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Biochemical changes in Bifidobacterium bifidum var. Pennsylvanicus after cell wall inhibition VII. Structure of the phosphogalactolipids
Biocizi/kcu ?’ Elsevicr
et Bioplrysicrr Ar,frr. 348 ( I 974) 370.-387 Scientific Publishing Company. Amsterdam
- Printed
in The Netherlands
BBA 56450
BIOCHEMICAL
CHANGES
PENNSYLVANICUS VII. STRUCTURE
(Received
December
AFTER
1N B~F~~~BACTERiU~ CELL WALL
B~Fi~~~~~~ VAR
1NHlBlTlON
OF THE PHOSPHOGALACTOLIPIDS
14th.
1973)
SUMMARY
The structures of two major phosphogaiactoiipids from BiJdohacreritml bifidum var. pennsylvanicus have been established by chemical analysis and chemical and enzymatical degradation procedures. The compounds were identified as 3-U-f6’-(snglycero-1 -phosphoryl)-/3-D-galactofuranosyI)-snI ,2-diglyceride and its lysoderivative. A third minor phosphogalactolipid was a diacyl derivative of the former compound. About 10-200,~ of these compounds appeared to be gala~topyranosyi derivatives. Phosphorylgalactosylglycerol and galactose phosphate were identified as one of the products of alkaline and acid hydrolysis, respectively. Marked differences were present in the fatty acid composition of monogala~tosyldigly~eride, phosp~latidylglycerol and the two main phosphogalactolipids.
INTRODUCTION
TWO unknown phosphate-containing lipids, earlier [I] designated as Compounds I 5 and I 7, have been detected in B#idobacterium hzji’dwu var. penns!.lvanic’lts. These compounds constituted a considerable amount of the total lipid radioactivity of cell wall synthesis by lack of human after incorporation of 32P. After inhibition milk, which contains essential growth factors [2] the galactose content of the total lipids, the ratio of digalactosylto monogaia~tosyldigiycerides and the percentages of these unknown phosphate-containing lipids were considerably decreased [i, 41. These unknown lipids were also detected in 14 B@dobacterium strains of various origin, but were not present in 9 ~ac~obucii~~~ strains [5]. Originally [I] the compounds were characterized as polyglycerof phosphohpids, because glycerophosphorylglycerol was detected as one of the deacylation products. However, after extensive purification of the unknown lipids, this compound proved to be absent after deacylation. Because further investigations led to an identification of galactose as a component, the unknown lipids could belong to the class of phosphoglycolipids [6, 71. The structure of the six major galactosyldiglycerides in 3. h13dt4m var. ~e~ln~~~7~~a~liclis was reported
371
earlier [S]. In this paper we report the elucidation of the structure of the two major phosphogalactolipids and give also some information about the structure of a third phosphogalactolipid, which was earlier [I] designated as Compound 6. The investigations were hampered by the fact that the compounds were present in both galactofuranoside and galactopyranoside forms which could not be separated. The composition of the intact lipids and a mild alkaline deacylation product was analyzed. Their structure was established by identification of the products obtained by chemical and enzymatical degradation procedures. MATERIALS
AND
METHODS
Cultivation of the organism in 8-1 cultures for I 6 h and extraction of lipids were performed as described previously [I]. Cells were washed twice with 0.2 M sodium acetate buffer (pH 5.0). Incorporation of 32Pi was made in 8oo-ml cultures by addition of I mCi of Na,H3’P04 and cultivation for 16 h. The lipids from these cultures were mixed with the non-radioactive lipids. Phospholipids were obtained by silicic acid fractionation of total lipids [I]. They were subfractionated on silicic acid columns with chloroform-methanol mixtures [I]. The purification of the two unknown compounds, phosphatidylglycerol and diphosphatidylglycerol was achieved by preparative thin-layer chromatography on silicagel G (Merck, Darmstadt, Germany) in the Solvent Systems I, chloroform-methanol-7 M ammonia (60:35: 5, by vol.) and 2, chloroform-methanol-acetic acid-water (125 : 37 : 9.5 : I .5, by vol.). For a fina. step silicagel HR with addition of 3 % magnesium silicate was used with Solvent System 3, chloroform-methanol-water (65 : 25 : 4, by vol.) as developer. After detection by autoradiography or scanning, the compounds were eluted from the silicagel with chloroform-methanol-water (IO : IO : I, by voi.). Lipids were stored under benzene-methanol (4:1, by vol.) at -20 “C. Hydrolysis procedures and isolation qf hydrolysis products Deacylation products of the phosphate-containing lipids were prepared according to a modified procedure of Dawson [9]. Before addition of the reagents the lipids were dissolved in 0.2 ml chloroform. The hydrolysis was repeated once. Paper electrophoresis for 2 h at 30 V/cm in Solvent System 4, pyridine-acetic acid-water (I :IO: 89, by vol.) (pH 3.5) was used for separation of the deacylation products. After detection by autoradiography they were eluted with water and purified by repeated electrophoresis or paper chromatography in the Solvent System 5, r-propanol-conc. ammonia-water (6 : 3 : I, by vol.). Acid hydrolysis of the deacylation products was performed by heating for 2 h at IOO “C in 2 M HCl. Alkaline hydrolysis was carried out by heating for 3 h at IOO “C in I M NaOH. Hydrolysis products were isolated by preparative paper electrophoresis in Solvent System 4. The on the origin remaining galactosylglycerol compounds were eluted and separated by paper chromatography in Solvent System 6, butanol-pyridine-water (6:4: 3, by vol.). Detection was carried out by scanning of radioactivity, autoradiography or staining with one of the reagents described previously [I, 81. 32P. a-glycerophosphate, glycerophosphorylglycerol, galactose 6-phosphate, glycerol, galactose and /I-galactosylglycerol compounds prepared from the monogalactosyldiglycerides [8] were used as reference substances for the qualitative identification of
