Dipartimento di Chimica Organica e Biochimica, Universitá di Napoli Federico II, via Cynthia 4, 80126 Napoli, Italy
Received on March 26, 2004; revised on May 12, 2004; accepted on May 12, 2004
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Abstract |
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Key words: Agrobacterium tumefaciens / lipid A / lipopolysaccharide / MALDI-TOF mass spectrometry / NMR spectroscopy
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Introduction |
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The external layer of the outer membrane of A. tumefaciens, like all Gram-negative bacteria, is largely composed by LPSs. These are amphiphilic macromolecules possessing a hydrophilic heteropolysaccharide (formed by core oligosaccharide and O-specific polysaccharide or O-chain) covalently linked to a lipophilic moiety named lipid A, which anchors them to the lipid bilayer. Rough-form LPSs do not possess an O-specific polysaccharide and are frequently named lipooligosaccharides (LOSs). LOSs may occur in both wild and laboratory strains possessing mutations in the genes encoding the O-specific polysaccharide biosynthesis or transfer. LPSs are also known as endotoxins because they play a key role in the pathogenesis of Gram-negative infections in animal and plant hosts. In particular, the lipid A component is the effective toxic part of LPSs and is the primary immunostimulator center of Gram-negative bacteria. In animals, lipid A promotes the activation of the innate immune system by the induction of cytokine and other endogenous mediators. Toll-like receptor (TLR4)MD2 complexes present on the surface of immune cells exclusively recognizes lipid A, detecting the presence of a Gram-negative infection (Alexander and Rietschel, 2001; Medzhitov, 2001
). An uncontrolled and massive immune response due to high levels of endotoxins leads to the septic shock.
The biological activity of lipid A is strictly dependent on its primary structure (Seydel et al., 2000). Few nontoxic lipid A species and chemically synthesized analoges can antagonize the action of LPS on human cells, such as the one from Rhodobacter capsulatus (Christ et al., 1995
). Thus the study of lipid A molecules from nonhumanpathogen bacteria is extremely important to identify novel lipid A molecules that in human cells display inhibitory activity on the production of proinflammatory agents stimulated by LPS/lipid A of toxic Gram-negative bacteria.
From a structural point of view, lipid A is a glycolipid with a highly heterogeneous but rather conservative structure, typically composed of a 2-deoxy-2-amino-glucose (glucosamine, GlcN) disaccharide backbone, phosphorylated at position 1 and 4' (Raetz et al., 2002; Zähringer et al., 1999
). Other substituents can be linked to phosphate or replace it. In addition, the disaccharide backbone is acylated by 3-hydroxy fatty acids at position 2, 3, 2', and 3' of both proximal and distal glucosamine (GlcN I and GlcN II, respectively). These fatty acids (primary) can be further esterified at their 3-hydroxy group by other fatty acids (secondary).
Herein, the structure of the lipid A components derived from A. tumefaciens strain C58 is determined.
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Results |
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The O-linked fatty acid analysis revealed the presence of 3-hydroxy-butyroyl, 14:0 (3-OH), 28:0 (27-OH), and, in very minor amount, 26:0 (25-OH) fatty acid residues. The position of the hydroxyl group in the long-chain acyl moieties was inferred by comparison of gas chromatography (GC)-MS fragmentation peaks of the O-acetyl methyl ester derivative with those of an authentic sample from Rhizobium etli CE3 LPS (Que et al., 2000). Actually, the EI spectrum of O-acylated 28:0 (27-OH) residue showed diagnostic fragments at m/z 453 (M-43), 436 (M-60), 404 (M-32), 481 (M-15), and 87 [CH3-CH-OAc]+, the last two indicative of the location of the hydroxy group. An analogous fragmentation pattern was present for 26:0 (25-OH) derivative.
The total fatty acid analysis showed, besides the O-linked acyl chains, 16:0 (3-OH) and 18:1 (3-OH) residues as amide-linked fatty acids, as definitely established by the fatty acid analysis on the de-O-acylated lipid A obtained by anhydrous hydrazine treatment (see later discussion).
