Báo cáo khoa học: Detailed structure of lipid A isolated from lipopolysaccharide from the marine proteobacterium Marinomonas vaga ATCC 27119T pot

Detailed structure of lipid A isolated from lipopolysaccharide
from the marine proteobacterium
Marinomonas vaga
ATCC 27119
T
Inna N. Krasikova
1
, Natalie V. Kapustina
1
, Vladimir V. Isakov
1
, Andrey S. Dmitrenok
2
, Pavel S. Dmitrenok
1
,
Natalie M. Gorshkova
1
and Tamara F. Solov’eva
1
1
Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Vladivostok, Russia;
2
Suntory Institute for Bioorganic Research, Osaka, Japan
The c hemical s tructure of a novel lipid A, the major com-
ponent of the lipopolysaccharide from the marine gamma-
proteobacterium Marinomonas vaga ATCC 27119
T
,was
determined by compositional analysis, NMR spectroscopy,
and MS. It was found to be b-1,6-glucosaminobiose

1-phosphate acylated with (R)-3-[dodecanoyl(dodece-
noyl)oxy]decanoic acid {C10 : 0 ( 3O-C12 : 0 [3O-C12 : 1]) }
or (R)-3-(decanoyloxy)decanoic acid [C10 : 0 (3O-C10 : 0)],
(R)-3-hydroxydecanoic acid [C10 : 0 (3OH)], and (R)-3-
[(R)-3-hydroxydecanoyloxy]decanoic acid (C10 : 0 {3O-
[C10 : 0 (3OH)]}) at the 2, 3, and 2¢ positions, respectively. It
showed low lethal toxicity, which is probably related to
specific structural attributes. T he absence of a fatty a cid at
the 3 ¢ position and a phosphoryl group at the 4¢ position and
also the p resence of an amide-linked (R)-3-hydroxyalkanoic
acid th at is further O-acylated w ith a n other ( R)-3-hydroxy-
alkanoic acid, distinguish M. vaga lipid A from other such
molecules.
Keywords: fast-atom bombardment MS (FAB-M S); lipid A;
marine proteobacteria; Marinomonas vaga;NMR.
Gram-negative bacteria, along with classical membrane
lipids based on glycerol, contain an unusual glycophos-
pholipid known a s lipid A. Functioning as a lipid anchor for
lipopolysaccharide (LPS), it is one of the m ain c omponents
of the outer membrane of bacteria [1], making it important
for maintenance of the normal physiology and growth of
micro-organisms [2]. LPSs (O-antigens and endotoxins
of Gram-negative b acteria) constitute a specific class of
biopolymers that h ave a wide spectrum of biological
(endotoxic) activity i n m ammals [3] i ncluding pathophysio-
logical effects such as endotoxemia and septic shock [4].
There i s compelling e vidence that the endotoxic activity of
LPS is expressed by a lipid fragment of its molecule [3–5].
Therefore, an intensive search for potential endotoxin
antagonists on the basis of lipid A is now being carried

out [6].
A structure common to a number of lipid A molecules
is b-1,6-
D
-glucosaminyl-
D
-glucosamine, which has a-glyco-
sidic (1 position) and nonglycosidic (4¢ position) phosphate
groups and i s acylated w ith a mide linked ( at positions 2
and 2 ¢) and ester linked (at positions 3 and 3¢)(R)- 3-
hydroxy and (R)-3-acyloxy fatty acids [7]. On the other
hand, according t o data available so far, lipid A structural
variants displaying high endotoxin antagonism have a
disaccharide or monosaccharide backbone, mainly one
phosphate group, and low degree of acylation [8–10].
Lipid A acylation patterns, which are important in
binding bacteria to, and activation of, h ost cells [3,5] a re
known to d epend strongly on the growth conditions [11–
13]. We hypothesize that mar ine bacteria, w hich inhabit a
specific environment (low t emperature, high h ydrostatic
pressure, and high salinity [14]), may produce lipid A
molecules o f unusual structure and, possibly, of pharma-
cological i nterest.
However, very little i s known abo ut the structure and
function of lipid A from marine proteobacteria. Although
some have been examined [15,16], only their fatty acids
were identified. More recent studies have revealed some
peculiarities of lipid A molecules from marine bacteria,
the most pronounced being a penta-acyl-type structure
[17,18].

We carried out extensive structural analysis of lipid A
from the Marinomonas vaga AT CC 27119
T
LPS. M. vaga
(formerly known as Alteromonas vaga) was first isolated
from sea water off the coast of the Hawaii an archipelago
in 1972 [ 19]. I n 1983, toget her wit h Alt eromonas com mu-
nis, i t formed a separate genus named Marinomonas [20].
M. vaga belongs t o the gamma subclass of Proteobacte-
ria. It is an aerobic, rod-shaped, polarly flagellated
bacterium with psychrophilic and m oderately halophilic
properties. For growth, it requires Na
+
,Mg
2+
,and
Ca
2+
in concentration s found in sea w ater [21]. Lipid A
from this b acterium aroused our interest because it has a
penta-acyl and monophosphoryl type of structure, con-
tains s hort-chain (R)-3-hydroxydecanoic acid as t he main
acyl residue [17], and is theoretically an endotoxin
antagonist.
Correspondence to I. Krasikova, Pacific Institute of Bioorganic
Chemistry, Far Eastern Branch of the Russian Academy of Sciences,
159, 690022, Vladivost o k-22, Russia. Fax: + 7 4232 31 40 50,
E-mail:
Abbreviations: CAD, collision-activated dissociation; FAB-MS, fast-
atom bombardment mass spectrometry; GlcN, glucosamine; HSQC,

