227

Biochimica et Biophysics Acta, 380 (1976) 227-244 @ EIsevier Scientifk F’ubIisbing Company, Amsterdam - Printed in The Netherlands

BRA 56660

GLYCEROPHOSPHORYL PHOSPHATIDYL KOJIBIOSYL DIACYLGLYCEROL, A NOVEL PHOSPHOGLUCOLIPID FROM STREPTOCOCCUS

FAECALIS

WERNER FISCHER and H. ROBERT LANDGRAF Institute of Physiological Chemistry, Wasertutmstr. 6 (C. F. R.)

Univeniiy Erlangen-Niimbcrg,

D-8620 Erlangen.

(Received September 3rd. 1974)

1. From Smptococcus fizecald a third novel phosphoglucolipid w88 isolated. It contained D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 2 : 3 : 4 : 2. 2. The structure was shown to be 3( 1)-O-[6”(sn-glycerol-1-phoephoryl), 6’(1,2 diacyl-sn-glycerol-3-phoephoryl)-2’-O-(a-D-glucopyranosyl)u-D~ucopyranosyll-1(3),2&acylglycerol. The novel lipid is related to the earlier described phosphatidyl and glycerophosphoryl kojibiosyl diacylglycerol from Streptococcus fuecaZisby combining their substituents in its structure. 3. The structure of the deacylated core was accomplished by analyses of the breakdown products obtained on Smith degradation and strong alkaline hydrolysis. The location of the acyl groups was achieved by degradation of the intact lipid with 60% HF (w/v) which resulted in the formation of inorganic phosphate, glycerol, diacylglycerol and diglucosyl diacylglycerol. The snglycerol-1-phosphoryl and 3sn-phosphatidyl substituents were located by hydrolysis of the lipid with 98% acetic acid (v/v) and subsequent analysis of the resultant phosphatidyl diglucosyl diacylglycerol. 4. The composition and positional distribution of the constituent fatty acids at the two diacylglycerol portions was studied by combination of chemical and enzymatic degradations. The fatty acids are the same as with the other polar lipids of S. faecalis, they are present in nearly the same proportions and display the same non-random distribution pattern. 5. A possible relationship between sn-glycerol l-phosphate containing phosphoglucolipids and lipoteichoic acid will be discussed.

l

pun of thk p-et Sepbmberl973.

to-

firrt

presented

without

publiutlon

at Harden

Conference on F'hOmhOli~idr.

228

Introduction During the last years phosphoglycolipids derived from mono- or dihexosyl diacylglycerols have been found mostly in gram-positive bacteria [l-7] . For biosynthetical reasons they can be divided according to the nature of their glycerophosphate residue into compounds with (i) 3-sn-phosphatidyl [ 1,2,4,6], (ii) sn-glycerol-&phosphoryl [ 31, and (iii) sn-glycerol-l-phosphoryl [ 5,7] as the substituent on their basic unit. In previous work we have shown that strains of Streptococcus foe&is contain two of the forementioned types, namely phosphatidyl and sn-glycerol-1-phosphoryl diglucosyl diacylglycerol [ 5,6] . In the present communication we describe a novel phosphoglucolipid from S. faecafis with the structure 3(l)- 0- [ 6”-(sn-glycerol-1-phosphoryl)-6’(1,2-diacyl-snglycerol-3-phosphory1)-2’-O-a-D-glucopyranosyl)-cu-D-glucopyranosyl]-1(3),2 diacylglycerol. It can be classified as a fourth type because it carries the substituents of both the other phosphoglucolipids at exactly the same positions of the basic diglucosyl diacylglycerol. Experimental General The materials used were essentially the same as in previous work [ 51.

Phospholipase AZ (EC 3.1.1.4) from hog pancreas and sn-l-lecithin was a gift from Professor G.H. de Haas. Phosphatidylglycerol was isolated from a Streptococcus (serological Group B) by column chromatography on DEAEcellulose, and was analyzed as described earlier [8] . Glycolphosphorylglycerol and glycolphosphorylglycol were prepared from phosphatidylglycerol and its deacylation product respectively using a modified version of Smith degradation (see below). Glycerophosphorylglycerol and en-glycerol-l-phosphorylcholine were obtained by deacylation of the corresponding lipids. Methods used in thin-layer, column and gas-liquid chromatography were mainly the same as described previously [5]. Fatty acids were methylated with ethereal diaxomethane [9] for analysis by gas-liquid chromatography [ 51. Polyolphosphates were trimethylsilylated for gas-liquid chromatography and mass spectral analysis (Varian MAT CHS) according to the method of Sherman et al. [lo] . Polyolphosphate dies&s must be analyzed within a few hours after trimethylsilylation, otherwise cleavage occurs with the formation of trimethylsilyl polyolphosphate monoesters. Carboxylic esters, carbohydrate, D-glucose, glycerol, sn-glycerol 3-phosphate, glycolaldehyde, glyceraldehyde, phosphorus, periodate, and formaldehyde were determined by established procedures. (For the original references to the methods see refs 5 and 6.) Lipids The presently described phosphoglucolipid stems from the same lipid extracts of S. faecilis var. faecaiis 7064 and S. faecalis var. zymogenes 20672

which were used in previous work for the isolation of glycerophosphoryl [5] and phosphatidyl diglucosyl diacylglycerol [6]. On purification of the former compound the new phospholipid was obtained in an almost pure form. (For

