospray mass spectrometry
(CE-MS). Mass spectrometric experiments were conduc-
ted by using a Q-Star Quadropole/time-of-flight instrument
(Applied B iosystems/Sciex, Concord, ON, Canada). B riefly,
samples were analyzed on a crystal Model 310 CE
instrument (ATI Unicam, Boston, MA, USA) coupled to
a Q-Star via a microIonspray interface. A sheath solution
(isopropanol/methanol, 2 : 1, v/v) was delivered at a flow
rate of 1 lLÆmin
)1
to a low dead volume tee. The separation
was obtaine d on a bare fused-silica capillary, o f  90 cm
length, u sing 10 m
M
ammonium acetate/amm onium
hydroxide in deionized water, pH 9.0, co ntaining 5% (v/v)
methanol. A voltage of 25 kV was typically applied at the
injection. Mass spectra were acquired w ith dwell times of
2.0 s per scan i n positive ion detection mode. Fragment ions

formed by collision activation of selected precursor ions
with nitro gen in the RF-only q uadrupole c ollision ce ll, were
recorded by a t ime-of-flight mass analyzer. Collision
energies were typically 120 eV (laboratory f rame of refer-
ence).
Hydrolysis. Hydrolysis was p erformed with 4
M
CF
3
CO
2
H
(110 °C, 3 h), monosaccharides were conventionally con-
verted into the alditol acetates and analysed by GLC on an
Agilent 6850 chromatograp h equipped with a DB-17
(30 m · 0.25 mm) fused-silica column using a temperature
gradient of 180 °C(2 min) fi 240 °C, at 2 °CÆmin
)1
.GC-
MS was performed on the Varian Saturn 2000 system with
an ion-trap mass spectral detector using the same column.
Gel chromatography. Gel chromatography was carried out
on Sephadex G-50 (2.5 · 95 cm) and Sephadex G -15
columns (1.6 · 80 cm) in pyridinium-acetate buffer,
pH 4.5 (4 mL of pyridine and 10 mL of AcOH in 1 L of
water), and the eluate was monitored by a refractive index
detector.
Configuration experiments
For determining the absolute configuration of t he mono-
saccharides, product 2 (1 mg) was treated with (S)-2-

butanol/AcCl (0.25 mL, 10 : 1, v/v) for 2 h at 85 °C, dried
under t he stream of air, acetyl ated and t hen analysed b y GC
in comparison with authentic standards, prepared from the
respective monosaccharides with (S)- an d (R)-2-butanol.
For determination of the configuration of N-acetylgalac-
tosaminuronic acid (GalNAcA), a sample (2 mg) of LGL
B
was treated with 1
M
HCl in methanol (100 °C, 4 h), dried,
and t hen the product was peracetylated by Ac
2
O i n pyridine
(0.5 + 0.5 mL, at 85 °C for 30 min) and reduced with
excess NaBD
4
in96%(v/v)ethanol(1 mL)at40°C. Acetic
acid (1 mL) was added, the product was dried under a
4686 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004
stream of air a nd then dried twice with the a ddition of 1 mL
of meth anol to remove boric acid. (R)-2-BuOH ( 0.5 mL)
and AcCl (0.08 mL) were added to the dry residue, the
mixture was incubated for 4 h at 85 °C, filtered, dried,
acetylated by Ac
2
O in p yridine ( 0.5 + 0.5 mL, at 85 °Cfor
30 min), dried and analyzed by GC-MS with the standards
prepared from
D
-GalN and (R)- and (S)-2-BuOH.

The absolute configuration of
L
-aspartic acid was deter-
mined by chiral HPLC of the oligosaccharide hydrolysate
on a Chirex D p enicillamine column (250 · 4.6 mm;
Phenomenex) in 15% (v/v) methanol containing 2 m
M
CuSO
4
, with UV detection at 2 54 nm.
Fatty acid methyl esters (FAMEs) were generated from
1 mg s amples of LGL
B
by the addition of 1 mL o f 3
M
HCl
in methanol (Alltech Associates, Inc., Deerfield, I L, USA)
and incubation at 100 °C for 18 h. Following liberation of
the FAMEs, the hydrolysates were neutralized with 0.46 g
of silver carbonate and doped with 204.5 lg of tridecanoic
acid (in n-pentanol) as an internal standard. The samples
were centrifuged and the FAMEs were resolved by
PerkinElmer Sigma 3 gas chromatography, equipped with
a glass column [3.05 m · 2 mm internal diameter, packed
with 3% (w/v) SP-2100 DOH, 100/120 Supelcoport w ith
carrier gas (N
2
)], at a flow rate of 50 mlÆmin
)1
. The oven was

