An aldehydophosphoglycolipid from Acholeplasma granularum

419

Biochimica et Biophysics Acto, 617 (1980) @ Elsevier/North-Holland Biomedical Press

419-429

BBA 57521

AN ALDEHYDOPHOSPHOGLYCOLIPID

FROM ACHOLEPLASMA

GRANULARUM

P.F. SMITH, K.R. PATEL Department (Received

and A.J.N. AL-SHAMMARI

of Microbiology,

July 24th,

University of South Dakota, Vermillion, SD 57069

(U.S.A.)

1979)

Key words: Aldehydophosphoglycolipid;

Lipid structure;

(A. granular-urn)

Summary A novel phosphoglycolipid and a triglucosyl diacylglycerol were found among the lipids of Acholeplusma grundarum. The tentative structure of the phosphoglycolipid was determined to be a phosphotriester, [O-D-glucopyranosyl-( 1 + 1)-X-glycerol-3-O-I [D -glyceraldehyde-3-0] [ 1,2-diacyl-X-glycerol-3-0kojibiosyl-6-0-Iphosphate. The structure assigned to the triglucosyl lipid was 0-D -glucopyranosyl-( 1 + 3)-a-D -glucopyranosyl-( 1 + 2)-0-D -glucopyranosyl(1 + 1)-diacylglycerol.

Introduction Acholeplasma granular-urn is considered to be a non-pathogenic saprophytic species belonging to the wall-less procaryotic class, Mollicutes [ 1,2]. Examination of the lipids of other Acholeplasma species has revealed a variety of novel lipid structures [ 3,4] some of which exhibit immunological specificity [ 51 and impart important properties to the unprotected acholeplasmal membrane [3]. The incomplete serological relatedness of A. granular-urn to other Acholeplasma species [2] suggested the existence in this organism of glycolipids with unknown structures. This paper presents data demonstrating a novel phosphoglycolipid containing an aldehyde radical and a triglucosyl diacylglycerol in A. granularurn as well as lipids common to other acholeplasmas.

Materials and Methods Growth of the organism. A granularurn strain BTS39 was obtained from Joseph G. Tully, National Institutes of Health, Bethesda, MD. Cultivation of the organism was carried out in liquid medium of the following composition: 20 g tryptose (Difco Laboratories, Detroit, MI, U.S.A.); 5 g yeast extract

420

(Difco), 5 g NaCl, in a total volume of 1 1. After adjustment to pH 8.0 with 10 M NaOH, the medium was autoclaved. Glucose and, in some instances, PPLO (pleuropneumonia-like organism) serum fraction (Difco) were added aseptically to give final concentrations of 0.25% (w/v) and 0.5% (v/v), respectively. The usual batch of organisms consisted of 100 1 of a 24 h culture started from a 10% inoculum grown in the same medium for multiple transfers. Incubation was carried out statically at 37°C in 3-1 flasks. Prior to harvesting, cultural and microscopic checks were performed to ensure purity of cultures. The organisms were harvested by concentration in a Sharples centrifuge followed by sedimentation at 25 000 X g and 0°C in a Sorvall RCBB centrifuge. The yellow sediment was washed in cold 0.2 M phosphate buffer (pH 7.5). When isotopically labeled lipids were desired, lOO-ml cultures were grown in the afore-mentioned medium supplemented with 1 mCi carrier-free HJ3’P04, 2.5 /..&i potassium [l-‘4C]oleate (spec. act. 30 Ci/mol), 2.5 PCi [2-14C]mevalonic acid (spec. act. 40 Ci/mol) or 50 I.tCi sodium [2-14C]acetate (spec. act. 25 Ci/mol). Extraction and purification of lipids. Lipids were extracted from freeze-dried organisms by stirring for 1 h at room temperature with 30 or more ~01s. of CHC13/CH30H (2 : 1, v/v). After two additional extractions, the pooled extracts were dried in vacua at 40°C. Isotopically labelled organisms were extracted as a wet sediment. Removal of non-lipid contaminants was accomplished by passage through Sephadex G-25 in CHC13/CH30H/Hz0 (60 : 30 : 4.5, v/v) [6]. The lipids were separated into major classes by passage through a silicic acid column (2 X 8 cm of Unisil, Clarkson Chemical Co., Williamsport, PA, U.S.A.). The neutral lipids were eluted with 150 ml CHC13, the glycolipids with 150 ml acetone and the polar lipids with 200 ml CH30H. Since incomplete separation of glycolipids from polar lipids was achieved by silicic acid chromatography, additional separation was accomplished by passage of the acetone and methanol elutes through a DEAE-cellulose (acetate form) column (2.5 X 22 cm). Glycolipids were eluted with (5 bed ~01s.) CHC1JCH30H (7 : 3, v/v) and acidic lipids were eluted with CHC1JCH30H/NH40H (specific gravity 0.88) (70 : 30 : 2, v/v) containing 0.4% ammonium acetate. Further separation and purifications were carried out by thin-layer chromatography on 1 and 0.25 mm layers of Silica Gel H activated by heating at 130°C for 1 h. All solvents were redistilled in glass vessels before use. Radioautography of lipids. Radiolabeled lipids were located by exposure for 24-48 h of Kodak Blue X-ray film to thin-layer plates developed in various solvents. Chromatographic procedures. The products of acid and alkaline hydrolyses, periodate oxidation and enzymatic hydrolysis were examined by ascending chromatography on cellulose sheets (Eastman Chromagram Cellulose without Fluorescent Indicator, No. 13255, Eastman-Kodak, Rochester, NY, U.S.A.). The solvent systems used were n-propanol/NH40H (specific gravity 0.88)/water (6 : 3 : 1, v/v) and n-butanol/pyridine/H20 (6 : 4 : 3, v/v). In addition, polyols were examined by thin-layer chromatography on 0.25 mm layers of Silica Gel H using the CHC13/CH30H/HCOOH (65 : 25 : 10, v/v). Phosphorus was detected using the molybdate spray of Hanes and Isherwood [ 71; the polyols by alkaline AgNOJ and the periodate-Schiff reaction [ 81; organic acids with bromocresol purple.