229

details see ref. 5). Minor contaminations were removed by column chromatography on NaI-ICQ -treated silic acid [ll] or preparative thin-layer chromatography on silica gel plates Woelm (chloroform/methanol/water(65 : 35 : 8, by vol.). S. faecalis var. liquefaciens 7415 and S. hemolyticus D-58 were grown as described earlier [5,12]. The lipids were extracted according to Prottey and Ballou [13]. Membranes of S. faecalis ATCC 9790 were a gift from Dr H. Miildner (Gijttingen). They were extracted by a modified Bligh and Dyer method [14,15] . S. faecalis ATCC 9790 was harvested in the logarithmic growth phase, the other strains in the stationary phase. Alkaline and acid hydrolyses

Deacylation of phospholipids and their lipid breakdown products was carried out by mild alkaline hydrolysis [6]. Strong alkaline hydrolysis for cleavage of phosphodiester bonds was performed in 0.1 M NaOH at 100°C for 24 h in screw-capped Teflon vessels. Acid hydrolysis was done in 2 M HCl at 100°C for 2 h and at 125” C for 48 h, respectively [5] . Further treatment of the hydrolysates for the various analyses was essentially the same as described before [5], but the strong alkaline hydrolysate was neutralized by passage through a column of cation exchange resin (Merck, S 1080), NH,, *-form, the effluent of which was taken to dryness in vacua. Smith degradation

The procedure used was an improved version of the previous one [5,6]. Deacylated lipid (3-4 pmol) was oxidized with 0.1 M NaIO,, (0.5 ml) at roomtemperature for 12-14 h. The remaining NaIO,, was converted into NaIOJ by addition of a slight excess of glycol. Absolute ethanol (0.9 ml) was added in an ice bath. The precipitated NaIOJ was removed by filtration through &nixed glass wool and was washed with 80% ethanol (v/v, 4 ml) at +4”C. Most of the ethanol was removed from the combined filtrates on a rotavapor: NaBH., or NaB’ H4 (10 mg) was added in ice. After standing overnight at +4”C the excess of borohydride was destroyed by addition of acetone (0.6 ml) in ice. In order to convert sodium borate into the ammonium salt the reaction mixture was passed through a column of cationexchange resin (Merck S 1080), NH4 *-form. Ammonium borate and the rest of the added glycol was removed by taking the effluent to dryness followed by repeated evaporations with methanol. Controls running with diglucosylglycerol and glycerophosphorylglycerol have shown that neither acetal bonds nor phosphodiester linkages were broken throughout this procedure. The recovery of phosphorus was’95-100%. The reduced oxidation product was subjected to mild acid hydrolysis (0.1 M HCl, 37”C, 24 h). Glyceraldehyde, glycolaldehyde and glycerol were determined by chemical or enzymatic procedures, and the results were related to the phosphorus content. In another aliquot of the hydrolysate, which was neutralized with ionexchange resin as described before [5,6], glycol was determined by gas-liquid chromatography on Chromosorb 102 [5,6] using the released glycolaldehyde and glyceraldehyde as internal standards. In order to characterize the phosphorus-containing product of mild acid hydrolysis, the reaction mixture was passed through a column of anion exchange resin (Merck,

230 h4P 508O), acetate form. Polyols and aldehydes were washed out with water, the phosphorus-containing product was eluted with 2 M acetic acid. The effluent was taken to dryness at a bath temperature of 40°C with repeated addition of carbon tetrachloride. The residue was trimethylsilylated and analyzed by gas-liquid chromatography [5] . For quantitative determination known amounts of glycolphosphorylglycol and glycerophosphorylglycerol were added to a sample of the reduced oxidation product prior to mild acid hydrolysis; the standard mixture was subjected to the same treatment.

Degradation with HF Degradation of the phosphoglucolipid was carried out with 60% HF (w/v) as described before [5,6], but after neutralization with LiOH the mixture was centrifuged, the supernatant removed, and the residue washed with water once more. The supematants were combined for analysis of water-soluble breakdown products. The lipids formed on degradation were extracted from the LiF residue with chloroform/methanol (1 : 1 and 2 : 1; by vol.) Degradation with acetic acid [16,17] Degradation of phosphoglucolipids was performed in 98% acetic acid (v/v) at 100°C for 45 min in glass stoppered tubes. After cooling, acetic acid was removed in vacua with several additions of carbon tetrachloride. The watersoluble and lipid breakdown products were separated by Folch partition. Degradation of phosphoglucolipids remained rather incomplete after heating for 15 and 30 min, whereas heating for more than 45 min enhanced the formation of partially acetylated byproducts (see Results). Increasing water content up to 20% gave no improvement but slowed down phosphodiester cleavage and caused some hydrolysis of glycosidic and fatty acid ester linkages. Enzymatic hydrolysis Treatment of water-soluble phosphate esters with alkaline phosphatase (EC 3.1.3.1) was carried out in 1 M glycine buffer (pH 9.5). For incubations with acidic phosphatase (EC 3.1.3.2) 0.1 M citrate buffer (pH 5.3) was used. Incubation with cr-glucosidase (EC 3.2.1.20) and lipase (EC 3.1.1.3) from Rhizopus arrhizus were done as described before [ 5,181. Hydrolysis of the phosphoglucolipid with phospholipase AZ (EC 3.1.1.4) from hog pancreas was carried out as follows: lipids (0.5-2 pmol) were dissolved in 1 ml diethyl ether; enzyme (200 units for Lipid I, 750 units for Lipid III) was added in 0.6 ml 0.1 M Tris buffer (pH 7.3, containing 30 mM CaC& ). The mixture was shaken at room temperature. Complete conversion of Lipid I and Lipid III into the corresponding lyso compounds was achieved within 1 and 12-18 h, respectively. Fatty acids and lyso compounds were obtained by Folch partition using 0.2 M CaCl, with a few drops of acetic acid as water phase. Results Occurrence and properties S. faecalis contains five phosphoglycolipids