372
the hydrolysis products by paper efectrophoresis in System 4 and paper chromatography in the Systems 5,6 and 7, ethylacetate-pyridine-water (10:4:3, by vol.). Preparations used for analytical determinatiolls, mass spectrometry and proton magnetic resonance spectroscopy were purified additionally by chromatography on acid washed Schfeicher and Schiifl paper No. 2045~ in Solvent System 6. Enzymatic hydrol.vsis procedures Enzymes were obtained from Boehringer and Soehne, Mannheim, unless otherwise indicated. Hydrolysis by phospholipase A, (EC 3. I. 1.4). An amount of I .o jlmole (30.000 cpm) [3zP]phosphogafactolipid, [32P]phosphatidyfgfycerof or egg lecithin was dispersed in I .5 ml of diethyf ether and shaken with 0.2 ml of o. I M Tris-mafeate buffer (pH 7.0) containing 0.5 hlmofe CaCf, and 6-10 units phosphofipase A, (Crofalus adamaffte~s, Sigma Chemical Co., St. Louis MO. or Crotalus terr. fur) for 2-4 hat 37 “C. Incubation of phosphogalactol~pids was also performed in 5 ml of 0.1 M Tris-HCI buffer (pH 8.0) containing 2 mg sodium deoxychotate, 12.5 /lmofes CaCf, and 6 units phosphofipase A,, the activity of which was checked using egg yolk lipoprotein [IO]. If_ydrolysis by phospholipase C (EC 3.1.4.3). Incubation of I jtmofe (30000 cpm) [32P]phosphogafactofipid, [32P]phosphatidyfgfycerof or egg lecithin was carried out with 0.3 ml of 0.2 M Tris-mafeate buffer (pH 7.4), containing 4 Itmoles CaCfz and 2 units of phospholipase C from Bacillus cereus for 4 h at 37 ‘C. The phosphofipase C preparation was obtained from Dr R. Zwaaf, Department of Riochemistry, University of Utrecht. The activity of the enzyme was measured by continuous titration of the acid produced by hydrolysis of egg yolk lipoprotein [I I 1. Hydrolysis by p~~~sp~?o~~~ase D (EC 3.1.4.4). Amounts of I /[mole [“2P]phosphate containiilg lipids were dispersed in 2 ml diethyf ether and shaken with 0.8 ml of 0.05 M sodium acetate buffer (pH 5.6) containing 4.5 pmofes CaCf, and 2.8 mg (I .4 units) phospholipase D from cabbage. Incubation was performed for 4-16 h at 37 “C.
Hydrolysis b.v pancreatic lipase (EC 3. I. 1.3). E3’PIPhosphate-containing lipids were sonicated with a Bransson sonifier Model Br2 for 30 s in an amount of I jtmole with 5 ml of 0.05 M Tris-HCl buffer (pH 8.0) containing 2 mg sodium deoxycholate and 12.5 [rmofes CaCI,. 1.2 ml of these emulsions were incubated with 25 /moles CaCf, and 50 pg pork pancreatic fipase for 2 h at 23 “C. The pure lipase preparation was obtained from Dr R. Verger, Department of Biochemistry, University of Utrecht. The activity of the preparation was checked using tributyrine as substrate [12]. ~~~dro~?tsisby p~?os~~l~odiesteras~ (EC 3.1.4.1). Incubation of water-soluble phosphodiesters was performed in 0.1 M Tris-HCI buffer (pH 8.9) for 3 h at 37 ‘C with IO units phosphodiesterase from Russel’s viper venom (B grade, Calbiochem). When the phosphogalactofipids were used as substrate, an equal volume diethyl ether was added and the incubation mixture shaken violently for 4 h at 30 C. NydroEysis by phosphomonoesterases. Incubation with alkaline phosphatase (EC 3.1.3. I) from calf intestine was performed in a 0.1 M Tris-HCI buffer (pH 8.2), from potatoes was used in a O. I ?W citrate containing 1 mM Mg” +. Acid phosphatase buffer (pH 5.6). Incubations were performed for 4 h at 37 “C. Hydrolysis by gaiac’tosidases. The water-soluble hydrolysis products were incubated with cc-galactosidase from coffee beans and with P-galactosidase from
373
Escherichiu coli for 3-16 h according previously described procedures [8]. The same check procedures were followed. After incubation with lipolytic enzymes or phosphodiesterase the procedure of Bligh and Dyer [13] was followed to separate the lipid-soluble and the water-soluble fractions. The amount and the identity of the (radioactive) products were determined as described below. Periodate oxidation
Periodate oxidation was always performed at 22 “C. Lipids were dissolved in chloroform-ethanol (I : 3, by vol.) and treated with 0.25 vol. IO mM NaIO, at pH 5.0 for 24 h. The isolated deacylation and hydrolysis products were oxidized in 10-20 mM NaIO, at pH 6.0. Mild periodate oxidation was performed in 5 mM NaTO, for 15 min. Smith degradation of the phosphogalactolipid was performed in chloroformethanol (I : 3, by vol.) after addition of 0.25 vol. 0.05 M NaIO, for 72 h at 23 “C. The oxidation mixture was treated with ethyleneglycol and KBH, [8]. Thereafter the lipid was deacylated in 0.1 M methanolic NaOH. The inorganic ions were separated and the oxidation product hydrolyzed in 2 M HCI for 2 h at IOO “C. For Smith degradation of water-soluble compounds 0.05 M periodate (pH 6.0) was used for 16 h. After addition of ethyleneglycol the oxidation products were reduced with KBH, for at least 16 h at 4 “C. The residual KBH, was destroyed with acetic acid and K+ and borate ions eliminated [8]. The products were partially used for studies by chromatography or electrophoresis. Another part was hydrolyzed for 2 h in 2 M HCI at IOO“C or for 72 h in 6 M HCI at 120 “C. The thus obtained products were analyzed by paper electrophoresis and chromatography or by gas-liquid chromatography together with reference compounds. Treatment with HF
Degradation of lipids and their deacylation products was performed with 60 % HF according to the procedure of Toon et al. [14] for 16-24 h at 4 “C. The solutions were neutralized with a LiOH solution and freeze dried. Lipids were extracted with chloroform-methanol (2:1, by vol). The detection of glycerol was performed as described by Fischer et al. [I 51. Analytical procedures