421

Analytical

TLC was carried out on 0.25 mm layers of Silica Gel H activated at 130°C for 1 h. The following solvent systems were employed: for neutral lipids, the two solvent system of Skipski (91, isopropyl ether/acetic acid (96 : 4, v/v) followed by light petroleum/diethyl ether/acetic acid (90 : 10 : 1, v/v); for glycolipids, CHCl&H30H (9 : 1, v/v); for polar lipids, CHCW CH30H/H20 (65 : 25 : 4, by vol.). Spots were detected either by charring with 50% methanolic H#O+ periodate-Schiff reaction, iodine vapor, the phosphate reagent of Vaskovsky and Kostetsky [lo], or 1% ninhydrin in water-saturate n-butanol). Gas chromatography was performed on a Hewlett-Packard model 402 biomedical gas chromatograph with hydrogen flame ionization detectors. Retention times and peak areas were recorded with a Hewlett-Packard model 3370A digital electronic integrator. All analyses were performed on glass columns, 1.8 m X 0.6 cm, packed either with SE-30 (5.5%) on 100-120 mesh Gas Chrom Q (Applied Science Laboratories, State College, PA, U.S.A.) or methyl phenyl silicone OV-17 (7%; Ohio Valley Specialty Chemical Co., Marietta, OH, U.S.A.) on Gas Chrom Q. Nominal gas flow rates were: helium, 60 ml/min; hydrogen, 80 ml/min, and oxygen, 200 ml/min. Methyl esters of fatty acids were prepared by refluxing in 10% HC10&!H30H and analysed at an oven temperature of 160°C with flash heater and detector block temperatures of 220°C and 28O”C, respectively. Sugars, glycosyl glyceroIs and polyols were converted to their trimethylsilyl derivatives by incubation of the dry samples for 1 h in a mixture of pyridine/hexamethyldisilazene/trimethylchlorosilane-N,O-bis(trimethylsilyl)/trifluoroacetamide (2 : 2 : 1 : 1, v/v). Reagent was removed under a stream of nitrogen and the residues reconstitu~d in CHCl, for injection. Programmed temperature chromato~aphy was performed from an initial temperature of 19O”C, held for 12 min, followed by a programmed increase of 5 C deg./min to 350°C. Isothermal gas chromatography was performed at 130°C for shortchain polyols, 190°C for free monosaccharides and methylglycosides, and at 260°C for mono- and diglycosyl glycerols. A Cl9 fatty acid and inositol were used as internal standards for qu~ti~tive analyses of fatty acids and polyols, respectively. Relative molar response factors were determined experimentally. Degmdatiue procedures. Deacylation by alkaline methanolysis was carried out either in 0.2 M sodium methoxide at room temperature for 1 h or in 1 M KOH/CH,OH at 37°C for 10 min. Reaction mixtures were neutralized with Dowex 50X-8 (hydrogen form) ion-exchange resin. After addition of water, fatty acid esters were extracted into hexane, and the aqueous methanol phase was evaporated to dryness either in vacua or under a stream of nitrogen, with warming. Strong alkaline hydrolysis was conducted in 1 M NaOH at 100°C for 1 h in a sealed tube under an atmosphere of nitrogen. Acid methanolysis was carried out in anhydrous 0.5 M HCl/CH30H at 100°C for 16 h under N2. Acid hydrolysis was carried out with 1 M HCl at 100°C for 24 h. Fatty acids were extracted into hexane. Aqueous phases from both types of solvolysis were evaporated to dryness without neutralization. Residual HCl was removed by repeated evaporation with Hz0 and finally with CH30H. Strong acid hydrolysis for detection of possible phosphonates used 6 M HCl at 100°C for 72 h. HF hydrolysis was carried out in 70% HF at 0°C for 4 h. Cold CHCl~/CH~OH

