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: innakras@piboc.dvo.ru 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). References 1. Lugtenberg, B. & van Alphen, L. (1983) Molecular architecture and functioning of t he outer membrane of Escherichia coli and other Gram-negative bacteria. Biochim. Biophys. Acta 737,51– 115. 2. Galloway, S.W. & R aetz, C.R.H. (1990) A m utant of Escherichia coli defective in the first step of endotoxin biosynthesis. J. B iol. Chem. 265, 6394–6402. 3. Rietschel, E.T., K irikae, T., Sc hade, F .U., Mamat, U., Schm idt, G., L oppnow, H., Ulmer, A.J., Za ¨ hringer, U., Seydel, U., Di Padova, F., Schreier, M. & Brade, H. (1994) Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 8, 217–225. 4. 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(2002) Determination of the structure of the lipid A from the lipopolysaccharide o f Pseudomonas cichorii by means of NMR and M ALDI-TOF mass spectrometry. Eur. J. Org. Che m. 18, 3119–3125. 2904 I. N. Krasikova et al.(Eur. J. Biochem. 271) Ó FEBS 2004 . Detailed structure of lipid A isolated from lipopolysaccharide from the marine proteobacterium Marinomonas vaga ATCC 27119 T Inna N. Krasikova 1 , Natalie. 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