(Fig. 1). Lipid I and II have

231

been previously chara+ixed

as 8(1)-0+%(1,2

d&q&mglycero3phosphoryl)-2~ 2diacylglycerol[6] and 1(3)0-[6”-(sn-giyc~l-phoephoryl)-2’-0-(a-D-glycopyranosyl)a~D-glucopyranosyl]diacylglycerol [6]. The presently described Lipid III, although a minor component like Lipid II, is clearly visible on thin-layer chromatograms of crude lipid extracts from S. faecalis (Fig. 1). The fact that Lipid III can be detected together with the other polar lipids in extracts of cytoplasmic membranes (Fig. 2a (2)) suggests that it may also be a membrane constituent. The occurrence of Lipid III seems to be confined to strains of S. faecalis (Group D) since it has not been found in S. hemolyticus D-63 (Group A) nor in S. Zuctis (Group N) in spite of the fact that these Streptococci are able to synthesize the related Lipids I and II like group D Streptococci (Fig. 2b). Lipid III has been isolated from the same extracts of S. faecalis var. fueculis and S. faecalis var. zymogenes which were used in previous work [ 5,6] . The purified Lipid III was about 0.8% (w/w) of the total lipids from S. faecalis var. faecalis whereas Lipid I and II accounted for 6 and 1.6% (w/w), respectively [5,6]. It should be noted, however, that phospho- and glycolipids together account for only 40-50% of the total lipid weight in all Streptococci so far studied. O-(a~D~u~p~OSyI)a-DglU~p~nO~l]-l(~),

PYLLTWOdfwdoarl-8X~ Tho~~~~llrd*)ruwrltbahlaroform~o~ol/~~~65: dkectioa

of crud8

8.

fwwuallDM8

on 8 Dkte of duc.8 gsl (Mack). 25: 4. by vol.), and the second

with chloforoxm/uwto~/md~~acmtic ‘kcidlW8ter (50 : 20 : 10 : 10 : 5. by vol.). GarbobY~emum=mvlamumd wm c*ruoh*ol/H~so4. MGDG - monoghlco8Yl diacYlglycffoL DGDG - d@ueo@ dla~axol. T8DQ - ~ucoml dka~Woe?ol. Lipid I - phorphntidyl d&lucoayl dlac~~~osrol. LtDld II - m~ceroi-l&orphoryl d&lucosyl d&~l&~erol. L&id III = presently derodbed phogh4ucoYDid (the double rpot my be due to different dtr presentin the CNde extracts) X1. X2 - ph~hoducolipldr which have not yet been Lok$ed. Compound X1 ir chromatogmphically identical with phwhetidyl moaxulucoal dkc~lgl~wrol which hr been imlated from S. hcmolyticur D58 Wscba. W.. unpuM). Contrary to 9. hemolytfcur. no podtionti iwmmr of Lipid I with pboaphatidyl lInkad to the outer Jucow k present(Fkhcr. W.. unpublished).

232

Fig. 2. Thin-Jayer cbromatograms of crude lipid8 from various Streptococci. Abbreviationa III in FJg. 1. (a) Lipid extra& from: 1. whole cella of S. la.ecaJ& vu. faecolb 7064: 2, cytoplasmic membrane of S. laecolb ATCC 9790. (b) Lipid extracts from: 1. S. laecoUa var. foecal& 7064: 2, S. faeccJJa var. zymogened 20672; 3. S. faecalir var. Jiquefaciens 7416; 4. S. hemolyficur D-68; 5, S. Joctis. Development by chloroform/ acetone/methanol/acetic acid/water (b0 : 20 : 10 : 10 : 6, by vol.). Detection with a-naphtbollH2904.

On thin-layer chromatograms purified Lipid III had the staining properties of a ninhydrin-negative phosphoglycolipid, and revealed like Lipid II 1,2glycolgroups on treatment with periodate/Schiff reagent [19]. It showed a curious chromatographical behaviour : on plates of silica gel Woelm it migrated much

FU. 8. TUHAY~I CMOIPILOEUIU of purlfled phophoelucollpidr cdate of silks cl. Merck (b). 6uwlea: 1. Lit&l I from 8. faeeollr vu. faecaJ*: 3. Lipid III from S. fcvc& vu. zymcwena; 4. Dwdo~ment ritb cblorofoxm/meth~~ol/w~tor (65 : 86 : 8. by acid.

on plate of dliee sol. Woehn (a) md on var. ftwcslb; 2. LipId III from 8. ~aecaJ& LipId II !rom- S. faeceJ& vu. faecoJJ#. VOL). Deteation by or~htbol/aM