programmed as f ollows: 150 °C f or 5 min; followed by 150°
to 230° Cat8° CÆmin
)1
. Data analysis was conducted by
using the
PEAKFIT
Ò v. 4.11 software package (Systat
Software Inc., Richmond, CA, USA). Comparison of
FAME retention times with those of a Bacterial Acid
Methyl Esters CP
TM
mix (Matreya, Inc., Pleasant Gap, PA,
USA) permitted tentative FAME identifications to be
made. The latter were confirmed b y G LC-MS a nalysis
(Analytical Services, Queen ’s University, K ingston, ON,
Canada).
LGL
B
was O-deacylated by hydrazinolysis, as described
by Gu et al. [25]. Briefly, 40 mg o f LGL
B
was i ncubated
with anhydrous hydrazine for 3 h at 37 °C, w ith occasion al
mixing. The mixture was then chilled to )20 °Candan
equal volume of chilled acetone was added dropwise. The
product was recovered by centrifugation, washed once w ith
chilled acetone, dried and weighed.
Separation of oligosaccharides 1 a nd 2 was performed
by ion-exchange chromatography on a Hitrap Q anion-
exchange column containing 5 mL of Q-Sepharose Fast

Flow (Amersham Pharmacia Biotech) in a gradient of
water/1
M
NaCl over 1 h with UV detection at 220 nm. The
products were desalted by gel chromatography on a
Sephadex G-15 column.
Biological assays
LAL a ssays were conducted by the Associates of Cape Cod,
Inc. (Cape Cod, MA, USA), by using the gel-clot method,
and the number of endotoxin units (EU) was compared
with control standard endotoxin from Escherichia coli
O113. The activation o f TLRs w as measur ed by quantifying
the production of tumour necrosis factor-a (TNF-a)by
whole blood cells, in response to a panel of TLR agonists, a s
described by Tapping et al. [26]. Briefly, whole blood from
healthy donors was collected into tubes containing heparin
and d iluted 1 : 4 in RPMI 1640. Sa mples were a liquoted
into 96-well plates, agonist was added, and incubatio n was
carried out at 37 °C in an atmosphere of 5% carbon dioxide
for 6 h. Cell supernatants were removed a nd assayed for
cytokine production by standard sandwich ELISA in
96-well Immunlon plates (Dynatech L aboratories, Chant-
illy, VA, USA). The TNF-a ELISA was p erformed by using
mAbs 68B6A3 or 68B2B3 for capture and t he biotinylated
mAb 68B3C5 (Biosource International, Camarillo, CA,
USA), followed by streptavidin-conjugated horseradish
peroxidase (HRP), for d etection. ELISAs were d eveloped
by using o-phenylenediamine as a substrate, and the
absorbance was measured at 490 nm by using a Spectramax
plate reader and software (Molecular Devices, Sunnyvale,

CA, USA). All values were interpolated from either a log-
log or a four-parameter fit of a curve generated from
appropriate standards. Agonists examined were the
S. aurant ia LGL
B
(5 lgÆmL
)1
), zymosan (5 · 10
9
particles
per mL; Molecular Probes, Eugene, OR, USA), heat-killed
Staphylococcus aureus (2.5 · 10
6
particles per mL; Molecu-
lar Probes), PolyIC (50 lgÆmL
)1
; Sigma Genosys, The
Woodlands, TX, USA), E. coli Re595 LPS (20 ngÆmL
)1
;
repurified as decribed previously [26]), R848 (1 lgÆmL
)1
;
Invivogen, San Diego, CA, USA) and CpG Oligo (2 l
M
;
Sigma Genosys).
Results
Darveau-Hancock extraction o f stationary phase S. auran-
tia cells gave a white powdery substance i n a yield of 15.6%