heteronuclear single quantum correlation; LPS , lipopolysaccharide.
(Received 2 5 December 2003 , revised 29 April 2 004,
accepted 11 May 2004)
Eur. J. Biochem. 271, 2895–2904 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04212.x
Materials and methods
Growth of bacteria and isolation of LPS
M. vaga ATCC 27119
T
cells were cultured at 8–10 °Con
rotary shakers in 1 -L conical fl asks filled with 500 mL liquid
nutrient m edium c ontaining (g per liter of 50% sea water,
pH 7.5–7.8): b actopeptone ( Difco) (5); casein hydrolysate
(Merck) ( 2); y east extract ( Merck) (2) ; glucose (1); KH
2
PO
4
(0.2); MgSO
4
(0.05). At the stationary phase of growth
(5 days), the bacteria were harvested by centrifugation
(300 g) and washed consecutively with distilled water,
acetone, ethanol, hexane, and chloroform/methanol (2 : 1,
v/v, twice) to yield d efatted cells (0.6 8 gÆL
)1
). LPS [25 mgÆ
(g dry cells)
)1
] was obtained by extraction w ith h ot ph enol/
water according to the conventional procedure [22] and
purified from nucleic acids by precipitation with 40% ( v/v)

trichloroacetic acid [23].
Isolation of lipid A
To obtain lipid A , LPS ( 300 mg) was hydrolysed in 1%
(v/v) aqueous acetic acid for 3 h a t 100 °C. Chloroform
solution of the sediment obtained b y centrifugation o f t he
LPS hydrolysate was w ashed with distilled water (three
times), dried with anhydrous Na
2
SO
4
, and precipitated with
acetone t o yield crude lipid A (0.25 mgÆmg LPS
)1
). Lipid A
was further purified by Sephadex LH20 and silica gel
column chromatography.
Analytical methods
To determine sugar composition, lip id A was converted into
free monosaccharides by hydrolysis in 6
M
HCl for 24 h at
100 °C. Hexosamines were qualitatively and quantitatively
estimated u sing an LKB Biochrom 4251 A lpha Plus system
(Cambridge, UK) amino-acid analyser. The absolute con-
figuration of glucosamine (GlcN) was determined by GLC
of its acetylated 2-octyl glycoside according to the method
of Leontein et al . [24] with some modifications.
Total phosphorus generated after lipid A charring with
HClO
4

was determined by the ammonium molybdate
method as described previously [25].
Free total fatty acids were obtained by alkaline hydrolysis
of lipid A (6
M
NaOH, 4 h, 100 °C). The ester-bound fatty
acids w ere released b y mild alkali treatment o f lipid A (1 mg)
with 12% aqueous NH
4
OH (200 lL) for 1 8 h at 20 °Cas
described [26]. The released fatty acids were converted into
methyl esters with ether s olution of diazome thane and
identified by GLC and GLC/MS. Before hydrolysis, the
calculated amount of pentadecanoic acid was added to the
lipid A as internal standard. The position of the double
linkage in the unsaturated acid was d etermined b y GLC/MS
of its p yrrolidide d erivative [27]: difference s o f 12 a tomic
mass units between homologous ions at m/z 140 (C4) and
m/z 152 (C5) indicated a double bond at C5.
Determination of the (
R, S
) configuration
of 3-hydroxy fatty acid
To isolate 3-hydroxyalkanoic acid, total fatty a cids (22 mg)
obtained by alkaline hydrolysis of the defatted M. vaga
ATCC 27119
T
cells were fractionated on the silica gel
column with the following solvent s ystems: hexane; hexane/
ether, 1 : 1; hexane/ether/acetic acid, 1 : 1 : 0.1 (by vol.).

Using the PerkinElmer 141 polarimeter, the specific rotation
of the fraction corresponding to 3-hydroxy fatty acid was
determined to be [a]
D
¼ )14.1 ° (0.5, CHCl
3
).
Chromatography
Gel-permeation chromatography was performed on a c ol-
umn (560 · 15 mm) of Sephadex LH20 in c hloroform/
methanol (3 : 1, v/v). Silica-gel column chromatography was
performed with chloroform/methanol in various ratios. TLC
was carried out on ready-made aluminium-backed Sorbfil
(Sorbpolymer, Krasnodar, Russia) plates using the following
systems: chloroform/methanol/water (10 : 7.5 : 1.5, by vol.)
or chloroform/methanol/water/conc. ammonia (10 : 5.0 :
0.8 : 0.4, by vol.). Bands were visualized by heating the plates
for 1 0 m in at 130 °C a fter spraying with 10% ( v/v) H
2
SO
4
in
methanol. N inhydrin reagent was used to detect free amino
groups in lipid A. GLC analyses of f atty acids (methyl esters)
were performed w ith a Shimadzu G C9A c hromatograph
(fused-silica gel Supelcowax 10 and SPB-5 columns,
30 m · 0.25 mm) at a temperature of 200 and 210 °C,
respectively. For determination of monosaccharide, absolute
configuration a nd analysis of fatty acid pirrolidides, an
Agilent 6850 series GC system chromatograph with a