233 TABLE

I

ANALYSIS

OF PURIFIED

LIPID III

Valuer are given as mol pet 2 mol phO#phONa. Lipid III from

S.faecali# var.faecolis s. faecoli# “ar.rymoge”es

Cub* hydrate

DGlucoa

1.97

1.83 1.92

-

Glycerol

2.94 2.66

Acyl mUPS

PllwphONs

3.86 4.02

2.00 2.00

faster than Lipid II (Fig. 3a) whereas this difference disappeared largely on Merck plate8 (Fig. 3b). A similar behaviour of cardiolipin relative to phosphatidylglycerol suggested fiit that Lipid III might also contain two phosphodiester groups. The analytical data of Lipid III (Table I) are near the values expected for a compound containing D-glucose, glycerol, fatty acid8 and phosphorus in molar ratios of 2 : 3 : 4 : 2. Most of the following studies have been performed on the preparation from S. faecalis var. faecalis. Structure of the deacyfated core

After deacylation by mild alkaline hydrolysis SW%of the lipid phosphorus was recovered in the water phase. A single water-soluble compound was found which moved on partition chromatography more elowly than deacylated Lipid I and II (Fig. 4) and stained positively with phosphate- and periodate/Schiff reagents. The deacylation product was attacked neither by phosphomonoe&erases nor by a-glucosidase. The degradative steps performed for structural analyeis and the quantitative results obtained thereby are summarized in Fig. 5. Partial acid hydrolysis

nr. 4. Thin-lw~ c-matouam of deawkted phorpho6lucoltpida from S. faecolb vu. fawafla on platea of cehloaa (WaW. S=pbr: 1. dwhhd I&id I; deacyM.ed Lipid III:3. dacylated Lipid II. Development with propan-2-01/26% ammonk/water (3.5 : 1.0 : 1.0. by vol). Detection by perIoda Sclw! reagent.

2.

234

DIGLUCOSYLGLYCEROL

?x,/J- GLYCEROPHOSPHATES

0.1 M Na SH 100'C, 2L h

j H2OH

\

/:O;“z.“:h

CL- Glucosldase No

REACTION

-

Oxldatlon

Phosphomonoesterase

0.1 M HCL 2L h

.

mole NaIOL

3.14

mole CH20

mole

GLYCOL

0.8L

mole

GLYCOLALDEHYDE

0.88

mole

GLYCERALDEHYDE

o.a7

37’C.

5.92

, a4 mO,eGLYCOLPHOSPHORYLGLYCEROL Fig. 6. Degradative sequences establishing the structure of the deacylated core. Quantitative resulti are given in mol/mol deacvlation product, corresponding to 2 mol phosphorus. (For details of Smith degradation and determiaation of the breakdown products, see Experimental.)

(2 M HCl, lOO”C, 2 h) liberated glucose almost completely and about one third of glycerol; continued hydrolysis under more drastic conditions which are known to cleave also phosphomonoesters [20], released the rest of the glycerol and the phosphorus as inorganic phosphate. Strong alkaline hydrolysis which breaks phosphodiester bonds gave diglucosylglycerol, and released nearly all of the phosphorus as glycerophosphates. Only negligible amounts of free glycerol (0.08 mol/mol P) were found. Glucose was not released before subsequent treatment of the alkaline hydrolysate with cu-glucosidase which liberated also the third glycerol. From these results it must be concluded that the core consists of a digiucosylglycerol with two e-linkages, and of two glycerophosphate residues which are linked to the diglucosylglycerol by diester bonds. On oxidation with NaI04 (0.01 M) at 37” C the core reduced per mol5.81 and 5.92 equivalents of periodate within 48 and 72 h, respectively, with the liberation of about 3 mol proportions formaldehyde. For definitive linkage analysis another sample was subjected to Smith degradation. The reduced oxidation product (Fig. 5) gave on mild acid hydrolysis about 1 mol proportion of each glycol, glycolaldehyde and glyceraldehyde, and about 2 mol proportions glycolphosphorylglycerol which was identified as described below. Glycerol was released in negligible amounts (0.06 mol/mol P) which were in the same range as with authentic glycolphoaphorylglycerol under the same conditions. On strong acid hydrolysis (2 M HCl, 125”C, 48 h) 0.98. mol glycerol and 1.00 mol inorganic phosphate were released per mol phosphorus.

235 TABLE

II

IDENTIFICATION

OF THE PHOSPHORUS-CONTAINING

SMITH DEGRADATION

FROM DEACYLATED

BREAKDOWN

LIPID III BY GAS-LIQUID

Column: 210 cm X 4 mm; 2% OV 17 on chromosorb WA W; temp. 166-196’C TrimethylsiIyl derivatives

Authentic samples glycolphoephorylglycol ~ycolphorphoryl6lycerol ~ycerophosphoryl6lycerol Breakdown product

PRODUCT

OBTAINED

ON

CHROMATOGRAPHY (2’Clmin).

