Báo cáo khóa học: Characterization of Mesorhizobium huakuii lipid A containing both D-galacturonic acid and phosphate residues ppt

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Báo cáo khóa học: Characterization of Mesorhizobium huakuii lipid A containing both D-galacturonic acid and phosphate residues ppt

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Eur J Biochem 271, 1310–1322 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04038.x Characterization of Mesorhizobium huakuii lipid A containing both D-galacturonic acid and phosphate residues Adam Choma1 and Pawel Sowinski2 Department of General Microbiology, Maria Curie-Sklodowska University, Lublin, Poland; 2Intercollegiate NMR Laboratory, Department of Chemistry, Technical University of Gdansk, Poland The chemical structure of the free lipid A isolated from Mesorhizobium huakuii IFO 15243T was elucidated Lipid A is a mixture of at least six species of molecules whose structures differ both in the phosphorylation of sugar backbone and in fatty acylation The backbone consists of a b (1¢fi 6) linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharide that is partly substituted by phosphate at position 4¢ The aglycon of the DAG-disaccharide has been identified as a-D-galacturonic acid All lipid A species carry four amidelinked 3-hydroxyl fatty residues Two of them have short hydrocarbon chains (i.e 3-OH-i-13:0) while the other two have longer ones (i.e 3-OH-20:0) Distribution of 3-hydroxyl fatty acids between the reducing and nonreducing DAG is symmetrical The nonpolar as well as (x-1) hydroxyl long chain fatty acids are components of acyloxyacyl moieties Two acyloxyacyl residues occur exclusively in the nonreducing moiety of the sugar backbone but their distribution has not been established yet The distal DAG amide-bound fatty acid hydroxyls are not stoichiometrically substituted by ester-linked acyl components Lipopolysaccharides (LPS) are characteristic components of the outer leaflet of the outer membranes of Gram-negative bacteria Those glycoconjugates have a common general architecture They contain three distinct regions: lipid A, a nonrepeating oligosaccharide core and an O-polysaccharide composed of a varying number of repeating units The O-polysaccharide chain is the major target of animal immune responses, thus it is also referred to as the O-antigen The core oligosaccharide is a spacer between the O-chain and lipid A and is linked to the latter by an acid labile ketosidic bond Lipids A in many Gram-negative bacteria (especially in animal pathogens) have a conserved structure In the majority of cases, their backbones are composed of a b-1,6-D-glucosamine disaccharide with two phosphate residues attached at positions and 4¢ Up to four fatty acids are bound by ester or amide linkages to the backbone glucosamines Lipid A is responsible for the endotoxic properties of lipopolysaccharide The structure of lipid A seems to be essential in maintaining outer membrane integrity and flexibility and is crucial for bacterial cell viability [1–3] Lipopolysaccharide is important in the process of symbiotic interaction between Rhizobium and the host plant [4,5] Environmental conditions (in planta and ex planta) as well as plant-derived molecular signals induce entire LPS modifications in Rhizobium [6] The structures of Rhizobium lipid A indicate great variation in the glycosyl component of its backbone as well as the acylation pattern The lipid A backbone of Sinorhizobium is similar to that from enteric bacteria [7,8] Lipids A from Rhizobium etli and biovars of Rhizobium leguminosarum have identical and unusual structures R etli lipids A are devoid of phosphate groups [9–11] and a galacturonic acid residue replaces the 4¢-linked phosphate in the lipid A backbone The distal part (distant from the reducing end of the backbone) of lipid A is almost the same for all lipid A species isolated The proximal glucosamine is partly oxidized to 2-aminogluconate [12,13] A specific deacylase removes the ester-linked fatty acids from the C-3 position of the lipid A precursor, thus this hydroxyl is only partially substituted by an acyl residue in the matured lipid A [14] The symbiont of Sesbasnia, Rhizobium sp Sin-1 [15], has lipid A composed of b-D-glucosamine attached to 2-aminogluconate by (1fi 6) glycoside linkage When compared with R etli this lipid A lacks galacturonic acid at position 4¢ [16] In contrast to the above mentioned lipid A structures, the mesorhizobial and bradyrhizobial lipids A have not been fully chemically characterized to date Bradyrhizobium lipid A backbones are composed exclusively of 2,3-diamino-2,3-dideoxyglucose with mannose as a subsituent in some of them [4,5,17,18] No data about Allorhizobium (renamed Rhizobium undicola [19]) and scant information about Azorhizobium [20,21] lipopolysaccharides and lipids A are available Mesorhizobium loti lipids A contain DAG and phosphate residues [22,23] and M huakuii also possesses DAG-type lipid A [24] Mesorhizobium lipids A are known to carry a number of b-hydroxyl fatty acids accompanied by small amounts of 4-oxo fatty acids Correspondence to A Choma, Department of General Microbiology, Maria