by heating

422

(1 : 1, v/v) was added to extract the lipid product, the lower organic phase removed, washed with cold saturated NaHC03 and dried under a stream of N2. This method cleaves phosphodiester bonds without cleaving glycosidic and most acyl ester bonds. The consumption of NaI04 was measured spectrophotometrically at 226 nm [ 111. The intact phosphoglycolipid or its water-soluble deacylation products (0.5-2.0 pmol phosphorus) were suspended in 2 ml water. Use of aqueous 0.1 M NaHC!03 as suspending medium gave spurious results. Suspension of the intact lipid was aided by sonic treatment at 20 Hz for a few seconds. After the addition of 3.0 ml of 0.01 N NaIO,, 100~~1 samples were diluted to 5.0 ml with water at 0,15, and 30 min, and 1, 2,4, 6 and 24 h and read at 226 nm in a Beckman DU spectrophotometer. At 0 and 4 h, 1.0 ml samples were removed, the reaction terminated by the addition of 1.0 ml 25% (w/v) NaHS03 and formaldehyde determined by the chromotropic acid. assay as described by Kiyasu et al. [12]. The disodium salt of rat-glycerol l-phosphate was used as a standard. A reagent control was employed to monitor the decomposition of NaI04 over the reaction period. Other end products of complete periodate oxidation were determined by gas-liquid chromatography. Excess NaIO, was destroyed by the addition of NaHSO,. 100 mg NaBH, was added, and the mixture was allowed to stand at room temperature overnight to effect reduction of the aldehydes. After acid hydrolysis, the trimethylsilyl derivatives were formed and examined. Smith degradation was carried out by a procedure similar to that of Fischer and Landgraf [13]. Intact phosphoglycolipid or its deacylation products (5 E.tmol phosphorus) were incubated in a 15-fold molar excess of aqueous 0.1 M NaIO, at room temperature overnight, with shaking. An amount of aqueous ethylene glycol, calculated to equal unreacted NaIO,, was added to consume residual periodate. After 30 min incubation, ethanol was added to a final concentration of 65% (v/v) to precipitate NaI03. After centrifugation, the precipitate was washed with CHC13, again with CHC1&H30H (1 : 1, v/v) and finally with 65% ethanol. The combined washings were dried in vacua, redissolved in 65% ethanol and treated overnight with 50 mg NaBH,. Excess borohydride was destroyed by the addition of 0.5 ml acetone, followed by 1 h incubation. The reaction mixture was passed through a 0.2 X 10 cm column of Dowex 50X-8 (hydrogen form) ion-exchange resin and eluted with 5 ml water. The eluate was dried in vacua and the borate removed by repeated evaporation from methanol. The residue was taken up in 0.1 N HCl and held for 24 h at 37°C. The acid was neutralized with Biorex AG-1 (hydroxide form) resin and the eluate treated with 50 mg NaBH, for 24 h at room temperature. The reaction was stopped with acetone and the borate removed by ion exchange and repeated evaporation from methanol. The final product was examined by TLC on cellulose and by GLC. Controlled periodate oxidation was accomplished by exposure of the phosphoglycolipid and its deacylation products to a 3-fold molar excess of NaIO, for l-5 min. Processing of the reaction mixture was identical to that used for Smith degradation except acid hydrolysis was performed with 1 N HCl at 100°C for 1 h. Cr03 oxidation [14-161 was performed on the triglucosyl diacylglycerol