The purity of the isolated phospholipids and phosphogalactolipids were checked in at least three systems by thin-layer chromatography in Solvent Systems I, 2, 3 or 8, chloroform-methanolconc. ammonia (70: 20: 2, by vol.) or by chromatography on silicagel impregnated paper (Whatman S G 81) in System 9, chloroformmethanoldiisobutylketone-acetic acid-water (45 : I 5 :30 : 20: 4, by vol.) [I]. They were also checked on their deacylation products. The purity of the isolated deacylation products and the hydrolysis products was checked by paper electrophoresis in System 4 and by paper chromatography in the Systems 5, 6 and 7. The identity of the hydrolysis products was established by analysis and comparison with reference compounds in the same systems. Staining procedures have been given previously [I, 81. Analytical methods for galactose, glycerol, formaldehyde, periodate, ester
374
groups, phosphorus and total hexose have been described previously [I, X]. strGlycerol j-phosphate was determined with glycerol-j-phosphate dehydrogenase (EC 1.1.1.8) [r6]. Gas chromatography of trimethyl-silylated degradation products from lipids and their deacylation products was performed on the same columns and under the same conditions as previously used [8]. Preparation and gas chromatography of fatty acid methyl esters were also described before [t7]. Nonadecanoic acid methyl ester was used as an internal standard for quantitative analysis. The radioactivity of an aliquot of the organic phase after Bligh and Dyer [I 31 extraction was measured after evaporation of the solvent in IO ml toluene, containing 4 g Omnifluor (NEN Chemicals GmbH, Dreieichenhain, W. Germany) per 1. An aliquot of 0.5--1.0 ml of aqueous solutions was counted in to ml Aquasol (Merck, Darmstadt). Radioactive compounds on thin-layer plates or chromatograms were located by scanning or by exposure for 24-96 h of Kodak X-ray film. The radioactivity of the spots was measured by liquid-scintillation counting [I]. Optical rotations were measured with a Perkin-Elmer Model 141 polarimeter. Glycosides were dissolved in water. Pertrimethylsilylation of the galactosylglycerol compounds followed by mass spectrometry and PMR spectroscopy were performed as described by Kamerling and co-workers [I 8, I 91. RESULTS
The purification of the unknown phosphate-containing lipids in B. h$k/um var. pennsvl~~akx.tsproved to be rather complicated. Silicic acid column fractionation and thin-layer chromatography in 3 solvent systems were necessary to separate them from all contaminating lipids. Their Iocation on a two-dimensional thin-layer chromatogram is given in Fig. I. The unknown phosphate-containing lipids designated as 6, 15, 17 and 18 turned out to belong to the phosphogalactolipids. Results on the structure of the more apolar Lipid 6 will be described at the end of this section. Lipid 18 proved to be another salt form of Lipid 17, because it did only separate after extraction on the first thin-layer chromatogram. After elution it migrated together with Lipid 17. The compounds did not stain with ninhydrin. The presence of sulphate in the lipids could be excluded by lack of [3”S]sulphate incorporation into the lipids. Acid hydrolysis of the pure compounds in 2 M HCI at IOO “C for 2 h yielded glycerol, glycerol phosphate, galactose and galactose phosphate as constituents. The identity of these compounds was established by paper chromatography in 3 solvent systems (5, 6 and 7), that of glycerol and galactose also by gas chromatography. The results on the structure of galactose phosphate will be given in a separate paragraph. Quantitative analyses of the pure Lipids I 5 and I 7 demonstrated a molar ratio for hexose : glycerol: phosphate of about 1: 2 : I (Table I). Both lipid compounds were rapidly periodate-Schiff positive and yielded I mole formaldehyde per mole phosphate, indicating the presence of one 1,2-glycol group. The results of the fatty acid ester assays by the alkaline hydroxamate method and by gas chromatography showed both the presence of 2 moles fatty acids per mole phosphate in Lipid 15 and one in Lipid 17. A survey of the main chemical and enzymatical degradation procedures is
375
Fig. I. Autoradiogram of a two-dimensional thin-layer chromatogram on silicagel G of a lipidextract from cells grown with 32Pi. The chromatogram was developed in Direction I with chloroformmethanol-7 M ammonia (60: 35:5, by vol.) and in Direction II with chloroform-methanol-acetic acid-water (125: 37:9.5:1.5, by vol.). Identity of spots: I, diphosphatidylglycerol; z, acylphosphatidylglycerol; 4, triacyl-bis-(glycerophosphoryl)glycerol; 6, phosphogalactolipid together with phosphatidylglycerol, salt form; 7 and 8, phosphatidylglycerol; 9, alanylphosphatidylglycerol; IO, diacylbis-(glycerophosphoryl)glycerol; I I, monoacyl-bis-(glycerophosphoryl)glycerol ; I 3. not identified ; I 5, phosphogalactolipid; 17 and 18, phosphogalactolipid; 19, not identified. The spot at the upper right side of the autoradiogram originates from a marker. TABLE
I
ANALYTICAL Molar values
DATA
ON LIPIDS
15 AND
17 AND
THEIR
DEACYLATION
PRODUCT
ratios to phosphate are given as the mean of duplicate analyses on at least 2 preparations. of periodate rapidly used reflect the amounts used within 5 min. N.D., not determined. Compound
Phosphate Hexose Glycerol Galactose Formaldehyde Periodate rapidly used Periodate total used Acyl groups
I5
Compound
1.00 0.88 2.05
I .oo 0.69
0.54 0.76 0.90
0.51 0.74
1.02
1.88
I.95 N.D. 1.23 1.15
17
Deacylation
The
product
I .oo I .05 1.90 0.60 2.07
1.87 3.30
given in Fig. 2. Autoradiograms of an electropherogram and a paper chromatogram of 32P-containing degradation products are reproduced in Figs 3 and 4, respectively. The common mild alkaline hydrolysis product of the phosphogalactolipids (MPi 0.52 and RF in Solvent System 5 0.44) contained nearly all the lipid phosphorus, hexose
376
.“M .
c
: ‘i
ri
,A i.
