Primary Structure of a New Phosphocholine-containing Glycoglycerolipid of Mycoplasma fermentans
1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês
10.1074/jbc.272.42.26262
ISSN1083-351X
AutoresUlrich Zähringer, Frauke Wagner, Ernst Rietschel, Gil Ben‐Menachem, Joseph Deutsch, Shlomo Rottem,
Tópico(s)Toxoplasma gondii Research Studies
ResumoThe chemical structure of a novel phosphocholine-containing glycoglycerolipid, the major polar lipid in the cell membrane of Mycoplasma fermentans PG18, was investigated by chemical analyses, gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as one- and two-dimensional homo- and heteronuclear NMR spectroscopy and identified as 6′-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-α-d-glucopyranosyl-(1′→3)-1,2-diacyl-glycerol (MfGL-II). Palmitate (16:0) and stearate (18:0), in a 3.6:1 molar ratio, constitute the major fatty acids present. MALDI-TOF mass spectrometry revealed two major pseudomolecular ions atm/z 1049.5 [MI + H]+and 1077.3 [MII + H]+ representing a dipalmitoyl as the major component and a palmitoyl-stearoyl structure as a minor component. This is the first report of 2-amino-1,3-propanediol-1,3-bisphosphate present in a natural product. This glycoglycerolipid is the second phosphocholine-containing glycoglycerolipid found in M. fermentans. The chemical structure of a novel phosphocholine-containing glycoglycerolipid, the major polar lipid in the cell membrane of Mycoplasma fermentans PG18, was investigated by chemical analyses, gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, as well as one- and two-dimensional homo- and heteronuclear NMR spectroscopy and identified as 6′-O-(3"-phosphocholine-2"-amino-1"-phospho-1",3"-propanediol)-α-d-glucopyranosyl-(1′→3)-1,2-diacyl-glycerol (MfGL-II). Palmitate (16:0) and stearate (18:0), in a 3.6:1 molar ratio, constitute the major fatty acids present. MALDI-TOF mass spectrometry revealed two major pseudomolecular ions atm/z 1049.5 [MI + H]+and 1077.3 [MII + H]+ representing a dipalmitoyl as the major component and a palmitoyl-stearoyl structure as a minor component. This is the first report of 2-amino-1,3-propanediol-1,3-bisphosphate present in a natural product. This glycoglycerolipid is the second phosphocholine-containing glycoglycerolipid found in M. fermentans. The human pathogen Mycoplasma fermentans PG18 was isolated from the urogenital tract several decades ago (1Wise K.S. Kim M.F. Theiss P.M. Lo S.-C. Infect. Immun. 1993; 61: 3327-3333Crossref Google Scholar). Because of reports indicating its possible role as a cofactor accelerating the progression of human immunodeficiency virus disease, its significance as a pathogen in other immunocompromised patients (2Blanchard A. Montagnier L. Annu. Rev. Microbiol. 1994; 48: 687-712Crossref PubMed Scopus (99) Google Scholar), and its role in the pathogenesis of rheumatoid arthritis, interest in M. fermentans has recently increased (3Slomiany B.L. Slomiany A. Glass G.B.J. Eur. J. Biochem. 1977; 78: 33-39Crossref PubMed Scopus (35) Google Scholar). Although little is known of the molecular mechanisms underlying M. fermentanspathogenicity (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar), it has been shown that human immunodeficiency virus-associated cytopathic effects could be increased by the presence of M. fermentans (2Blanchard A. Montagnier L. Annu. Rev. Microbiol. 1994; 48: 687-712Crossref PubMed Scopus (99) Google Scholar) and that M. fermentans is capable of fusing with T-cells and peripheral lymphocytes (5Franzoso G. Dimitrov D.S. Blumenthal R. Barile M.F. Rottem S. FEBS Lett. 1992; 303: 251-254Crossref PubMed Scopus (32) Google Scholar). It is reasonable to assume that Mycoplasma membrane components are involved in the attachment and fusion of the microbe with eukaryotic host cells. Salman et al. (6Salman M. Deutsch J. Tarshis M. Naot Y. Rottem S. FEMS Microbiol. Lett. 1994; 123: 255-260Crossref PubMed Scopus (23) Google Scholar) isolated an unusual phospholipid from the cell membranes of M. fermentans and showed that this material (compound X) was capable of enhancing the fusion of small, unilamellar vesicles with MOLT-3 lymphocytes in a dose-dependent manner. Matsuda et al. (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar) isolated two glycoglycerolipids (GGPL-I 1The abbreviations used are: GGPL, glycoglycerophospholipid; MfGL, M. fermentans glycolipid; GLC-MS, gas-liquid chromatography-mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MALDI-LIN-TOF, matrix-assisted laser desorption/ionization time-of-flight recorded in the linear mode; EI-MS, electron impact-mass spectrometry; CI-MS, chemical ionization-mass spectrometry; COSY, correlated