HP 1 MS 5% phenylmethylsiloxane capillary column
(30 m · 250 lm · 0.25 lm)wasusedoverthetemperature
gradient 160 –250 °C(5°CÆmin
)1
).
MS
GLC/MS analyses were carried out on a Hewlett–Packard
model 6890 gas chromatograph equipped with HP 5 MS
5% phenylm ethylsiloxane capillary column (30 m ·
250 lm · 0.25 lm) and conn ected to a Hewlett–Packard
model 5973 mass spectrometer. Samples were injected in the
split mode with a split ratio of 1 : 15 at the injector
temperature 250 °C. The oven temperature was pro-
grammed t o increase from 150 °Cto210°Catarateof
5 °CÆmin
)1
. Helium was used as carrier gas.
Fast-atom bombardment (FAB)-MS spectr a were
obtained using a high resolution mass spectrometer
(AMD 604S) with Cs
+
energy of 8 kV. Lipid A was
dissolved in chlo roform/methanol (2 : 1, v/v) a t a concen-
tration of 10 mgÆmL
)1
,and0.5lL sample solution was
mixed with 0.5 lL matrix solution of glycerol. The 0.5 lL
aliquot of this mixture was d eposited on a metallic sample
holder and analysed immediately after being dried in a
stream of air.

NMR spectroscopy
The
1
H,
13
C, and
31
P NMR spectra of lipid A were recorded
at 317 K in CDCl
3
/CD
3
OD (4 : 1, v/v) or at 298 K in
CD
3
OD/CDCl
3
/D
2
O (3 : 2 : 1, by vol.) mixtures at 500,
125, and 202.5 MHz, respectively, using a Bruker DRX-500
spectrometer equipped w ith a reverse probe.
13
Cand
1
H
chemical shifts were expressed in d relative to trimethylsilane
(d
H
0.00, d

C
0.00).
31
P chemical s hifts were measured relative
to 85% o rthophosphoric acid (d
P
0.00). 2D spectra [DQ F
2896 I. N. Krasikova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
COSY, TOCSY, and heteronuclear single quantum corre-
lation (HSQC)] were measured using a standard Bruker
program.
Samples of lipid A for NMR analysis were p repared as
described [ 28]. Lipid A was dissolved i n ultrapure d eionized
water, with 0.36
M
triethylamine added in the cold. Insol-
uble remnant was separated by centrifugation (12 000 g,
1 m in) in an Eppen dorf centrifuge and discarded. Water
supernatant was a cidified with 1
M
HCl to p H  1. The
sediment of lipid A obtained was separated b y centrifuga-
tion and dissolved in chloroform. To make the solution
transparent, some drops of methanol were added. The
solution obtained was washed with deionized water three
times and evaporated. Lipid A (4 mg for
13
CNMRor
 0.5 mg for
1

H NMR) was dissolved in 0.6 mL of the
corresponding solvent mixtures.
Toxicity
The toxicity o f M. vaga lipid A w as teste d in outb reed ( wild-
type)
D
-galactosamine-sensitized mice (16–18 g) by the
method of Galanos et al.[29].
D
-Galactosamine hydrochlo-
ride (16 mg per animal) and different a mounts of lipid A
(0.004–4 lg) were injected intraperitoneally as mixtures in
0.4 m L phosphate buffered saline into groups of four
animals. A control group of four mice was injected with
saline only. Deaths were monitored for 48 h. Toxicity
was defined as LD
50
calculated by the method of Nowotny
[30]. E xperime nts were performed in a ccordance with the
Pacific Institute of B ioorganic Chemistry Policy o n Human
Care and Use of Laboratory Animals.
Results
Isolation and characterization of lipid A from
M. vaga
Extraction of defatted M. vaga ATCC 27119
T
cells with
phenol/water [22] yi elded LPS in the water phase. Accord-
ing to the TLC data, the crude lipid A i solated from LPS by
hydrolysis with 1% aqueous acetic acid with high yield

(25% of dry LPS weight) is quite homogeneous with the
prevalence of one of the components (Fig. 1, lane 1).
After gel filtration on a Sephadex LH20 column
followed by silica-gel column chromatography, purified
lipid A (Fig. 1, lane 2), which consisted of GlcN,
phosphorus and fatty acids in the amounts shown in
Table 1 , was obtained. The absolute configu ration of
GlcN was shown to be
D
. No free amino groups were
detected with ninhydrin, indicating that no phosphoryl-
ethanolamine, aminoarabinose, or other unsubstituted
amino compounds were present in the M. vaga lipid A.
On a molar basis, there are one phosphate gro up a nd five
fatty acid r esidues p er two r esidues o f G lcN.
Analysis of
M. vaga
lipid A backbone
A combination of 1D and 2D N MR experiments was used
to determine the structure of the M. vaga lipid A b ackbone
(Table 2). In the
1
HNMR s pectrum, the resolved doublet-
doublet at 5.42 p.p.m. (J
H1, H2
¼ 3.6 Hz) and doublet
signal at 4.71 p.p.m. (J
H1¢,H2¢
¼ 7.8 Hz) were assigned to
the proton at the glycosidic position and to the anomeric

