Archaea Contain a Novel Diether Phosphoglycolipid with a Polar Head Group Identical to the Conserved Core of Eucaryal Glycosyl Phosphatidylinositol*

The structure of a major ether polar lipid of the methanogenic archaeon Methanosarcina barkeri was identified as glucosaminyl archaetidylinositol. This lipid had archaeol (2,3-di-O-phytanyl-sn-glycerol) as a core lipid portion, and the polar head group consisted of 1 mol each of phosphate, myo-inositol and D-G~cN. The polar head group was identified by means of chem- ical degradations, phosphatidylinositol-specific phospholipase C treatment, permethylation analysis, and fast atom bombardment-mass spectrometry as glucos-aminylinositol phosphate, which was linked to the glycerol backbone via a phosphodiester bond. The stereochemical configuration of the phospho-myo-inositol residue of glucosaminyl archaetidylinositol was determined to be ~-D-myo-inosito~ 1-phosphate by measur- ing optical rotation of phospho-myo-inositol prepared by nitrous acid deamination and alkaline hydrolysis from the lipid. ‘H NMR of the intact lipid showed that GlcN was linked to C-6 position spectra were obtained with a data size of 1024 X 2048 and spectral width of 2500 X 5000 Hz. The mixing time in the latter was 50 ms. Materials-Phytane, biphytane, archaeol, caldarchaeol, hydroxyar-chaeol, archaetidyl-myo-inositol, and archaetidic acid were prepared from total lipids of M. barkeri (Nishihara and Koga, 1991) or Meth-anobacterium thermoautotrophicum (Nishihara et al., 1989). Phos- phatidylinositol-specific phospholipase C from Bacillus thuringiensis was purchased from Funakoshi Co., Ltd. (Japan). Chitohiose was the product of Wako Pure Chemical Industries Ltd. (Japan).

The structure of a major ether polar lipid of the methanogenic archaeon Methanosarcina barkeri was identified as glucosaminyl archaetidylinositol. This lipid had archaeol (2,3-di-O-phytanyl-sn-glycerol) as a core lipid portion, and the polar head group consisted of 1 mol each of phosphate, myo-inositol and D-G~cN. The polar head group was identified by means of chemical degradations, phosphatidylinositol-specific phospholipase C treatment, permethylation analysis, and fast atom bombardment-mass spectrometry as glucosaminylinositol phosphate, which was linked to the glycerol backbone via a phosphodiester bond. The stereochemical configuration of the phospho-myo-inositol residue of glucosaminyl archaetidylinositol was determined to be ~-D-myo-inosito~ 1-phosphate by measuring optical rotation of phospho-myo-inositol prepared by nitrous acid deamination and alkaline hydrolysis from the lipid. 'H NMR of the intact lipid showed that GlcN was linked to C-6 position of myo-inositol as an a-anomer. It is, finally, concluded that the complete structure of this lipid is 2,3-di-O-phytanyl-sn-glycerol-phospho-l'[6'-0-(2"-amino-2"-deoxy-cw-~-glucopyranosyl)]-1'-D-myo-inositol. This lipid has a hybrid nature of an archaeal feature in alkyl glycerol diether core portion and an eucaryal feature in the polar head group identical to the conserved core structure (GlcNp(~l-6)-myo-inositol 1-phosphate) of glycosylated phosphatidylinositol which serves as a membrane protein anchor in eucaryal cells.
Comparison of small subunit ribosomal RNA base sequences (Woese et al., 1990;Winker and Woese, 1991) and phylogenetic trees of duplicated genes (Iwabe et al., 1989) show that all life on the earth divides into three primary groupings, the Bacteria (formerly eubacteria), the Archaea (formerly archaebacteria), and the Eucarya (formerly eukaryotes) (Woese et al., 1990;Winker and Woese, 1991). The unique biochemical properties of each primary group support * 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.