based upon the cell dry wieght. T his h igh y ield is not
unexpected as the surface to volume ratio of this bacterium
is 13.6Ælm
)1
, approximately 3 .5 times higher t han that of
E. coli or Sal. enterica sv. typhimurium (3.9Ælm
)1
). The
Darveau-Hancock procedure does not discriminate between
high (ÔsmoothÕ)orlow(ÔroughÕ)molecularmassLPS,
provides a high yield of product and should apply equally to
polysaccharides or glycolipids [22]. Potential complex
glycolipids were separated from previously characterized
glycogen storage granules by size exclusion chromatography
with examination of the fractions for carbohyd rates and
hexosamines [27]. A low molecular mass carbohydrate-
containing material (LGL
B
) was isolated, and when
examined by Tricine–SDS/PAGE [24] , demonstrated mobil-
ity s imilar t o the rough LPS of a Sal. enterica sv. typhimurium
TV 119 Ra mutant (Fig. 1). Another material, LGL
A
,was
identified as a larger glycolipid and is thought to contain
O-antigen like r epeats, contributing to the b anding pattern
observed in crude S. aurantia extract ( data not shown).
Preliminary colorimetric analysis [28] indicated that
LGL
B

did not contain any 2-keto-3-deoxy-
D
-manno-oct-2-
ulosonic acid (Kdo). The material was subjected to
methanolysis [29], and the fatty acid methyl esters were
analyzed by GLC, revealing fi ve major acyl constituents,
none of which were the characteristic hydroxylated fatty
acids of LPS (Table 1).
LGL
B
was O-deacylated by t reatment with anhyd rous
hydrazine, and the oligosaccharides were separated by
anion-exchange chromatography to give two main compo-
nents (1 and 2), which differed by one monosaccharide
residue. Their structure was determined by NMR spectros-
copy, MS and chemical analysis. Monosaccharide analysis
(GC of alditol acetates or acetylated products of acidic
methanolysis) of both products showed that their compo-
Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL
B
(Eur. J. Biochem. 271) 4687
sition was similar, comprising glycerol, xylose, mannose,
glucose, galactose, and 3-amino-3,6-dideoxyhexose in a
ratio o f 1 : 2.5 : 1 : 1.6 : 1 : 1.5, and additionally nonquan-
tified glucuronic, galactosaminouronic, and galacturonic
acids. The presence of excess glucose (glucitol) in alditol
acetate analysis is a r esult of the reduction o f glucurono-
lactone. The 3-amino-3,6-dideoxyhexose is quantified
approximately b ecause of the lack of a quantitative
standard compound.

NMR spectra of both oligosaccharides were completely
assigned by using 2 D N MR techniques ( Figs 2–4, Table 2).
Monosaccharides were identified o n the basis o f vicinal
proton coupling constants and
13
C NMR chemical shifts.
Anomeric configurations were deduced from the J
1,2
coupling constants and chemical shifts of H-1, C-1 and
C-5 signals. The position of C-6 signals of uronic acids was
found from HMBC correlations to H-5 protons. Connec-
tions between monosaccharides were identified on the basis
of NOESY ( Fig. 3) and HMBC correlations. The following
inter-residual NOEs were observed in oligosaccharides 1
and2:P1G4(in1),C1G4(in2),andG1A2,G1A1,A1G5,
A1L4, A1L3, L1E4, E 1F4, F1I4, I1D4, D1N3, D1N4,
N1Q1, B1K4, K1E3, O1I3, and M1D3. These correlations
include several contacts to nontransglycosidic protons next
to the linkage position, and between H-1 o f a monosac-
charide a nd H-5 of a glycosylating residue in the e vent of an
a-(1–2)-linkage. R espective H MBC c orrelations between
H-1 and a carbon at the transglycosidic position were
identified for all linkages. Amide linkage between C-6 of
residue E and an amino group of the aspartic acid was
identified on t he basis of the HMBC correlation b etween
H-2 of aspartic acid and C-6 of the GalA E, thu s showing
that aspartic acid is amide linked through i ts amino group to
C-6 of galacturonic acid E (Fig. 4).
Absolute configuration o f the monosaccharides was
determined by GC analysis of acety lated 2-butyl glycosides.