proton H1¢ of the nonreducing glucosamine residue,
respectively. In the
1
H,
13
C HSQC s pectrum, these proton
signals c orrelated with t wo
13
C a nomeric signals at 95.35
and 1 01.26 p .p.m. Such carbon and proton shifts together
with the J
H1,H2
verify the a-pyranoside a nd b-pyr anoside
forms for the proximal and distal glucosamine r esidues in
lipid A of M. vaga.
The coupling of resonance at 5.42 p .p.m. (J
H1, P
¼
6.2 Hz) and a shift of the C1 signal to lower field were
observed, indicating that position 1 of GlcN I was esterified
by the phosphate group. In addition, the carbon signal at
95.35 p .p.m. w as a doublet (J
C1, P
¼ 6.7 Hz), also pointing
to the presence of phosphate at C1. In accord with this, a
signal was observed at  )2 p.p.m. in the
31
P-NMR
spectrum of M. vaga lipid A (Table 2).
The resonance fi eld of C6 atoms of glucosaminobiose was

represented by only one signal (61.86 p.p.m). The resonance
of the second C6 (68.60 p.p.m) determined by the HSQC
Fig. 1. TLC of M. vaga ATCC 27119
T
lipid A b efore (1) an d after ( 2)
fractionation.
Table 1. C hemical c omposition of M. vaga ATCC 27119
T
lipid A. All
the d ata are the mean of three or more independent assays and the
range o f experimental e rror was less than 5%.
Constituent
Amount of constituent
lmolÆmg
)1
lmolÆ(2 lmol GlcN)
)1
Glucosamine 1.208 2.00
Total phosphate 0.687 1.14
C10 : 0 0.206 0.34
C12 : 0 0.228 0.38
C12 : 1 0.291 0.48
C10 : 0 (3OH) 2.409 3.99
Ó FEBS 2004 Marinomonas vaga lipid A structure (Eur. J. Biochem. 271) 2897
and DEPT-135 experiments was shown to be shifted to a
lower field ( )8 p.p.m), demonstrating b-1,6-linkage in
backbone of M. vaga lipid A.
From the anomeric protons at 5.42 and 4.71 p.p.m., the
signals of the H2-H6 protons of the reducing and
nonreducing glucosamine residues were assigned using 2D

COSY and TOCSY spectra (Table 2 ). The signals of H2
and H2¢ appeared at 4.14 and 3.62 p .p.m., respectively,
confirming N-acyl substitutions [31]. Accordingly, two C2
signalsthatcorrelatedwiththeH2andH2¢ protons were
present i n the
1
H,
13
C H SQC spectrum. In line with the well
established principle that O-acylation at C3 shifts the
Table 2.
1
H,
13
C, and
31
P-NMR data for M. vaga ATCC 27119
T
lipid A . Spe ctra were recorded at 317 K in CDCl
3
/CD
3
OD (4 : 1 , v/v) (
13
C,
31
P)
and at 298 K i n CD
3
OD/CDCl

3
/D
2
O(3:2:1,byvol.)(
1
H). Chemical shifts are expressed in d relative to trimethylsilane (d
H
0.00, d
C
0.00) or 8 5%
orthophosphoric acid ( d
P
0.00). Comparisons with spectra of Escherichi a coli lipid A [31] and b-1,6-linked di-N-acetate of glucosamine disaccharide
[32] and a combination of 1D a nd 2D NMR experiments were used to assign the M. vaga ATCC 271 19
T
lipid A NMR signals. d, Doublet; dd,
doublet-doublet;m,multiplet;t,triplet.
Position
E. coli lipid A M. vaga lipid A
dH dC dP dC dH (J, Hz) dC dP
GlcN I
1 5.455 94.6 )0.191 5.42 (dd, 3.6, 6.2) 95.35 d )2.0
2 4.147 52.7 4.14 51.90 d
3 5.213 74.7 5.22 (dd, 9.5, 10.5) 74.62 d
4 3.679 68.0 3.61 (t, 9.4) 68.85 d
5  4.03 72.2 4.09 m 73.47 d
6a 4.103 68.6 4.06 m 68.60 t
6b 3.847 3.89 m
GlcN II
1¢ 4.163 103.0 )1.937 102.20 4.71 (d 7.8) 101.26 d

2¢  3.83 54.6 56.23 3.62 m 56.61 d
3¢ 5.18 74.2 74.49 3.56 m 74.84 d
4¢ 4.166 71.8 70.70 3.38 m 71.18 d
5¢ 3.460 76.3 76.44 3.36 m 76.49 d
6¢a  3.94 60.8 61.36 3.89 m 61.86 t
6¢b 3.774 3.73 m
Fatty acids (signals of C¼O atoms were at 171.30; 172.70; 172.90; 173.00; 173.80; 174.00)
NHCO-R (2)
2 2.51 m 41.64
3 5.16 t 71.38
4 1.56 34.48
5 1.29 26.10
NHCO-R (2¢)
2 2.65 m 41.14
3 5.29 t 72.24
4 1.69 34.48
5 1.29 27.90
OH (3, 2¢¢)
2 2.52 42.70, 42.53
3 4.04 68.85, 68.79
4 1.50 37.54, 37.46
Normal
2 2.28 t 34.23 t
3 1.62 m 25.62
4 1.30 m 29.78
(CH
2
)
n
1.30 m 29.78–29.94