Curie-Sklodowska University, 19 Akademicka St., 20–033 Lublin, Poland Fax: + 48 81 5375959, Tel.: + 48 81 5375981, E-mail: achoma@biotop.umcs.lublin.pl Abbreviations: DAG, 2,3-diamino-2,3-dideoxyglucose; LPS, lipopolysaccharides (Received 12 September 2003, revised February 2004, accepted 16 February 2004) Keywords: Mesorhizobium huakuii; lipid A; 2,3-diamino2,3-dideoxy-D-glucose; MALDI-TOF; 2D-NMR Ó FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur J Biochem 271) 1311 Numerous ester-linked nonpolar and (x-1) hydroxyl long chain fatty residues were found in those preparations [25,26] In this report, we describe the structural investigation of a unique lipid A isolated from Mesorhizobium huakuii We show that DAG-type lipid A backbone is double decorated: (a) nonstoichiometrically, with phosphate at position 4¢ of the distal DAG, and (b) with a-linked galacturonic acid at position of the proximal unit Phosphorylated and nonphosphorylated lipid A preparations are a mixture of three subfractions differing in acylation patterns Experimental procedures Bacterial strain, growth, and isolation of lipopolysaccharide and lipid A Mesorhizobium huakuii IFO15243T strain was obtained from the Institute for Fermentation, Osaka, Japan Bacteria were grown at 28 °C in liquid mannitol/yeast extract medium 79CA [27] and were aerated by vigorous shaking Cells were centrifuged at 10 000 g, washed twice with saline and once with distilled water The wet bacterial paste was extracted by the modified hot phenol/water procedure [28] The water layer was dialysed firstly against tap water, then against distilled water The crude LPS was purified by repeated ultracentrifugation at 105 000 g for h The LPS solution (5 mgỈmL)1) in aqueous 1% (v/v) acetic acid was kept at 100 °C for h The lipid A precipitate was collected by centrifugation, washed twice with hot distilled water and lyophilized mass spectrometry For this analysis, lipid A samples were methanolysed (1 M HCl, 80 °C, 18 h), N-acetylated and trimethylsililated [29] The content of phosphorus in lipid A was determined according to Lowry [30] Chemical modification of lipid A Subfractions of lipid A (about mg) were dephosphorylated in 48% (v/v) aqueous HF at °C for 48 h [31] HF was removed by evaporation in the stream of nitrogen with cooling in an ice bath De-O-acylation of lipid A subfractions was performed according to modified procedure of Haishima and coworkers [31] Preparations were treated with anhydrous hydrazine at 37 °C for h The reaction mixtures, after cooling, were poured into cold acetone The resulting lipid A precipitates were collected, washed twice with acetone and then gently dried in the stream of nitrogen Gas chromatography-mass spectrometry GC-MS was carried out on a Hewlett-Packard gas chromatograph (model HP5890A) equipped with a capillary column (HP-5MS, 30 m · 0.25 mm) and connected to a mass selective detector (MSD model HP 5971) Helium was the carrier gas The temperature program for fatty acid methyl esters and for alditol acetates analysis was as follows: initially 150 °C for min, then raised to 310 °C at a ramp rate of °C min)1, final time 20 The temperature program for trimethylsililo derivatives of methyl glycosides was, accordingly: initially 80 °C for min, then raised to 310 °C at a ramp rate of °C min)1, final time Purification and separation of lipid A species Crude lipid A was purified and separated into subfractions according to a modified procedure described by Que and coworkers [9] Briefly, lyophilized lipid A ( 30 mg) was dissolved in 20 mL of CHCl3/methanol/H2O (2 : : 1; v/v/v) and loaded onto a DEAE column (1 cm · cm, Whatman DE23) The column was washed with 30 mL of the same solvent and that eluate was collected as a single fraction Next, the lipid material was eluted by a two step gradient of ammonium acetate: first with 30 mL of CHCl3/ methanol/250 mM NH4Ac (2 : : 1; v/v/v), and then with 30 mL of CHCl3/CH3OH/500 mM NH4Ac (2 : : 1; v/v/v) The presence of organic substances in the eluate was monitored by spotting 10 lL of each fraction on a silica plate and visualized by spraying the plate with 10% (v/v) sulfuric acid in methanol followed by charring Separated fractions were converted to two-phase Bligh–Dyer system by adding the appropriate amount of water and chloroform Water layers were discarded and organic layers were supplemented with fresh portions of the upper phase of a freshly prepared two-phase Bligh–Dyer mixture The washed organic layers were separated by centrifugation and dried Preparations were stored at )20 °C in CHCl3/ methanol (1 : 1; v/v) Mass spectrometry Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was performed on a Voyager-Elite (PE Biosystems) instrument using delayed extraction, in both positive and negative ion modes The samples were desorbed with a nitrogen laser and extraction voltage of 20 kV Lipid A samples were dissolved in CHCl3/ CH3OH (2 : 1; v/v) The analysed compounds (0.5 lL) were mixed with 50% (v/v) 2,5-dihydrobenzoic acid in acetonitril as matrix Each spectrum was the average of about 256 laser shots Liquid matrix-assisted secondary ion mass spectrometry (LSIMS) was performed using AMD 604 (AMD Intectra