spectroscopy; ROESY, rotating frame Overhauser enhancement spectroscopy; HMQC, heteronuclear multiple-quantum coherence; Gro, glycerol; Glc, glucose; AP, 2-amino-1,3-propanediol; Cho, choline; HF, hydrofluoric acid. and GGPL-III) fromM. fermentans. GGPL-I structure was shown to be 6′-O-phosphocholine-α-d-glucopyranosyl-1,2-diacyl-sn-glycerol (7Matsuda K. Kasama T. Ishizuka I. Handa S. Yamamoto N. Taki T. J. Biol. Chem. 1994; 269: 33123-33128Abstract Full Text PDF PubMed Google Scholar) as elucidated by mass and NMR spectroscopy. It was later shown (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar) that the structure of a more polar glycolipid (GGPL-III) isolated from the same strain of M. fermentans was very similar to that of GGPL-I. The chemical structure of GGPL-III, however, has so far remained obscure. The only distinguishing structural feature known is that it differs from GGPL-I in having an additional amino residue (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar). Both GGPLs were shown to be species-specific major lipid antigens ofM. fermentans (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar). Here we describe the structural analysis of a new type of polar lipid isolated from M. fermentans, and we present the complete structural analysis of MfGL-II. 2Unfortunately there exists a confusing terminology in the literature concerning these biologically significant polar glycolipids. We propose replacing the equivalent terms GGPL-I (also called lipid v (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar)) with MfGL-I and replacing the equivalent terms GGPL-III = lipid vi (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar) = compound X (6Salman M. Deutsch J. Tarshis M. Naot Y. Rottem S. FEMS Microbiol. Lett. 1994; 123: 255-260Crossref PubMed Scopus (23) Google Scholar) with MfGL-II. The Roman numerals indicate the sequence of their discovery and structural description. Furthermore, we show that both glycolipids of M. fermentans, GGPL-I and GGPL-III, share the basic structure of 6′-O-phospho-α-d-glucopyranosyl-(1′→3)-1,2-diacyl-glycerol (7Matsuda K. Kasama T. Ishizuka I. Handa S. Yamamoto N. Taki T. J. Biol. Chem. 1994; 269: 33123-33128Abstract Full Text PDF PubMed Google Scholar) but differ in their polar head groups. Cultures of M. fermentansstrain PG18 and strain Incognitus (provided by S.-C. Lo, Armed Forces Institute of Pathology, Washington, D.C.) were grown in a modified Channock medium (8Deutsch J. Salman M. Rottem S. Eur. J. Biochem. 1995; 227: 897-902Crossref Google Scholar) inoculated with a 48-h culture at an inoculum level of 2% and incubated statically at 37 °C. After 68 h the cells were harvested, washed twice, and freeze-dried as described previously (8Deutsch J. Salman M. Rottem S. Eur. J. Biochem. 1995; 227: 897-902Crossref Google Scholar) with yields ranging from 130 to 160 mg dry weight per liter of medium. Freeze-dried cells were suspended in 25 mm Tris/HCl, pH 7.5, containing 0.25m NaCl to a final concentration of 25 mg of cells per ml. Lipids were extracted from cell suspensions by the method of Bligh and Dyer (9Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43103) Google Scholar) and concentrated to near dryness on a rotary evaporator. Quantitative separation of MfGL-II was achieved by silica gel column chromatography. Total lipid was redissolved in 2 ml of chloroform and loaded onto a silica gel column (1.5 × 3 cm; Kieselgel 60, 230–400 mesh, Merck), equilibrated with chloroform and sequentially eluted with four bed volumes of chloroform (fraction 1), chloroform/methanol, 1:4 (v/v, fraction 2), chloroform/methanol/water, 1:4:0.7 (fraction 3), chloroform/methanol/water, 1:4:1 (fraction 4), chloroform/methanol/water, 1:4:1.5 (fraction 5), chloroform/methanol/water, 1:8:3 (fraction 6), methanol (fraction 7), methanol/water, 7:3 (fraction 8), and methanol/water, 6:4 (fraction 9). Fractions were vacuum evaporated to dryness, redissolved in 0.5 ml of elution solution, and analyzed by thin layer chromatography. Since fractions 4–7 contained pure MfGL-II they were combined and dialyzed in 10 mm EDTA against water for 4 days at 4 °C. MfGL-II (370 μg) was dissolved in 0.5m HCl/MeOH and incubated at 85 °C for 40 min. The solvent was removed under a stream of nitrogen, and the liberated fatty acid methyl esters were analyzed by GLC and GLC-MS. Following mild methanolysis, samples were dissolved in 2 m HCl/MeOH, incubated at 85 °C for 16 h, and peracetylated with acetanhydride/pyridine (1:2, v/v) for 60 min at 85 °C. Carbohydrates and other components of MfGL-II were analyzed as their peracetylated derivatives by GLC and GLC-MS. MfGL-II (290 μg) and derived