Primary Structure of a New Phosphocholine-containing Glycoglycerolipid of Mycoplasma 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 (1). 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 (2), and its role in the pathogenesis of rheumatoid arthritis, interest in M. fermentans has recently increased (3). Although little is known of the molecular mechanisms underlying M. fermentans pathogenicity (4), it has been shown that human immunodeficiency virus-associated cytopathic effects could be increased by the presence of M. fermentans (2) and that M. fermentans is capable of fusing with T-cells and peripheral lymphocytes (5).
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. (6) 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. (4) isolated two glycoglycerolipids (GGPL-I 1 and GGPL-III) from M. fermentans. GGPL-I structure was shown to be 6Ј-O-phosphocholine-␣-D-glucopyranosyl-1,2-diacyl-sn-glycerol (7) as elucidated by mass and NMR spectroscopy. It was later shown (4) 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 (4). Both GGPLs were shown to be species-specific major lipid antigens of M. fermentans (4).
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. 2 Furthermore, we show that both glycolipids of M. fermentans, GGPL-I and GGPL-III, share the basic structure of 6Ј-O-phospho-␣-D-glucopyranosyl-(1Ј33)-1,2-diacyl-glycerol (7) but differ in their polar head groups.

MATERIALS AND METHODS
Growth of the Organism-Cultures of M. fermentans strain PG18 and strain Incognitus (provided by S.-C. Lo, Armed Forces Institute of Pathology, Washington, D.C.) were grown in a modified Channock medium (8) 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 (8) with yields ranging from 130 to 160 mg dry weight per liter of medium.
Lipid Extraction and Purification-Freeze-dried cells were suspended in 25 mM Tris/HCl, pH 7.5, containing 0.25 M 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 (9) 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 ϫ 3 cm; Kieselgel 60, 230 -400 mesh, Merck), equilibrated with * This work was supported by the German-Israeli Foundation for Scientific Research and Development, the Fonds der Chemischen Industrie (EThR), and the Deutsche Forschungsgemeinschaft Grants SFB 367, B2; SFB 470, B4 and B5. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Mild Methanolysis-MfGL-II (370 g) was dissolved in 0.5 M 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.
Strong Methanolysis and Peracetylation-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.
Dephosphorylation-MfGL-II (290 g) 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.
Methylation Analysis-MfGL-II (1.3 mg, 1.2 mol) was per-N,Oacetylated with acetanhydride/pyridine (1:2, v/v) at room temperature for 16 h in the dark followed by O-deacylation with 0.3 ml of 0.25 M NaOCH 3 (75 mol) 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 (CH 2 N 2 ) 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 (10). 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 M trifluoroacetic 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 (NaBD 4 ) at room temperature overnight in the dark. The resulting product was peracetylated and analyzed by GLC-MS.
O-Deacylation-MfGL-II (12.4 mg, 11.8 mol) was O-deacylated with 0.33 ml of 0.25 M NaOCH 3 (82.5 mol) 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 ϭ 0.38) and no lysoforms (R F ϭ 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 ϫ 112 cm) and eluted with water. The fractions were analyzed by the anthrone test (11), and positive fractions were combined, freeze-dried (yield 6.7 mg, quantitative), and analyzed by NMR.
Thin Layer Chromatography-For TLC aluminum sheets of precoated silica gel 60 F 254 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 (12) 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.
Gas-Liquid Chromatography-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-Mass Spectrometry-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. EImass spectra were recorded at 70 eV and CI-mass spectra were obtained with ammonia as reactant gas.
Nuclear Magnetic Resonance (NMR) Spectroscopy-NMR spectra were obtained with a Bruker AM-360 spectrometer. Spectra of native MfGL-II (20 mg) were recorded in 10:10:3 (CDCl 3 :MeOD:D 2 O, v/v, 0.5 ml) and referenced to ␦ H ϭ 7.26 ppm (CDCl 3 ) and ␦ C ϭ 77.0 ppm (CDCl 3 ), respectively. 1 H NMR spectra were measured at 360 MHz ( 13 C NMR, 90.5 MHz); 31 P NMR spectra were measured over a spectral range of 20,000 Hz with both 1 H broad band coupling and decoupling. The spectrometer frequency was 145.78 MHz. 31 P NMR signals were referenced to an 80% (mass/ volume) solution of H 3 PO 4 as an external standard. The spectra of Odeacylated MfGL-II (6.7 mg) were recorded in 0.5 ml of D 2 O and referenced to ␦ H ϭ 2.225 ppm and ␦ C ϭ 31.45 ppm (external acetone). One-and two-dimensional homonuclear ( 1 H, 1 H COSY, ROESY, and relayed COSY) and two-dimensional H-detected 1 H, 13 C HMQC and 1 H, 31 P HMQC experiments were performed using standard Bruker (Rheinstetten, Germany) software (XWINNMR, version 1.1). The coupled and decoupled 31 P NMR spectra and the 1 H, 31 P HMQC spectrum were recorded with O-deacylated MfGL-II in D 2 O at room temperature.

Isolation of MfGL-II-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 ϫ 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 of M. 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.
Fatty Acid Analysis-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 (8). 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-Oacetyl-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.