For 3-amino-3,6-dideoxygalactose (Fuc3N), the O-specific
polysaccharide from Proteus penneri 16wasusedasa
source of reference
D
-Fuc3N, where its
D
-configuration was
determined earlier [30]. For determining the configuration
of GalNAcA, an LGL
B
sample was treated with HCl in
1
5 4 3 2 [ppm]
2
Fig. 2.
1
H NMR spectra of oligosaccharides
1and2.
15
234
Fig. 1. Visualization of LGL
B
by Tricine–SDS/PAGE and silver
staining rev eals that this material c o-electrophoreses with the Ra form of
lipopolysaccharide (LPS) from Salmonella enterica sv. typhimurium.
Lane 1, wild-type Sal. enterica sv. typhimurium LPS, 30 lg; lane 2,
Spirochaeta aurantia crude LGL, 20 lg; lane 3, S. aurantia LGL
B
,
15 lg; lane 4, Sal. enterica sv. typhimurium TV119 ( Ra mutant), 2 lg;

lane 5, Sal. enterica sv. minnesota R5 (Rc m u tant), 2 lg.
Table 1. Fatty acid methyl e ster (FAME) analysis, GLC and GLC-MS
indicated that the majority of fatty acids contained in Spirochaeta
aurantia LGL
B
are either branched or unsaturated. Values stated a re the
average n molÆmg
)1
with standard deviations (±) obtained from
quantifying and averaging areas under specific peaks from GLC
analysis of four separate samples of LGL
B
.
Identity of fatty acid LGL
B
(nmolÆmg
)1
)
Tetradecanoic acid (C14:0) 34.9 ± 1.3
13-Methyltetradecanoic acid (iC15:0) 224.1 ± 9.0
15-Methylpentadecanoic acid (iC16:0) 117.6 ± 12.4
9-Hexadecenoic acid (C16:1
9
) 155.3 ± 10.5
9-Octadecenoic acid (C18:1
9
) 58.3 ± 5.6
4688 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004
methanol, a nd the product w as peracetylated in order to
reacetylate free amino groups; it was checked by GC-MS

for the presence of methyl ester of m ethyl G alNAc. The
product w as reduced with NaBD
4
in 96% (v/v) ethanol at
40 °C,treatedwithHCl-(R)-2-BuOH, acetylated and ana-
lysed by GC-MS. A total ion chromatogram showed no
well pronounced peaks; however, a fragmentogram for t he
expected glycosyl cation of m/z 332 contained peaks with
the same retention time as that obtained f rom
D
-GalN with
(R)-2-BuOH; mass spectra of the products obtained from
LGL
B
wereshiftedtohighmassbytwounitsowingto
a double deuteration at C-6. Thus, G alNA had the
D
-configuration.
These data, taken together, allow u s to p ropose the
structures of oligosaccharides 1 and 2, as presented in
Scheme 1.
The o ligosaccharides were further analyzed by CE-MS
and by CE-MS/MS (Figs 5 and 6). The mass spectra
obtained in positive ion detection mode for oligosaccharide
1 showed a m ajor do ubly charged ion at m/z 1180.25
(observed molecular mass: 2358.50 Da; calculated exact
molecular mass for C
85
H
130

O
72
N
4
: 2358.6633 Da). The
MS for oligosaccharide 2 showed a m olecular mass of
2375.56 Da (calculated exact mass for C
85
H
129
O
74
N
3
:
2375.6422). In addition, an ammonium adduct of com-
pound 2 with m/z 1197.25 was observed as the most
abundant ions (observed molecular mass: 2392.50 Da). The
composition details, as well as some sequence information
of those two major c omponents w ith m/z 1180.24 and
1197.25, were further characterized by tandem mass
spectrometry (MS/MS). The fragmentation of cationic
oligosaccharides typically proceeds by cleavage at the
glycosidic bonds, which provides sequence and branching
information [31]. The charge state of a fragment ion is then
identified by u sing the isotope profile, owing to the high
resolution provided by the TOF mass analyser. The
product-ion s pectrum ( MS/MS s pectrum), obtained from
a doubly charged ion a t m/z 1197.25, is illustrated in Fig. 6.
This spectrum revealed two major doubly charged ions at

m/z 503.63 and 672.67, which corresponds to the single
charged ion at m/z 1006.27 and 1344.37, respectively. In
addition, a series of single charged ions was generated via
the formation of complementary fragment pair of B and Y
ions. A s i ndicated i n F ig. 6 , t he fragment ion a t m/z 18 76.56
corresponded to the loss of the nonreducing end C-G-A
unit and ammonium from the molecular ion. Further
fragmentation gave the fragment ion at m/z 1436.42, owing
to the loss o f the branching xylose residues M and O, a nd of
GlcA residue L. The remaining linear sequence, consisting
of K-B-F-I -D-N-Q, was confirmed by t he observation of
5.6 5.4 5.2 5.0 4.8 4.6 ppm 5.6 5.4 5.2 5.0 4.8 4.6 ppm
5.2
4.7
4.2
3.7
ppm
TOCSY NOESY
A12
B12,14
B13
C12
C13
C15
C14
D12,13
D15
D14
E15
E14