phosphomethyl ester were dephosphorylated by treatment with 48% aqueous HF at 4 °C for 36 h. Following solvent removal in vacuo over KOH, the product was peracetylated and analyzed as described earlier. d-Serine (Sigma) was methylated by acidic methanolysis (0.5 mHCl/MeOH, 85 °C, 40 min), and the resulting methyl ester was reduced with NaBH4 in methanol/water (1:1, v/v) to the corresponding 2-amino-1,3-propanediol which was then peracetylated and analyzed by GLC-MS. MfGL-II (1.3 mg, 1.2 μmol) was per-N,O-acetylated with acetanhydride/pyridine (1:2, v/v) at room temperature for 16 h in the dark followed byO-deacylation with 0.3 ml of 0.25 mNaOCH3 (75 μmol) in absolute methanol at 37 °C for 3 h prior to methylation analysis. The pH was adjusted to 4 with 0.1 m HCl/MeOH, and the phosphates were transformed to methyl esters with ethereal diazomethane (CH2N2) treatment at room temperature for 15 min. The solvents were removed under a stream of nitrogen, and the product was washed three times with ether/n-hexane (10:90, v/v) to remove liberated fatty acids. The sample was dried in vacuo and methylated (10Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3216) Google Scholar). The permethylated sample was extracted with water/chloroform (7 ml, 6:1, v/v); the aqueous layer was washed twice with 1 ml of chloroform, and the combined organic phases were washed again with 100 ml of water and taken to dryness under a stream of nitrogen. After methylation the product was dephosphorylated as described earlier, and an aliquot was directly peracetylated (see above) and analyzed by GLC-MS. The phosphate position in the glucose moiety was determined with another aliquot. MfGL-II was hydrolyzed (4 mtrifluoroacetic acid, 100 °C, 4 h), and the solvent was removed on a rotary evaporator. The product was redissolved in water/methanol (10:1, v/v), and the pH was adjusted to neutral with NaOH. The sample was then reduced (NaBD4) at room temperature overnight in the dark. The resulting product was peracetylated and analyzed by GLC-MS. MfGL-II (12.4 mg, 11.8 μmol) wasO-deacylated with 0.33 ml of 0.25 mNaOCH3 (82.5 μmol) in absolute methanol at 40 °C. The reaction course was followed by TLC (chloroform/methanol/water, 100:100:30, v/v). After 1 h no MfGL-II (R F = 0.38) and no lysoforms (R F = 0.16) were found, and a single spot remained at the origin. The solvent was reduced to near dryness under a stream of nitrogen, and 2 ml of chloroform/water (1:1, v/v) were added. The organic layer was washed twice with 2 ml of water, and the combined aqueous phases were loaded onto a Sephadex G-10 column (2 × 112 cm) and eluted with water. The fractions were analyzed by the anthrone test (11Shields R. Burnett W. Anal. Chem. 1960; 32: 885-886Crossref Scopus (155) Google Scholar), and positive fractions were combined, freeze-dried (yield 6.7 mg, quantitative), and analyzed by NMR. For TLC aluminum sheets of pre-coated silica gel 60 F254 thickness 0.2 mm (Merck) were used. The plates were developed at room temperature with chloroform/methanol/water, 100:100:30 (v/v). Glycolipid spots were detected by dipping the plates into a solution of 15% sulfuric acid in ethanol and heating to 110 °C for 2 min. Phospholipid spots were detected by the Dittmer-Lester reagent (12Dittmer J.C. Lester R.L. J. Lipid Res. 1964; 5: 126-127Abstract Full Text PDF PubMed Google Scholar) diluted with acetone (1:20, v/v). Amino groups containing lipids were detected by dipping the plates into a solution of 0.2% ninhydrin in acetone followed by heating to 60 °C for 15 min. GLC was performed with a Varian model 3700 chromatograph equipped with a capillary column of SPB-5 using a temperature gradient from 150 to 320 °C with a temperature rise of 5 °C/min. GLC-MS was carried out with a Hewlett-Packard model 5989 equipped with a capillary column (HP-5) under the same conditions as GLC. The ion source temperature was 200 °C. EI-mass spectra were recorded at 70 eV and CI-mass spectra were obtained with ammonia as reactant gas. MALDI-TOF mass spectra of positive ions were recorded with a Reflex II, Bruker-Franzen (Bremen, Germany) spectrometer in the linear mode (MALDI-LIN-TOF) at 28.5 kV acceleration voltage using 2,5-dihydroxybenzoic acid as matrix. The spectra were obtained from 0.5 μl of a 1:1 (v/v) mixture of aqueous matrix solution (25 mg of 2,5-dihydroxybenzoic acid in 500 μl) and native MfGL-II (1 μg/μl in chloroform/methanol, 1:1, v/v). NMR spectra were obtained with a Bruker AM-360 spectrometer. Spectra of native MfGL-II (20 mg) were recorded in 10:10:3 (CDCl3:MeOD:D2O, v/v, 0.5 ml) and referenced to δH = 7.26 ppm (CDCl3) and δC = 77.0 ppm (CDCl3), respectively. 