MS analysis of native lipid A fraction
The negative ion matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum of native lipid A from A. tumefaciens revealed a pattern of molecular ion peaks representative of a complex mixture consisting of penta-acylated and tetra-acylated species. In particular, according to compositional analysis, the penta-acylated species A1 and A2 (Table I, Figure 1) were both consistent with a bis-phosphorylated disaccharide backbone carrying two 14:0 (3-OH), one 28:0 (27-OH), and one 3-hydroxy-butyroyl residues. Species A2 carried a second 16:0 (3-OH) residue, whereas species A1 (m/z 26) carried a 18:1 (3-OH) residue. Penta-acylated species B1 and B2 (
m/z 26) lacked the 3-hydroxy-butyroyl residue with respect to A1 and A2 species.
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MS analysis of the de-O-acylated lipid A fraction
An aliquot of the lipid A fraction was de-O-acylated with anhydrous hydrazine in Tetrahydrofurane (THF). The negative ion MALDI-TOF mass spectrum (Figure 2) showed only two ions at m/z 1032.5 and 1006.5 attributed to di-acylated species. The ion at m/z 1006.5 was consistent with bis-phosphorylated diglucosamine backbone carrying two 16:0 (3-OH) residues, whereas the ion at m/z 1032.5 possessed one 18:1 (3-OH) residue and one 16:0 (3-OH) residue. The positive ion MALDI-TOF mass spectrum (data not shown) revealed oxonium ions from GlcN II at m/z 496.4 and 522.5(m/z 26) deriving from the cleavage of the glycoside linkage and indicative of the presence of a 16:0 (3-OH) or a 18:1 (3-OH) residues on the distal amide-linked residue. According to acyl composition of native lipid A (Table I), only one 18:1 (3-OH) residue can be present in the molecule, and thus, when present, it is logical to locate it as amide-linked fatty acid to GlcN II.
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Analysis of the dephosphorylated lipid A fraction
The full fatty acid distribution was established by MS analysis of dephosphorylated lipid A fraction. The positive ion MALDI-TOF mass spectrum exhibited pseudomolecular ions [M + 23]+ with the same acylation pattern revealed by the mass spectrum of intact lipid A. The absence of phosphate and the presence of species still bearing the butyroyl residue excluded a linkage of this last group via phosphate to the disaccharide backbone. Furthermore, the spectrum comprised oxonium ions at m/z 1149.3 and 1175.3 both deriving from the nonreducing unit, consistent with a GlcN ion carrying one O-linked 14:0 (3-OH) and one N-linked 16:0/18:1 (3-OH), esterified by a 28:0 (27-O-butyrate). The absence of an oxonium ion couple lacking a 14:0 (3-OH) allowed to conclude that this fatty acid was nonstoichiometrically present on GlcN I.
NMR spectroscopy of the lipid A fraction
A combination of homo- and heteronuclear 2D nuclear magnetic resonance (NMR) experiments were performed to confirm the previous MS suggestions and eventually characterize the lipid A family from A. tumefaciens LPS.
Although there is abundant literature (Brecker, 2003 and references therein; Ribeiro et al. 1999
; Wang and Hollingsworth, 1996
) concerning NMR experiments realized on lipid A indicating several combinations of solvents for obtaining NMR spectra with a good resolution, we intentionally chose dimethyl sulfoxide (DMSO)-d6 at 343 K as a suitable system. Actually, in these conditions, the N-H protons of monosaccharides are visible as well and represent a valuable alternative initial point to assign proton resonances (Brecker, 2003
; Molinaro et al., 2002
; Silipo et al., 2002b
).
A combination of homo- and heteronuclear 2D NMR experiments (correlation spectroscopy [COSY], total correlation spectroscopy [TOCSY], rotating frame Overhauser enhancement spectroscopy [ROESY], and heteronuclear single quantum coherence [HSQC]) were performed to assign of the fully acylated lipid A mixture signals (Table II). The spectra showed signals relative to the more abundant carbohydrate backbone (Table II) besides species present in minor amounts. Starting from the anomeric and/or the amide protons in the TOCSY and COSY spectra, all the carbohydrate spin system could be attributed. The coupling constants and the chemical shift values were in agreement with the presence of two 2-amino-2-deoxy residues in gluco configuration and in 4C1 conformation.