423

and its water-soluble deacylation product. The component to be examined was peracetylated in acetic anhydride/dry pyridine (1 : 1, v/v) at room temperature for 24 h. Reagents were removed under a stream of nitrogen. Approximately 10 mg peracetylated sample and 100 mg Cr03 were suspended in 1 ml glacial acetic acid and sonicated for 3 h at 55°C in a sonic cleaning bath. The reaction mixture was extracted twice with 2 ml CHCIJ after the addition of 2 ml water. Pooled chloroform phases were reextracted with water, then dried over Na,SO, and evaporated to dryness. The residue was subjected to mild deacylation and the water-soluble products examined by gas-liquid chromatography of their trimethylsilyl derivatives. Degradation with alkaline phosphatase (EC 3.1.3.1) from Escherichia coli was performed in 0.1 M Tris buffer (pH 8.0) at 37°C for 3 h. The reaction was stopped and protein precipitated by the addition of 3 ~01s. of absolute ethanol. After centrifugation, the supernatant fluid was dried in vacua and examined by TLC and GLC. Enzymatic hydrolysis with a-glucosidase (EC 3.2.1.20) was carried out by incubating 5-10 mg water-soluble deacylation product with 1 mg enzyme in 1 M phosphate buffer (pH 7.5) overnight at 37°C. The same conditions were used for P-glucosidase (EC 3.2.1.21) except that 1 M acetate buffer (pH 5.0) served as buffer. The reactions were stopped by addition of 3 ~01s. of ethanol. The precipitated protein was removed by centrifugation and the supernatant fluid dried in vacua. Chemical analyses. Phosphorus was determined by the method described by Ames [ 171. Glycerol was assayed enzymatically by measuring the reduction of NAD’ by a-glycerophosphate dehydrogenase (EC 1.1.1.8) after glycerol phosphorylation by glycerate kinase (EC 2.7.1.31) [X3]. D- and L-lactic acids were assayed enzymatically using D-lactate dehydrogenase (EC 1.1.1.28) [ 191 and L-lactate dehydrogenase (EC 1.1.1.27) [20], respectively, and measuring the reduction of NAD’. Glucose was measured by the phenol-sulfuric acid method [21], the anthrone reaction [22] and enzymatically with glucose oxidase. Reducing activity was measured by the method of Park and Johnson [23]. Examination of the phosphoglycolipid for the possible presence of uranic acid residues was performed using the carbazole method [ 241; for amino sugars by the Elson-Morgan reaction [25]; for ketohexoses by the cysteine-sulfuric acid method [26]; deoxypentoses and 3,6-dideoxy hexoses by the arsenite-thiobarbituric acid procedure [27]. Fatty acid esters were quantitated by the hydroxamate method [28] as well as by GLC. Results 50 g of freeze-dried organisms were obtained from 6 100-l batches. Lipids, extractable with CHCI&H~OH accounted for 12% of the cell dry weight. These lipids were comprised of neutral lipids (5%), glycolipids (38%) and polar lipids (57%). All of the lipids from these three classes could be identified with lipids found in other Acholeplasma species, except for two. One of these was observed in the acidic lipid fraction, migrating with an RF of 0.25 on Silica Gel H plates developed with CHC13/CH30H/Hz0 (65 : 25 : 4, by vol); the other in the glycolipid faction, migrating with an RF of 0.32 in the same solvent.

424

Structure

of the phosphoglycolipid

The unknown acidic lipid, which accounted for 25% of the total lipid phosphorus, was observed to contain sugar and phosphorus by use of spray reagents and to react positively with the periodate-Schiff reagent or the Schiff reagent alone. Plasmalogens were ruled out since no change in migration pattern occurred upon exposure to HCL fumes prior to TLC. Analyses of the composition of the intact lipid revealed the presence of fatty acid esters, glucose, glycerol and phosphorus in a molar ratio of 2 : 3 : 2 : 1. Mild deacylation produced one phosphorus-containing product migrating with an RF of 0.25 in n-propanol/NH,/H,O (6 : 3 : 1, by vol.), identical to the RF of sn-glycerol 3-phosphate. However, this spot on the thin-layer chromatogram was periodate-Schiff negative, unlike that for sn-glycerol 3-phosphate. In fact, another spot was revealed by this reaction with an R, of 0.53. The Hanes, Isherwood phosphate reagent also revealed a third spot, albeit not of the typical blue of phosphorus compounds, with an R, of 0.72. These results suggested an impure starting compound. However, repeated purification on thin-layer plates and two-dimensional TLC did not result in the detection of any impurity. Likewise, repeated washing with water of a solution of the lipid in CHC1JCH30H/0.1 N HCl neither removed nor altered the lipid in the organic phase. Strong alkaline hydrolysis yielded the same products as mild deacylation, i.e., the phosphoruscontaining fragment, the rapid periodate-Schiff-reactive component and the fast-migrating fragment exhibiting a rapidly appearing greyish-blue color with the Hanes-Isherwood reagent, hereinafter designated fractions 1, 2 and 3, respectively. Each of these fragments was isolated by TLC, eluting from cellulose with warm water (Fig. 1). Fraction 1, upon acid hydrolysis, yielded glucose, glycerol and phosphorus in a molar ratio of 1.0 : 0.8 : 1.0. Its non-reactivity with the periodate-Schiff reagent suggested that the glycerol was not a terminal radical. Mild acid hydrolysis resulted in the formation of a mixture of cr- and /3-glycerophosphates. Enzymatic removal of phosphate using alkaline phosphatase yielded a glucosyl glycerol which was indistinguishable by GLC from authentic ~-D-glucopyranosyl-(1 + l)-glycerol (retention times, 5.61 and 5.69, respectively; (Y-D-gluco-