E12
E13
F12
F13
G15
F14
I12
I13
I14
E45
G12
G14
G13
K12
K13
K14,15
L12
L13,15
L14
F45
I45
D45
I24
E34
E24
I34
D34
N14
I23
F34

N12
N13
M15
O15
M14
M13
M12
M15
O12
O15
O13
O14
F24
A1:G1
A1:G5
A12
A1:L4
A1:L3
B12
B1:K4
C1:G4
C12
D12
D1:N4
D1:N3
G12
F12
E12
G1:A2
E35

E5:F2
E1:F4
F1:I4
E45
I1:D4
I12
K1:E3
K15
K12
F45
O1:I3
M1:D3
I34
I35
F34
N15
N13
N1:Q1
M15
O15
O13
Fig. 3. Fragments of TOCSY (left) an d NOESY (right) s pectra o f oligosaccharide 2. Intraresidual correlations are labeled with a letter designation o f
the m o nosaccharide residue and numbers o f the c orrelating protons. Inte r-residual correlations a re labeled withlettersforbothmonosaccharides.
Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL
B
(Eur. J. Biochem. 271) 4689
fragment ions at m/z 292.06, 468.09, 613.1 6, 789.21, 1006.27,
1182.24, and 1344.37, respectively. The fragment ion at m/z
556.15 corresponds to the unit I-D-N, which might result
from the loss of Q from I-D-N-Q (m/z 648.20) o r from the

loss of F f rom F -I-D-N (m/z 732 .18). However, many o ther
combinations of fragments are also possible, because of the
existence of branches i n t he molecule. Similarly, the t andem
MS was conducted for the doubly charged ion at m/z
1180.25 (data not shown) and the mass spectral data f ully
agree with the sequence determined by NMR.
Knowledge of the deacylated oligosaccharide structures
allowed analysis of intact glycolipids by NMR. Spectra of
reasonable quality were obtained at 60 °C in the presence
of 5% fully deuterated SDS. All monosaccharides present
in the products 1 and 2 were identified, a nd the ratio of
structures 1 a nd 2 w as c lose to 1 : 1. All chemical shifts
remained m ostly unchanged with the e xception of H/C-2
and H /C-3 of the glycerol residue. P roton signals were
strongly downfield shifted owing to acylation ( Table 2);
13
C signals also experienced downfield substitution effects.
No data regarding attachment of particular acyl groups at
O-2 and O-3 of the glycerol residue was obtained. Several
attempts to obtain a mass spectrum of the LGL
B
by using
CE-MS, ESI-MS and MALDI were unsuccessful, prob-
ably because this compound is not soluble without a
detergent. These results show that no additional acylation,
except at the glycerol residue, is present in the oligosac-
charides.
LGL
B
does not activate any Toll-like receptor

The gelation of LAL is a standard assay based on the
nonspecific immune response of the horseshoe crab, and is
used to assess the endotoxic potential of various substances
[32]. LGL
B
displayed a 100- fold less endot oxic potential,
registering 2.5 · 10
5
EUÆmg
)1
when compared to an E. coli
O113 LPS control (1 · 10
7
EUÆmg
)1
) in a LAL gel clot
assay.
LGL
B
was also examined for its ability to act as a TLR
agonist. Attempts to measure a reaction f rom cells trans-
fected specifically with human TLR2 or TLR4 were
unsuccessful, regardless of the concentration of LGL
B
examined (data not shown). T he whole blood assay u ses
fresh human blood (which contains a variety of Toll
receptors) and measures the total r elease of TNF-a by
ELISA [26]. Cells were stimulated with defined TLR
agonists (zymosan a nd heat-killed Staph. aureus for
TLR2; PolyIC for TLR3; E. coli Re595 LPS for TLR4;