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The sequence of the carbohydrate residues was gained by the ROESY and HSQC spectra. The ß-(16) linkage between the GlcN residues was proven by either the strong interresidue NOE correlation of the proton H-1 B (4.47 ppm) with H-6a A (3.64 ppm) and the down-field displacement of C-6 A (69.3 ppm).
A 31P-1H HSQC allowed to establish the location of the phosphate groups. In the spectrum two plain correlations were observed, the 31P chemical shifts were both characteristic of phosphate monoester groups. In the first cross-peak, a 31P signal at 1.20 ppm correlated to a H-1 A signal at 5.23 ppm, whereas the second signal at 1.08 ppm correlated to a proton signal at 4.01 ppm attributed to H-4 B. Thus the phosphate groups were linked to O-1 of residue A and to O-4 of residue B. These data allowed us to define the archetypal lipid A disaccharide backbone [P4-ß-D-GlcpN-(1
6)-
-D-GlcpN-1
P], bearing acyl groups at position 2 and 3 of both GlcN residues.
Going in depth in the 2D NMR spectra, an alternative carbohydrate spin system was identified (C) (Figure 4b). In the anomeric region of the HSQC spectrum, C-1 C carbon resonance at 91.7 ppm correlated to proton H-1 at 4.90 ppm, and both these high-field chemical shift values were indicative of a free reducing end. The H-2 and H-3 chemical shifts of spin system C showed a significant down-field displacement in agreement with N- and O-acylation at these positions. The C-2 C carbon signal resonated at 52.6 ppm, proving the presence of 2-acylamido-2-deoxy-hexose and all of other resonances were in good agreement with a -GlcN I, which was evidently lacking the phosphate at O-1. In fact, no cross-peaks were present in the 31P-1H HSQC spectrum for the H-1 C signal. The existence of a different GlcN residue lacking phosphate was already suggested by the MS analysis that showed the existence of monophosphorylated species.
2D NMR spectroscopy was even more remarkable for fatty acid analysis; indeed, nine spin systems attributable to the acyl moieties were assigned.
The high-field region of the NMR spectra (Figures 5 and 6) showed various signals attributable to acyl moieties. The HSQC spectrum showed three cross-peaks at 3.82/67.5, 3.79/67.5 and 3.70/67.9 ppm attributable to ß H/C resonances of 14:0 (3-OH), 16:0 (3-OH), and of 18:1 (3-OH). Furthermore, by COSY and TOCSY spectra it was also possible to assign the resonances of the diastereotopic and of the
methylene protons of these acyl chains. In the HSQC spectrum, a cross-peak of a down-field shifted signal at 5.05/71.1 ppm was attributed to ß H/C of primary fatty acids 16:0/18:1(3-O-R) when acylated at O-3.
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Concluding structural remarks
Using the results obtained from all the analyses, it is now possible to fully assign all ion peaks of the negative ion MALDI-TOF mass spectrum of the intact lipid A fraction derived from A. tumefaciens strain C58 (Figure 1).
The lipid A fraction from A. tumefaciens strain C58 possesses the basic bis-phosphorylated disaccharide backbone [P4-ß-D-GlcpN-(1
6)-
-D-GlcpN-1
P] and consisted in a mixture of differently acylated species. The main penta-acylated species was acylated at position N-2 and O-3 of both GlcN residues, the only existing acyloxyacylamide moiety was located on the nonreducing GlcN and in turn could be substituted by a 3-hydroxy-butyroyl residue at the (
1) position (Figure 7).