O-C"

?”

p-C"-C"2-

Cl-P-0-CC*-

I

Fig.

1. Structure

of aldehydophosphoglycolipid.

?” ;; CH-C"

425

pyranosyl-(1 -+ 1)-glycerol, 4.82 min). Treatment of either fraction 1 or its dephosphorylated product with &glucosidase liberated greater than 90% of the glucose, whereas cu-glucosidase treatment produced no free glucose. From these results fraction 1 was identified as P-D -glucopyranosyl-( 1 -+ I)-X-glycerol 3-phosphate, Fraction 2 was indistinguishable from diglucosyl glycerol on thin-layer chromatograms developed in the three different solvent systems. Dephosphorylation of the intact phosphoglycolipid with 70% HF yielded one product in the organic phase which migrated on thin-layer chromato~~s as diglucosyl diacylglycerol. The water-soluble mild deacylation product of this fragment and fraction 2 possessed identical retention times on GLC as a-D-glucopyranosyl-(1 -+ 2)-cu-D-glucopyranosyl-(1 -+ 1)-glycerol (11.1, 11.1, 11.0 min, 1 -+ I)-glycerol, respectively; p-D-glucopyranosyl-( 1 + 6)-P-D-glucopyranosyl-( 9.6 min). Free glucose was liberated by treatment of fraction 2 with cu-glucosidase but none upon treatment with @-glucosidase. Controlled periodate oxidation (1 min at 23°C) followed by borohydride reduction and acid hydrolysis yielded only glucose and ethylene glycol as products. Periodate oxidation resulted in the formation of 1 mol of formaldehyde/2 mol of glucose. Acid hydrolysis of fraction 2 yielded glucose and glycerol in a molar ratio of 2.2 : 1 .O. Fraction 2 was identified as a-D -glucopyranosyl-( 13 2)~-D-glucopyr~osyl-( 1 --f l)-sn-glycerol. Fraction 3 contained no phosphorus in spite of the blue-gray color given with the Hanes-Isherwood reagent. It was periodate-Schiff negative but stained with alkaline AgN03. Examination of a variety of sugars, glycols, aldehydes and short-chain mono- and dicarboxylic acids by thin-layer cellulose chromatography revealed that all carboxylic acids produced a blue-gray color with the Hanes-Isherwood reagent. The RF of fraction 3 most closely approximated the RF values of glycerol and lactic acid, Examination of acetylated and trimethylsilylated fraction 3 by GLC resulted in a peak with a retention time closely approximating that of lactic acid. Assay of fraction 3 with D- and L-lactate dehydrogenase showed the presence of both D- and L-lactic acids in a molar ratio of 4 : 1. The amount of lactic acid present in the unfractionated strong alkaline hydrolysate of the intact phosphoglycolipid was approximately equivalent to the amount of phosphorus, i.e., molar ratio of D- + L-lactic acid : phosphorus, 0.7 : 1.0. Thus fraction 3 identified as a mixture of D- and L-lactic acids. None of these three fragments of alkaline hydrolysis, as identified, could explain the Schiff reactivity and the alkaline lability of the intact phosphoglycolipid. The intact lipid exhibited one reducing group for each mol of phosphorus and 3 mol of glucose. Borohydride reduction of the intact lipid, followed by complete acid hydrolysis yielded 3 mol of glycerol per mol of phosphorus in contrast to a molar ratio of glycerol to phosphorus of 2 : 1 in the non-reduced lipid. Furthermore, strong alkaline hydrolysis of the reduced lipid no longer yielded the fraction 3. These results suggested that the Schiff reactivity of the intact lipid was due to a free aldehyde group and that this group was the precursor for the lactic acid. It appeared most probable that this aldehyde group was glyceraldehyde attached to the lipid molecule through the 3-position. If, in all likelihood, the glyceraldehyde was linked to the phosphate