Compositional Analysis of MfGL-II by GLC and GLC-MS
Methylation analysis of native MfGL-II (N-acetylation, deacylation, methylation (10), dephosphorylation (48% HF), hydrolysis (trifluoroacetic acid), and reduction (NaBD 4 )) 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 fragmen-tation pattern corresponding to 1,2-di-O-methyl-3-O-(O-acetyltri-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 at m/z 163 (spectra not shown)) indicating that glucose was substituted with one phosphate. In addition, 1,3-di-Oacetyl-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.
Because the phosphocholine moiety in native MfGL-II was not accessible by methylation analysis and GLC-MS spectrometry other analytical procedures were used.  (Fig. 3) with various pseudomolecular ions having Na ϩ and K ϩ attached to M I and M II , respectively. The difference between the molecular weights (⌬m/z ϭ 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 M I ([M I ϩ H] ϩ ϭ 1049.5) is consistent with the formula C 49 H 99 O 17 N 2 P 2 and also with the structure shown in Fig. 8.
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 (8).
NMR Spectroscopy-The structure of the MfGL-II was further elucidated by different NMR experiments on native as well as on O-deacylated MfGL-II. The results are summarized in Table I for 1 H signals and in Table II for 13 C signals. The assignment of various signals produced by one-and two-dimen-sional homo-and heteronuclear NMR spectroscopy (Fig. 4, Fig.  5) were in good agreement with those of the diacyl-(glycosyl-6phosphate)glycerol moiety recently identified in M. fermentans by Matsuda et al. (7).
In the O-deacylated MfGL-II, the resonances of H-1a Gro , H-1b Gro , and H-2 Gro were shifted upfield (Table I), whereas the positions of resonances of the other glycerol proton signals (H-3a Gro and H-3b Gro ) were unchanged. These findings indicated that the glycosyl moiety is linked to O-3 Gro as is found in an aminophosphoglycolipid of Clostridium innocuum (13).
The ␣-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 ␣-glucopyranoside configuration. The chemical shifts of C-2 Glc , C-3 Glc , C-4 Glc , C-5 Glc , and C-6 Glc as well as the coupling constant J 6a,6b were similar to those of ␣-glucose 6phosphate (14), 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-1 Glc and both H-3a Gro and H-3b Gro indicating a close proximity of these protons consistent with an ␣-(1Ј33) linkage (Fig. 6).
The assignment of the 1 H NMR and 13 C NMR signals was done by 1 H, 1 H COSY and 1 H, 13 C COSY experiments in which  the assignment of C-1 AP and C-3 AP may be interchanged because it was not possible to distinguish clearly between these carbon signals. The C-2 AP signal in the native MfGL-II was a doublet of doublet with two similar heteronuclear coupling constants of J C-2,P 7.1 Hz and J C-2,P Ј 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-3 AP and H-3a,b AP , 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 ␣-galactopyranosyl moiety (13). The differences for C-3 AP and H-3a,b AP 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-1 Cho signal at 4.21 ppm coupled with the H-2 Cho signal appeared as a superposition with other signals. The -N ϩ (CH 3 ) 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 ␣-glucosyl moiety (7).
The 1 H and 13 C NMR signals of the ester-linked fatty acids,  Table I.  summarized in Tables I and II, were in the expected range (13). Also present in the 1 H 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 31 P NMR experiments of native MfGL-II measured in 100:100:30 (CDCl 3 :MeOD:D 2 O, v/v) gave no satisfactory resolution for the 31 P signals. Therefore, the phosphate linkages were determined with O-deacylated MfGL-II. The broad band-decoupled 31 P NMR spectrum of the Odeacylated MfGL-II showed two singlets at 1.09 and 0.08 ppm, respectively (spectrum not shown). However, in the protoncoupled gated 31 P NMR spectrum, the two phosphate groups gave identical multiplets due to the coupling with two struc-  Tables I and II. 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-1,3propanediol, and one from the Glc residue. The 1 H, 31 P HMQC spectrum (Fig. 7) revealed unambiguously the linkage of the phosphate groups. Both 31 P signals (P-1 and P-2) showed highly complex cross-peaks. The 31 P signal at 1.09 ppm (P-1) correlated with a multiplet of H-1a,b AP (ϳ3.96 ppm) and with the H-6a Glc and H-6b Glc signals (ϳ4.08 and ϳ4.12 ppm, respectively). The other 31 P signal (P-2) at 0.08 ppm showed crosspeaks with a multiplet of H-3a,b AP (ϳ3.96 ppm) and with the H-1a,b Cho signal (4.33 ppm).

DISCUSSION
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 (5). It has been suggested that Mycoplasma com- ponents released to the lymphocyte upon Mycoplasma-lymphocyte fusion adversely affected lymphocyte function (6). The fusogenicity of M. fermentans cells is correlated with a membrane-associated MfGL-II (6); 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 (6,8) or GGPL-III (7).
MfGL-II shows high structural homology to GGPL-I (MfGL-I), a phosphocholine-containing glyceroglycolipid from M. fermentans (7). 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. (4) 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 (4). Our data and those presented by Matsuda et al. (4) 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 the M. 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 (15), of lipoteichoic acid (16), and of the cell wall associated teichoic acid of Streptococcus pneumoniae (17). In Haemophilus influen-  Table I. zae phosphocholine was also identified on the surface and was found to be attached to the lipopolysaccharide (18,19). 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 phosphocholinecontaining pneumococcal C-polysaccharide (20). 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. (13) 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 to Mycoplasma (21), 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 (13). 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 an O-antigen component of the lipopolysaccharide of Vibrio cholerae H11. Here, however, this moiety is linked by an amide bond to the carboxyl group of D-galacturonosyl residues (22).
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.