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The main lipid A species were also isolated in small amount by preparative thin-layer chromatography (TLC) and analyzed only via gas-liquid chromatography (GLC)-MS and MALDI-TOF MS. Three main products were obtained, and their structure was confirmative of the previous structural analysis. The slower migration product was composed of the major bis-phosphorylated penta-acylated species (A1 and A2), and the other two products consisted of monophosphorylated tetra- and penta-acylated lipid A species (C and E).
The high recovery after hydrolysis at pH 4.5 of lipid A species still bearing acid-labile substituents (long chain fatty acid, O-linked primary fatty acids) suggests that monophosphorylated lipid A species are true LPS component.
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Discussion |
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The LPS fraction of A. tumefaciens is composed by two main lipid A species (Figure 7), the first one is bearing (1) two unsubstituted fatty acids 14:0 (3-OH) in ester linkage; (2) two 16:0 (3-OH) or 16:0 (3-OH)/18:1 (3-OH) pair in amide linkage; (3) a long chain fatty acid, 28:0 (27-OH) linked to 16:0 (3-OH) on GlcN II; and (4) a 3-hydroxy-butyroyl residue linked to 28:0 (27-OH). The second species, present in minor amounts, is missing a 14:0 (3-OH) on GlcN I. Other species are deriving from these two and lack phosphate or ß-OH-butyrate.
From a structural point of view, the lipid A structure from A. tumefaciens appears as a hybrid lipid A that on one hand retains the typical acylation pattern of other Rhizobium lipid A species (Bhat et al., 1994; Gudlavalleti and Forsberg, 2003
; Jayaretnam et al., 2002
). Namely, it is characterized by the presence of long chain fatty acids (i.e., C28:0 (27-OH), C26:0 (25-OH)) that can be further substituted at
-1 position by a unit of 3-hydroxy-butyroyl residue. As in the hybrid lipid A from Sinorhizobium LPS, this acyl moiety is present on the distal GlcN. In accordance, it presents a low degree of O-acylation at C-3 of GlcN I and shows heterogeneity in the amide-linked fatty acid composition.
On the other hand, the lipid A disaccharide skeleton of R. etli and R. leguminosarum are characterized by an atypical trisaccharide containing a distal galacturonic acid in -1,4 linkage to a GlcN, which is in turn (ß-1,6)-linked to a proximal 2-aminogluconic acid residue. The lipid A of from A. tumefaciens possesses the standard disaccharide backbone and polar heads of Enterobacteriaceae LPS, for example, Escherichia coli, and also found in the LPS of Sinorhizobium sp. NGR234 (Gudlavalleti and Forsberg, 2003
).
The lipid A component of A. tumefaciens LPS shows some general similarities to the lipid A structures of the endosymbiont bacterium Sinorhizobium sp. NGR234 LPS (Gudlavalleti and Forsberg, 2003). The carbohydrate backbone and O-linked fatty acids 14:0 (3-OH) are shared by both species, and the N-linked fatty acids are partially different because in A. tumefaciens LPS is present the 16:0 (3-OH)/18:1 (3-OH) pair, and this was not previously found in reliable amounts. In general, with respect to Sinorhizobium sp. NGR234 lipid A, the one from A. tumefaciens presents a lower degree of heterogeneity in the acylation pattern, particularly in N-linked fatty acids.
The structural information can be helpful in considering the biological action of A. tumefaciens. In Rhizobium, the peculiar acylation of lipid A (long chain fatty acid, low acylation pattern, low phopshorylation) is deemed as a strategy of the bacterium to escape or attenuate the plant response to fruitfully establish the symbiosis (Basu et al., 1999). Analogously, in this plant pathogen bacterium, these structural features could be a chemical camouflage of the bacterium to evade the defense-related plant responses by masking itself as a symbiotic bacterium. For example, in Rhizobium, the occurrence of such a long chain fatty acid is seen as a necessary means to establish a successful symbiotic infection. It exerts a stabilizing effect on bacterial outer membrane during the stressful events connected with the early stages of symbiosis, namely, the close interaction with plant cell wall. In addition, the hypothesis that this fatty acid could be an instrument to disrupt plant membrane cannot be ruled out (Kannenberg and Carlson, 2001
). Undoubtedly, the presence of the long chain fatty acid in pathogen Agrobacterium LPS would assume another meaning, for example, it stabilizes the bacterial outer membrane, thus becoming a useful instrument to connect and attack plant cells.