GmbH) mass spectrometer operated in the negative ion mode with primary ion beam of Cs+ Samples were mixed with a matrix of meta-nitrobenzyl alcohol (m-NBA) Lipid A was analysed by ESI-MS using Finnigan Mat TSQ 700 mass spectrometer operated in the negative ion mode The samples were dissolved in a CHCl3/CH3OH (2 : 1; v/v) mixture supplemented with 0.1% (v/v) concentrated ammonia and introduced into electrospray source at a flow rate of lLỈmin)1 NMR spectroscopy Glycosyl composition analysis Lipid A samples were analysed for fatty acids and aminosugars as described previously [24] Neutral and acidic sugars were determined by gas-liquid chromatography and H-NMR experiments were performed in CDCl3/dimethylsulfoxide-d6 (2 : 1; v/v) mixture 2D (DQF COSY, TOCSY, NOESY) 1H-NMR and 1H/13C as well as 1H/31P-HSQC experiments were carried out on Varian Unity plus 500 1312 A Choma and P Sowinski (Eur J Biochem 271) Ó FEBS 2004 Fig GC-MS profile of trimethylsilyl ether derivatives of N-acetylated methyl glycosides and fatty acid methyl esters obtained by methanolysis of dephosphorylated lipid A from Mesorhizobium huakuii IFO 15243T Peaks were identified by their mass spectra and by comparison of retention times with standards GalA, galacturonic acid; DAG, 2,3-diamino-2,3-dideoxyglucose; p.e impurity-ester of phtalic acid; *, unidentified compound For more details about the fatty acid composition of IFO 15243T see [24] instrument at 48 °C using standard VARIAN software 1D 31 P-NMR spectra were registered on a Bruker 300 spectrometer, operating at 121.58 MHz at 40 °C For this analysis, the lipid A was dissolved in D2O containing 2% deoxycholate and mM Na2EDTA The pH of lipid A solutions was adjusted with NaOH to 7.3 and 10.6, respectively Phosphorous chemical shifts were measured relative to an external standard of 85% (v/v) phosphoric acid at 0.00 p.p.m Results Chemical analyses The compositional analysis of crude lipid A preparation obtained from M huakuii IFO 15243T LPS revealed the presence not only of 2,3-diamino-2,3-dideoxyglucose (DAG) and a complex set of fatty acids (both ester and amide bound), as described previously [24], but also galacturonic acid and phosphate residues The presence of GalA was unequivocally confirmed by GC-MS analysis of trimethylsilil ethers of methyl glycosides liberated from lipid A by methanolysis (Fig 1) The 31P-NMR spectrum of the crude lipid A revealed a prominent signal with chemical shift of 1.71 p.p.m observed at neutral pH This signal was shifted to 4.71 p.p.m when the pH of the lipid A suspension was raised to 10.6 (Fig 2) These properties are indicative of phosphomonoesters other than glycosyl1-phosphate The location of the phosphate was directly determined by two-dimensional heteronuclear magnetic resonance (see below) On the basis of chemical shift value and lack of the cross peak with protons from the lipid A backbone on the 31P/1H-HSQC spectrum, the weak signal at 1.88 p.p.m was attributed to inorganic phosphate impurities of the lipid A preparation The results of quantitative measurements of phosphorus and DAG content showed that no more than half of the lipid A molecules bear phosphate residues Fatty acids found in the IFO 15243T lipid A (Fig 1) can be divided into two groups The first one, easily liberated by mild alkali or acid solvolysis, contains all saturated and unsaturated nonpolar as well as (x-1) hydroxyl, and (x-1) Fig 31P-NMR spectra of the crude lipid A from Mesorhizobium huakuii IFO 15243T The signal at 1.71 p.p.m (A) recorded at pH 7.3, shifted to 4.71 p.p.m (B) at pH 10.60, and represents ester-bound monophosphate residue oxo long chain fatty acids These are ester-linked to the lipid A The second group of fatty acids needs strong liberation conditions [32] This group comprises all 3-hydroxyl and 4-oxo fatty acids, which are connected directly to the lipid A backbone via amino groups [24] The molar proportions among fatty acids isolated from lipid A were almost the same as described earlier for the total LPS [24] The main amide-bound fatty acids identified were as follows: 3-OH-12:0, 3-OH-i-13:0, 3-OH-20:0 and 3-OH21:0 Among them, 3-OH-i-13:0 and 3-OH-20:0 clearly predominated The types of ester-bound fatty acids were also numerous, but only four of them, namely i-17:0, 20:0, 22:1 and particularly 27-OH-28:0 fatty acid, predominated (Fig 1, [24]) The calculated proportion between Ó FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur J Biochem 271) 1313 amide- and ester-linked fatty acids was approximately : Therefore, one can expect that DAG type lipid A could contain not more than six acyl residues The complex mixture of the lipid A preparation was separated into two fractions, based on DEAE gravity column chromatography The first fraction (designated lipid A–P), which was eluted with solvent containing 250 mM ammonium acetate, was devoid of phosphate, as shown by 31P-NMR The phosphate was detected in +P the second fraction (named lipid A ), successively eluted with a solvent mixture containing 500 mM NH4Ac MALDI-TOF analysis of lipid A preparations Both subfractions of lipid A were investigated by mass spectrometry Ions representative of each species of lipid