x 0.89 t 14.08
x-1 1.3 m 22.73
x-2 1.27 m 32.03
CH¼CH 5.35 m 131.22, 128.44
CH
2
-CH¼CH 2.05 m 26.67, 27.35
2898 I. N. Krasikova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
resonance of C2 in N-acylated glucosamine [33], the C2
signal (51.90 p.p.m) of G lcN I, found in a higher fi eld in
comparison with the C2 signal (53.9 p .p.m) in a-met hyl-O-
{2-deoxy-2-[(R,S)-3-hydroxytetradecanoylamino]-b-D-
glucopyranosyl}-(1 fi 6)-2-(2-deoxy-2-[(R,S)-3-hydroxy-
tetradecanoylamino]-
D
-glucopyranoside 6-diphenylphos-
phate [34], indicates that the reducing end of the M. vaga
lipid A backbone has a substituent at t he C3 atom. The
hydroxy group at C3 ¢ of GlcN II is free, as evidenced by the
nonreducing end C2¢ chemical-shift value (56.61 p.p.m)
which c oincides with the nonreducing end C2¢ chemical-shift
value ( 56.6 p.p.m) in the above a-methyl b-1,6-diglucosa-
mine 6-diphenylphosphate and b-1,6-linked di- N-acetate of
glucosamine d isaccharide, which h ave no substituent a t C3¢
[32,34]. The low-field position of the H3 resonance
(5.22 p .p.m.) in the M. vaga lipid A
1
Hspectrumclearly
indicates acylation of the hydroxy group at the C3 position,
and t he chemical-shift value ( 3.56 p.p.m.) of H3¢ demon-

strates that the hydroxy g roup at the C3¢ position is f ree.
The methine proton signals of H4 and H4¢,and
methylene proton s ignals of H6¢, which correlated with
C4, C4¢,andC6¢ resonances at 68.85, 71.1 8 and
61.86 p .p.m., respectively, were found between 3 and
4 p .p.m., suggesting the a bsence of acyl or phosphoryl
residues at the hydroxy groups of these positions.
Together, these data demonstrate that the M. vaga lipid
A backbone is composed of two b-1,6-linked
D
-GlcN
residues and a phosphate group at p osition 1. Both amino
groups an d t he hydroxy group at C3 are substituted, and the
hydroxy groups at C3¢,C4,C4¢,andC6¢ are f ree.
Characterization of the fatty acid moiety of
M. vaga
lipid A
Fatty acid analysis of the lipid A studie d reve aled the
presence of decanoic acid (C10 : 0 ), dodecanoic a cid
(C12 : 0), dodecenoic a cid (D
5
-C12 : 1), a nd four (R)-3-
hydroxydecanoic acids [C10 : 0 (3OH)] (Table 1). In
accord with this, the resonance field of C2 atoms of
3-hydroxyalkanoic acids (41–44 p.p.m. [35]) was represen-
ted by four signals (Table 2). On the other hand, only two
signals (at 37.54 a nd 37.46 p.p.m.) were present in the C4
resonance field of C10 : 0 (3OH). Resonation of C4 atoms
for two other C10 : 0 (3OH) molecules was shifted to a
lower field (34.48 p.p.m., signal of double intensity)

because of t he substitution of their hydroxy groups with
other f atty acid residues. Two t ypes of 3-acyloxyalkanoic
acids, ester a nd amide linked, may be p resent in lipid A
[7,36]. For ester-linked ones, there must be a characteristic
C2 signal at 38–39 p .p.m. [35]. In the
13
C-NMR s pectrum
of M. vaga lipid A, such a signal was absen t, s uggesting
that both 3-acyloxydecanoic acids were amide-bound. It
should be also noted that trans-D
2
-unsaturated acids,
characteristic for many enterobacterial lipid A hydro-
lysates [37] and pointing to the presence of acyloxy esters,
occurred only in trace amounts in the M. vaga lipid A.
This is circumstantial evidence that the lipid A studied
does not contain ester linked 3-acyloxyacyl residues. On
the other hand, the lack of
13
C resonance at  44 p.p.m.,
characteristic of C2 of amide- linked 3-hydroxy ac ids
[34,35], confirms the a bsence of nonsubstituted N-linked
3-hydroxy a cids in this molecule.
As GLC and GLC/MS data show, the M. vaga lipid A
contains unsaturated D
5
-C12 : 1 acid (Table 1). In
accordance with this, two carbon signals, at 131.22 and
128.44 p.p.m., and two multiplets, at 5.35 and 2.03 p .p.m.,
were present i n t he

13
Cand
1
H NMR spectra (Table 2),
confirming the p resence of fatty acids with a double bond.
The multiplets gave a coupling cross-peak in the
1
H,
1
H
COSY spectrum a nd were a ssigned to vinylic and allylic
protons.
The g eometry of the double bond was established by
analysis of chemical shifts of neighboring (in relation to
double bond) carbons. It i s known t hat carbons adjacent to
trans double bonds have chemical shifts in the range of
d 29.5–38.0 p.p.m., w hereas those adjacent to cis double
bonds have d va lues of 26.0–28.5 p.p.m. [ 38]. From
1
H,
13
C
HSQC and proton decoupled
13
C spectra, the NMR signals
of related carbon atoms of M. vaga lipid A were found at
26.67 and 27.35 p.p.m., indicating a cis configuration for the
double bond in unsaturated fatty acid.
Acyl distribution on the backbone of
M. vaga