In line with these speculations, the low negative charge (anomeric phosphate lacking) and low degree of acylation of this lipid A could be a strategy to attenuate host defense response.
From a biochemical point of view, the lack of acyloxyacyl esters and the presence of a long chain fatty acid suggest that a lipid A biosynthetic pathway similar to of Rhizobium is present in Agrobacterium as well. Namely, all the enzymatic systems requested for the synthesis, transport, and linkage of the long chain fatty acid and de-O-acylation of lipid A are also present in the biosynthetic pathway of Agrobacterium lipid A and therefore can be a useful target for antibiotic strategy.
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Materials and Methods |
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The cells were extracted three times with a mixture of aqueous 90% phenol/chloroform/petroleum ether (2:5:8 v/v/v) as described (Galanos et al., 1969). After removal of organic solvents under vacuum, the LOS fraction was precipitated from phenol with water; the precipitate was washed with aqueous 80% phenol and then three times with cold acetone and lyophilized (30 mg, yield: 0.7% of the bacterial dry mass).
The LOS fraction was detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis (12%) (Tsai and Frasch, 1982), and the gel was stained with silver nitrate for detection of LPSs/LOSs as described (Kittelberger and Hilbink, 1993
).
LOS was further purified using a column (100 x 3 cm, eluent 50 mM NH4CO3) of Sephacryll S-300 (Pharmacia, Uppsala, Sweden), and the resulting fraction represented the pure lipooligosaccharide.
Preparation of lipid A, de-O-acylated lipid A, and dephosphorylated lipid A
Free lipid A was obtained by hydrolysis of the LOS with 10 mM sodium acetate buffer containing 1% sodium dodecyl sulfate, pH 4.4, 100°C, 3 h.
De-O-acylated lipid A was obtained by treatment with anhydrous hydrazine in THF at 37°C for 1.5 h, followed by precipitation with cooled acetone and centrifugation (5000 x g, 15 min, 4°C). Alternatively, a mild de-O-acylation was performed by treatment of lipid A (200500 µg) with 32% ammonium hydroxide (200 µl, 20°C, 16 h) (Silipo et al., 2002b). Lipid A was dephosphorylated with fluoridic acid (HF) 48% (4°C, 48 h).
TLC of lipid A were carried out on 20 x 20 cm Silica Gel 60 TLC plates (Merck, Darmstadt, Germany; 0.25 µm thickness) with CHCl3/MeOH/H2O (100:75:15 by volume) + acetic acid 0.01%. The spots were visualized by spraying the plate with 10% (v/v) ethanolic H2SO4 and charring.
Alternatively, TLC was performed using chloroform/methanol/water/triethylamine (30:12:2:0.1), and bands were visualized by spraying with water and marked with a pencil. The fraction visualized were scraped, eluted with chloroform/isopropyl alcohol/water 5:3:0.25 (v/v), and filtered on a small column of Sephadex LH-20 with the same solvent system as described (Gudlavalleti and Forsberg, 2003).
General and analytical procedures
Monosaccharides were identified as acetylated O-methyl glycosides derivatives. After methanolysis (2 M HCl/MeOH, 85°C, 24 h) and acetylation with acetic anhydride in pyridine (85°C, 30 min) the sample was analyzed by GC-MS. The absolute configuration of the monosaccharides was obtained according to the published method (Leontein and Lönngren, 1978).
Methylation analysis was carried out on dephosphorylated product: briefly, the sample (1 mg) was kept at 4°C, 48 h, in HF 48% (200 µl) and then evaporated under a stream of nitrogen. Methylation was performed with methyl iodide as published (Ciucanu and Kerek, 1984). The hydrolysis of the methylated sugar backbone was performed with 4 M trifluoracetic acid (100°C, 4 h), and the partially methylated product, after reduction with NaBH4, was converted into alditol acetates with acetic anhydride in pyridine at 80°C for 30 min and analysed by GLC-MS as described shortly.