Relative retention lime

1.00 1.69 2.97 1.60

The trimethylsilylderivative of the phosphorus-containing product obtained on mild acid hydrolysis gave on gas-liquid chromatography a single peak with the retention time of trimethylsilyl-glycolphosphorylglycerol (Table II). Its structure and origin was established by mass spectral analysis. Fig. 6 shows the spectrum of nondeuterated authentic tetrakis (trimethylsilyl)-glycolphosphorylglycerol (a) and that of the corresponding Compound (b) obtained on Smith degradation of deacylated Lipid III using NaB2Ho for reduction. The fragmentation patterns are similar to each other and to those recorded for related compounds [21-231. The ions m/e 491 (M-15), 416 (M-TMSOH), 415 (M-TMSOD) suggest

b)

% 6. The 7O-eV MU rpectra of (a) tati &utwated umIo6uue obtained on Smith ddtion

(trlmcthyldlyl)3ycolphorphoryWyeaol, of deacylated Lipid III.

and (b) the

236

that the parent compound (b) contains two deuterium atoms. A monodeuteroted glycerophosphate moiety with deuterium in Position 1 is reflected by the peaks m/e 462, 446 and 402 (M-TMSOCHD). The ion m/e 463 may be explained by transfer of the trimethylsilyl group and * H, instead of the trimethylsilyl group and H (m/e 462) onto the tris-(trimethylsilyl)-glycerophosphoryl portion (cf. [21] ) since a corresponding pair of m/e 461 and 462 was found with tetrakis (trimethylsilyl)~glycolphosphorylglycerol deuterated in Position I of the glycol moiety. Finally, in Compound (b) a monodeuterated glycol is indicated by the ions m/e 402, 360,372 and 272. Thus, both polyols of glycolphosphorylglycerol must have been formed from oxidized precursors on Smith degradation of deacylated Lipid III indicating that the glycol moiety is derived from a glycerol and the glycerol portion from C-4 ‘through C-6 of a glucopyranosyl residue. This fact together with the quantitative data of Smith degradation products (Fig. 5) proves that in the parent molecule both glycerophosphates are a-isomers and bound to C-6 of the glycosyl residues which are in the pyranose form. The non ionic hydrolysis products (Fig. 5) indicate further that the two glucosyl moieties are linked to a (1 + 2)-bound disaccharide which in turn is attached to the a-position of the third glycerol. The liberation of 1 mol proportion glycol proves independently that none of the glycerophosphates is linked to the glycerol of diglucosylglycerol. The periodate consumption and formaldehyde production is in accordance with this overall structure. Stereochemical configumtion of the glycerophosphates For stereochemical analysis of the both a-glycerophosphates in the deacylated core the glycerophosphates released on strong alkaline hydrolysis were analyzed as given in Table III. The results obtained with reference compounds confirm our earlier observations [5,6,24] that the liberated a-glycerophosphate preserves largely the original stereoconfiguration. Since deacylated Lipid III contains two a-glycerophosphate residues (cf. Fig. 5) the data in Table III indicate unequivocally that one of them must be sn-glycerol 3-phosphate, the other one the unusual sn-l-isomer. At this stage it is still open which of the enantiomeric a-glycerophosphates is linked to the outer and inner glucose (cf. Fig. 5) or whether each glucose carries a mixture of them. TABLE

III

ANALYSIS OF GLYCEROPHOSPHATES OBTAINED LIPID III AND OF SOME REFERENCE COMPOUNDS

ON STRONG

ALKALINE

HYDROLYSIS

OF

Glycerophorphates and a-dycerophorphate ware astimated as trimetbyldlyl-darivatives by -liquid chromatW?aphy udn8 Jycolphosphete as an internal standard. m-Glycerol 3.phosphate and free dycarol were Uuyed by succesdve nactlon with glycerophosphate dehydrogenw and 8lycerokinasc [ 61. Values are given as mol/mol phosphorus. Alkaline hydrolysate of

PhorphoNs

Free dyccrol

Glycerophosphates

cr_Glycerophosphate

Lipid III 3-m-Pbosphatidylchde 1-•-PbosphatldylchoUoa 3-•-Phorphatldyl-l’~nOycsml

i.00

0.08 0.01 0.03 0.98

0.92 0.91 0.98 0.98

0.38 0.39 0.40 0.43

1.00 1.00 1.00

m-Glycerol S-phosphate 0.20 0.31 < 0.01 0.22

237

Location of the acyl group8 and the enantiomeric glycwophoephates The degradative stepa which were used to locate the four acgl groups and the enantiomeric giycerophoephates are outlined in Fig. 7. Lipid III gave on oxidation with NaI04 1.00 mol proportion formaldehyde which indicatea that one of the glycerols is not esterif&. After degradation with 60% HF (w/v) the main products were: glycerol (0.95 mol/mol), inorganic phosphate (1.7 mol/ mol), a mixture of 1,2- and 1,3diglycerides, and a phosphate-free glycolipid which cochromatographed with authentic a-kojibiosyl diacylglycerol. Thus, two acyl groups are apparently located at the glycerol of the basic kojibiosylglycerol; the other pair of acyl groups, released as diacylglycerols, must have been linked to one of the glycerophosphates whereas the second glycerophosphate portion which gave raise to free glycerol cannot have been substituted. The proportions of liberated glycerol and inorganic phosphate indicate that the glycerophosphate moiety was almost completely, but the phosphatidyl residue incompletely split off. Accordingly, among the breakdown products a phosphoglucolipid could be detected with the chromatographic properties of Lipid I (Fig. 8a). This gave first evidence that the phosphatidyl residue may be linked to the inner glucose,

60 *I. HF

ooc, 24 h Phospholipose

0.5 CH20/

-No1044

R-cc-oR-co-0,

H2

I

98%

A2

P

CH3COOH

100° C, 45 min

WI H2f -042

+

GLVCEROPHOSPHATES

H2 “2 &lk

H H

Fig.