A, recorded on the negative and positive ion MALDI-TOF and negative ion ES-MS spectra, their corresponding composition and the theoretically calculated masses are listed in Table +P Lipid A is a complex mixture of individual molecules The two major species (Z and Y) could be easily distinguished from mononegatively charged pseudomolecular ions on MALDI-TOF spectrum (Fig 3A) The third (X) cluster of ions, which were less intensive, was visible between m/z at 1600 and 1700 All those ions correspond to lipid A that posseses a backbone with a monophosphate residue accompanied by six, five and four acyl moieties, respectively Dephosphorylation procedure, to which +P lipid A was submitted, led to a downshift of each molecular ion by 80 mass units The spectrum of the +P dephosphorylated lipid A is almost identical to that –P obtained for the lipid A preparation (Fig 4) Moreover, the MALDI-TOF spectrum of lipid A–P treated with 48% HF did not change significantly when compared with the unprocessed preparation (data not shown) +P Species Z of lipid A (Fig 3A) contained ions within the range from 2287 to 2478 mass units Those ions correspond to lipid A molecules composed of two DAG, one of which is phosphorylated, one GalA, four 3-hydroxyl fatty acids, one (x-1) hydroxyl long chain fatty acid and one a nonpolar fatty acyl residue The most intense ion in this cluster (m/z at 2357) could be attributed to the molecules of lipid A containing two 3-OH-i-13:0 and two 3-OH-20:0 acids, as well as two ester-bound acids (e.g 20:0 and 27-OH-28:0) This is merely one possible explanation due to the fact that numerous combinations of fatty acids different to those found in lipid A exist However, taking into consideration the quantities of lipid A fatty acids this proposition seems to be the most probable The amide-bound fatty acids isolated from M huakuii IFO15243T lipid A and from other mesorhizobia can be separated into two clusters [24–26] The first contains short chain fatty acids, mainly 3-OH-12:0 and 3-OH-i-13:0, whereas the second is represented by 3-OH-20:0 and other fatty acids similar in length For correct calculation of the pseudomolecular ion masses found on the MALDI-TOF spectra it is necessary to take into account the masses of two 3-OH short chain fatty acyls (e.g 3-OH-i-13:0) and two longer 3-OH fatty acyl residues (e.g 3-OH-20:0) The ions from species Y are usually 295 mass units lighter than the respective ions from species Z That corresponds to a loss of eicosanoyl residue from hexaacyl lipid A Therefore, the Y species comprise ions representing lipid A molecules carrying five acyl residues (four 3-OH fatty acids and one (x-1) hydroxyl long chain fatty acid) The ion at m/z 1640 and those close to m/z 1640, designated as species X, correspond to tetraacyl lipid A molecules with all acyl residues directly linked to the sugar backbone by amide bonds De-O-acylation of lipid A fractions led to decay of species Z and Y and resulted in increase of signals corresponding to ions of species X (data not shown) The total decrease of mass due to de-Oacylation of phosphorylated as well as of nonphosphorylated lipid A was the same and equalled 717 Da (loss of both 294 and 423 mass units) The positive ion MALDI-TOF mass spectra of the +P lipid A (Fig 3B) showed two additional species generated after laser-induced cleavage of glycosidic linkages between 2,3-diamino-2,3-dideoxyglucoses within the lipid A backbone The first species [B1+(Z)] of oxonium ions originated from hexaacyl phosphorylated lipid A (prominent ions at m/z 1481 and 1508) The second species [B1+(Y)] consist of ions with masses close to that at m/z 1187 Those ions are made up of DAG, two 3-hydroxyl fatty acyl moieties and (x-1) hydroxyl long chain fatty acid Those B1+ fragment ions support the conclusion that the 27-hydroxyoctacosanoic acid and eicosanoic acid, when present, are located on the distal diaminoglucosyl residue of the lipid A Moreover, the sugar component of B1+ lacks hydroxyl groups suitable for attachment of these fatty acids by ester bonds The appropriate hydroxyls are located at positions of amide linked acyl of the distal DAG Therefore, both 27-OH-28:0 and 20:0 fatty acids are components of acyloxyacyl residues The predicted ions for the third type of oxonium ions composed of DAG and two amide acyl residues have not been registered, due to the fact that the spectra were usually recorded from m/z 1000–3000 The correct calculation of masses for B1+ type ions requires taking into account the appropriate amidelinked fatty acids That group of acyl residues consists of fatty acid pairs The first acid in each pair is shorter (e.g 3-OH-i-13:0) while the second one is longer (e.g 3-OH20:0) Analysis of lipid A by means of LSI mass spectrometry revealed negative ions m/z at 862.7, 876.8 and 890.7 (data not shown) The most intensive ion (m/z at 876.8) corresponds to a lipid A fragment composed of DAG, GalA, 3-OH-i-13:0 and 3-OH-20:0 A similar ion was observed for P gingivalis and F meningosepticum lipids A on negative ion FAB-MS/MS spectra [33,34] In conclusion, these data point to the symmetrical localization of amide-bound acyl residues in M huakuii lipid A The 