426

through an ester bond, an extremely alkaline-labile phosphotriester would exist. Glyceraldehyde would yield lactic acid by a p-elimination reaction. Both the intact lipid and its water-soluble deacylation products consumed 6 mol of periodate/mol of phosphorus. Only the deacylation mixture yielded formaldehyde in the amount of 1 mol per mol of phosphorus. The Smith degradation products consisted of only ethylene glycerol and glycerol. Formic acid was not assayed. Both fatty acids residues were found associated with the diglucosyl diacylglycerol isolated in the organic phase of 70% HF hydrolysis. The correct composition of the phosphoglycolipid was shown to be fatty acid esters, glucose, glycerol, glyceraldehyde and phosphorus in a molar ratio of 2 : 3 : 2 : 1 : 1. Its most probable structure is a glucosyl glycerophosphoryl diglucosyl diacylglycerol with a glyceraldehyde attached through a phosphoester bond forming a phosphotriester lipid. Structure

of the triglucosyl

diacylglycerol

The unidentified glycolipid in A. granularum, upon total acid hydrolysis, yielded fatty acids, glucose and glycerol in a molar ratio of 3.0 : 2.9 : 1.0. The trimethylsilyl derivative of the water-soluble deacylation product was not detectable by GLC, undoubtedly due to its very long retention time. Exposure of this product to a-glucosidase did not result in the liberation of glucose. However, /3-glucosidase action resulted in the formation of equivalent molar quantities of glucose and a diglucosyl glycerol indistinguishable by GLC from Q-Dglucopyranosyl-( 1 + 2)-CY-D -glucopyranosyl-( 1 + l)-glycerol. Treatment with P-glucosidase followed by a-glucosidase resulted in the appearance of glucose and a monoglucosyl glycerol indistinguishable by GLC from cy-D-glucopyranosyl( 1 + 1)-glycerol. As incubation time was increased, the monoglucosyl glycerol disappeared with the concomitant appearance of glucose. The fi-configuration of the terminal glucose was confirmed by CrO, oxidation, whereby fl-glucosidic linkages are preferentially attacked. The major product of Cr03 oxidation was shown by TLC and GLC to be identical to ol-D-glucopyranosyl(1 + 2)-a-D -glucopyranosyl-( 1 + 1)-glycerol. The nature of the glycosidic linkage of the P-linked glucose was determined by periodate oxidation. The watersoluble deacylation product (3 pmol) was incubated with NaIO, (300 pmol) for 24 h at room temperature and the reaction stopped with a calculated amount of ethylene glycol. Following reduction with 50 mg NaBH4 overnight and removal of the borate ions, the product was hydrolyzed in 1 N HCl for 1 h at lOO”C!, neutralized and subjected to another course of borohydride reduction. Upon examination of the trimethylsilyl derivatives by GLC, glycerol and sorbitol only could be identified as products. Since sorbitol could arise only from an internal glucose in which carbon-3 is occupied, the only tenable structure for the deacylation product is p-D-glucopyranosyl-(1 -+ 3)-a-D-glUCOpyraThe exact location of the nosyl-( 1 --f 2)-a-D -glucopyranosyl-( 1 + 1)sn-glycerol. fatty acid residues on the intact lipid was not resolved. Identification

of the remaining

lipids

The other lipids found in A. granularum were identifiable with lipids previously reported to occur in other Acholeplasma species [ 3,4]. Identification was achieved by comparison of their chromatographic properties, ratios of compo-

427 TABLE

I

LIPIDS

OF GRANULARUM

BTS39

Lipid

Content

Neutral lipids Triacylglycerol 1,2-Diacylglyeerol P,3-Diacylglycerol Car&maids (newosporene,

5% of total lipids

hydroxyneurosporene) % total [‘4Cloleic acid in glycolipids

Glycolipids -D-Glucopyranosyl-(1 -D-Glucopyranosyl-(1 -D-Glucopyranosyl-(1 D-glueopyranosyl-(1

--Z l)-2,3-diacyl glycerol -+ 2)-cu-D-glucopyranosyl-(1 3 3)+glucopyranosyl-(1 -+ 1)_2,3_diacylglyceroI

--f I)-2.3-diacylglycerol -+ 2)*-

12.5 83.0

38% of total lipids

%4iZtal 32P Polar lipids Acylated cardiolipin Diphosphatidyl glycerol Phosphatidylglycerol Phosphoglycolipid Unidentified fpolyprenol-P)