1H NMR spectra were measured at 360 MHz (13C NMR, 90.5 MHz);31P NMR spectra were measured over a spectral range of 20,000 Hz with both 1H broad band coupling and decoupling. The spectrometer frequency was 145.78 MHz. 31P NMR signals were referenced to an 80% (mass/volume) solution of H3PO4 as an external standard. The spectra ofO-deacylated MfGL-II (6.7 mg) were recorded in 0.5 ml of D2O and referenced to δH = 2.225 ppm and δC = 31.45 ppm (external acetone). One- and two-dimensional homonuclear (1H,1H COSY, ROESY, and relayed COSY) and two-dimensional H-detected1H,13C HMQC and 1H,31P HMQC experiments were performed using standard Bruker (Rheinstetten, Germany) software (XWINNMR, version 1.1). The coupled and decoupled31P NMR spectra and the 1H,31P HMQC spectrum were recorded with O-deacylated MfGL-II in D2O at room temperature. The MfGL-II of M. fermentans(Incognitus strain) was obtained from 1.2 g of dry cells after Bligh and Dyer extraction. It was purified to homogeneity by silica gel column (1.5 × 3 cm, Kieselgel 60, Merck) eluted stepwise with mixtures of chloroform/methanol/water and methanol/water with increasing polarity (fractions 4–7). The yield of MfGL-II from 134 mg of total lipids was 21 mg of MfGL-II (15.7%) and, thus, the MfGL-II is a major lipid in the membrane ofM. fermentans. High levels of MfGL-II were also found with the PG18 strain. As the yields of biomass from the PG18 strain were much higher, all subsequent experiments were performed with this strain. The MfGL-II was found to be degraded by mild acidic methanolysis indicating that the fatty acids were ester-linked. Methyl palmitate (16:0) and methyl stearate (18:0) were identified as the major fatty acids in a molar ratio of 3.6:1 similar to that described previously (8Deutsch J. Salman M. Rottem S. Eur. J. Biochem. 1995; 227: 897-902Crossref Google Scholar). Strong methanolysis (2 m HCl/MeOH, 85 °C, 16 h), dephosphorylation (48% HF), and peracetylation revealed, upon GLC-MS analysis, methyl hexose with the same retention time and fragmentation pattern (EI-MS) as peracetylated methyl α-d-glucose. In addition to the glucose, anN-containing substance as deduced from its odd-number molecular mass, M r = 217, in CI-MS ([M + H]+ = 218, [M + NH4]+ = 235) expressed a retention time and molecular weight being compatible with 1,3-di-O-acetyl-2-acetamido-propanediol. Since 2-amino-1,3-propanediol was not yet identified inMycoplasma, we tried to corroborate this analysis by a simple chemical synthesis for reference material starting fromd-serine which was methylated, reduced, and per-O-acetylated (see "Materials and Methods"). With GLC-MS analysis the resulting compound showed identical retention time and fragmentation pattern (EI-MS (Fig. 1) as well as CI-MS (spectra not shown)), thus substantiating the original assignment. GLC-MS analysis after dephosphorylation and peracetylation revealed a structure with a molecular weight of 506 (CI-MS) corresponding to glucose with glycosidically linked glycerol, thus indicating that glucose is directly bound to glycerol. The 2-amino-1,3-propanediol moiety was found as 1,3-di-O-acetyl-2-acetamido-propanediol derivative identical to that of the synthetic 2-amino-1,3-propanediol described above, thus indicating that 2-amino-1,3-propanediol is involved in the structure of MfGL-II as a phosphoester. Methylation analysis of native MfGL-II (N-acetylation, deacylation, methylation (10Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3216) Google Scholar), dephosphorylation (48% HF), hydrolysis (trifluoroacetic acid), and reduction (NaBD4)) was done in two steps. First, after methylation and dephosphorylation an aliquot of the sample was peracetylated and subjected to GLC-MS analysis. The EI-MS spectra revealed a fragmentation pattern corresponding to 1,2-di-O-methyl-3-O-(O-acetyl-tri-O-methyl-glucopyranosyl)glycerol (peaks at m/z 247 assigned to the glucopyranoside moiety and at m/z 103 assigned to the glycerol moiety with the typical McLafferty rearrangement atm/z 163 (spectra not shown)) indicating that glucose was substituted with one phosphate. In addition, 1,3-di-O-acetyl-2-(N-methylacetamido)propanediol was detected suggesting that the 2-amino-1,3-propanediol moiety was symmetrically substituted with two phosphates (spectra not shown). To identify the position of the phosphate residue on the glucopyranosyl moiety, the sample was hydrolyzed and reduced yielding 1,5,6-tri-O-acetyl-2,3,4-tri-O-methyl-glucitol (spectra not shown), indicating the presence of a 6-phosphate on the glucopyranosyl moiety. These findings suggested a linear structure of diacyl-glyceryl-glucopyranoside with 6′-O-(2"-amino-1",3"-diphospho-1",3"-propanediol)-α-d-glucopyranosyl-(1′→3)-glycerol