Tt
.4 u
and glycerol, which indicated that these lipids contained only ester bonds. The deacylation product showed the same hexose: glycerol: phosphate ratio as the intact lipids, but an additional r,z-glycol group (Table I). The amounts of sugar estimated by the total hexose assay (anthron method) and the specific galactose determination in an acid hydrolyzate showed a ratio of about two. However another hexose did not appear to be present in the lipids. The
377
Fig. 3. Autoradiogram of an electropherogram of 32P-containing degradation products of Lipid 15 and phosphatidylglycerol. Water-soluble phosphate esters were separated by paper electrophoresis for zh at 30 V/cm in a pyridine-acetic acid-water buffer (I :IO: 89, by vol.) (pH 3.5). Identity of spots: I, Pi; 2, the mild alkaline deacylation product; 3. the deacylation product after periodate oxidation and reduction; 4. the same after addjtjonal acid hydrolysis; 5, the products after acid hydrolysis of the deacylation product; 6, the products after strong alkaline hydrolysis of the deacylation product; 7, glycerophosphorylglycerob The spots on the start line are only markers.
Fig. 4. Autoradiogram of a paper chromatogram of 32P-containing degradation products of Lipid 15, phospbatidylgIycero1 and diphosphatidylgIycero1. The chromatogram was developed with n-propanolcont. ammonia-water (6: 3:1, by vol.). The start line is marked by two dark spots. The following fractions were separated: I, the deacylation product of Lipid 15; 2, the deacylation product after oxidation and reduction; 3, the same after additional acid hydrolysis; 4, the products after acid hydrolysis of the deacylation product; 5, the products after strong alkaline hydrolysis of the deacylation product; 6, glycerophosphorylglycerol; 7, bis-(glycerophosphoryl)glycerol.
ratio became one, when galactose was assayed in an acid hydrolyzate, treated with alkaline phosphatase. These results suggested that a binding of phosphate to D-galactose was still present after acid hydrolysis. The detection of galactose phosphate as a component of the phosphogalactolipids excluded the possibility that the unknown lipids were glycerophosphorylglycerol derivatives. The earlier [I] reported observation of glycerophosphorylglycerol as one of the deacylation products could be explained by the presence of a small amount of lysophosphatidylglycerol in the preparations of the isolated phosphogalactolipid. The glycerophosphorylglycerol structure as a base for a phosphatidylglycerylgalactose was also disproven by the following observation. No glycerol could be detected after periodate oxidation of the deacylation product of the phosphogalactolipid, when the oxidation was only followed by addition of arsenite to destroy the excess of periodate, and acid hydrolysis. The same procedure applied on bis-(glycerophosphoryl)glycerol as a check yielded glycerol. The unknown lipids and their common deacylation product were not affected by treatment with phosphomonoesterases, suggesting a phosphodiester linkage. Phosphodiesterase from Russell’s viper venom did not split this linkage. This does not exclude a phosphodiester linkage in a phosphoglycolipid, because diglycosylphosphorylglycerol was also inert to this enzyme [20]. Alkalir~ehydrolysis c~fthe deacylatiotl product Strong alkaline hydrolysis (I M NaOH at IOO ‘C for 3 h) of the deacylation product yielded besides glycerol and glycerophosphate a polyol and a polyolphosphate MP, 0.58, (Fig. 3). The polyol contained equimolar amounts of glycerol and galactose, the polyolphosphate glycerol, galactose and phosphate in the same proportions. The relative amounts of glycerophosphate and the polyolphosphate agree with the amounts of galactosyiglycerol and glycerol, respectively (Table 11). Both phosphates were hydrolyzed by alkaline phosphatase, indicating that two monoesters were formed by alkaline hydrolysis from a diester. The polyol showed in the Solvent Systems 5 and 6 two spots, with R, values identical to the furanose and the pyranose TABLE
II
RELATIVE
AMOUNTS
OF DEGRADATION
PRODUCTS
OFTHE
DEACYLATtD
The deacylation product was hydrolyzed for z h in 2 M HCI or for 3 h Degradation products were separated by paper electrophoresis. The j*P containing products was measured and given in percentages of total 32P experiments. The ratio of glycerol and galactosylglycerol was determined and after acid hydrolysis of the neutral products after alkaline hydrolysis. the glycerophosphate isomers were assayed as described in the text. Product
Acid hydrolysis
Galactose phosphate Glycerophosphate Phosphorylgalactosylglycerol Glycerol Galactosylglycerol sn-Glycero I -phosphate s,Kilycero j-phosphate Glycero z-phosphate
33 63
Alkaline hydrolysis
s4 46 45 55
‘I 1 80 20 ~._~_~__
149 5’ ~_~
45 4
LIPID
15
in I M NaOH at loo C. activity of the phosphatractivity as the mean of z by a glycerol assay before The relative amount? of
379
isomers of galactosylglycerol, prepared from the monogalactosyl lipids of B. b$dum var. ~e~nsylva~icus [8]. The ratio of the furanose form (Iz, 0.43) and the pyranose form (RF 0.27) was determined after separation in Solvent System 6. Galactose assay after elution and acid hydrolysis resulted in a ratio of 81 :rg. Gas chromatographic analysis of the trimethylsilyl derivatives of the polyol gave also two compounds with retention times identical on two columns to those of the monogalactosylglycerol compounds derived from the gaiactosyldiglycerides. The main one (86%) proved to cochromatograph with 3-U-~-D-gaIactofuranosyl-~~-glycero1 from B. bl~d~rn var. ~e~~sy~vanic~s, the other (14 %) with the 3-~-~-D-galactopyranosyl-so-glycerol from spinach and B. bl$dum var. pennsylvanicus. The polyolphosphate appeared to be a mixture of two compounds with R, values of 0.3 I and 0.25, respectively in Solvent System 5. After phosphatase action on the polyolphosphate the same two forms of galactosylglycerol were detected as above. Thus the polyolphosphate appeared to be phosphory~galactosyig~ycerol, present in both furanose form (RF 0.31) and pyranose form (RF 0.~5). The appearance of phosphoryIgalactosylglycero1 at alkaline hydrolysis of the deacyiation product can be explained by the fact that cyclic phosphodiesters can be formed on the galactose molecule, in the case of the furanose form between carbon atoms 5 and 6, in the case of the pyranose form between carbon atoms 4 and 6. Only these carbon atoms are mentioned, because the phosphate group is linked to carbon atom 6 in the intact deacylation product, as will be shown in the next paragraph. The formation of phosphorylgaIactosylglycero1 is remarkable, because phosphoglucohpids do not yield any detectable product in which the phosphate-glucose linkage is retained, like phosphoryldiglucosylglycerol (7, 151. Phosphoglucolipids can not easily form a cyclic diester on the glucose molecule at alkaline hydrolysis, because the glycerophosphate is linked to carbon atom 6 of a gIucopyranoside with a trans hydroxyIgroup at Position 4. The cis- or trans-orientation of the neighbouring hydroxyl groups inff uences markedly the products of alkaline hydrolysis, as was established by Brown et al. [21 J. Acid hydrolysis