4.0 21.5 49.4 24.3 0.8

57% of total lipids

nent parts, enzymic hydrolyses and, in the case of carotenoid pigments, spectral characteristics. Table I lists these lipids and their contribution to the total lipids of the organism, When this organism was grown in a medium containing cholesterol, two additional lipids appeared. One was identified as cholesterol by GLC. The second lipid migrated with an RF of 0.8 on Silica Gel H plates developed with CHC13/CH30H (9 : 1, v/v). After alkaline hydrolysis, its RF changed to 0.5, identical to authentic cholesteryl monoglucoside. Acid hydrolysis yielded equimolar quantities of glucose and cholesteryl as determined by GLC. This lipid was identified as an acyl cholesteryl glucoside. Over 90% of the fatty acids in all individual lipids could be accounted for by palmitic, stearic and oleic acids. Discussion Two of the lipids found in A. ~a~ular~~ represent structures heretofore unreported in nature. The triglucosyl diacylglycerol is a variation of the structure of other triglucosyl diacylglycerols [29-311, but is unique in that the trisaccharide moiety contains only glucose in two different anomeric configurations. The structure proposed for the phosphoglycolipid is distinctive, not only by being a phosphotriester but also by containing a free aldehyde group. Structural dete~ination was complicated by the appearance of more than one product, even by mild alkaline hydrolysis. Compositional data initially suggested a structure such as a glycerophosphoryl triglucosyl diacylglycerol. This structure should yield glycerophosphate and a triglucosyl glycerol upon strong alkaline hydrolysis. In actuality only a diglucosyl glycerol was obtained. Furthermore, such a structure would not give a rapid Schiff reaction without prior

428

oxidation with periodate. The only structure compatible with the extreme alkaline lability is a phosphotriester in which one of three available oxygens on the phosphors is occupied by a molecule containing a free aldehyde group in the &position and an hydroxyl group in the cr-position to the carbon atom linked to the phosphorus [ 32,331. Glyceraldehyde fulfils these requirements. Exposure to alkali, even under mild conditions, would result in a &elimination reaction giving rise to lactic acid, one of the products identified. Further evidence that gly~eraldehyde is the parent molecule was the appearance of a third mole of glycerol in the intact lipid upon reduction with borohydride and the loss of lability to mild alkali of this reduced product. Both the intact lipid and its deacylation products consumed 6 mol of periodate per mol of phosphorus. The absence of any increase in amount of periodate consumed by the deacylated lipid would be expected. One pair of vicinal hydroxyl groups would be unmasked on the glycerol containing the fatty acid esters, while the adjacent hydroxyl and carbonyl groups of the glyceraldehyde would be lost by its conversion to lactic acid. Alkali hydrolysis of such a phosphotriester also would release the diglucosyl glycerol with the phosphorus remaining attached to the glycerol of glucosyl glycerol. The fatty acid esters were assigned to the glycerol moiety of the diglucosyl glycerol group, since treatment with HF yielded only one component in the organic phase of the reaction products and this component was indistinguishable from diglucosyl diacylglycerol. Furthermore, the 1 mol of formaldehyde produced by periodate oxidation of the deacylation products could arise only from the glycerol of this fragment, i.e. fraction 2. Periodate consumption, the nature of the products of Smith degradation and alkaline lability suggest that the glucosyl glycerophosphate group is attached to carbon atom 6 of the second glucosyl unit of diglucosyl diacylglycerol. Hence, all of the data support the structure shown in Fig. 1. Glycerophosphoglycolipids have been shown to occur in most Gram-positive bacteria [34,35]. There appears to be a metabolic relationship between the occurrence of these lipids and the presence of lipoteichoic acids. Fischer and coworkers [13,34] have suggested that their unusually low concentration in bacteria points to their role as metabolic intermediates rather than as membrane constistuents. On the other hand phosphatidyl glycolipids rarely occur in the free form in bacteria but exist as lipid anchors for lipoteichoic acids [36, 371. Among the class Mollicutes which consists of the wall-less procaryotes, phosphoglycolipids have been found in Acholepluasma laidlawii [ 38,391, Thermop~usma acidophi~um [40] and, now, A. gra~~~ururn. In contrast to the walled Gram-positive bacteria, these phosphoglycolipids occur as one, if not the major, constituent of the total lipid fraction. This fact lends support to Fischer’s hypothesis, since none of the Mollicutes appears capable of the biosynthesis of teichoic or lipoteichoic acids. The large amounts of phosphoglycolipids points to a possible pile up of a metabolic intermediate resulting from the inability of the organisms to synthesize wall polymers. The present findings of a phosphoglycolipid containing a reactive free aldehyde group lends even more credence to a metabolic role for this type of lipid.