2,3-diacylamido-2,3-dideoxyglucose, obtained by mild solvolysis [35] followed by mild hydrolysis of the dephosphorylated lipid A, was reduced with NaBD4, than subjected to Smith oxidation, again reduced with NaBH4 and after acetylation, the four-carbon fragments of DAG carrying amide-bound fatty acids were analysed by means of GC-MS Preliminary data from those experiments indicate that N-2 position in distal and proximal DAG is occupied mainly by 3-hydroxyleicosanoic acids The shorter acids were found to be bound at N-3 position of the amino sugar ring The fatty acid distribution will be verified during further studies MALDI-TOF (ion type and m/z-value) MALDI-TOF (ion type and m/z-value) – + B1 1108 + B1 1400 [M + Na]+ 2004 – – – Unphosphorylated lipid A fraction – [M + Na]+ 2299 + B1 1187 – [M-H]– 1640 [M + Na]+ 1663 + B1 1481 [M-2H])2 1030.7 [M-H]– 2063 [M + Na]+ 2085 – – – – – – [M-2H])2 nd [M-2H])2 1177.9 Phosphorylated lipid A fraction [M-H]– [M + Na]+ 2380 2357 ES-MS (ion type m/z-value) Negative mode MS Positive mode MS – – – – – – – 2063.4 2357.8 molecular mass calculated from ES-MS value 1 2 1 2 DAG 0 0 1 1 P Composition 1 1 1 1 GalA 2 4 2 4 3-OHfatty acids 1 1 1 1 (x-1)-OH -fatty acids 1 0 Nonpolar fatty acids 61 81 94 114 61 81 66 94 114 Total number of fatty acid carbon atoms Backbone, · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0, 20:0 Backbone, · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0, 20:0 DAG · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0, 20:0 DAG · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0 Backbone*, phosphate, · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0, 20:0 Backbone, phosphate, · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0 Backbone, phosphate, · 3-OH-20:0, · 3-OH-i-13:0 P-DAG, · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0, 20:0 P-DAG, · 3-OH-20:0, · 3-OH-i-13:0, 27-OH-28:0 Proposed composition of the molecule 1106.774 1401.297 1982.896 2277.419 1186.753 1481.276 1640.137 2062.875 2357.398 Predicted molecular mass Table Data from mass spectrometry analyses Positive and negative ions derived from phosphorylated and nonphosphrylated lipid A fractions of Mesorhizobium huakuii IFO 1243T; their compositions and proposed structures Backbone*, trisaccharide of b-D-DAG-(1 fi 6)-a-D-DAG-(1fi1)-a-D-GalA; P, phosphate residue; nd, not determined 1314 A Choma and P Sowinski (Eur J Biochem 271) Ó FEBS 2004 Ó FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur J Biochem 271) 1315 Fig Negative (A) and positive (B) ion MALDI-TOF mass spectra of the phosphorylated subfraction of lipid A from M huakuii IFO 15243T Lipid A yields three ion clusters (Z, Y, X) They differ by the degree of acylation Species X contains four amide-bound fatty acids Species Y is pentaacyl lipid A (with 27-OH-28:0 fatty acid residue) Species Z is hexaacyl lipid A The proposed formulas and masses of pseudomolecular ions ([M ) H]– and [M + Na]+) are summarized in Table The individual ions in the clusters differ by 14 units (acyl chain length differences) Positive ion spectrum contains two B+1 type ion clusters derived from cleavage of the glycosidic linkage in lipid A Unidentified ions are marked with asterisks (*) + +P The B1 ions from lipid A (e.g m/z at 1187 and 1508, Fig 3B) differed by 80 mass units from those originating from lipid A–P (e.g m/z at 1108 and 1428, Fig 4) Comparing Figs 3B and 4, it is easy to notice that the phosphate deprived lipid A appears to have a higher number of connected fatty acids On the spectrum, shown in Fig 4, the signals for hexaacyl lipid A are considerably more intensive than others Pentaacyl lipid A molecules dominate in the case of the phosphorylated lipid A preparation Possibly, a weak acid hydrolysis (the procedure used for lipid A liberation) causes a partial de-O-acylation of the native lipid A molecules In contrast to R etli, R leguminosarum and S melilotii [8–10], we did not find lipid A molecules containing 3-hydroxylbutyrate or 3-metoxylbutyrate 1316 A Choma and P Sowinski (Eur J Biochem 271) Ó FEBS 2004 Fig Positive ion MALDI-TOF mass spectrum of unphosphorylated subfraction of lipid A from M huakuii IFO 15243T This lipid A subfraction yields the three ion clusters X1, Y1 and Z1 They differ in the degree of acylation pattern and contain four, five and six acyl residues, respectively The spectrum contains two B+1 type ion clusters derived by cleavage of the glycosidic linkage in lipid A NMR spectroscopy of lipid A preparations +P De-O-acylated lipids A and lipid A–P were dissolved in a mixture of dimethylsulfoxide (DMSO-d6) and chloroform (CDCl3) for NMR experiments Figure shows the one+P dimentional proton spectrum of de-O-acylated lipids A H and 13C chemical shift assignments were based on 2D homonuclear experiments: DQF-COSY (Fig 6), TOCSY (Fig 7) and 1H/13C heteronuclear single quantum coherence (HSQC) experiments The values of carbon and proton chemical shifts are summarized in Table Three signals were identified in the anomeric region of 13 C-NMR chemical shifts for both lipid A fractions These data suggested that the lipid A backbone contains three sugar residues Four signals were found between 50 and 55 p.p.m for