R848 for TLR7; and CpG O ligo ( 2 l
M
) for TLR9), and the
production of TNF-a was q uantified (Fig. 7). Even when
5.5
5.0 4.5 4.0 3.5 3.0
100
80
60
40
178
174
170
Asp2
Asp3
Asp13;Asp34
E56
Asp2:E6
Asp12;Asp24
HSQC
HMBC
E5
Fig. 4. Fragments of HSQC and HMBC
spectra of compound 1. Labels illustrate
assignment of the amide linkage between the
amino group of Asp and the carboxyl group of
GalA residue E.
Scheme 1. The structures of o ligosaccharides 1 and 2. Oligosaccharide 1, R ¼ a-Fuc3N (P); o ligosaccharide 2, R ¼ a-Glc (C).
4690 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004
high concentrations of LGL

B
were added, no production of
TNF-a was detected, showing t hat this large glycolipid
cannot stimulate TLR2, -3, -4, -7 or -9.
Discussion
Although s ome s tructural i nformation has been obtained
from other spirochetes, the complete elucidation o f t he
LGL
B
from S. aurantia represents the first complete
structure o f a large glycolipid from these bacteria. The
dodecasaccharide LGL
B
is anchored by a diacyl glycerol.
A glycolipid containing a single sugar, BbGL-II, and also
anchored on a g lycerol, has been identified in B. burg-
dorferi [13]. I t i s s urface localized, and antibodies to this
molecule were detected in patients with Lyme disease. A
diacyl glycerol anchor has also been purposed for the
glycolipids of T. denticola, T. maltophilum,andT. brenn-
aborense [6,12]. A glycolipid identified in T. pectinovorum
contained glycerol, and the majority of fatty acids were
branched, although on the basis of detection of Kdo in
this material, the authors designated it LPS. A diacyl
glycerol anchor may substitute for lipid A, an observa-
tion supported by the absence of any homologs to genes
involved in lipid A biosynthesis in the completed ge-
nomes of B. burgdorferi, T. pallidum or T. denticola
[9,10,33].
All o f t he treponemal g lycolipids i dentified have either

fully saturated o r branched fatty acids , in contrast to the
Table 2. NMR data for compounds LGL
B
, 1 and 2. Data refer to both compounds, except where indicated. N-Acetyl at I2: H-2/C-2 2.12/23.8,
C-1176.2 p.p.m.
Unit, compound 1 2 3 4 5 (5eq) 6 (5ax) 6b
A, a-Man
1
H 5.71 4.12 3.99 3.92 3.72 3.90 3.90
13
C 99.8 81.2 71.5 67.7 74.1 61.5
B, a-Fuc3N
1
H 5.58 4.01 3.68 4.02 4.18 1.27
13
C 98.6 66.3 53.6 69.4 67.7 16.6
P, a-Fuc3N (1)
1
H 5.58 3.99 3.65 4.01 4.16 1.25
13
C 98.6 66.3 53.6 69.4 67.7 16.6
C, a-Glc (2)
1
H 5.55 3.59 3.76 3.47 3.78 3.86 3.86
13
C 99.6 73.0 74.0 70.5 73.0 62.4
D, a-GalA
1
H 5.32 4.22 4.23 4.69 4.65
13

C 96.5 68.7 79.3 79.4 72.4 175.6
E, a-GalA6Asp
1
H 5.20 4.12 4.20 4.70 5.05
13
C 100.8 68.8 79.4 79.4 72.2 170.6
F, a-GalA
1
H 5.18 3.89 4.21 4.50 4.79
13
C 99.3 68.9 69.6 80.3 72.8 176.0
G, a-GlcA (1)
1
H 5.19 3.69 4.06 3.83 4.30
13
C 101.6 73.1 75.0 77.3 73.7 177.2
G, a-GlcA (2)
1
H 5.19 3.69 4.06 3.82 4.30
13
C 101.6 73.1 75.0 77.8 73.7 177.2
I, a-GalNAcA
1
H 5.11 4.46 4.10 4.69 4.76
13
C 99.3 49.8 77.3 77.6 72.9 176.0
K, b-GlcA
1
H 4.80 3.50 3.86 3.91 3.90
13