§ To whom all correspondence should be addressed. Tel.: 81-93-603-1611 (ext. 2242); Fax: 81-93-602-5482. the concept of the three domains (a new rank proposed by Woese et al. (1990)). Recent phylogenetic studies have revealed a closer relatedness of the Archuea to the Eucarya than to the Bacteria (Iwabe et al., 1989;Woese et al., 1990), and this has been supported by several aspects of biochemical features. The polar lipid of archaea is one of the representative features which distinguishes archaea from the other organisms. The structures of the core lipids (di-0-radyl glycerol portion) of archaeal ether polar lipid are quite different from those of ester lipids of bacteria and eucarya (Langworthy et al., 1982). On the other hand, most of the polar head groups of the ether lipids are common to that of ester lipids of the bacteria and eucarya, e.g. phosphoethanolamine, phosphoserine, phospho-myo-inositol, or disaccharides (Nishihara et al., 1989;Nishihara and Koga, 1991). These facts bring up the interest in more detailed analyses of complete structures of ether lipids and in search of a phylogenetic relationship of archaeal and other organisms' lipids.
Methunosarcina barkeri is an archaeon (archaebacteria) which produces methane from acetate, methanol, or Hz + COz. Several major polar lipids of M. barkeri were found by thin-layer chromatography (TLC) (Nishihara and Koga, 1991). Four of these structures were determined as hydroxyarchaetidylserine, hydroxyarchaetidyl-myo-inositol (major polar lipids), which have a new ether core lipid (hydroxyarchaeol), and standard archaetidylserine and archaetidyl-myoinositol (minor polar lipids). We have determined the complete structure of the most predominant lipid (designated as PNLG in Nishihara and Koga (1991)) and report it here. The structure of the polar head group was identical to that of the common part of the eucaryal glycosylated phosphatidylinosi-to1 (GPI)' membrane anchor while the core lipid was typical of archaeal lipids. This is the first report that shows the apparent relationship in the membrane lipids between the Archaea and the Eucarya. The nomenclature of archaeal lipids proposed by us (Nishihara et al., 1987) and the expression of absolute stereochemical configurations of phosphoinositol recommended by IUPAC-IUB (1974) are used throughout in this paper.

Growth of Organism, Extraction and Purification of Lipids-".
barkeri (DSM 800) was grown as reported previously (Nishihara and Koga, 1991). Extraction (Nishihara et al., 1987) and DEAE-cellulose column fractionation of total lipid of M. barkeri were carried out as described previously (Nishihara et al., 1989). PNLG was eluted from the column by methanol and further purified by TLC with solvent A (see below).
Shimadzu GC 9A gas-liquid chromatograph equipped with flame ionization detectors. Hydrocarbons, acetylated or trimethylsilylated derivatives of inositol, were analyzed as previously described (Nishihara et al., 1989). GlcN was chromatographed as an acetylated derivative under the same conditions as those for inositol acetate except that the temperature was 240 "C. Partially methylated alditol acetates were analyzed with a bonded DB-225 column (30 m; film thickness, 0.25 mm; J & W Scientific, USA) with a temperature program of 160-220 "C a t 2 "C/min. Chitobiose (GlcN(P1-4)GlcN) was used as the GlcN linkage standard. The amino compound in an aqueous fraction of methanolysate or the hydrolysate of PNLG was identified and determined by an amino acid analyzer (model 835, Hitachi, Japan).
