Intact LGLB contained two fatty groups at O-2 and O-3 of the glycerol residue.. However, even large amounts of LGLB were unable to stimulate any Toll-like receptor TLR examined, includin
Trang 1The structure and biological characteristics of the
Evgeny Vinogradov1, Catherine J Paul2, Jianjun Li1, Yuchen Zhou2, Elizabeth A Lyle3, Richard I Tapping3, Andrew M Kropinski2and Malcolm B Perry1
1
Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada;2Queen’s University, Kingston, ON, Canada;
3
University of Illinois, Urbana, IL, USA
In an attempt to isolate 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
molecular mass (LGLA) and low molecular mass (LGLB)
fractions Here we present the results of the analysis of the
glycolipid LGLB Deacylation of LGLBwith hydrazine and
separation of the products by using anion-exchange
chro-matography gave two major products Their structure was
determined by using chemical methods, NMR and mass
spectrometry All monosaccharides had theD-configuration,
and aspartic acid had the L-configuration Intact LGLB
contained two fatty groups at O-2 and O-3 of the glycerol residue Nonhydroxylated C14 to C18 fatty acids were identified, which were predominantly unsaturated or bran-ched LGLBwas able to gel Limulus amebocyte lysate, albeit
at a lower level than that observed for Escherichia coli O113 lipopolysaccharide However, even large amounts of LGLB were unable to stimulate any Toll-like receptor (TLR) examined, including TLR4 and TLR2, previously shown
to be sensitive to 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 bacterium is a free-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 implicated in periodontal disease
[5–7] Although classified as Gram-negative, controversy
exists over the existence of lipopolysaccharide (LPS) in the
outer membranes of spirochetes Clear genetic and
bio-chemical evidence exists for the presence of LPS in
Leptospira[8] and for its absence in T pallidum and Borrelia
[9,10] Limited structural analysis suggests that several oral
treponemes (T brennaborense and T maltophilium [6],
T medium [11], and T denticola [12]) possess 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 receptors (TLR) are 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 bacteria [15–17] Although LPS derived from enteric bacteria is a potent agonist for TLR4, other nonenteric bacterial LPS, 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, and T maltophilum appear to have functional similarity to LPS in that they possess some ability to gel Limulusamebocyte 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] The strict correlation between the structure of the LPS molecule with that of TLR specificity remains undefined but it is clear that TLR2 is capable of recognizing a wider range of potential lipid A structures than TLR4 [21]
S aurantiahas simple growth requirements that facilitate studies otherwise limited by the amount of cell mass, a problem often limiting studies on other spirochetes [2] We describe here the structural characterization of the carbo-hydrate skeleton and fatty acids of one of its glycolipids, LGL In addition we present evidence which suggests that
Correspondence to E Vinogradov, Institute for Biological Sciences,
National Research Council, 100 Sussex Dr., Ottawa, ON, Canada
K1A 0R6 Fax: +1 613 952 9092, Tel.: +1 613 990 0832,
E-mail: evguenii.vinogradov@nrc-cnrc.gc.ca
Abbreviations: EU, endotoxin units; FAME, fatty acid methyl esters;
GalNAcA, N-acetylgalactosaminuronic acid; GSL,
glycosphinogo-lipids; Fuc3N, 3-amino-3,6-dideoxygalactose; Kdo,
2-keto-3-deoxy-D -manno-oct-2-ulosonic acid; LAL, 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)
Trang 2while superficially resembling other spirochetal glycolipids,
LGLB is a multisaccharide glycolipid and is unable to
stimulate any TLR examined
Experimental procedures
Bacterial strain and growth conditions
The S aurantia strain, M1, used 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 30C with gentle aeration (30 r.p.m.; orbital
shaker; Forma Scientific, Marietta, OH, 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 from a total of 55 L of SGM and the combined cell
pellet was extracted following the method of Darveau &
Hancock [22] The final product was extracted once with
cold 95% (v/v) ethanol and twice with
chloroform/meth-anol (2 : 1, v/v) to remove phospholipids and carotenoids
The residue was resuspended in distilled water, and
contaminating protein was removed by treatment with