lipid A
The d istribution p attern o f acyl residues in M. vaga lipid A
was d etermined using negative a nd positive mode FAB-MS
and collision-activated dissociation (CAD) experiments.
The negative-ion FAB-MS spectrum ( Fig. 2A) revealed two
peaks of equal intensity at m/z 1281.39 and 1253.67 as the
highest mass ions, demonstrating the presence of two
molecular forms of the lipid A studied.
Fig. 2. (A) N e gative-ion and (B) positive-ion FAB mass spectra of
M. vaga ATCC 27119
T
lipid A demonstrating the acyl distribution
between the two glucosamine units. See Table 3 for peak identification.
Ó FEBS 2004 Marinomonas vaga lipid A structure (Eur. J. Biochem. 271) 2899
On the basis of the overall chemical compositio n
(Table 1), the peaks were attributable to two types of
molecular-ion species [M-H]
–
of penta-acylated lipid A
containing two g lucosamines, one phosphate group, four
3-hydroxydecanoic a cids, and one dodecanoic or decanoic
acid (Table 3). Using the monoisotopic mass for each atom,
molecular masses of 1282.8044 and of 1254.77 Da were
calculated for the formulae C
64
H
119
O
21
N

2
Pand
C
62
H
115
O
21
N
2
P, which are well c o-ordinated to signals at
m/z 1281.39 and 1253.67. Unfortunately, the intensity of the
peak at m/z 1281.39 and resolution of the mass spectrometer
were too low to detect the signal of the molecular-ion species
with C12 : 1 a cid. However, it should be noted that, d espite
the penta-acyl structure of M. vaga lipid A (Table 1,
Fig. 2A), six signals due to ester carbonyl and amide
carbonyl carbons were observed in i ts
13
C-NMR spectrum
(Table 2). We reason that this is c aused by the presence of
the third (with unsaturated D
5
-C12 : 1 acid) molecular
species in the p reparation studied. The low intensity of the
signals at 174.00 and 173.80 p.p.m. in contrast with the four
others demonstrates that they belong to saturated ( C10 : 0
and C12 : 0 with total a mount of 0.72 lmol per 2 m ol
GlcN, Table 1) and unsa turated (D
5

-C12 : 1, 0 .48 lmol per
2 m ol GlcN) fatty acids.
Along with the initial penta-acylated lipid A, de-O-
acylated, tetra-acyl (at m/z 1099.83), triacyl (at m/z 929.5)
and diacyl (at m/z 75 8.05) derivatives were detected in the
spectrum. According to the TLC data presented in Fig. 1
(lane 2), the sample of lipid A used for MS did not contain
de-O-acylated species (penta-, tetra-, tri-, and di-acylated
derivatives of lipid A have different chromatographic
mobility). So, they are not intact molecular species of
Table 3. Interpretation of signals (m/z) in FAB-MS spectra for M. vaga ATCC 27119
T
lipid A. Spectra were recorded in chlorof orm/methan ol
(2 : 1 , v/v).
FAB/MS in the negative mode
Disaccharide derivatives
1281.39 (I) [M
I
(2 GlcN +4 3OH (C10 : 0) +1 C12 : 0 + H
3
PO
4
)–H]
–
1253.67 (II) [M
II
(2 GlcN +4 3OH (C10 : 0) +1 C10 : 0 + H
3
PO
4

)–H]
–
1099.83 [M
I
–H]
–
– C12 : 0 (C12 : 1)
[M
II
–H]
–
– C10 : 0
929.5 [M
I
–H]
–
– C12 : 0 – 3OH (C10 : 0)
[M
II
–H]
–
– C10 : 0 – 3OH (C10 : 0)
758.05 [M
I
–H]
–
– C12 : 0 – 2 3OH (C10 : 0)
[M
II
–H]

–
– C10 : 0 – 2 3OH (C10 : 0)
Monosaccharide derivatives
780.05 [M
I
–H]
–
– GlcN ) 2 · 3OH (C10 : 0)
752.5 [M
II
–H]
–
– GlcN ) 2 · 3OH (C10 : 0)
598.4 [M
I
–H]
–
– GlcN ) 2 · 3OH (C10 : 0) – C12 : 0
[M
II
–H]
–
– GlcN ) 2 · 3OH (C10 : 0) – C10 : 0
580.7 [M
I
–H]
–
– GlcN ) 2 · 3OH (C10 : 0) – C12 : 0 – H
2
O

[M
II
–H]
–
– GlcN ) 2 · 3OH (C10 : 0) – C10 : 0 – H
2
O
428.31 [M
I
–H]
–
– GlcN ) 3 · 3OH (C10 : 0) – C12 : 0
[M
II
–H]
–
– GlcN ) 3 · 3OH (C10 : 0) – C10 : 0
410.92 [M
I
–H]
–
– GlcN ) 3 · 3OH (C10 : 0) – C12 : 0 – H
2
O
[M
II
–H]
–
– GlcN ) 3 · 3OH (C10 : 0) – C10 : 0 – H
2

O
CAD on m/z 929.5
758.05 929.5 – 3OH (C10 : 0)
741.02 929.5 – 3OH (C10 : 0) – H
2
O
CAD on m/z 598.4
428.31 598.4 – 3OH (C10 : 0)
410.92 598.4 – 3OH (C10 : 0) – H
2
O
FAB/MS in positive mode
520.56 [M
I
– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C12 : 0]
+
[M
II
– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C10 : 0]
+
502.59 [M
I

– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C12 : 0 – H
2
O]
+
[M
II
– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C10 : 0 – H
2
O]
+
332.41 [{M
I
– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C12 : 0] ) 3OH (C10 : 0) – H
2
O]
+
[{M
II

– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C10 : 0] ) 3OH (C10 : 0) – H
2
O]
+
314.41 [{M
I
– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C12 : 0] ) 3OH (C10 : 0) ) 2 · H
2
O]
+
[{M
II
– GlcN – H
3
PO
4
) 2 · 3OH (C10 : 0) – C10 : 0] ) 3OH (C10 : 0) ) 2 · H
2
O]
+
2900 I. N. Krasikova et al.(Eur. J. Biochem. 271) Ó FEBS 2004
M. vaga lipid A but arise f rom fragmentation duri ng F AB-

MS. The peak at m/z 1099.83 was interpreted to correspond
to a loss of C12 : 0 or C10 : 0 residues from the initial
molecular species, and those at m/z 929.5 a nd m/z 758.05
were obtained through additional loss of one or two
residues of C10 (3OH), respectiv ely. These data indicate that
nonhydroxy fatty acids and two residues o f C10 (3OH) of
M. vaga lipid A were attached to glucosaminobiose via
rather labile ester linkages. On the o ther hand, the ion at m/z
758.05 was confirmed as consisting of monophosphorylated
GlcN backbone with two a mide-linked C10 (3OH) r esidues.
The CAD experiment with a fragment ion at m/z 929.5
(Fig. 3 A, Table 3) causing an additional (in comparison
with the ion at m/z 1099.83) loss o f C10 (3OH) confirmed
that lipid A of M. vaga contains two amide-linked and two
ester-linked C10 (3OH) fatty acids.
In addition, a s et of other f ragment-ion signals derived
from the c leavage of th e glycosidic linkage were seen in the
negative-ion FAB mass spectrum (Fig. 2A, Table 3). They
corresponded t o monophosphorylated monoglucosamine
derivatives substituted w ith one (peaks at m/ z 428.31 and
410.92) or two [peaks at m/z 59 8.4 (high intensity) and m/z
580.7] units of C10 : 0 ( 3OH) and w ith one of nonhydroxy
acids(C10:0andC12:0atm/z 752.5 and 780.5,
respectively; low intensity). According t o t he above com-
positional a nalysis a nd NMR spectroscopy data (Tables 1
and 2), the M. vaga lipid A contains only one phosphate
group, which is located at C1 of the proximal G lcN I . Thus
these signals were attributable to the reducing end of
glucosaminobiose, which brings three fatty acid units, two
of which are C10 : 0 (3OH). T he ion with m/z 428.31

consisting of glucosamine, a phosphate group and C10 : 0
(3OH) confirms that one residue of this fatty acid is
N-linked. The second unit of C10 : 0 (3OH) of GlcN I is
ester-linked. CAD of the fragment ion at m/z 598.4 (Fig. 3B,
Table 3) gave ions at m/z 428.3 [a loss of a C10 : 0 (3OH)
residue] and m/ z 410.9 [an additional l oss of H
2
O(D m/z
18)], thus confirming the presence of both ester-linked and
amide-linked C10 : 0 (3 OH) f atty acids in GlcN I. Taking
into consideration NMR data (Table 2), the este r-linked
C10 : 0 (3OH) is thought to be attached to C3 of M. vaga
lipid A.
The position of t he secondary nonhydroxy fatty acids on
the proximal G lcN I was determined by treatment of lipid A
with weak alkali (12% aqueous NH
4
OH). As shown by
Silipo et al. [ 26], this mild procedure i s a ble t o s plit the acyl
and acyloxy esters selectively, leaving the acyl and acyl-
oxyacyl amides unaffected. In line with this, weak alkali
hydrolysis of M. vaga lipid A released C10 : 0 (3OH), but
C10 : 0, C12 : 0 and C12 : 1 f atty acids w ere present on ly in
trace amounts, and acyloxyacyl derivatives were completely
lacking. Subsequent hydrolysis of de-O-acylated lipid A
with a stronger alkali gave C10 : 0 (3OH), C10 : 0, C12 : 0
and C12 : 1. T ogether w ith F AB-MS d ata t hese findings
confirm that nonhydroxy fatty acids must b e in the
secondary position at the N-linked C10 : 0 (3OH) a cid of
GlcN I.

In the FAB-MS spectrum (Fig. 2B, Table 3), measured
in the positive mode, diagnostic peaks important for
structure elucidation, were observed at m/ z 520.56, 502.59,
332.41, and 31 4.41. The first t wo ions consisted of gluco-
samine and two residues of C10 : 0 (3OH) and were
assigned to the o xonium ions produced by cleavage of
glycosidic linkage. The oxonium ions arise from GlcN II, so
the M. vaga lipid A nonreducing g lucosamine unit b ears
two C 10 : 0 (3OH) fatty acids, which are loc ated at the C2 ¢
position [according to NMR data (Table 2), hydroxy
groups a t the C3 ¢,C4¢,andC6¢ positions of GlcN II are
free]. One C 10 : 0 (3 OH) fatty acid has an amide-type o f
linkage [signals at m/ z 332 and 314.4, which result f rom t he
loss of the C10 : 0 (3OH) residue from the f ragment ions at
m/z 5 20.59 and 5 02.56, are in g ood agr eement with such
assumption], and the other one is the secondary fatty acid at
C3 of the amide-linked C10 : 0 (3OH).
Based on the above r esults together with the analyses of
chemical composition and NMR data, the s tructure of
M. vaga lipid A i s proposed as t he structure illustrated in
Fig. 4.
Toxicity
The toxic properties of M. vaga lipid A were examined in
outbreed
D
-galactosamine-sensitized mice by the method of
Galanos et al. [ 29]. I t a ppears that the lipid A s tudied has a
significantly higher lethal dose (1.46 lg) than the Yersinia
pseudotuberculosis O:1b LPS, the LD
50