each preparation They were assigned to the C-2 and C-3 carbon atoms linked directly with the amino groups The remaining sugar ring carbon signals were observed in the region from 60 to 78 p.p.m TOCSY and DQF-COSY spectra revealed three glycosyl ring systems The anomeric proton (HA-1) at 4.98 p.p.m was assigned to a-linked galacturonic residue Its spin system (A) consists of five protons for which all the cross peaks have been traced and marked on Fig and resulting chemical shifts listed in Table Analysis of the sugar proton system B (Fig 7) was initiated at the anomeric proton (HB-1, dH ¼ 4.87 p.p.m., J1,2 ¼ 2.8Hz) That proton showed an evident correlation to HB-2 (dH ¼ 3.84 p.p.m.), which showed a strong correlation to HB-3 (dH ¼ 4.08 p.p.m.) Furthermore, HB-3 showed a coupling with HB-4 (dH ¼ 3.48 p.p.m.) The remaining glycosyl proton cross-peaks were observed at following chemical shifts: 3.48 p.p.m./3.97 p.p.m (HB-4/ HB-5), 3.97 p.p.m./3.60 p.p.m (HB-5/HB-6a), 3.60 p.p.m./ 3.89 p.p.m (HB-6a/HB-6b) The proton chemical shifts for both sugar ring systems (A and B) were similar to those published for A pyrophilus lipid A [36] Chemical shifts of +P the distal aminosugar (sugar ring system C) in lipid A were in good agreement with those from A pyrophilus lipid A distal DAG, however, two shift exceptions (for HC-4 and HC-3) were observed The HC-4 signal appeared at Fig Proton NMR spectrum of de-O-acylated lipids A+P fraction The sample was dissolved in DMSO-d6/CDCl3 (1 : 2, v/v) The spectrum was recorded at 500 MHz, at 48 °C Some signals from sugar backbone are indicated The letters refer to the carbohydrate spin systems as was described in the text and shown in Table The numerals next to the letters indicate the protons in the respective residues Signal positions from olefinic protons, terminal methyl protons, bulk methylene protons and protons from a, b and c positions of 3-hydroxy fatty acids are marked with; -CH ¼ CH-, -CH3, and -CH2-, a, b and c, respectively CHCl3, DMSO and H2O represent signals from solvents and absorbed water Ó FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur J Biochem 271) 1317 Fig A partial DQF-COSY spectrum of de-O-acylated phosphorylated subfraction of lipid A The spectrum was recorded at 500 MHz, at 48 °C The letters refer to the carbohydrate spin systems as was described in the text and shown in Table The numerals next to the letters indicate the protons in the respective residues Fig A partial TOCSY spectrum of de-O-acylated phosphorylated subfraction of lipid A The spectrum was recorded at 500 MHz, at 48 °C The letters refer to the carbohydrate spin systems as was described in the text and shown in Table The numerals next to the letters indicate the protons in the respective residues dH ¼ 4.01 p.p.m., which was about 0.3 p.p.m downfield from the A pyrophilus lipid A equivalent signal and about 0.5 p.p.m downfield from the H-4 signal characteristic of DAG with unsubstituted hydroxyl group at C-4 carbon atom (dH for HB-4, Table 2) The downfield shift of HC-4 was caused by the presence of ester-bound phosphate residue Analysis of carbon chemical shifts led to the same conclusions, since CC-4 (dC ¼ 71.9 p.p.m) appeared down- field compared to the proximal CB-4 unsubstituted by phosphate (dB ¼ 67.5 p.p.m) The location of phosphate substituent on CC-4 was established upon HC-4/31P (4.01 p.p.m./1.35 p.p.m) correlation observed in 1H/31P HSQC spectrum The sequence of the monosaccharides was established by NOESY experiment (Fig 8) A strong interresidue NOE signal was observed between HA-1 of GalA and Ó FEBS 2004 1318 A Choma and P Sowinski (Eur J Biochem 271) Table 1H- and 13C-NMR chemical shifts and coupling constants of sugar backbones of lipid A fractions DAG-I proximal 2,3-diamino-2,3dideoxyglucose moiety in the lipid A from M huakuii IFO 15243T, DAG-II distal 2,3-diamino-2,3-dideoxyglucose moiety in the lipid A from +P M huakuii IFO 15243T; lipid A , phosphorylated fraction of lipid A; lipid A–P, unphosphorylated lipid A; nd, not determined; J, coupling constant Spectra were recorded at 500 MHz (1H) and 125.7 MHz (13C) in DMSO-d6/CDCl3 (2 : 1, v/v) DAG-II (C) Residue (spin system) DAG-I (B) GalA (A) d (J,[Hz]) 13 d d (J,[Hz]) 13 d H-1 4.39 (8.2) 3.72 3.94 4.01 3.23 3.53 3.79 7.23 7.26 C-1 103.1 H-1 C-1 92.8 H-1 C-2 C-3 C-4 C-5 C-6 54.5 54.7 71.9 77.8 60.8 4.87 (2.8) 3.84 4.08 3.48 3.97 3.60 3.89 7.26 7.24 C-2 C-3 C-4 C-5 C-6 52.0 51.3 67.5 71.8 68.7 H-2 H-3 H-4 H-5 C-1 102.8 C-1 92.4 H-1 C-2 C-3 C-4 C-5 C-6 53.8 53.8 68.8 nd 61.4 C-2 C-3 C-4 C-5 C-6 52.0 51.7 70.3 71.8 68.8 H-2 H-3 H-4 H-5 H C H C H d (J,[Hz]) 13 4.98 (2.8) 3.78 3.94 4.08 4.42 C-1 94.9 C-2 C-3 C-4 C-5 C-6 67.9 68.8 70.8 71.1 nd C-1 94.5 C-2 C-3 C-4 C-5 C-6 68.2 71.7 70.5 71.0 nd C d Lipid A+P H-2 H-3 H-4 H-5 H-6a H-6b NH-2 NH-3 H-2 H-3 H-4 H-5 H-6a H-6b NH-2 NH-3 Lipid A–P H-1 H-2 H-3 H-4 H-5 H-6a H-6b NH-2 NH-3 4.35 ( 8) 3.73 3.77 3.34 3.22 3.58 3.71 7.46 7.38 H-1 H-2 H-3 H-4 H-5 H-6a H-6b NH-2 NH-3 4.89 ( 2) 3.86 4.13 3.52 3.93 3.62 3.93 7.42 7.41 5.02 ( 3) 3.78 3.95 4.12 4.42 Fig A partial NOESY spectrum of deO-acylated phosphorylated subfraction of lipid A The spectrum was recorded at 500 MHz and at 48 °C The