qf the deacylation product
Acid hydrolysis (2 M HCl, IOO“C, 2 h) of the deacylation product gave as products glycerophosphate and galactose phosphate (Table II), together with free glycerol and galactose. The identification of the galactose phosphate was accomplished by quantitative analysis, by paper chromatography and by periodate oxidation studies. Quantitative analysis showed after phosphatase action a galactose phosphate ratio of 1.0: 1.2. The galactose phosphate showed the same RF value as galactose 6phosphate in 2 solvent systems (5 and 6) and stained with ani~inephtaIate reagent. After treatment with KBHS and phosphatase no galactose was found with the gafactose dehydrogenase assay. Only galactitof could be detected by paper chromatography in this case. These observations and the relative good acid stability of the phosphate excluded the existence of a galactose r-phosphate structure. Results of periodate oxidation of the deacylation product excluded a location of phosphate on carbon atoms 2 and 3 (Table I). In the intact deacylation product ga~actopyranosylgIycero1 coufd be bound to glycerolphosphate at carbon atom 4 or 6 of galactose, galactofuranosylglycerol at atom 5 or 6. Acid hydrolysis of the pyranose isomer of the deacylation product could yield both galactose 4-phosphate and 6-phosphate, because formation of a cyclic
380
ester between atoms 4 and 6 of galactopyranose is possible, like Burger and Glaser [22] suggested at acid hydrolysis of polygalac~osy~glycerophosphate. The phosphodiester linkage of the furanose compound may be cleaved by forming a cyclic ester between carbon atoms 5 and 6 of galactose. In this way- phosphate migration can take place. Another possibility is that the glycosidic linkage is split earlier than the phosphodiester. In this case galactose 5- and 6-phosphate can also be expected as products of acid hydrolysis and phosphate migration. Galactose [32P]phosphate obtained by acid hydrolysis of the deacylation product was subjected to periodate oxidation and reduction with KBH,. The products were separated by paper electrophoresis and by paper chromatography in solvent system 5.49 “/, of the radioactivity was found as ethyleneglycol phosphate (MP, 0.93 and RF 0.29), 45 y0 as glycerophosphate. The formation of these two products can be explained by phosphate migration during the acid hydrolysis of the deacylation product, resulting in a mixture of galactose 5- and &phosphates. Galactose [32P]phosphate prepared from phosphorylgalactosylgly~ero~ by acid hydrolysis was also subjected to periodate oxidation. After reduction and electrophoresis the ratto of the 32Pactivities of glycol phosphate and glycerol phosphate was 1.5, which indicates a prevalence of galactose 6-phosphate. The linkage of the phosphate to galactose in the deacylation product could not definitively be established by these procedures. When the deacylation product was hydrolyzed under mild acid conditions another diester with an MP, value of 0.62 and a R, of 0.22 (Solvent System 5) could be isolated. Analysis gave for this compound a ratio phosphate: galactose: glycerol: r,2-glycoi of I .o: I .03:1.19:1.30 with an use of 5.02 moles periodate per mole phosphate. Only ethyleneglycol [32P]phosphate was observed as a phosphate-containing product after periodate oxidation, reduction and acid hydrolysis. These results give evidence for the structure of this diester as 6-~-gly~erophosphorylgalactose and establish thus the linkage of the phosphate to the carbon atom 6 of galactose in the deacylation product. From the acid hydrolysis experiments w e may conclude that the acid labile galactofuranosidic linkage is split first. The resulting dies&r glycerylphosphorylgalactose yields at further hydrolysis a mixture of both glycerol phosphate and galactose phosphate, The deacytated core of the phosphoglucolipids contains gly~opyranosidic linkages, which appear more acid-stable than the phosphodiester. The formation of a cyclic diester on the glucose moiety does not appear possible when the phosphate is linked at the 6-position and the glycosidic linkage is intact. Therefore phosphoglucolipids do not give any glucose phosphate at acid hydrolysis [7, 151. Though it seems possible, that the galactopyranoside isomer of our phosphogalactolipids yields also galactose phosphate at acid hydrolysis, we have no experimental evidence for this. We could not separately hydrolyze the furanose and pyranose isomers of the deacylation product, because they could not be isolated as pure compounds. Conjiguration qf’ the glyccrophosphate moiety The stereochemical configuration was determined on the glycerophosphate isolated after alkaline hydrolysis of the deacyIation product. The ratio of glycerol 3tl)-phosphate ~~-glycerophosphate) and glycerol 2-phosphate (~-glycerophosphate~ obtained after hydrolysis was investigated by periodate oxidation, followed by NaBH, reduction and paper electrophoretic separation of the resulting glycol phoswas phate and the residual glycerophosphate. The 32P activity of these compounds
381
determined. The ratios of both a-glycerophosphate and P-glycerophosphate obtained after acid or alkaline hydrolysis of the deacylation product (Table II) agree with those in the literature on hydrolysis of other glycerophosphodiesters [r5, 22-261. As the glycerophosphate moiety is originally present as a-isomer, which will be shown below, the isomerization observed indicates again a phosphodiester, which is known to be split via a cyclic intermediate. With stereospecific glycerophosphate dehydrogenase [I 61 we could identify after alkaline hydrolysis only 4.2 y< of the total glycerophosphate as sn-glycerol 3phosphate. This results in a value for st-glycerol I-phosphate of 45%. Under the same conditions glycerophosphorylglycerol gave after alkaline hydrolysis, as expected, 56 % glycerol 2-phosphate, 24 % sn-glycerol f-phosphate and 20 % sn-glycerol 3-phosphate. After alkaline hydrolysis a-glycerophosphate is mainly found in its original stereochemical configuration, like Baer and Kates [22] showed. Therefore these results give evidence that the deacylation product of