pronase (25 lgÆmL)1) for 18 h at 37C A final extraction
by chloroform/methanol (2 : 1, v/v) was followed by
dialysis against distilled water using Slide-a-lyzer 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 dry
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 mM Tris/HCl, pH 8; 50 mM
EDTA; 10% w/v) SDS] and fractionated on a 5.5· 40 cm
column of Sephacryl S-300 HR (Sigma-Aldrich) at 25C
[column buffer: 10 mM Tris/HCl, pH 8; 10 mM EDTA;
0.2MNaCl; 0.3% (w/v) SDS] Fractions of 2.1 mL were
collected at an average flow rate of 1.5 mLÆmin)1 The
fractions containing the low molecular mass material
(LGLB), as determined by standard SDS/PAGE with silver
stain [23], were pooled, precipitated with cold 0.375M
MgCl2in 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; 10% (w/v) spacer gel; 4.5% (w/v) stacking
gel) was used to examine the low molecular mass portions of
LPS and LGL [24] LPS from Salmonella enterica sv
typhimuriumwild type, Sal enterica sv typhimurium TV 119
(Ra mutant) and Sal enterica sv minnesota R5 (Rc mutant)
were purchased from Sigma-Aldrich Products in
acryl-amide gels were visualized by silver staining [23]
NMR spectroscopy and general methods NMR spectra were recorded at 25C in D2O on a Varian UNITY INOVA 600 instrument using acetone as the external reference (1H, 2.225 p.p.m., 13C, 31.45 p.p.m.) Varian standard programs COSY, NOESY (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 Biosystems/Sciex, Concord, ON, Canada) Briefly, 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)1to a low dead volume tee The separation was obtained on a bare fused-silica capillary, of 90 cm length, using 10 mM ammonium acetate/ammonium hydroxide in deionized water, pH 9.0, containing 5% (v/v) methanol A voltage of 25 kV was typically applied at the injection Mass spectra were acquired with dwell times of 2.0 s per scan in positive ion detection mode Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell, were recorded by a time-of-flight mass analyzer Collision energies were typically 120 eV (laboratory frame of refer-ence)
Hydrolysis Hydrolysis was performed with 4MCF3CO2H (110C, 3 h), monosaccharides were conventionally con-verted into the alditol acetates and analysed by GLC on an Agilent 6850 chromatograph equipped with a DB-17 (30 m· 0.25 mm) fused-silica column using a temperature gradient of 180C (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 the mono-saccharides, product 2 (1 mg) was treated with (S)-2-butanol/AcCl (0.25 mL, 10 : 1, v/v) for 2 h at 85C, dried under the stream of air, acetylated and then analysed by GC
in comparison with authentic standards, prepared from the respective monosaccharides with (S)- and (R)-2-butanol For determination of the configuration of N-acetylgalac-tosaminuronic acid (GalNAcA), a sample (2 mg) of LGLB was treated with 1MHCl in methanol (100C, 4 h), dried, and then the product was peracetylated by Ac2O in pyridine (0.5 + 0.5 mL, at 85C for 30 min) and reduced with excess NaBD4in 96% (v/v) ethanol (1 mL) at 40C Acetic acid (1 mL) was added, the product was dried under a
Trang 3stream of air and then dried twice with the addition of 1 mL
of methanol 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 85C, filtered, dried,
acetylated by Ac2O in pyridine (0.5 + 0.5 mL, at 85C for
30 min), dried and analyzed by GC-MS with the standards
prepared fromD-GalN and (R)- and (S)-2-BuOH
The absolute configuration ofL-aspartic acid was
deter-mined by chiral HPLC of the oligosaccharide hydrolysate
on a Chirex D penicillamine column (250· 4.6 mm;
Phenomenex) in 15% (v/v) methanol containing 2 mM
CuSO4, with UV detection at 254 nm
Fatty acid methyl esters (FAMEs) were generated from
1 mg samples of LGLBby the addition of 1 mL of 3MHCl
in methanol (Alltech Associates, Inc., Deerfield, IL, USA)
and incubation at 100C 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 with
carrier gas (N2)], at a flow rate of 50 mlÆmin)1 The oven was
programmed as follows: 150C for 5 min; followed by 150
to 230 C at 8 CÆmin)1 Data analysis was conducted by
using the PEAKFIT v 4.11 software package (Systat
Software Inc., Richmond, CA, USA) Comparison of
FAME retention times with those of a Bacterial Acid
Methyl Esters CPTMmix (Matreya, Inc., Pleasant Gap, PA,
USA) permitted tentative FAME identifications to be
made The latter were confirmed by GLC-MS analysis
(Analytical Services, Queen’s University, Kingston, ON,
Canada)
LGLBwas O-deacylated by hydrazinolysis, as described
by Gu et al [25] Briefly, 40 mg of LGLBwas incubated