C 104.8 74.7 77.4 77.4 78.0 176.3
L, b-GlcA (1)
1
H 4.81 3.34 3.75 3.83 3.77
13
C 104.4 74.9 77.5 77.4 78.1 176.4
L, b-GlcA (2)
1
H 4.81 3.34 3.75 3.83 3.74
13
C 104.4 74.9 77.5 77.4 78.1 176.4
M, b-Xyl
1
H 4.62 3.39 3.46 3.72 3.33 3.98
13
C 106.5 74.4 77.3 70.5 66.4
N, b-Gal
1
H 4.55 3.72 3.91 4.29 3.80 3.88 3.88
13
C 104.1 70.4 78.6 66.4 76.2 62.3
O, b-Xyl
1
H 4.47 3.28 3.42 3.69 3.33 3.98
13
C 106.6 73.9 77.1 70.5 66.4
Asp
1
H 4.42 2.93
2.93

13
C 178.8 52.3 39.1 178.8
Q, Gro, 1 and 2
1
H 3.85 4.03 3.68
4.00 3.76
13
C 72.1 71.7 63.7
Q, Gro, LGL
B
1
H 3.84 5.32 4.15
3.84 4.50
13
C 69.8 72.0 64.8
Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL
B
(Eur. J. Biochem. 271) 4691
1000 1100 1200 1300 1400
m/z
1197.25
1189.28
1180.30
1116.27
1131.28
1000 1 100 1200 1300 1400
1180.25
1114.29
1
2

Fig. 5. CE-MS spectra of oligosaccharides 1 and 2.
200 400 600 800 1000 1200 1400 1600 1800 2000
m/z
613.16
556.15
468.09
672.67
146.08
503.63
732.18
648.20
218.06
789.21
714.18
292.06
824.24
321.08
1006.27
339.09
1199.29
663.67
807.22
1023.26
376.09
1344.37 1744.531436.42
1182.24
1876.56
K
B
F

I
1612.48
D NQL OM
C-G-A,NH
3
Fig. 6. MS/MS spectrum obtained from a doubly charged ion at m/z 1197.25 of o ligosaccharide 2.
4692 E. Vinogradov et al. (Eur. J. Biochem. 271) Ó FEBS 2004
unsaturated acyl group of BbGL-II. Schultz et al.
indicated that the presence of fatty acid branching in
T. denticola is analogous to adap tations in Gram-positive
bacteria to alter membrane fluidity [12]. Gram-negative
bacteria are known to m odify the d egree of saturation in
their fatty acids to modulate membrane fluidity [34,35].
LGL
B
contained both unsaturated and branched fatty
acids (i.e. C14:0, iC15:0, C16:1), t he only spirochete
glycolipid identified, to date, with both of these modi-
fications, suggesting LGL
B
may form highly fluid mem-
branes.
S. aurant ia LGL
B
comprises 15% lipid by mass, corres-
ponding well with the p roportion of fatty acids in the
glycolipid OML521 (10.7%) from T. denticola, a glycolipid
that is also estima ted t o be similar in size to Ra LPS [12]. Ra
LPS is t he minimum L PS unit required for efficient and
proper folding, and functioning, of porin [36]. T. denticola

and S. aurantia possess two of the largest porins yet
discovered in Gram-negative bacteria: given the absence
of LPS i n t hese bacteria, OML251 and LGL
B
may function
in place of Ra LPS, and contribute to the folding or
stabilization of porin [37,38].
While S. aurantia stains Gram -negative and possesses
an outer membrane containing porin, phylogenetically it is
not closely related to the bacterial phylum (Proteobacte-
ria) that contains the typical Gram-negative cells, such as
E. coli. Other nonproteobacterial organisms in which
glycolipids replace LPS include Chloroflexus aurantiacus
and Fibrobacter succinogenes. The former bacterium is
thought to contain outer membrane galactolipids [39],
while the latter c ontains a low molecular mass glycolipid
with glycerol anchor and man y charged groups in the
oligosaccharide p art, which makes it, in overall d esign,
similar t o S. aurantia LGL
B
. I nterestingly F. succinogenes
also has a capsular polysaccharide with a lipid anchor
[40].
Even within the Proteobacteria, one finds examples where
LPS has been replaced by glycolipids. Sphingomonas pau-
cimobilis and N ovosphingobium capsulatum contain glyco-
sphinogolipids (GSLs) [41–43] in lieu of LPS.
Although there is no similarity at a structural level to
LPS, studies investigating treponemal glycolipids have
shown that most are able to gel LAL and stimulate