Analytical Methods and Degradatiue Procedures-Phosphorus was determined by the method of Bartlett (1959). myo-Inositol and GlcN were estimated by GLC after acetylation with hexacosane as an internal standard. Hydrocarbon chains were prepared from the lipids by hydroiodic acid degradation followed by LiAIH, reduction as previously reported (Nishihara et al., 1989). Core lipid of PNLG was prepared by splitting off the polar head group by acid methanolysis (5% HCl/methanol at 100 "C for 3 h). The polar head group obtained in the aqueous fraction of acid methanolysis of PNLG was further hydrolyzed by strong acid hydrolysis (6 M HCI, 100 "C, for 18 h). GlcN moiety of PNLG was purified from the strong acid hydrolysate by paper chromatography with solvent F. The GlcN eluted from the paper was dissolved in 0.085 M HCl for measuring optical rotation. Authentic GlcN HC1 salt was used as the standard of optical rotation. Dephosphorylation with HF was performed as described by Morii et al. (1986). Nitrous acid deamination of PNLG was carried out as follows. To the lipid (10-20 mg) dried up in a test tube, 0.6 ml of acetic acid and 1.8 ml of 2 M aqueous NaN02 solution were added, and the mixture was incubated for 4 h at 58 "C. The chloroformsoluble product was recovered by partitioning with 4 ml of chloroform and methanol (l:l, by volume). Inositol phosphate moiety was prepared from the archaetidyl-myo-inositol (nitrous acid deamination product of PNL6) by alkaline hydrolysis (Pizer and Ballou, 1959) with minor modifications (Nishihara et al., 1989). Hydrolysis of PNLG with phosphatidylinositol-specific phospholipase C was carried out as described (Taguchi et al., 1980). Permethylation and preparation of partially methylated alditol acetates were performed by the method of Yang and Hakomori (1971) after acetylation of a free amino group (McConville and Bacic, 1989).
Physical Measurements-IR spectrum was recorded as a thin film using a Shimadzu IR spectrometer IR450S. Optical rotations were measured at 589 nm with a high sensitivity polarimeter (PM-201, Otsuka Electronics, Japan). Fast atom bombardment-mass spectrometry (FAB-MS) was carried out in a positive mode with a matrix of rn-nitrobenzyl alcohol by using a JMS DX-300 mass spectrometer (Japan Electron Optics Laboratory, Japan). For 'H NMR, PNLG was dissolved in chloroform-dl/methanol-d3/water-d2 (5:104 by volume) or chloroform-dl/methanol-d3 (2:l by volume). NMR spectra were recorded by using a Bruker AM 400 spectrometer. Two-dimensional double quantum filtered correlation spectroscopy (2D-DQFCOSY) and total correlation spectroscopy (TOCSY) spectra were obtained with a data size of 1024 X 2048 and spectral width of 2500 X 5000 Hz. The mixing time in the latter was 50 ms.
Structure of the Core Lipid-GLC of hydrocarbon chain prepared from PNLG showed only one peak which coincided with phytane. A chloroform-soluble product of acid methanolysis of PNLG was cochromatographed with archaeol by TLC with solvent E. The specific optical rotation, [ a ]~, of the product was +8.50", which was identical to that (+8.43") of authentic archaeol. This result confirmed that the core lipid of PNLG had the stereochemical configuration of 2,3-di-Ophytanyl-sn-glycerol which was common to archaeal polar lipids.
Structure of the Polar Head Group-Acid methanolysis completely cleaved the polar head group, and all of the phosphorus was recovered as organic phosphate in the methanol/ water phase after the Bligh and Dyer partition (1959). Analyses of the methanol/water-soluble product by GLC (after acetylation) under the conditions described under "Materials and Methods" and by an amino acid analyzer showed no peak on the chromatograms. When the product was further hydrolyzed with 6 M HCl at 100 "C for 18 h, free GlcN was detected by an amino acid analyzer, and myo-inositol and GlcN were found by GLC after acetylation or trimethylsilylation. The molar ratio of inorganic phosphate, myo-inositol, and GlcN in these products was 1:1.00:0.86. The optical rotation [ a ]~ of GlcN from PNLG was +71", which was identical to that of D-GlcN (+73"). The fact that the acid methanolysis completely degraded PNLG to yield archaeol suggested that archaeol and inositol were linked via phosphodiester linkage because the glycosyl bond of the glucosamine was resistant to acid hydrolysis due to the presence of the NH: group. To determine the sequence of the constituents of the polar head group, the following analysis by chemical reactions was carried out. Dephosphorylation with HF yielded archaeol as the major chloroformsoluble product accompanied with a small amount of archaetidic acid (phosphomonoester of archaeol). The chloroformsoluble product of NaN02 treatment gave a single major spot on TLC that comigrated with authentic archaetidylinositol on TLC with solvents A, B, or C. The positive ion FAB-MS spectrum of the product showed the molecular ion peak of m/ z 917 (M + Na)+ which was consistent with the molecular weight of archaetidylinositol (M = 894). Phosphatidylinositolspecific phospholipase C treatment of intact PNLG yielded archaeol as a chloroform-soluble product. N-Acetylated PNLG showed mobilities relative to PNLG of 1.55 and 0.80 on TLC with solvents A and D, respectively. Partially methylated alditol acetate of GlcNAc prepared from PNLG cochromatographed on GLC with 2-N-methyl acetoamido-3,4,6-tri-Omethyl-1,5-di-O-acetyl-2-deoxyl glucitol prepared from Nacetylated chitobiose. This indicated that the GlcN is the pyranose form and present as the terminal end of the polar head group linking at the C-1 position. These results established that D-G~cN was directly linked to my-inositol to which archaeol was linked via a phosphodiester linkage. Inositol phosphate was obtained from archaetidyl-myo-inositol prepared from PNLG by removal of GlcN with NaN02. The specific optical rotation ([aID) of the resultant inositol 1phosphate was -9.9", which coincided to 1-D-1-phospho-myoinositol (-9.8") of soybean phosphatidylinositol (Pizer and Ballou, 1959;Ballou and Pizer, 1960).