as the hydrophilic backbone (Fig. 2). Because the phosphocholine moiety in native MfGL-II was not accessible by methylation analysis and GLC-MS spectrometry other analytical procedures were used. The molecular size of underivatized MfGL-II was investigated by MALDI-LIN-TOF mass spectrometry in the positive ion mode. Native MfGL-II showed two major pseudomolecular ions (m/z 1049.5 [MI + H]+ and m/z 1077.3 [MII + H]+) (Fig. 3) with various pseudomolecular ions having Na+ and K+ attached to MI and MII, respectively. The difference between the molecular weights (Δm/z = 28) suggested the presence of two molecular species expressing variability in the fatty acid components, representing a dipalmitoyl derivative of MfGL-II as the major component and a palmitoyl-stearoyl derivative as a minor component, respectively. The molecular weight of MI([MI + H]+ = 1049.5) is consistent with the formula C49H99O17N2P2and also with the structure shown in Fig. 8.Figure 8Structure of the main fraction of native MfGL-II.6′-O-[(3"-Phosphocholine-2"-amino-1",3"-propanediol)-αd-glucopyranosyl](1′→3)-1,2-dipalmitoyl-sn-glycerol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The presence of different fatty acid residues in the glycerol moiety was further supported by detecting methyl palmitate and methyl stearate in the GLC-MS analysis (see above). These findings were in good agreement with previous reports on fatty acid composition of MfGL-II (8Deutsch J. Salman M. Rottem S. Eur. J. Biochem. 1995; 227: 897-902Crossref Google Scholar). The structure of the MfGL-II was further elucidated by different NMR experiments on native as well as onO-deacylated MfGL-II. The results are summarized in TableI for 1H signals and in TableII for 13C signals. The assignment of various signals produced by one- and two-dimensional homo- and heteronuclear NMR spectroscopy (Fig.4, Fig. 5) were in good agreement with those of the diacyl-(glycosyl-6-phosphate)glycerol moiety recently identified inM. fermentans by Matsuda et al. (7Matsuda K. Kasama T. Ishizuka I. Handa S. Yamamoto N. Taki T. J. Biol. Chem. 1994; 269: 33123-33128Abstract Full Text PDF PubMed Google Scholar).Table I1 H chemical shifts of native and O-deacylated MfGL-IIResidueGlucoglycerolipidChemical shifts [ppm] and coupling constants [Hz]H-1aH-1bH-2H-3(a)H-3bH-4H-5H-6aH-6bGro (A) Native3.991-aNonresolved multiplet.4.355.203.563.72(J 1a,1b12.1)(J 1b,22.4)(J 3a,3b10.8)(J 3b,2 5.5)O-Deacylated3.601-aNonresolved multiplet.3.693.961-aNonresolved multiplet.3.503.83(J 1a,1b11.7)(J 1b,2 4.4)(J 3a,22.5)(J 3a,3b 10.5)(J 3b,23.7)Glc (B) Native4.743.373.573.433.553.904.08(J 1,23.4)(J 2,3 9.7)(J 3,49.2)(J 4,5 9.3)(J 5,6a5.1)(J 6a,6b 11.5)(J 5,6b7.4)O-Deacylated4.943.593.753.503.814.081-aNonresolved multiplet.4.121-aNonresolved multiplet.(J 1,24.0)(J 2,3 9.5)(J 3,49.5)AP (C) Native3.971-bAssignments in one line may have to be reversed.3.971-bAssignments in one line may have to be reversed.3.604.031-bAssignments in one line may have to be reversed.4.031-bAssignments in one line may have to be reversed. O-Deacylated3.961-aNonresolved multiplet.,1-bAssignments in one line may have to be reversed.3.961-aNonresolved multiplet.,1-bAssignments in one line may have to be reversed.3.353.961-aNonresolved multiplet.,1-bAssignments in one line may have to be reversed.3.961-aNonresolved multiplet.,1-bAssignments in one line may have to be reversed.Cho (D) Native4.211-aNonresolved multiplet.4.211-aNonresolved multiplet.3.561-aNonresolved multiplet.3.121-cSinglet −N+(CH3)3, (integral 9H).O-Deacylated4.331-aNonresolved multiplet.4.331-aNonresolved multiplet.3.681-aNonresolved multiplet.3.231-cSinglet −N+(CH3)3, (integral 9H).For solvents and references see "Materials and Methods." For fatty acid residues the chemical shifts (in ppm) are as follows: H-2, 2.24; H-3, 1.52; H-4 to H-15, 1.18; and H-16, 079.1-a Nonresolved multiplet.1-b Assignments in one line may have to be reversed.1-c Singlet −N+(CH3)3, (integral 9H). Open table in a new tab Table II13 C chemical shifts of native and O-deacylated MfGL-IIResidueGlucoglycerolipidChemical shifts [ppm] and (heteronuclear coupling constants [Hz])C-1C-2C-3C-4C-5C-6Gro (A)Native62.4369.5965.69O-Deacylated63.5971.8270.22Glc (B)Native98.9571.1372.4368.3670.7063.38O-Deacylated100.0172.6174.0970.2871.9565.40AP (C)Native62.012-aAssignments may have to be reversed.51.2161.822-aAssignments may have to be reversed.