7. Degrsdstiw

-O-CO-R

4 -O-CO-R

and the rn~tlorn~~c ~y~erophorphah. and the uhos~~o(lucolWd obtained on dewtion tith

mauencea for the loutton of swl growa

(For analmm of the Jmarouhorphbr acid ma TabM IV and Vh

metie

238

Fig. 8. Thin-layer chromatograms of carbohydrate containing breakdown products obtained from Lipid III by degradation with (a) 60% HP (w/v) and (b) with phospholfpw AZ. Samples: (a) 1. positional isomer of Lipid I; 2. breakdown products of Lipid III: 3, Lipid I. Breakdown products of Llpid III (from top to bottom): kofihioayl diac~lgl~cerol. Ltpid I. lyso compound of kojibiosyl dlacylglycerol. lyso compounds (2) of Lipid I, Lipid II. In contrast to the other products, kojibiosyl dlacylgIycero1 and its bso cqmpound did not stain with phosphate reagent. No intact Lipid III could be detected (control with tica gel plates, Merck). (b) 1. Lipid I; 2. Lipid I, after treatment with phosphollpase Aj; 3. Lipid III; 4, Lipid III. after treatment with phospholfpase AZ. Plates of sfllca gel &VoeIm). Development with chloroform/methanol/water (65 : 35 : 8, by vol.). Detection with ~c-~phthol/H2SO4.

since Lipid I and it8 positional isomer (phosphatidyl reeidue on the outer glucose) can be clearly distinguished on thin-layer chromatography (Fig. 8a). On treatment with phospholipase A2 (hog pancreas) Lipid III was completely converted into a lyso compound (Fig. 8b). According to the stereochemical specificity of this enzyme [ 26 ] the snglycerol Z&phosphate must be involved in the acylated glycerophosphate portion. The location and stereochemical configuration of the phosphatidyl residue was fully established by degradation of Lipid III with 98% acetic acid (v/v) and by subsequent analysis of the main lipid breakdown product. From its known mechanism [ 16,171 this hydrolysis was expected to eliminate the non-substitued glycerophosphate from Lipid III. A8 shown in Fig. 9 degradation of Lipid III resulted indeed in the formation of a phosphoglucolipid (Lane 6) with the chromatographic behaviour of Lipid I. Accordingly, controls running with Lipid II gave cu-kojibiosyl diglyceride (Lane 2), whereas Lipid I proved to be stable (Lane 7). As can also be seen from Fig. 9 all of the glycolipids studied formed byproducts which moved faster on thin-layer chromatography than the parent compounds. Most probably they are partially acetylated derivative8 because on deacylation they gave the same product8 a8 their parent compounds. Transacylation is less probable since acetic acid treatment of kojibiosyl-glycerol resulted also in the formation of faster running derivatives. On treatment of Lipid III .36% of phosphorus became water soluble, indicating a cleavage of 72% of the starting compound. Another treatment of the surviving Lipid III (isolated by thin-layer chromatography) with acetic acid resulted in the 8ame breakdown, and therefore in an almost complete degradation of the starting lipid.

239

Fig. 9. Thin-layer chromatognun of products obtained !rom ~IUCO- end phoevhoslucoHptda on treatment with 98% ace& add (V/V). Sem~lee: 1. Lipid II: 2, Lipid II, treated; 3. koitbioeyl dtaHclycero1. tmated; 4, kojibioeyl diecylgiycerol; 5, Upid Ill; 6. Lipid III. treated; 7. I&id I. tieted: 8. Llptd I; 9. tiodttonal homer of lApId I. Pkta of dltca gel (Merck). Development wltb chloroform/ecetone/memethnol/ece~c wzid/water (60 : 20 : 10 : 10 : I. by vol.). Detection by u_nrphtboI/H~804.

The water phase contained no glycerol but only glycerophosphatee euggesting that, like on alkaline degradation, the phosphodiester is split by cyclization at the glycerophosphate moiety. The glycerophosphates released from Lipid III and from 8ome reference compound8 were analyzed a8 is ehown in Table IV. It became apparent that, in contra8t to alkaline degxadation, the composition of the a-glycerophosphates liberated by acetic acid doe8 not allow to draw any conclusion concerning the stereochemical configuration of the glycerophosTABLE

IV

ANALYSIS OF GLYCEROPHOEPHATEE ENCE COMPOUNDS WITH 98% ACETIC

OBTAINED ACID

ON DEGRADATION

OF LIPID Ill AND

REFER-

In no cue glycerol WM liberetad. Noa-cyclIc dycerophoephete’ wea aneyed u glycerol efter txeetment of the degcadetton mixture wtth phoephomonaataue. totd dycerophoephate after consecutive treatment wtth NeOIi (0.06 M. 37%. 12 h) end phoetahomoiioeeterw. cr-G1ycerophorphat.e.awere determined by. perlodete chromotropic ecid procedure. anOycero1 Sphwhate enzymettcelly. Valuea ere given ea mol/ mol phoephorua. Phosphorus

Weter-soluble phorphoma from lipid llI m-Glyccrd-1-phaphorlchollne BGlycerophoghate

1.00 1.00 1.00

TOW

Non cycle

IdYcerophorphate

awere phosphate*

0.96 0.98 1.02

0.66 0.69 0.64

a_Glycemphorphete

m-Glycerol 3-phorphete

0.60 0.61 0.64

0.26 0.26 0.28

* The difference between total end non cyclic glycerophmhate is tantettvely ueumsd to be cyclic dycerophorphate. This fraction wee not further rtudied: en acetylated derivative ten therefore not be excluded.