'H NMR of PNLG was carried out to determine the configuration of GlcN and position of the inositol moiety a t which GlcN was linked. When the spectrum was recorded as the solution of chloroform-dl/methanol-d3/water-d2, all the resonances from the polar head group were clearly separated and assigned (Fig. 1) by decoupling and 2D-DQFCOSY and TOCSY spectra. The coupling pattern of H-1 of the inositol residue confirmed that the phosphate group is linking to the C-1 position. The chemical shift of the anomeric proton of GlcN (5.57 ppm, Jl,z = 3.8 Hz) indicates that it is linked as an a-isomer. Since the linking position of GlcN is C-1, H-1 of GlcN was irradiated to examine the nuclear Overhauser effect (NOE). However, interresidual NOE was scarcely ob-  served in this solvent (data not shown). On the other hand, a strong NOE was observed between H-1 of GlcN and H-6 of the inositol residue ( Fig. 2 A ) , while the spectrum was not so clearly resolved (Fig. 2B) when the spectrum was recorded in a solvent of chloroform-dl/methanol-d3. The difference in the strength of NOE in two solutions may be ascribed to the difference in conformations of the inositol-GlcN moiety in these solvents. The NOE observed shows that H-1 of GlcN is close to H-6 of the inositol residue. From the model building of the glucosaminyl-myo-inositol moiety, the linkage of C-1 of GlcN to either C-1, C-5, or C-6 of the inositol residue may be consistent with the NOE observation. The possibility of the linkage to H-1 of inositol was, however, excluded because of the coupling between H-1 of inositol and phosphorus ( Fig.  1) and the optical rotation of the phospho-myo-inositol indicating the presence of phosphate at the C-1 position. Then, the 'H NMR spectrum of archaetidyl-myo-inositol prepared from PNLG by the NaN02 deamination was recorded and compared with that of PNLG (Table I). The difference in the chemical shifts was largest at H-1 and H-6. Consequently, it can be concluded that C-1 of GlcN was linked to the C-6 position of myo-inositol. Finally it is concluded that the complete structure of PNLG is 2,3-di-O-phytanyl-sn-glycerol-phospho-l'-[6'-0-(2"-amino-2"-deoxy-a-D-glucopyranosyl)]-1'-D-myo-inositol (glucosaminyl archaetidyl-myo-inositol) (Fig. 3). DISCUSSION The present study establishes the structure of glucosaminyl archaetidylinositol of M. barkeri. This is a novel lipid which consists of archaeol and glucosaminyl-(phospho)-myo-inositol. The core portion, archaeol (2,3-di-O-phytanyl-sn-glycerol) is typical of archaeal polar lipids. However, the structure of the polar head group is noteworthy. Glucosaminyl inositol is distinctively unique because of its identical structure to the polar head group of glucosaminyl phosphatidylinositol in eucarya. The position of myo-inositol at which GlcN is bound and the anomeric configuration (GlcN(a1-6)inositol) of M.