with anhydrous hydrazine for 3 h at 37C, with occasional
mixing The mixture was then chilled to)20 C and an
equal volume of chilled acetone was added dropwise The
product was recovered by centrifugation, washed once with
chilled acetone, dried and weighed
Separation of oligosaccharides 1 and 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/1MNaCl 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 assays 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 of TLRs was measured by quantifying
the production of tumour necrosis factor-a (TNF-a) by
whole blood cells, in response to a panel of TLR agonists, as
described by Tapping et al [26] Briefly, whole blood from
healthy donors was collected into tubes containing heparin
and diluted 1 : 4 in RPMI 1640 Samples were aliquoted
into 96-well plates, agonist was added, and incubation was
carried out at 37C in an atmosphere of 5% carbon dioxide for 6 h Cell supernatants were removed and assayed for cytokine production by standard sandwich ELISA in 96-well Immunlon plates (Dynatech Laboratories, Chant-illy, VA, USA) The TNF-a ELISA was performed by using mAbs 68B6A3 or 68B2B3 for capture and the biotinylated mAb 68B3C5 (Biosource International, Camarillo, CA, USA), followed by streptavidin-conjugated horseradish peroxidase (HRP), for detection ELISAs were developed
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 aurantiaLGLB(5 lgÆmL)1), zymosan (5· 109particles per mL; Molecular Probes, Eugene, OR, USA), heat-killed Staphylococcus aureus(2.5· 106particles 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 lM; Sigma Genosys)
Results
Darveau-Hancock extraction of stationary phase S auran-tiacells gave a white powdery substance in a yield of 15.6% based upon the cell dry wieght This high yield is not unexpected as the surface to volume ratio of this bacterium
is 13.6Ælm)1, approximately 3.5 times higher than that of
E coli or Sal enterica sv typhimurium (3.9Ælm)1) The Darveau-Hancock procedure does not discriminate between high (smooth) or low (rough) molecular mass LPS, 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 carbohydrates and hexosamines [27] A low molecular mass carbohydrate-containing material (LGLB) was isolated, and when examined by Tricine–SDS/PAGE [24], demonstrated mobil-ity similar to the rough LPS of a Sal enterica sv typhimurium
TV 119 Ra mutant (Fig 1) Another material, LGLA, was identified as a larger glycolipid and is thought to contain O-antigen like repeats, contributing to the banding pattern observed in crude S aurantia extract (data not shown) Preliminary colorimetric analysis [28] indicated that LGLBdid 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 five major acyl constituents, none of which were the characteristic hydroxylated fatty acids of LPS (Table 1)
LGLB was O-deacylated by treatment with anhydrous 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
Trang 4compo-sition was similar, comprising glycerol, xylose, mannose,
glucose, galactose, and 3-amino-3,6-dideoxyhexose in a
ratio of 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 result of the reduction of glucurono-lactone The 3-amino-3,6-dideoxyhexose is quantified approximately because of the lack of a quantitative standard compound
NMR spectra of both oligosaccharides were completely assigned by using 2D NMR techniques (Figs 2–4, Table 2) Monosaccharides were identified on the basis of vicinal proton coupling constants and13C NMR chemical shifts Anomeric configurations were deduced from the J1,2 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 and 2: P1G4 (in 1), C1G4 (in 2), and G1A2, G1A1, A1G5, A1L4, A1L3, L1E4, E1F4, 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 of a monosac-charide and H-5 of a glycosylating residue in the event of an a-(1–2)-linkage Respective HMBC correlations 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 the basis of the HMBC correlation between H-2 of aspartic acid and C-6 of the GalA E, thus showing that aspartic acid is amide linked through its amino group to C-6 of galacturonic acid E (Fig 4)
Absolute configuration of the monosaccharides was determined by GC analysis of acetylated 2-butyl glycosides For 3-amino-3,6-dideoxygalactose (Fuc3N), the O-specific polysaccharide from Proteus penneri 16 was used as a source of referenceD-Fuc3N, where itsD-configuration was determined earlier [30] For determining the configuration
of GalNAcA, an LGLB sample was treated with HCl in
1
2
Fig 2. 1H NMR spectra of oligosaccharides
1 and 2.