letters refer to the carbohydrate spin systems as was described in the text and shown in Table The numerals next to the letters indicate the protons in the respective residues The inter- and intraresidue signals are labeled starting from anomeric protons Diagnostic interresidue cross peaks are underlined Ó FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur J Biochem 271) 1319 HB-1 of the proximal DAG Both sugars possess a anomeric configurations that are reflected in the small values of J1,2 coupling constants and the appropriate values of chemical shifts The downfield shift of carbon CB-6 from the proximal DAG and strong cross peak HC-1/HB-6a (4.39 p.p.m./3.60 p.p.m), as well as less intensive cross peak at 4.39 p.p.m./3.89 p.p.m (HC-1/ HB-6b) on NOESY spectrum, unequivocally indicate the presence of (1fi 6) glycosidic linkage between the two DAG residues Chemical shifts: CC-1 (103.1 p.p.m), HC-1 (4.39 p.p.m) and large ( 8Hz) coupling constants J1,2 measured for the distal DAG confirmed its b-anomeric configuration Putting all the presented data together, we propose the +P chemical structures for lipid A (species Z, Y, X) as shown in Fig Fig Tentative structures of lipid A species from Mesorhizobium huakuii IFO 12543T The proposition of the positions of 3-hydroxyl acyls is based on preliminary chemical degradation of lipid A The predicted positions of ester bound fatty acids were elicited from literature data and specificity of LpxXl acyltransferase [46] The proposed structures corresponds to [M + Na]+ ions at m/z 2380 (Z), 2085 (Y), 1663 (X) in Fig 2B and to ions at m/z 2299 (Z1), 2004 (Y1), 1583(X1) in Fig 3B 1320 A Choma and P Sowinski (Eur J Biochem 271) Lipid A–P is deprived of phosphate The 1D 31P-NMR spectrum contained only a trace signal, which in the 1H/31PHSQC experiment gave weak intensity correlation peak +P with chemical shifts almost identical as for lipid A –P Therefore, one ought to conclude that lipid A preparation was contaminated with traces of the phosphorylated lipid A variety Chemical shifts (for protons and carbons) assigned to lipid A–P were almost identical to those of lipid A+P (Table 2) with the exception of H-4 and C-4 in distal DAG The absence of phosphate caused an upfield shift of both proton HC-4 and carbon CC-4 These values of chemical shifts became similar to the corresponding values from the proximal DAG (Table 2) Three 13C signals in the range of 50–55 p.p.m were observed Two of them were assigned to CB-2 and CB-3 (52.0 p.p.m and 51.7 p.p.m., respectively) in the proximal DAG The third signal was found to represent CC-2 and CC-3 overlapped resonances of the distal DAG Similarly, chemical shifts of HC-2 and HC-3 of the distal DAG (lipid A–P) were almost identical They were distinguished upon two separate cross peaks with different amide protons (HC-2/NHC-2; 3.73 p.p.m./ 7.46 p.p.m and HC-3/NHC-3; 3.77 p.p.m./7.38 p.p.m.) observed in DQF-COSY spectrum The chemical shifts and cross peak positions derived from remaining N-acyl residue protons are not being discussed in this paper NMR data of those components for M huakuii IFO15243T lipid A are very similar to the data published earlier for A pyrophilus [36], R etli CE3 [10], Rhizobium sp Sin-1 [16] and other lipid A preparations Discussion Mesorhizobium huakuii IFO 12543T lipopolysaccharide appears to posses a unique lipid A Two backbone species have been identified The complete structure of the first species was characterized as (HO)2PO2-b-D-DAG(1fi6)-aD-DAG(1fi1)-a-D-GalA Thus, the diaminosugar backbone is flanked at both sides with the negatively charged residues D-GalA and phosphate In relation to this structure, the second species of lipid A is devoid of phosphate and therefore possesses nonsymmetrically located and weaker negative charge Lipid A with DAG disaccharide backbone, without phosphate at position has originally been identified in the extremely termophilic bacterium A pyrophilus Its backbone is substituted from both sides (at position and 4¢) with a-D-GalA [36] Within the Rhizobiaceae, other lipids A without phosphate were isolated from R etli, R leguminosarum biovars and Rhizobium sp Sin-1 Negative charges in those molecules originated from galacturonic acid attached at 4¢ and/or from a proximal glucosamine oxidized to the form of 2-aminogluconic acid [10,16] Membrane associated oxidase responsible for this process has been recently detected within OM preparation from R leguminosarum [12,13] DAG–DAG disaccharide has been found in the lipid A from Bradyrhizobium sp (Lupinus) [17], M loti [22], A pyrophilus [36], B abortus [37], L pneumophila [38,39] and other bacteria, but structural studies have been performed only for a few lipid A preparations [36,40,41] Here, we describe the first complete structure of 2,3-diamino-2,3-dideoxyglucose backbone lipid A from bacteria belonging to the Rhizobiaceae Ó FEBS 2004 According to the current knowledge, the first steps of lipid A biosynthesis seem to be the same in all Gramnegative bacteria and lead to a 1,4¢-bisphosphorylated aminosugar disaccharide acylated at positions 2, 3, 2¢, 3¢ by 3-hydroxyl fatty acids Kdo disaccharide occupies position O-6¢ on lipid A [9,42,43] Usually, UDP-glucosamine is a precursor for this pathway, but it is well known that the same or very similar pathway exists within bacteria synthesizing mixed or DAG-type lipid A [44] Thus, common lipid A precursor molecules (Kdo)2lipidIVA, are the point at which the biosynthesis pathways for enterobacteria and rhizobia diverge In Mesorhizobium cells, the common lipid A precursor should be processed by 1-phosphatase, following galacturonic acid transferase, before maturation The predicted 4¢-O phosphatase would carry out its partial dephosphorylation A computer analysis of M loti MAFF303099 genome sequence (http://www.kazusa.or.jp/ rhizobase/), a bacterium closely related to M huakuii [19,45], revealed sequences (mll1545, mll0630, mlr8270) with high homology to the genes encoding key enzymes in the lipid A biosynthesis pathway (lpxC, lpxB and lpxK, respectively) Basu and coworkers [46] described a kb DNA fragment from R leguminosarum that encodes C28 acyltransferase (LpxXL) and related proteins that may participate in the biosynthesis of (x-1)-28:0 and similar fatty acids Among the genes identified, a structural gene encoding a highly specific acyl carrier protein (acpXL) [42,46] capable of long chain fatty acid (C28–C30) incorporation as the secondary substituent of amide-bound 3-hydroxyl fatty acyls has been found Chromosomal fragments from M loti MAFF 303099 have very similar sequence to genes encoding those enzymes [46] Moreover, another DNA fragment from M loti contains a sequence corresponding to lpxE of R leguminosarum This gene, when expressed in E coli, yields a product with a 1-phosphatase activity [47] Therefore, it is very likely that lpxE gene is present within M huakuii that produces lipid A where phosphate at position is entirely replaced by galacturonic acid Long chain fatty acids with a hydroxyl group at the (x-1) position are present in lipids A of almost all bacteria from the Rhizobiaceae [18,25] and in lipids A of facultative intracellular pathogens [48–50] Low endotoxin activity of lipopolysaccharides from those bacteria has been observed For example, the lethal toxicity in galactose-sensitized mice was 1000-fold lower for M loti LPS when compared to S typhimurium LPS [23] It was shown that the lipopolysaccharides from R etli, R galegae, and Rhizobium sp Sin-1 display a largely reduced capacity to induce TNFa in human monocytes [51] Moreover, Sin-1 LPS preparation antagonized an E coli-induced synthesis of TNFa by these monocytes The inhibition of the plant innate immune system should be achieved for the proper development of symbiosis The experimental data suggest that the presence of long chain (x-1) hydroxyl fatty acids influencing the immunogenicity of lipid A could be one of the factors responsible for such immune system response inhibition Those fatty acids seem to be important but not essential for symbiosis because the acpXL::kan mutant of R leguminosarum lacking the 27-OH-28:0 acid in its lipid A is still able to form nitrogen-fixing nodules [52] Lipid A is a valuable marker in chemotaxonomic studies of rhizobia It is well known that the chromosomal Ó FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur J Biochem 271) 1321 background of rhizobia varies considerably This is also reflected in rhizobial lipids A The diversity in lipid A structures indirectly confirms the hypothesis that symbiotic N2-fixing bacteria evolved from nonsymbiotic and nonrelated ancestors by horizontal transfer of symbiotic genes (nod, nif, fix) as symbiotic islands Similar conclusions were reached when rhizobial Nod factors and the symbiotic relationship between Rhizobium and the legumes were compared [53] Acknowledgements The authors are grateful to I Komaniecka for help with some experiments and to Dr T Urbanik-Sypniewska for critical reading of the manuscript and helpful discussion References 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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, 213–217 51 Vandenplas, M.L., Carlson, R.W., Jeyaretnam, B.S., McNeill, B., Barton, M.H., Norton, N., Murray, T.F & Moore, J.N (2002) Rhizobium Sin-1 lipopolysaccharide (LPS) prevents enteric LPSinduced cytokine production J Biol Chem 44, 41811–41816 52 Vedam, V., Kannenberg, E.L., Haynes, J.G., Sherrier, D.J., Datta, A & Carlson, R.W (2003) A Rhizobium leguminosarum acpXL mutant produces lipopolysaccharide lacking 27-hydroxyoctacosanoic acid J Bacteriol 185, 1841–1850 53 Broughton, W.J & Perret, X (1999) Genealogy of legumeRhizobium symbioses Curr Opin Plant Biol 2, 305–311 ... ions of species X (data not shown) The total decrease of mass due to de-Oacylation of phosphorylated as well as of nonphosphorylated lipid A was the same and equalled 717 Da (loss of both 294 and. .. impurities of the lipid A preparation The results of quantitative measurements of phosphorus and DAG content showed that no more than half of the lipid A molecules bear phosphate residues Fatty acids... were measured relative to an external standard of 85% (v/v) phosphoric acid at 0.00 p.p.m Results Chemical analyses The compositional analysis of crude lipid A preparation obtained from M huakuii

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