Total fatty acid content was obtained by acid hydrolysis lipid A. Briefly, lipid A was first treated with HCl 4 M (4 h, 100°C) and then neutralized with NaOH 5 M (30 min, 100°C). Fatty acids were then extracted in CHCl3, methylated with diazomethane, and analyzed by GLC-MS.
The ester-bound fatty acids were selectively released by base-catalyzed hydrolysis with NaOH 0.5 M/MeOH (1:1 v/v, 85°C, 2 h), then the product was acidified, extracted in CHCl3, methylated with diazomethane, and analyzed by GLC-MS. Fatty acids from both preparations were acetylated with acetic anhydride in pyridine (85°C, 30 min) further analyzed with GC-MS. The absolute configuration of fatty acids was determined as described (Rietschel, 1976).
All GLC analyses were performed on a Hewlett-Packard 5890 instrument, SPB-5 capillary column (0.25 mm x 30 m, Supelco, Bellefonte, PA), for sugar methylation analysis and O-acetylated methyl ester fatty acids the temperature program was 150°C for 2 min, then 2°C min1 to 200°C for 0 min, then 10°C min1 to 260°C for 11 min, then 8°C min1 to 300°C for 20 min; for absolute configuration analysis it was 150°C for 8 min, then 2°C min1 to 200°C for 0 min, then 6°C min1 to 260°C for 5 min. For fatty acids analysis, the temperature program was 80°C for 2 min, then 8°C min1 to 300°C for 15 min.
MS analysis
MALDI-TOF analyses were conducted using a Perseptive (Framingham, MA) Voyager STR instrument equipped with delayed extraction technology. Ions formed by a pulsed UV laser beam (nitrogen laser, = 337 nm) were accelerated through 20 kV. Mass spectra reported are the result of 128 laser shots. Insulin and myoglobin were used for external calibration. The dried samples was dissolved in CHCl3/CH3OH (50/50 v/v) at a concentration of 25 pmol ml1. The matrix solution was prepared by dissolving 2, 5-dihydroxybenzoic acid in CH3OH at a concentration of 30 mg ml1 or trihydroxyacetophenone in CH3OH/0.1% trifluoroacetic acid/CH3CN (7/2/1 by volume) at a concentration of 75 mg ml1. A sample/matrix solution mixture (1:10 v/v) was deposited (1 ml) onto a stainless steel gold-plated 100-sample MALDI probe tip and dried at 20°C.
NMR spectroscopy
1H and 13C NMR spectra of lipid A were measured on Varian INOVA 500 equipped with a reverse probe at 343 K in DMSO-d6. 13C and 1H chemical shifts are expressed in relative to DMSO (
H 2.49,
C 39.7). 31P-NMR spectra were measured on a Bruker DRX 400 spectrometer equipped with a reverse probe at 343 K in DMSO-d6. Aqueous 85% phosphoric acid was used as external reference (0.00 ppm) for 31P NMR spectroscopy.
NOESY and ROESY were measured using data sets (t1 x t2) of 4096 x 1024 points, and 16 scans were acquired. A mixing time of 300 ms was employed. Double quantum-filtered phase-sensitive COSY experiment was performed with 0.258 s acquisition time using data sets of 4096 x 1024 points, and 64 scans were acquired. TOCSY was performed with a spinlock time of 80 ms, using data sets (t1 x t2) of 4096 x 1024 points, and 16 scans were acquired. In all homonuclear experiments, the data matrix was zero-filled in the F1 dimension to give a matrix of 4096 x 2048 points and was resolution enhanced in both dimensions by a shifted sine-bell function before Fourier transformation. Coupling constants were determined on a first-order basis from 2D phase-sensitive double quantum-filtered COSY (Rance et al., 1983; States et al., 1982
). The HSQC experiment spectrum was measured in the 1H-detected mode via single quantum coherence with proton decoupling in the 13C (or 31P) domain, using data sets of 2048 x 512 points, and 64 scans were acquired for each t1 value. The experiments were carried out in the phase-sensitive mode according to the method of States et al. (1982)
, and the data matrix was extended to 2048 x 1024 points using linear prediction extrapolation (de Beer and van Ormondt, 1992
; Hoch and Stern, 1996
; Stern et al., 2002
).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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Basu, S.S., White, K., Que, N., and Raetz, C. (1999) A deacylase in Rhizobium leguminosarum membranes that cleaves the 3-O-linked beta-hydroxymyristoyl moiety of lipid A precursors. J. Biol. Chem., 274, 1115011158.