Toll-like r eceptors [12,20], suggesting t hat a t a functional
level they possess some similarity. LGL
B
was able to gel
LAL, but did not stimulate any TLR examined: this is
an unusual situation, paralleled in the spirochete litera-
ture only by the inability of the Borrelia glycolipids to
activate TLR2 o r -4 [13]. TNF-a release was measured
following the e xposure o f h uman mononuclear cells to
two diffe rent GSLs from S. paucimobilis: t he mono-
glycosylated GSL-1, and the tetraglycosylated GSL-4A.
GSL-1 was unable to activate the release of monokines,
in contrast to the larger GSL-4A, although induction was
still 10 000-fold below that of the LPS standard [44].
While this appears to be s imilar to the situation with the
monoglycosylated BbGL-II, the inability of LGL
B
to
stimulate TNF-a release precludes size as the only
explanation for the difference in biological activity
observed with GSLs.
Another oral spirochete implicated in periodontal dis-
ease, T. medium, contains the glycolipid, Tm-Gp, which
abrogates TLR activation through interactions with
LPS-binding protein (LBP) and CD14, two important
components of TLR-mediated innate immunity [45]. The
blocking by Tm-Gp was dependent on the lipid portion of
the molecule, but whether S. aurantia LGL
B
would block a

TLR response is unknown. Structural studies of Tm-Gp
have focused o n a tetrasaccharide repeating unit, likened by
Asai and colleagues to the repeating unit of the LPS
O-antigen [11]. The c haracteristic laddering pattern on SDS/
PAGE suggests that Tm-Gp is different from LGL
B
,
although t hey both contain an a spartic a cid residue. T he
structures of the bioactive portion of Tm-Gp, and of t he
other treponemal glycolipid TLR agonists, need to be
elucidated to begin to identify possible motifs involved in
modulating TLR activity. T his i s especially interesting w hen
one realizes that the existing literature does not contain any
direct demonstration of a ligand-type interaction between a
TLR and any glycoconjugate, LPS or otherwise. LPS has
been shown, however, to bind LBP [46]. Interestingly, a
decrease in the fluidity of Re LPS, instigated by a Zn
2+
-
induced increase in acyl chain order, elevated the production
of TNF-a from human monocytes. The increase in acyl
chain order increased the bond strength between Re LPS
and LBP, and was thought to increase the transport o f the
LPS to t he target membrane. LBP is an important precursor
in the TLR-dependent release of TNF-a and h as been
shown to interact with both the T. maltophilum and
T. brennaborense glycolipids to enhance their ability to
stimulate TLRs [6]. It is tempting to speculate that the
highly disordered acyl chains of LGL
B

could abrogate the
interaction with LBP and prevent any release of TNF-a in
the whole blood assay for TLR activation. Specific struc-
tural e ntities of LPS, producing certain biological effects,
have been extensively studied given the central role of this
molecule in pathogenesis and vaccine development. Char-
acterization of any biological activity o f spirochete glyco-
lipids is important for similar reasons, especially in the case
of B. burgdorferi BbGL-II, given the difficulties in develop-
ing an effective proteinaceous vaccine targeting this organ-
ism [13,47].
0
5
10
15
20
25
30
35
40
TNF-α (ng/mL)
No Zymosan HKSA PolyIC Re LPS R848 CpG LGL
B
Fig. 7. Tumour necrosis factor-a (TNF-a) production through activation
of Toll-like receptors (TLR) in the presence of different agonists. Con-
trols for different TLR were as follows: zymosan and h eat-killed
Staphylococcus aureus (HK SA) for TLR2; PolyIC fo r TLR3;
Escherichia coli Re595 lipopolysaccharide (LPS) for TLR4; R848 for
TLR7; and CpG Oligo for TLR9. Error bars represent the standard
deviation of cellular a ctivation e xperiments pe rforme d in t riplicate.

Ó FEBS 2004 Analysis of Spirochaeta aurantia glycolipid LGL
B
(Eur. J. Biochem. 271) 4693
Acknowledgements
This work was supported by a Natural Sciences and Engineering
Research Council of Canada (NSE RC) Disco very Grant to A .M.K.
C.J.P. was the recipient o f an N SERC studentship.
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