240 TABLE V ANALYSIS OF THE DEACYLATED LIPID OBTAINED AS THE FROM LIPID III ON TREATMENT WITH 96% ACETIC ACID (V/V)

MAINDEQRADATION

PRODUCT

mol/mol phosphoNs Phoeplloms Glucoee. liberated on treatment with a-glucoddase

1.00 1.01

Producb after rtronp alkaline hydrolyeia &‘cerOFhoQhetas a-glycerophoephete sn-&'Cerol %DhOrphate Alkaline hydrolyute.

0.98 0.39 0.36

treated wltb aqlucoddue

glucose 6lycerol

1.98 0.99

phate in the parent molecule. A8 shown with figlycerophosphate phosphoryl migration takes place even on the released glycerophosphates. Therefore, the phosphatidyl diglucosyl diacylglycerol (Fig. 9) which wa8 produced from Lipid III by loss of the glycerophosphate wa8 isolated by preparative thin-layer chromatography (chloroform/acetone/methanol/acetic acid/water/SO : 20 : 10 : 10 : 6, vol.). The lipid WBSdeacylated and analyzed as shown in Table V, The data establish: (i) it is a glycerophosphoryl digluco8yl glycerol, (ii) the inner glucose is substituted with the glycerophosphate and (iii) more than 9096 of the latter i8 sn-glycerol 3phosphate. In consequence, the 8n-glycerol l-phosphate has to be located at the outer glucose of the native lipid III. Summarizing the results, the complete structure of Lipid III can be given a8 3(l)-O-[6”-(on-glycerol l-phosphoryl), 6’-(1,2diacyl-snglycerol-3-phosphoryl), 2~O-(u-~-glucopyran~1yl)-o-~-glucopyrano8yl]-l(3),2 diacylglycerol. The glycerol may be substituted by the disaccharide at Position 3 as is the ca8e with kojibiosyl diacylglycerol from S. faecilis [ 261. Fatty acid composition and distribution

For analysis the fatty .acids were released from Lipid III by mild alkaline hydrolysis. The constituent fatty acid8 of the phosphatidyl and kojibiosyl diacylglycerol portion were obt&ned from the lipid products of HF degradation (cf. Fig. 7). To get the positional distribution at both diglyceride moieties Lipid III ~88 treated with phospholipase AZ . The resulting 1~80 compound wa8 degraded by acetic acid into lysophosphatidic acid and kojibioayl diacylglycerol which in turn wa8 hydrolyzed with the l-position specific lipase from Rhizopus arrhizue (cf. l’ef8 6,6,18). The results are 8ummarized in Table VI. The overall fatty acid composition is very similar to that of the other polar lipid8 from S. faecalis (cf. [5,6] ). The composition of both diacylglycerol portion8 is also similar apart from the content of C1 8 monoenic and C, 9 cyclopropane acid whose 8um8, however, are nearly the 8ame. A8 cyclopropane fatty acid8 were reported to be aynthesized from the corresponding monoenic acid8 on preformed lipid molecule8

241 TABLE VI FATTY

ACID COMPOSITION

The reactiona used to liberate

AND THEIR POSITIONAL

DISTRIBUTION

IN LIPID III

the fatty acida from the four poaitiona separately are ftiven in the text.

CY,

cyclopropane fatty acid. VaIuer are given as mol k. Component

Lipid III total

Macylglycerol

Phorphatidyl reddue Total

Positlon 1 Trace

residue

Podtion 2

TOW

Poettion 1 l

Position 2

14 : 0 16 : 0 16: 1 18 : 0 18: 1 19 cy

6.7 28.4 10.4 1.7 39.7 14.0

6.1 31.6 8.2 2.1 33.6 19.3

7.4 2.2 2.9 49.0 38.4

11.4 46.1 19.0 2.2 18.6 2.7

3.2 29.7 10.8 2.9 46.5 6.7

0.6 4.4 3.6 4.1 73.5 13.4

6.8 56.0 18.0 1.7 19.5 Trace

C14-C16 ClrC19

44.6 56.4

44.9 65.0

9.8 90.3

76.5 23.4

43.7 66.1

8.6 91.0

18.8 21.2

* Although the enzymatkaIIy reIewd fatty acids were anaIyzed. the composition o! Podtion 1 wu cakulated ae difference between the fatty acide of the diglucosyl diacylgIycero1 and those of the lyso compound to avoid erron cawed by acyl migration.