the phosphogalactolipids contains the sn-glycerol I-phosphate. Periodate oxidation of‘ the deacylation product The deacylation product was readily attacked by periodate with a total consumption of about 3 moles per mole phosphate (Table I). The yield of 2 moles formaldehyde and the rapid use of 2 moles periodate indicated that the phosphate and galactose moieties are linked to a C-atoms of the glycerol molecules. The perlodate oxidation had to be carried out at pH 6.0, because of the acid lability of the glycosidic linkage. Periodate oxidation of the deacylation product at pH 4.0 gave after reduction and acid hydrolysis only ethyleneglycol phosphate as a phosphate-containing product. This established again the location of phosphate to carbon atom 6 of galactose, because otherwise glycerophosphate would also be detected as a product. After periodate oxidation of the deacylation product at pH 6.0 and NaBH, reduction a compound was obtained, which showed a higher electrophoretic mobility (MP, 0.68) and a higher R, value in Solvent System 5 (RF 0.49 in stead of 0.44). Acid hydrolysis resulted in two main phosphate compounds. One had the electropholetic and chromatographic properties of ethyleneglycol phosphate. This confirmed again the attachment of the phosphodiester linkage to a r C-atom of the glycerol moiety. The second phosphate showed a electrophoretic mobility (MP, 0.72) and RF value in Solvent System 5 (0.20) between glycetophosphate and galactose 6-phosphate, comparable with threitolphosphate [27]. Phosphatase treatment of this compound liberated a polyol, which showed at gas chromatography retention times identical on two columns with threitol. Besides threitol a small amount of glycerol was then also observed. Threitolphosphate could be formed by oxidation of a galactofuranose unit at carbon atoms 2 and 3. This confirms that the deacylation product is mainly present as a galactofuranoside. The glycerol observed originates from the galactopyranoside isomer. Configuration qf the galactose moiety The acid lability of the deacylation product, paper and gas chromatography of galactosylglycerol preparations isolated and the products after periodate oxidation of the deacylation product gave already strong evidence that the galactose moiety is mainly present in the furanose form. Studies on the configuration of the glycosidic linkage were performed on the deacylation product and the isolated galactosylglycerol.
The deacylation product was stable against M- and /I-galactosidase. The deacylation products from other phosphoglycolipids were also not or only partially attacked by glycosidases [r5, 20, 261. The isolated galactosylglycerol was stable against a-galactosidase from coffee beans and only for 17% hydrolyzed by &galactosidase from Escherickia coli. This percentage agrees with the 14 and r9;:‘, fi-galactopyranosylglycerol detected by gas and paper chromatography, respectively. The stability of the /I-galactofuranosides to E. coli P-galactosidase was recently demonstrated [2X]. These results again indicate that the galactose residue is mainly present in the furanose form. The optical rotation values gave obvious evidence for a fi-furanose form. Two different preparations of the deacylation product showed a [a]n of -45.4 and -47.3 , respectively. The deacylation product was a mixture of both furanose and pyranose form, The purified galactofuranosylglycerol had a [u],, of - 102 “. Plackett [29] reported for a ~-~-galactofuranosylglycerol preparation a [~]u value of -80 ” f 13 , Reeves et al. [30] a value of -73 O. In analogy to that from the galactosyldigIycerides [8] the galactofuranosylglycerol preparation from the phosphogalactolipids of B. biju’lml var. permsvlwnicus is likely 3-0-/?-D-galactofuranosyl-sn_Rlycerol. The galactopyranosylglycerol had a [&I,, of -4. I ‘. Reported values for a 3-0-/?-D-galactopyranosyl-sr7-glycerol vary between -I- 7.5 and - 8.1 ” f8J. The mass spectrum of the trimethyIsilyfderivative of the isolated galactofurallosylglycero~ established its furanose form by the high ratios of the peaks nzje 217 and nlje 204 (217/204 = 19.8) and n?jr 205 and m/e 204 (205/204 = 1.8). Taking into account the mass spectral data, the PMR spectrum gave additional evidence for a fl configuration, because a coupling constant Jr .2 of o Hz was found (Kamerling, J. P., personal communications). Structure of’ the deacy1c;tio.n product From the analytical results on the intact compound and its degradation products we can conclude that the structure of the main deacylation product of Lipids 15 and 17 is: 3-0-[6’-sn-glycero-l-phosphoryl)-B_D-galactofuranosyl]-~~-glycerol. A minor amount of the galactopyranosyl isomer was also established. Location and compositim qf the fbrty ncids The intact phosphogalactolipids were completely resistant to the action of the phospholipases A, and C (Table 111) and II in contrast to phosphatidylglycerol and lecithin. Both Lipid Compounds 15 and 17 were attacked by pork pancreatic lipase, though Lipid 17 and the second ester bond of Lipid 15 were more slowly hydrolyzed like monoglycerides. These enzymatic hydrolysis experiments suggest that Compound 15 contains no phosphatidyl group, but a diglyceride unit linked to galactose. A free glycerophosphate molecule will be linked to galactose at the 6-position, because only this glycerol can contain the 1,2-glycol group, which is proven by the results of periodate oxydation. Both phosphogalactolipids gave a rapidly developing purple colour like phosphatidy~glycerol on periodate-Schiff staining at chromatograms and yielded one mole formaldehyde per mole phosphate (Table I). The presence of a non-acylated gfycerol molecule could also be established by its identification as a product after treatment of Lipid I 5 with 60 “/, HF. Other products of HF degradation were glycerophosphate, galactose and mono- and diglyceride. A lipid containing galactose could
383
TABLE
111
ACTION
OF PHOSPHOLIPASES
Extraction were
AND
15 and 17 were incubated
Lipids
according
separated
LIPASE
with lipolytic
enzymes
by thin-layer
activity over the fractions _. _ ._
chromatography
in Solvent
soluble
15 15
Lipid
I5 $
Lipid
15
Lipid Lipid
17
I
phospholipase lipase
Lipid t7llipasc _ -. _ -.-
and Methods.
distribution
of the 32P
_
soluble -.. 74
Lipid
73
78
21
28
72
18
79
6
94
9
91
5
95
64
36
-
-
-
-
of Lipid 15 -. ._ ._ 9 8
90
C
_
I. The
27 IO
A,
I 5T phospholipasc
System
.91 92
16
Lipid Lipid
Materials
Lipid 32P activity -. -. 15 Lyso-derivatives
Total 32P activity -.-_ -. -._ WaterLipid-
15
under
and lipid-soluble fractions. The lipids
is given in y,Lof the total activity as the mean of at least 2 experiments. -.-.-.