Fig 1 Visualization of LGL B by Tricine–SDS/PAGE and silver
staining reveals that this material co-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 mutant), 2 lg.
Table 1 Fatty acid methyl ester (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 are the
average nmolÆmg)1 with standard deviations (±) obtained from
quantifying and averaging areas under specific peaks from GLC
analysis of four separate samples of LGL B
13-Methyltetradecanoic acid (iC15:0) 224.1 ± 9.0
15-Methylpentadecanoic acid (iC16:0) 117.6 ± 12.4
Trang 5methanol, and the product was peracetylated in order to
reacetylate free amino groups; it was checked by GC-MS
for the presence of methyl ester of methyl GalNAc The
product was reduced with NaBD4in 96% (v/v) ethanol at
40C, treated with HCl-(R)-2-BuOH, acetylated and
ana-lysed by GC-MS A total ion chromatogram showed no
well pronounced peaks; however, a fragmentogram for the
expected glycosyl cation of m/z 332 contained peaks with
the same retention time as that obtained fromD-GalN with
(R)-2-BuOH; mass spectra of the products obtained from
LGLB were shifted to high mass by two units owing to
a double deuteration at C-6 Thus, GalNA had the
D-configuration
These data, taken together, allow us to propose the
structures of oligosaccharides 1 and 2, as presented in
Scheme 1
The oligosaccharides 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 major doubly charged ion at m/z 1180.25
(observed molecular mass: 2358.50 Da; calculated exact
molecular mass for C85H130O72N4: 2358.6633 Da) The
MS for oligosaccharide 2 showed a molecular mass of
2375.56 Da (calculated exact mass for C85H129O74N3:
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 components with 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 using the isotope profile, owing to the high resolution provided by the TOF mass analyser The product-ion spectrum (MS/MS spectrum), obtained from
a doubly charged ion at 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 As indicated in Fig 6, the fragment ion at m/z 1876.56 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 of the branching xylose residues M and O, and of GlcA residue L The remaining linear sequence, consisting
of K-B-F-I-D-N-Q, was confirmed by the 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
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) and NOESY (right) spectra of oligosaccharide 2 Intraresidual correlations are labeled with a letter designation of the monosaccharide residue and numbers of the correlating protons Inter-residual correlations are labeled with letters for both monosaccharides.
Trang 6fragment ions at m/z 292.06, 468.09, 613.16, 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) or from the
loss of F from F-I-D-N (m/z 732.18) However, many other
combinations of fragments are also possible, because of the
existence of branches in the molecule Similarly, the tandem
MS was conducted for the doubly charged ion at m/z
1180.25 (data not shown) and the mass spectral data fully
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 60C in the presence
of 5% fully deuterated SDS All monosaccharides present
in the products 1 and 2 were identified, and the ratio of
structures 1 and 2 was close to 1 : 1 All chemical shifts
remained mostly unchanged with the exception of H/C-2
and H/C-3 of the glycerol residue Proton signals were
strongly downfield shifted owing to acylation (Table 2);
13C 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 LGLBby 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
LGLBdoes 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] LGLB displayed a 100-fold less endotoxic potential, registering 2.5· 105EUÆmg)1when compared to an E coli O113 LPS control (1· 107 EUÆmg)1) in a LAL gel clot assay
LGLBwas also examined for its ability to act as a TLR agonist Attempts to measure a reaction from cells trans-fected specifically with human TLR2 or TLR4 were unsuccessful, regardless of the concentration of LGLB examined (data not shown) The whole blood assay uses fresh human blood (which contains a variety of Toll receptors) and measures the total release of TNF-a by ELISA [26] Cells were stimulated with defined TLR agonists (zymosan and heat-killed Staph aureus for TLR2; PolyIC for TLR3; E coli Re595 LPS for TLR4; R848 for TLR7; and CpG Oligo (2 lM) for TLR9), and the production of TNF-a was quantified (Fig 7) Even when
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 oligosaccharides 1 and 2 Oligosaccharide 1, R ¼ a-Fuc3N (P); oligosaccharide 2, R ¼ a-Glc (C).