(J C-1,P4.9)(J C-2,P7.1/7.7)(J C-3,P 4.4)O-Deacylated66.6551.8466.43(J C-1,P4.9)(J C-2,P8.2/7.7)(J C-3,P 4.4)Cho (D)Native58.9065.6153.36O-Deacylated60.6667.1955.16For solvents and references see "Materials and Methods." For fatty acid residues the chemical shifts (in ppm) are as follows: C-1, 173.72, and 173.47; C-2, 33.67 and 33.52; C-3, 24.37 and 24.28; C-4 to C-13, 28.79 to −28.54; ω-1, 22.02; ω-2, 31.29; and ω, 13.23.2-a Assignments may have to be reversed. Open table in a new tab Figure 5Two-dimensional H-detected1H,13C HMQC spectrum of the native MfGL-II. A, the whole spectrum with the assignment of the signals of the glycerol moiety (A) and fatty acids (FA);B, extension of the area shown in A by rectangle with the assignment of the signals of the glucosyl (B), 2-amino-1,3-propanediol (C), and choline (D) residues. The corresponding parts of the 1H and13C NMR spectra are displayed along thehorizontal and vertical axes, respectively. For numbering of atoms see Tables I and II.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For solvents and references see "Materials and Methods." For fatty acid residues the chemical shifts (in ppm) are as follows: H-2, 2.24; H-3, 1.52; H-4 to H-15, 1.18; and H-16, 079. For solvents and references see "Materials and Methods." For fatty acid residues the chemical shifts (in ppm) are as follows: C-1, 173.72, and 173.47; C-2, 33.67 and 33.52; C-3, 24.37 and 24.28; C-4 to C-13, 28.79 to −28.54; ω-1, 22.02; ω-2, 31.29; and ω, 13.23. In the O-deacylated MfGL-II, the resonances of H-1aGro, H-1bGro, and H-2Gro were shifted upfield (Table I), whereas the positions of resonances of the other glycerol proton signals (H-3aGro and H-3bGro) were unchanged. These findings indicated that the glycosyl moiety is linked to O-3Gro as is found in an aminophosphoglycolipid of Clostridium innocuum (13Fischer W. Hartmann R. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1994; 223: 879-892Crossref PubMed Scopus (13) Google Scholar). The α-anomeric configuration of the hexose was inferred by the coupling constant J 1,2 of 3.4 Hz. The other coupling constants of the hexose (J 2,3,J 3,4, and J 4,5) were larger than 8 Hz, indicating an α-glucopyranoside configuration. The chemical shifts of C-2Glc, C-3Glc, C-4Glc, C-5Glc, and C-6Glc as well as the coupling constant J 6a,6b were similar to those of α-glucose 6-phosphate (14Bock K. Pedersen C. Adv. Carbohydr. Chem. Biochem. 1983; 41: 27-66Crossref Scopus (1462) Google Scholar), providing evidence that a phosphate group was bonded to the O-6 hydroxyl of glucose. A ROESY experiment with O-deacylated MfGL-II showed nuclear Overhauser effects between H-1Glc and both H-3aGro and H-3bGro indicating a close proximity of these protons consistent with an α-(1′→3) linkage (Fig. 6). The assignment of the 1H NMR and 13C NMR signals was done by 1H,1H COSY and1H,13C COSY experiments in which the assignment of C-1AP and C-3AP may be interchanged because it was not possible to distinguish clearly between these carbon signals. The C-2AP signal in the native MfGL-II was a doublet of doublet with two similar heteronuclear coupling constants ofJ C-2,P 7.1 Hz and J C-2,P′ 7.7 Hz, respectively, indicating that the 2-amino-1,3-propanediol is symmetrically substituted by two phosphate residues. The chemical shifts, except those for C-3AP and H-3a,bAP, approximated the values reported for a phosphoglycolipid isolated from the taxonomically closely related C. innocuum, which contained a 1-phosphate-2-amino-1,3-propanediol at O-6 of α-galactopyranosyl moiety (13Fischer W. Hartmann R. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1994; 223: 879-892Crossref PubMed Scopus (13) Google Scholar). The differences for C-3APand H-3a,bAP indicated that in the MfGL-II the 2-amino-1,3-propanediol moiety is substituted at O-3, whereas in the phosphoglycolipid of C. innocuum it was not. The H-1Cho signal at 4.21 ppm coupled with the H-2Cho signal appeared as a superposition with other signals. The -N+(CH3)3 group was assigned to an intense signal (integral 9H) at 3.12 ppm. The chemical shifts were very close to the values reported for GGPL-I containing a choline phosphate group at O-6 of α-glucosyl moiety (7Matsuda K. Kasama T. Ishizuka I. Handa S. Yamamoto N. Taki T. J. Biol. Chem. 1994; 269: 33123-33128Abstract Full Text PDF PubMed Google Scholar). The 1H and 13C NMR signals of the ester-linked fatty acids, summarized in Tables I and II, were in the expected range (13Fischer W. Hartmann R. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1994; 223: 879-892Crossref PubMed Scopus (13) Google Scholar). Also present in the 1H NMR spectrum were signals of olefinic protons (5.25–5.26 ppm) corresponding to unsaturated fatty acids. This finding agrees well with data from GLC and GLC-MS indicating trace amounts of unsaturated fatty acids (18:1). Different experiments were performed to determine the position of the phosphate groups in MfGL-II. Various one-dimensional and two-dimensional 31P NMR experiments of native MfGL-II measured in 100:100:30 (CDCl3:MeOD:D2O, v/v) gave no satisfactory resolution for the 31P signals. Therefore, the phosphate linkages were determined withO-deacylated MfGL-II. The broad band-decoupled31P NMR spectrum of the O-deacylated MfGL-II showed two singlets at 1.09 and 0.08 ppm, respectively (spectrum not shown). However, in the proton-coupled gated 31P NMR spectrum, the two phosphate groups gave identical multiplets due to the coupling with two structurally related methylene protons (O-CH2Glc and O-CH2AP versus O-CH2AP and O-CH2Cho). The signal intensities upon integration matched precisely the theoretically expected binomial coefficient of 1:4:6:4:1 (found, 1.0:4.3:6.4:4.4:1.3). This splitting is typical for phosphate groups symmetrically substituted with two methylene groups. From these data it was concluded that in MfGL-II four methylene groups are attached to two phosphate residues: one from choline, two from the 2-amino-1,3-propanediol, and one from the Glc residue. The 1H,31P HMQC spectrum (Fig.7) revealed unambiguously the linkage of the phosphate groups. Both 31P signals (P-1 and P-2) showed highly complex cross-peaks. The 31P signal at 1.09 ppm (P-1) correlated with a multiplet of H-1a,bAP (∼3.96 ppm) and with the H-6aGlc and H-6bGlc signals (∼4.08 and ∼4.12 ppm, respectively). The other 31P signal (P-2) at 0.08 ppm showed cross-peaks with a multiplet of H-3a,bAP (∼3.96 ppm) and with the H-1a,bChosignal (4.33 ppm). Taken together these findings confirmed the structure of the MfGL-II as 6′-O-(3"-phosphocholine-2"-amino-1"-phospho1",3"-propanediol)-α-d-glucopyranosyl-(1′→3)-1,2-diacyl-glycerol. The results of the chemical analyses, GLC-MS analyses with synthetic reference compounds, MALDI-TOF mass spectrometry and various NMR experiments, described above were identical with those obtained by analyzing the MfGL-II of M. fermentans incognitus strain. The best structure suggested by these results is shown in Fig.8. The role of AIDS-associated M. fermentans in the pathogenesis of the disease has not been yet defined. An interesting hypothesis is based on the ability of M. fermentans to fuse with lymphocytes (5Franzoso G. Dimitrov D.S. Blumenthal R. Barile M.F. Rottem S. FEBS Lett. 1992; 303: 251-254Crossref PubMed Scopus (32) Google Scholar). It has been suggested that Mycoplasmacomponents released to the lymphocyte uponMycoplasma-lymphocyte fusion adversely affected lymphocyte function (6Salman M. Deutsch J. Tarshis M. Naot Y. Rottem S. FEMS Microbiol. Lett. 1994; 123: 255-260Crossref PubMed Scopus (23) Google Scholar). The fusogenicity of M. fermentans cells is correlated with a membrane-associated MfGL-II (6Salman M. Deutsch J. Tarshis M. Naot Y. Rottem S. FEMS Microbiol. Lett. 1994; 123: 255-260Crossref PubMed Scopus (23) Google Scholar); although several studies have investigated the chemical nature of this lipid, there is presently no consensus on the chemical structure. We have addressed this issue with the aim to establish unequivocally the primary structure of the MfGL-II previously designated as compound X (6Salman M. Deutsch J. Tarshis M. Naot Y. Rottem S. FEMS Microbiol. Lett. 1994; 123: 255-260Crossref PubMed Scopus (23) Google Scholar, 8Deutsch J. Salman M. Rottem S. Eur. J. Biochem. 1995; 227: 897-902Crossref Google Scholar) or GGPL-III (7Matsuda K. Kasama T. Ishizuka I. Handa S. Yamamoto N. Taki T. J. Biol. Chem. 1994; 269: 33123-33128Abstract Full Text PDF PubMed Google Scholar). MfGL-II was isolated from dried cells of M. fermentans PG18 using the extraction protocol described previously (8Deutsch J. Salman M. Rottem S. Eur. J. Biochem. 1995; 227: 897-902Crossref Google Scholar). Using various analytical procedures (GLC, MALDI-TOF mass spectrometry, and NMR spectrometry), we showed that while both compounds were isolated by identical procedures, the structure proposed (8Deutsch J. Salman M. Rottem S. Eur. J. Biochem. 1995; 227: 897-902Crossref Google Scholar) has to be revised. We have unequivocally identified the structure of the major polar membrane lipid of M. fermentans PG18 as 6′-O-(3"-phosphocholine-2"-amino1",3"-propanediol)-α-d-glucopyranosyl-(1′→3)-1,2-dipalmitoyl-glycerol (MfGL-II). MfGL-II shows high structural homology to GGPL-I (MfGL-I), a phosphocholine-containing glyceroglycolipid from M. fermentans (7Matsuda K. Kasama T. Ishizuka I. Handa S. Yamamoto N. Taki T. J. Biol. Chem. 1994; 269: 33123-33128Abstract Full Text PDF PubMed Google Scholar). The main difference between GGPL-I and MfGL-II is the presence of a 2-amino-1,3-propanediol moiety and an additional phosphate residue. Matsuda et al. (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar) described another phosphocholine-containing glycoglycerolipid which they termed GGPL-III and postulated to possess a structure very similar to GGPL-I but harboring an additional amino group (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar). Our data and those presented by Matsuda et al. (4Matsuda K. Harasawa R. Li J.