Bhat, U.R., Carlson, R.W., Busch, M., and Mayer, H. (1991) Distribution and phylogenetic significance of 27-hydroxy-octacosanoic acid in lipopolysaccharides from bacteria belonging to the alpha-2 subgroup of Proteobacteria. Int. J. Syst Bacteriol., 41, 213217.[Abstract]
Bhat, U.R., Forsberg, L.S., and Carlson, W. (1994) Structure of lipid A component of Rhizobium leguminosarum bv. phaseoli lipopolysaccharide. Unique nonphosphorylated lipid A containing 2-amino-2-deoxygluconate, galacturonate, and glucosamine. J. Biol. Chem., 269, 1440214410.
Brecker, L. (2003) Nuclear magnetic resonance of lipid Athe influence of solvents on spin relaxation and spectral quality. Chem. Phys. Lipids, 125, 2739.[CrossRef][ISI][Medline]
Christ, W.J., Asano, O., Robidoux, A.L., Perez, M., Wang, Y., Dubuc, G.R., Gavin, W.E., Hawkins, L.D., McGuinness, P.D., Mullarkey, M.A., and others. (1995) E5531, a pure endotoxin antagonist of high potency. Science, 268, 8083.[ISI][Medline]
Ciucanu, I. and Kerek, F. (1984) A simple method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209217.[CrossRef][ISI]
De Beer, R. and van Ormondt, D. (1992) Analysis of NMR data using time domain fitting procedures. NMR Basic Prin. Prog., 26, 201.
De Castro, C., Bedini, E., Nunziata, R., Rinaldi, R., Mangoni, L., and Parrilli, M. (2003) Elucidation of the O-chain structure from the lipopolysaccharide of Agrobacterium tumefaciens strain C58. Carbohydr. Res., 338, 18911894.[CrossRef][ISI][Medline]
Dow, M., Newman, M.A., and von Roepenack, E. (2000) The induction and modulation of plant defense responses by bacterial lipopolysaccharides. Annu. Rev. Phytopathol., 38, 241261.[CrossRef][ISI][Medline]
Galanos, C., Luderitz, O., and Westphal, O. (1969) A new method for the extraction of R lipopolysaccharides Eur. J. Biochem., 9, 245249.[ISI][Medline]
Gudlavalleti, S.K. and Forsberg, L.S. (2003) Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. J. Biol. Chem., 278, 39573968.
Hoch, J.C. and Stern, A.S. (1996) Linear prediction. In J.C. Hoch and A.S. Stern (Eds.), NMR data processing. Wiley, New York, pp. 77101.
Jayaretnam, B., Glushka, J., Kumar Kolli, V.S., and Carlson, R.W. (2002) Characterization of a novel lipid-A from Rhizobium species Sin-1. A unique lipid-A structure that is devoid of phosphate and has a glycosyl backbone consisting of glucosamine and 2-aminogluconic acid. J. Biol. Chem., 277, 4180241810.
Kannenberg, E.L. and Carlson, R.W. (2001) Lipid A and O-chain modifications cause Rhizobium lipopolysaccharides to become hydrophobic during bacteroid development. Mol. Microbiol., 39, 379391.[CrossRef][ISI][Medline]
Kittelberger, R. and Hilbink, F. (1993) Sensitive silver-staining detection of bacterial lipopolysaccharides in polyacrylamide gels. J. Biochem. Biophys. Meth., 26, 8186.[CrossRef][ISI][Medline]
Leontein, K. and Lönngren, J. (1978) Determination of the absolute configuration of sugars by gas-liquid chromatography of their acetylated 2-octyl glycosides. Methods Carbohydr. Chem., 62, 359362.