System
Lipid
as described
to Bligh and Dyer [13] gave water-soluble
_
._ _ -----
not be isolated. Presumably the furanosyl linkage is too labile in 60 % HF. This degradation method was not adequate therefore in this case to localize the fatty acids either both linked to glycerol or one to glycerol and one to galactose. The linkage of one fatty acid molecule of Lipid I 5 to galactose could also not definitively be excluded by periodate experiments, but periodate consumption of both Lipids 15 and 17 amounted only I mole per mole phosphate (Table I). Also higher concentrations up to 0.05 M did not result in a degradation of the galactose molecule. This could be proven by gas-chromatographic analysis after reduction with NaBH, and acid hydrolysis. However the organic solvent system and the steric inhibition 1311 by the diglyceride unit at C-I and the glycerophosphate group at C-6 may render the frans-glycol group of C-2 and C-3 inert to attack to periodate. The fatty acid composition of Lipid 15 showed marked differences with that of Lipid 17, monogalactosyldiglyceride and phosphatidylglycerol (Table IV). Lipid 17 can not be a I : I mixture of the two lyso-derivatives of Lipid 15. The markedly decreased content of octadecenoic and lactobacillic acid suggests a prevalence of the 2-acyl compound. These acids showed in phosphatidylglycerol from B. biJdunz var. TABLE
IV
FATTY
ACID
COMPOSITION
PHATIDYLGLYCEROLAND The percentage
OF
THE
PHOSPHOGALACTOLIPIDS
15 AND
of each fatty acid is given as the mean of duplicate
of each lipid. The fatty acid methyl esters arc designated
analyses of at least two preparations
by the number
of carbon atoms, followed
the number of double bonds. Fatty acid
14:o 16:o 16:1 18:o 18:1 Cycle- I9 : 0 -- .- .-
Monogalactosyl-
Phosphatidyl-
diglyccride --_-..___
glycerol
2. I 14.7
3.0 26.5
3.’ 25.7
3.8 23.0
45.6 6. I -. --
17, PHOS-
MONOGALACTOSYLDIGLYCERIDE
--
_..
36.5 8.7 _._ _._._..__
Compound -
_
__
5.1 33.0 5.4 20.5 28.6 5.7
I5
Compound
___._
_ I.5 55. ’ 3.0 28.0 II.9 _
_
I7
by
384
pennsylvanicus
degradation.
a strong preference for Position I, as was shown by phospholipase A In two strains of Streptococcus.faecalis the same preference was observed
[151. Structure
qf the more apolar phosphogalactolipid
Lipid 6 could not be isolated in sufficient amounts for analysis, because it amounts less than I y;‘, of total lipid phosphorus and is considerably contaminated with phosphatidylglycerol. Its deacylation product showed the same chromatographic and electrophoretic properties as the deacylation product of Compound 15. Analysis of the pure deacylation product gave a glycerol galactose phosphate ratio of 2: I : I. Alkaline hydrolysis gave the same products glycerol, galactosylglycerol, glycerophosphate and phosphorylgalactosylglycerol. The isolated glycerophosphate amounted 74 %, glycerol a-phosphate, 2 I “/;; sn-glycerol I -phosphate and 5 Y,,,al-glycerol J-phosphate. The configuration of the glycerophosphate unit is the same as in Lipids I 5 and I 7. Galactosylglycerol appeared to be about 90 “/I of the furanose form and IO )‘<:of the pyranose form. The mobility of Compound 6 on the thin-layer chromatograms points to the presence of four fatty acids. Between Lipids 6 and 15 another lipid was detected sometimes, which contained the same deacylation product and probably three fatty acids. The location of the fatty acids in Lipid 6 could not be established. A phosphatidy1 galactosyldiglyceride structure does not appear very likely, because the st7-Iphosphatidyl structure has been detected rarely in nature. Kates and coworkers [32-341 reported that the phytanyl ether analogs of phosphatidylglycerol and phosphatidylglycerophosphate and the sulphate ester of phosphatidylglycerol in Halobacterium cutirubum contained this unusual structure. The location of one or two acyl groups on the galactose molecule appears probable, because mono- and diacyl monogalactosyldiglycerides with a free carbon atom on Position 6 of galactose are present in B. b$dum var. pennsylvanicus [8]. Another argument for this location of the fatty acids is given in the next section. DISCUSSION
The unknown Lipids 6, 15 and J7 of B. b@dum var. pennsylvanicus proved to be phosphogalactolipids. The studies on their structure were hampered by the coexistence of furanose and pyranose forms and the acid lability of the galactofuranosyl linkage. The data obtained are compatible with the structure of Compound 15 (Fig. 5) as: 3-O-[6’-(sn-glycero-1-phosphoryl)-B-D-galactofuranosyl]-sn-I ,2-diglyceride. Lipid 17 has the same structure except one fatty acyl group. Lipid 6 is probably a diacyl derivative
Fig. 5. Structure
of the main phosphogalactolipid
of B. hifid~rm var. pennsqlwnicrrs
(Lipid
15).