-L. Kasama T. Taki T. Handa S. Yamamoto N. Microbiol. Immunol. 1995; 39: 307-313Crossref Scopus (15) Google Scholar) show that GGPL-III and the MfGL-II could be structurally identical compounds. It is of great interest that in both MfGL-I and MfGL-II the phosphocholine moiety is the terminal, exposed structural motif. As the two glycoglycerolipids constitute the major lipid fraction of theM. fermentans PG18 membrane, it appears likely that phosphocholine is a key structure in cellular adhesion of M. fermentans to host cells. Phosphocholine has recently been reported to be a constituent of the glycosphingolipid of the annelid Pheretima hilgendorfi(15Sugita M. Fujii H. Inagaki F. Suzuki M. Hayata C. Hori T. J. Biol. Chem. 1992; 267: 22595-22598Abstract Full Text PDF PubMed Google Scholar), of lipoteichoic acid (16Behr T. Fischer W. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1992; 207: 1063-1075Crossref PubMed Scopus (123) Google Scholar), and of the cell wall associated teichoic acid of Streptococcus pneumoniae (17Fischer W. Behr T. Hartmann R. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1993; 215: 851-857Crossref PubMed Scopus (170) Google Scholar). InHaemophilus influenzae phosphocholine was also identified on the surface and was found to be attached to the lipopolysaccharide (18Weiser J.N. Shchepetov M. Chong S.T.H. Infect. Immun. 1997; 65: 943-950Crossref PubMed Google Scholar,19Risberg A. Schweda E.K.H. Jansson P.-E. Eur. J. Biochem. 1997; 243: 701-707Crossref Scopus (44) Google Scholar). All bacteria having phosphocholine as part of their surface structures colonize the human nasopharynx. This observation supports the assumption that phosphocholine plays a key role in potentiating microorganism-host interaction. There are reports that the human C-reactive protein, an acute phase protein, has the ability to bind to phosphocholine-containing pneumococcal C-polysaccharide (20Volanakis J.E. Kaplan M.H. Proc. Soc. Exp. Biol. Med. 1971; 136: 612-614Crossref PubMed Scopus (330) Google Scholar). This complex activates the complement system. Thus, bacteria-associated choline appears to play an important role not only in cellular adhesion but also in subsequent inflammatory reactions. The 2-aminol-1,3-diphosphate-1,3-propanedio group has so far not been identified in nature. Fischer et al. (13Fischer W. Hartmann R. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1994; 223: 879-892Crossref PubMed Scopus (13) Google Scholar) described a 2-amino-1,3-propanediol-3-phosphate-carrying diradylglyceroglycolipid as a major membrane lipid of C. innocuum. Interestingly,Clostridia are taxonomically closely related toMycoplasma (21Johnston N.C. Goldfine H. Fischer W. Microbiology. 1994; 140: 105-111Crossref Scopus (29) Google Scholar), and it is tempting to speculate that in both organisms the 2-amino-1,3-propanediol is formed by transamination of dihydroxyacetone, a known intermediate of glycolysis (13Fischer W. Hartmann R. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1994; 223: 879-892Crossref PubMed Scopus (13) Google Scholar). This indicates that in M. fermentans glucose has at least two possible fates as follows: catabolic, for energy metabolism, and anabolic, for the biosynthesis of structural molecules. Recently 2-amino-1,3-propanediol has been detected as anO-antigen component of the lipopolysaccharide ofVibrio cholerae H11. Here, however, this moiety is linked by an amide bond to the carboxyl group of d-galacturonosyl residues (22Vinogradov E.V. Holst O. Thomas-Oates J.E. Broady K.W. Brade H. Eur. J. Biochem. 1992; 210: 491-498Crossref PubMed Scopus (78) Google Scholar). While studies are needed to elucidate the molecular basis for mycoplasmal adhesion to host cells, the finding of phosphocholine as a terminal structure represents an important step toward understanding the molecular mechanism of the pathogenicity of M. fermentans. We thank H. Moll for GLC-MS analyses and chemical synthesis, H.-P. Cordes for NMR analyses, and Dr. B. Lindner and H. Lüthje for measuring the MALDI-TOF spectra. We also thank Dr. E. Lüneberg (University of Würzburg) for help in cultivating M. fermentans. We are indebted to Drs. Y. A. Knirel and R. L. Pardy for critically reading the manuscript.
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