[27]the differences found may be explained by a low affinity of the diglucosyl diacylglycerol moiety of Lipid III and it8 biosynthetic precursor to cyclopropane synthetase. Both the phosphatidyl and diglyceride residue of Lipid III displayed a distinct preference of C; 4 through C1 6 fatty acids at Position 2 of their glycerol moieties whereas Position 1 was occupied preferentially by C, 8 monoenic and Cl9 cyclopropane acid. The same distribution pattern was found earlier with the other polar lipids from S. faecolis [5,6] suggesting that the same phosphatidic acid is the common biosynthetic precursor of both the phosphatidyl residue8 of phospholipids and the diacylglycerol moieties of glucolipids. The data obtained with Lipid III fit equally well into this picture. DiSCU88iOIl

From the present and previous studies [5,6] it became apparent that the cytoplasmic membrane of S. f4ecaZb contains five phosphoglycolipids. Lipid I-III (Fig. 10) are derived from kojibiosyl diacylglycerol which itself is an abundant component (Figs 1 and 2). In agreement, kojibiosyl diacylglycerol and the corresponding portions of Lipid I-III show the same positional distribution of their fatty acid8 (Table VI, cf. [5,6] ). Lipid III combine8 the substituents of both Lipid I and II, with the same location at the basic kojibiosyl diacylglycerol (cf. Fig. 10). In addition, the phosphatidyl portion8 of Lipid III and Lipid I display a nearly identical fatty acid composition (Fig. 11) and positional distribution (Table VI, cf. [6] ). The common structural features especially the location of the substituenta suggest that the biosynthesis of Lipid III should be closely related to that of Lipid I and II (Fig. 10). According to.Pieringer [28] Lipid I is formed by phosphatidyl transfer from diphoephatidyl- or phosphatidylglycerol to kojibiosyl diacylglycerol. As suggested earlier Lipid II could be synthesized by a corresponding transfer of sn-glycerol l-phosphate from phosphatidylglycerol [5]. Some

242

LIPID

III

H&O-CO--R

Fig. 10. StNCtUmS of Lipid I. II and III with proved and postulated biosyntietlc pathWaYs (see tert). DGDG = kojibfosyl dlacylglycerol, PG = phosphatidylglycerol, DPG = diphosphatidylglycerol.

evidence for this reaction was recently obtained in preliminary studies on the biosynthesis of sn-glycerol-phosphoryl galactosyldiglycerides in Bifidobacterium bifidum [7] . Furthermore an analogous reaction was observed in is Escherichiu coli where the sn-glycerol l-phosphate of phosphatidylglycerol transferred to a non lipid oligosaccharide [29,30]. 19cy

Mole % SO-

18:l 1s:o 16:l

LO.

30. 20.

lO:l/il

J

Cl G 0 G- PhosphotidylDGDGPhosphatidylResfdus Residue Residue Rosldue p w LIPID I LIPID 111 LIP10 II Fig. 11. Fatty acid composition of Lipid II, and both diglyceride moieties of Lipid III and I. (The data of Lipid I and II were taken from refr 6 and 6). Abbreviations as in F&. 10.

243

As in our studies no positional isomers of Lipid I and Lipid II have been found in S. faecalis (cf. Fig. 1 and ref. 5) it has to be concluded that the phosphatidyl transferase displays an absolute specificity to Position 6 of the internal glucose, whereas the proposed glycerophosphoryl transferase reacts just as specifically with Position 6 of the outer one. Consequently, the structure of Lipid III suggests that this lipid might be synthesized by the joint action of both these transferases. According to our previous studies [5,6] Lipid II differs from Lipid I as well as from the gluco- and phospholipids of S. faecalis by a lower content of C1 9 cyclopropane acid. Just the same has now been found for diglucosyl diacylglycerol moiety of Lipid III whereas its phosphatidyl moiety shows a normal content (Fig. 11). In the biosynthesis of Lipid III, therefore Lipid II should be the intermediate rather than Lipid I. Contrary to S. faecalis, some other Streptococci although able to synthesize Lipid I and II, can not form Lipid III (Fig. 2b) suggesting that their phosphatidyl transferase does not react with Lipid II. It is not yet known whether such minor components as Lipid III and Lipid II ‘have a function in the cytoplasmic membrane of S. fakalis. On the other hand it must be noted that by our extraction procedures only those lipids were picked up which occur in a free state. It could therefore be possible that larger amounts are present as covalently bound constituents of more complex structures. The lipoteichoic acids of gram-positive organisms which have polyglycerophosphate backbones are thought to be anchored in the cytoplasmic membrane by a covalent bond to a glycolipid of heretofore unknown structure [ 31-351. Evidence that the glycolipid moiety of S. faecafis lipoteichoic acid is a phosphatidyl a-kojibiosyl diacylglycerol has been afforded by degradation of the polymer with HF [32], but obviously thereby neither the location of the phosphatidyl residue nor that of the teichoic acid at the basic diglucosyl diacylglycerol could be determined. According to recent results with Staphylococcus aureus and S. sanguis lipoteichoic acid is synthesized from phosphatidylglycerol rather than from CDPglycerol [36,37]. If snglycerol l-phosphate can be proved to be transferred in this reaction the structure of Lipid III and II suggests that they could be involved in lipoteichoic acid, being either starting molecules of biosynthesis or enzymatic breakdown products. In this respect it might be relevant that similar to Lipid II and III the content of C19 cyclopropane acid was also found to be lower in lipoteichoic acid than in other membrane lipids of S. faecalis [ 321. Acknowledgements We wish to thank Professor G.H. de Haas for the gift of phospholipase and sn-l-lecithin, Dr H. Milldner for membrane lipids of S. faecalis, Professor-A. Tolle for supply of strains of Stteptococci, and Dr T. Tomita for mold lipase. We thank Mr R. Biermann for recording the mass spectra and Mr J. Herrmann for performance of part of gas-liquid-chromatographical analyses. The excellent asi&ance of Mrs Ursula Plotz is gratefulIy acknowledged. Uhich Fischer is thanked for his help. This work was supported by the Deutsche Forschungsgemeinschaft.

244

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