Medzhitov, R. (2001) Toll-like receptors and innate immunity. Nature Rev. Immunol., 1, 135145.[CrossRef][Medline]
Molinaro, A., Silipo, A., Lanzetta, R., Parrilli, M., Malvagna, P., Evidente, A., and Surico, G. (2002) Determination of the fine structure of the lipid A from the LPS of Pseudomonas cichorii by means of NMR and MALDI-TOF mass spectrometry. Eur. J. Org. Chem., 18, 31193125.[CrossRef]
Que, N.L.S., Lin, S.H., Cotter, R.J., and Raetz, C.R.H. (2000) Purification and mass spectrometry of six lipid A species from the bacterial endosymbiont Rhizobium etli. J. Biol. Chem., 275, 2800628016.
Raetz, C.R.H. and Whitfield, C. (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem., 71, 635700.[CrossRef][ISI][Medline]
Rance, M., Sørensen, O.W., Bodenhausen, G., Wagner, G., Ernst, R.R., and Wüthrich, K. (1983) Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem. Biophys. Res. Commun., 117, 47948.[ISI][Medline]
Ribeiro, A.A., Zhou, Z., and Raetz, C.R.H. (1999) Multi-dimensional NMR structural analyses of purified lipid X and lipid A (endotoxin). Magn. Reson. Chem., 37, 620630.[CrossRef][ISI]
Rietschel, E.T. (1976) Absolute configuration of 3-hydroxy fatty acids present in lipopolysaccharides from various bacterial groups. Eur. J. Biochem., 64, 423428.[Abstract]
Seydel, U., Oikava, M., Fukase, K., Kusumoto, S., and Brandenburg, K. (2000) Intrinsic conformation of lipid A is responsible for agonistic and antagonistic activity. Eur. J. Biochem., 267, 30323039.
Silipo, A., Lanzetta, R., Amoresano, A., Parrilli, M., and Molinaro, A. (2002a) Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of lipid A fatty acid distribution. J. Lipid Res., 43, 21882195.
Silipo, A., Lanzetta, R., Garozzo, D., Lo Cantore, P., Iacobellis, N.S., Molinaro, A., Parrilli, M., and Evidente A. (2002b) Structural determination of the lipid A of the lipopolysaccharide from Pseudomonas reactans. A pathogen of the cultivated mushrooms. Eur. J. Biochem., 269, 24982505.
Silverstein, R.M. and Webster, F.X. (1997) Spectrometric identification of organic compounds, 6th ed. John Wiley, New York.
States, D.J., Haberkorn, R.A., and Ruben, D.J. (1982) A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants. J. Magn. Reson., 48, 286292.[ISI]
Stern, A.S., Li, K.B., and Hoch, J.C. (2002) Modern spectrum analysis in multidimensional NMR spectroscopy: comparison of linear prediction extrapolation and maximum entropy reconstruction. J. Am. Chem. Soc., 124, 19821993.[CrossRef][ISI][Medline]
Tsai, C.M. and Frasch, C.E. (1982) A sensitive silver stain for lipopolysaccharides in polyacrylamide gels. Anal. Biochem., 119, 115119.[ISI][Medline]
Wang, Y. and Hollingsworth, R.W. (1996) An NMR spectroscopy and molecular mechanics study of the molecular basis for the supramolecular structure of lipopolysaccharides. Biochemistry, 35, 56475654.[CrossRef][ISI][Medline]
Zähringer, U., Lindner, B., and Rietschel, E.T. (1999) Chemical structure of lipid A: recent advances in structural analysis of biologically active molecules. In D.C. Morrison, H. Brade, S. Opal, and S. Vogel (Eds.), Endotoxin in health and disease. Marcel Dekker, New York, pp. 93114.