of which is 0.063 lg.
Discussion
This study is devoted to the structural elucidation of lipid A
from the marine proteobacterium M. vaga. To the best of
our knowledge, this is the second complete structure of lipid
A from a marine bacterium (lipid A from wild-type
Fig. 3. CAD exper iments o n ( A) 929.5 m/ z and (B) 598.4 m/z ions of
M. vaga ATCC 27119
T
lipid A. See Table 3 for peak identification.
Ó FEBS 2004 Marinomonas vaga lipid A structure (Eur. J. Biochem. 271) 2901
Pseudoalteromonas haloplanktis TAC 125 was the fir st [18]).
The data show that lipid A molecules from marine and
terrestrial b acteria both contain the same b-1,6-linked
glucosaminobiose backbone. However, M. vaga lipid A
has a number of unique chemical characteristics differenti-
ating it from enterobac terial lipid A. The first of these i s that
a short-chain 3-hydroxydecanoic acid is its main acyl
residue. It is found i n lipid A from s everal bacterial species,
such as Rhodocyclus (now Rubrivivax) gelatinosus [39],
Rhodospirillum tenue (now Rhod ocyclus tenuis)[40],Rhodo-
pseudomonas capsulata (now Rho dobacter capsulatus)[41],
Rhodopseudomonas (now Rhodobacter) sphaeroides [42],
some species of Pseudomonas [43], Sphaerotilus natans [44],
Comamonas testosteroni [45], and also Bordetella pertussis,
Chromobacterium sp. and Rhodopseudomonas sp.[36].Only
in four of them (R. gelatinosus [39], R. tenue [40], S. natans
[44], and C. testosteroni [45]) does 3-hydroxydecanoic acid
occur in both ester and amide linkages.
The second unusual structural c haracteristic of M. vaga

lipid A is that an ester-linked phosphate group attached to
position 4¢ of glucosaminobiose is completely lacking.
Similar structures were also found in lipid A isolated f rom
the anaerobic Bacteroides fragilis [46] and Porphyromonas
gingivalis [47], m icroaerophilic Helicobacter pylori strain
206–1 [48 ], and aerobic Flavobacterium meningosepticum
[49], a nd, p artly, in lipid A from Pseudomonas reacta ns and
Pseudomonas cichorii (lipid A from these bacteria has
nonstoichiometric substitution with the phosphate groups
at C4¢ [50,51]).
ThethirdspecificfeatureofM. vaga lipid A is that it has
a low degree of acylation and unusual d istribution o f a cyl
substituents over the backbone. O nly five fatty acid r esidues,
of which four were 3-hydroxydecanoic acids, were found in
its structure. In addition, 3-hydroxydecanoic acid acylated
only one hydroxy group of the d isaccharide. Three o ther
3-hydroxydecanoic acid residues (two w ith a mide linkages
and one as the s econdary acid) were located at C2 and C2¢.
The same acyl-deficient and p hosphate-deficient glucosam-
inobiose nonreducing end was also found in lipid A f rom
Helicobacter pylori strain 206-1, but its acyloxyalkanoic acid
contained n o h ydroxy acid [48]. As far as is known ( R)-3-
[(R)-3-hydroxydecanoyloxy]decanoic a cid i s t he first acyl-
oxyalkanoic acid formed by two 3-hydroxyalkanoic acid
residues, with the exception of lipid A from Vibrio cholerae,
in which the presence o f such an acid was not completely
proved [36].
Structural types of lipid A h arboring a smaller number of
fatty acids than endotoxically active molecules, including
penta-acyl lipid A, are of interest as potential endotoxin

antagonists [ 8–10]. Among the derivatives of lipid A that
can protect against Gram-negative septic shock, mono-
phosphoryl lipid A i s well known. The facts that a penta-
acyl monophosphoryl derivative of glucosaminobiose i s the
major s tructure type of M. vaga ATCC 27119
T
lipid A and
also that the a cyl residue at C3 ¢ of GlcN II is completely
missing in this lipid A suggest that it may show character-
istics of an endotoxin antagonist. T he rather low acute
toxicity of M. vaga lipid A correlates well with the above
suggestion. Further studies to examine this proposal are in
progress.
Acknowledgements
We are g rateful to Dr U. Z a
¨
hringer and Dr B. Lindner for useful
discussion on the performance of this work. We also thank Dr Yu.
A. Knirel and Dr A. V. Perepelov for t he opportunity to determine
the absolute configuration of
D
-glucosamine, and Dr O. P.
Moiseenko for G LC/MS measurements. Mrs N. M. Shepe tora for
fruitful assistance in preparing the English version. This work was
supported financially by the Russ ian Foundation for B asic Research
(grant 02-04-49 517), Progr am ÔPhysical and che mical biologyÕ for
Basic R esearch o f t he R ussian Academy of Science, Far East B ranch
of the Russian Academy o f Sciences (grants 03 -3-A-05-081 and
03-3-G-05-040).
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