Trang 7high concentrations of LGLBwere added, no production of
TNF-a was detected, showing that this large glycolipid
cannot stimulate TLR2, -3, -4, -7 or -9
Discussion
Although some structural information has been obtained
from other spirochetes, the complete elucidation of the
LGLB from S aurantia represents the first complete
structure of a large glycolipid from these bacteria The
dodecasaccharide LGLBis anchored by a diacyl glycerol
A glycolipid containing a single sugar, BbGL-II, and also
anchored on a glycerol, has been identified in B
burg-dorferi[13] It is surface 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, and T 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 of the treponemal glycolipids identified have either fully saturated or 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.
13
13
13
13
13
13
13
13
2.93
Q, Gro, LGL B
1
Trang 81000 1100 1200 1300 1400
m/z
1197.25
1189.28 1180.30 1116.27
1131.28
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
714.18 292.06 824.24
663.67807.22
1023.26 376.09 1182.241344.371436.42 1744.53
1876.56
K
B
F
I
1612.48
D N Q L O M C -G - A, NH3
Fig 6 MS/MS spectrum obtained from a doubly charged ion at m/z 1197.25 of oligosaccharide 2.
Trang 9unsaturated acyl group of BbGL-II Schultz et al.
indicated that the presence of fatty acid branching in
T denticolais analogous to adaptations in Gram-positive
bacteria to alter membrane fluidity [12] Gram-negative
bacteria are known to modify the degree of saturation in
their fatty acids to modulate membrane fluidity [34,35]
LGLB contained both unsaturated and branched fatty
acids (i.e C14:0, iC15:0, C16:1), the only spirochete
glycolipid identified, to date, with both of these
modi-fications, suggesting LGLB may form highly fluid
mem-branes
S aurantiaLGLBcomprises 15% lipid by mass,
corres-ponding well with the proportion of fatty acids in the
glycolipid OML521 (10.7%) from T denticola, a glycolipid
that is also estimated to be similar in size to Ra LPS [12] Ra
LPS is the minimum LPS 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 in these bacteria, OML251 and LGLBmay 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 contains a low molecular mass glycolipid
with glycerol anchor and many charged groups in the
oligosaccharide part, which makes it, in overall design,
similar to S aurantia LGLB Interestingly 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 Novosphingobium 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 receptors [12,20], suggesting that at a functional level they possess some similarity LGLB 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 or -4 [13] TNF-a release was measured following the exposure of human mononuclear cells to two different GSLs from S paucimobilis: the mono-glycosylated GSL-1, and the tetramono-glycosylated 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 similar to the situation with the monoglycosylated BbGL-II, the inability of LGLB 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 LGLBwould block a TLR response is unknown Structural studies of Tm-Gp have focused on a tetrasaccharide repeating unit, likened by Asai and colleagues to the repeating unit of the LPS O-antigen [11] The characteristic laddering pattern on SDS/ PAGE suggests that Tm-Gp is different from LGLB, although they both contain an aspartic acid residue The structures of the bioactive portion of Tm-Gp, and of the other treponemal glycolipid TLR agonists, need to be elucidated to begin to identify possible motifs involved in modulating TLR activity This is especially interesting when 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 Zn2+ -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 of the LPS to the target membrane LBP is an important precursor
in the TLR-dependent release of TNF-a and has 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 LGLBcould abrogate the interaction with LBP and prevent any release of TNF-a in the whole blood assay for TLR activation Specific struc-tural entities 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 of 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 targetdevelop-ing this organ-ism [13,47]
0
5
10
15
20
25
30
35
40
No Zymosan HKSA PolyIC Re LPS R848 CpG LGLB
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 heat-killed
Staphylococcus aureus (HKSA) for TLR2; PolyIC for TLR3;
Escherichia coli Re595 lipopolysaccharide (LPS) for TLR4; R848 for
TLR7; and CpG Oligo for TLR9 Error bars represent the standard
deviation of cellular activation experiments performed in triplicate.
Trang 10This work was supported by a Natural Sciences and Engineering
Research Council of Canada (NSERC) Discovery Grant to A.M.K.
C.J.P was the recipient of an NSERC studentship.
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