429

References 1 Switzer, W.P. (1964) in The Mycoplasmatales and the L-phase of Bacteria (Hayflick, L., ed.), PP. 612-614, AppletonCentury-Crofts, New York Tully, J.G. (1973) Ann. N.Y. Acad. Sci. 225. 7443 Smith, P.F. (1971) The Biology of Mycoplasmas, Academic Press, New York Smith, P.F., Langworthy. T.A. and Mayberry, W.R. (1973) Ann. N.Y. Acad. Sci. 225, 22-27 Sugiyama, T., Smith, P.F., Langworthy, T.A. and Mayberry, W.R. (1974) Infect. Immun. 10, 12731279 6 Wells, M.A. and Dittmer, J.C. (1963) Biochemistry 2, 1259-1263 7 Hanes, C.S. and Isherwood, F.A. (1949) Nature 164,1107-1110 a Shaw, N. (1968) Biochim. Biophys. Acta 164.435436 9 Skipski. V.P. and Barclay, M. (1969) Methods Enzymol. 14, 530-598 10 Vaskovsky, V.E. and Kostetsky, E.Y. (1968) J. Lipid Res. 9. 396 11 Dixon, J.S. and Lipkin. D. (1954) Anal. Chem. 26, 1092-1093 12 Kiyasu, J.Y., Pieringer, R.A., Paulus, H. and Kennedy, E.P. (1963) J. Biol. Chem. 238, 2293-2298 13 Fischer, W. and Landgraf, H.R. (1975) Biochim. Biophys. Acta 380, 227-244 14 Angyal, S.J. and James, K. (1970) Aust. J. Chem. 23, 1209-1221 15 Hoffman, J.. Lindberg, B. and Svensson, S. (1972) Acta Chem. Stand. 26. 661-666 16 Laine, R.A. and Renkonen, 0. (1974) Biochemistry 13, 2837-2843 17

ia 19 20 21 22 23 24 35 36 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Ames, B.N. (1966) Methods Enzymol. 8. 115-118 Wieland, 0. (1965) in Methods of Enzymatic Analysis (Bergmeyer. H.U.. ed.), 2nd edn., Academic Press, New York Gawehn, K. and Bergmeyer. H.U. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H.U.. ed.). 2nd edn.. Academic Press, New York Gutmann. I. and Wahlefeld, A.W. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H.U.. ed.), 2nd edn., Academic Press. New York Ashwell. G. (1969) Methods Enzymol. 8. 8545 Dische. Z. (1962) Methods Carbohydr. Chem. 1. 476461 Park, J.T. and Johnson, M.V. (1949) J. Biol. Chem. 181. 149-151 Dische. Z. (1955) Methods Biochem. Anal. 2. 313-358 Dittmer, J.C. and Wells, M.A. (1969) Methods Enzymol. 14. 483-530 Dische. Z.. Shettles, L.B. and Osnos, M. (1949) Arch. Biochem. Biophys. 22, 169-184 Keleti. G. and Lederer, W.H. (1974) Handbook of Micromethods for the Biological Sciences, Van Nostrand Reinhold Co., New York Rapport, M.M. and Alonzo. H. (1955) J. Biol. Chem. 217,193-198 Shaw, N. (1975) Adv. Microb. Physiol. 12. 141-167 Nakano, M. and Fischer, W. (1977) Hoppe-Seyler’s Z. Physiol. Chem. 356, 1439-1453 Nakano. M. and Fischer, W. (1978) Hoppe-Seyler’s Z. Physiol. Chem. 359, l-11 Brown. D.. Hayes, F. and Todd, A. (1957) Chem. Ber. 90. 936-941 Brown, D.M., Fried. M. and Todd, A.R. (1955) J. Chem. Sot. 2206-2210 Fischer, W.. Nakano, M.. Laine, R.A. and Bohrer, W. (1976) Biochim. Biophys. Acta 526, 268-297 Fischer. W., Laine, R.A. and Nakano, M. (1978) Biochim. Biophys. Acta 528. 298-308 Toon. P.. Brown, P.E. and Baddiley, J. (1972) Biochem. J. 127, 399-409 Ganfield, M-C.W. and Pieringer, R.A. (1975) J. Biol. Chem. 250. 702-709 Shaw, N., Smith, P.F. and Verheij. H.M. (1972) Biochem. J. 129. 167-173 Smith, P.F. (1972) Biochim. Biophys. Acta 260, 375-382 Langworthy, T.A., Smith, P.F. and Mayberry, W.R. (1972) J. Bacterial. 112. 1193-1200