385
of Compound 15, though its structure could not be completely established. About IO-2o;d of these compounds appeared to be present as galactopyranosyl compounds. Glycerylphosphoryl derivatives of galactosyldiglycerides have not been described earlier. The furanosyl structure of the sugar moiety is heretofore unique in bacterial phosphoglycolipids. We detected this form already in the acylated galactosyldiglycerides of B. bifidum var. pennsylvanicus [8]. It was also detected in the lipids of Mycoplasma mycoides [29], Bacteroides symbiosus [30] and Flavobacterium thermophi/urn [35]. Glycerylphosphoryldiglucosyl diglycerides have been isolated from a wide variety of bacteria [6, 7, I 5, 20, 261. A phosphatidyl derivative of monoglucosyldiglyceride was present in Pseudomonas diminuta [36]. Leuconostoc mesenteroides and Listeria monocytogenes contain glycerophosphorylgalactosylglucosyldiglycerides 171. Their structure has not yet been published. Phosphatidyldiglucosyldiglycerides have been shown to be present in Streptococci [IS, 371 and Acholeplasma laidlawii B [38]. The Phosphogalactolipids 6, I 5 and 17 from B. bQ?dum var. pennsylvanicus contain the unusual sn-glycerol r-phosphate like the glycerylphosphoryldiglucosyldiglyceride of Streptococci [15]. The enantiomorphous sn-glycerol 3-phosphate was present in the glycerylphosphoryldiglucosyldiglyceride of Acholeplasma laidlawii B [26] and the phosphatidylglucolipids of Srreprococci [IS, 371, A. laidlawii B [38] and Pseudomonas diminuta [36]. Phosphatidylglycerol and (acylated) monogalactosyldiglycerides can function as the precursors for the biosynthesis of the phosphogalactolipids of B. btjidum var. pennsylvanicus. Pieringer and co-worker gave evidence for his earlier suggestion [20,39) that a phosphoglucolipid in Streptococcus faecalis could be synthesized from diglucosyldiglyceride and phosphatidylglycerol and/or diphosphatidylglycerol [40]. Preliminary results with a particulate fraction from B. b$dum var. pennsylvanicus demonstrated that 32P activity from [32P]phosphatidylglycerol was incorporated into the Phosphogalactolipid Compound 6 and diphosphatidylglycerol. The incorporation into the phosphogalactolipid was increased by addition of diacyl monogalactosyldiglyceride. Radioactivity from [32P]diphosphatidylglycerol was not incorporated into Compound 6. The difference between phosphatidylglycerol and diphosphatidylglycerol in this system suggests that the srr-I-glycerophosphate unit is transferred in contrast to the phosphatidyi transfer in the S. faecufis system (4oJ. This agrees with a free sn-r-glycerophosphate group in Compound 6. Incorporation into Phosphogalactolipids 15 and 17 appeared to be absent in the used particulate system. A metabolic conversion from Lipid 6 by deacylation to Lipids 15 and 17 appears a real possibility in vivo because of the analogous stereoconfiguration of the glycerophosphate. Chase experiments with [32P]phosphate with intact cells sustain the precursor role of phosphatidylglycerol also for thesecompounds. The specific activities of phosphatidylglycerol and Lipids 15 and 17 changed with time in such a way that a real precursor-product relationship appears possible. Lipid 6 was present in a concentration too low for an investigation of its specific activity changes with time. The possible precursor role of the (acylated) monogalactosyldiglycerides will form an important point of further investigation. The differences in fatty acid composition between Lipid 15 and monogalactosyldiglyceride suggest that variations in fatty acid composition may play a role in the use of monogalactosyldiglyceride species for the biosynthesis of phosphogalactolipids. Contrary to our results the phosphoglucolipids. diglucosyldiglyceride and the other phospholipids from
Streptococci [37] showed the same fatty acid composition. Besides de novo synthesis a deacylation-reacylation cycle [4r] with lysoderivatives as intermediates may contribute to the final assessment of the fatty acid composition of our phosphogalactolipids like earlier was reported for the phospholipids in rat liver [42, 431. ACKNOWLEDGEMENTS
The authors are indebted to Mr E. Keizer for excellent technical assistance. Mass spectrometry and PMR spectroscopy were performed by Dr J. P. Kamerling, Department of Organic Chemistry, State University, Utrecht. Material support by the Netherlands Organization for the Advancement of Pure Research (ZWO-SON) is also gratefully acknowledged. REFERENCES I z 3 4
Exterkate, F. A. and Veerkamp. J. H. (1969) Biochim. Gybrgy, P., Norris, R. F. and Rose, C. S. (1954) Arch. Exterkate, F. A. and Veerkamp, J. H. (I 97 I) Biochim. Exterkate, F. A., Vrensen, G. F. J. and Veerkamp, J. 141.-154 5 Exterkate, F. A.. Otten, B. J.. Wassenberg, H. W. and
Biophys. Acta r76, 65-77 Biochem. Biophys. 48, 193-201 Biophys. Acta 23 I, 545-549 H. (1970) Biochim. Biophys. Acta Veerkamp,
J. H. f 1971) J. Bactcriol.
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If
Shaw, N. (1970) Bacterial. Rev. 34, 365- 377 Shaw. N. and Stead, A. ( 1972) FEBS Lett. 21. 249-253 Veerkamp J. H. (1972) Biochim. Biophys. Acta 273, 359-367 Dawson, R. M. C. (1960) Biochem. J. 75. 45-53 Uthe, J. F. and Magee, W. L. (1971) Can. J. Biochem. 49, 785-794 Zwaal. R. F. A., Roelofsen, B., Comfurius, P. and Van Deenen, L. L. M. (1971) Biochim.
Biophys.
12 I3 14 15
Acta 233, 474-479 Semeriva, M., Dufour, C. and Desnuelie, P. (1971) Biochemistry 10, 2143-2149 Blight. E. G. and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 91 r-91 7 Toon, P.. Brown, P. E. and Baddiley. J. (1972) Biochem. J. 127, 3999409 Fischer, W., Ishizuka, J.. Landgraf, H. R. and Herrmann, J. (1973) Biochim. Biophys.
Acta 296.
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28 29 30 31
5277545 Hohorst, H. J. (1970) in Methoden der enzymatischen Analyse, ~Bergmey~r. H. U., cd.) Vol. 11, pp. I 379-138 I, Verlag fhemie, Weinheim Veerkamp, J. H. (1970) Biochim. Biophys. Acta 210, 267-275 Kamerling. J. P., Vliegcnthart, J. F. G.. Vink, 3. and De Ridder, J. J. (1972) ‘Tetrahedron 27. 4275.-.4288 Vilkas, E., Amar, C., Markovits. J., Vliegenthart. J. F. G. and Kamerling. J. P. (1973) Biochim. Biophys. Acta 297, 423425 Ambron. R. 7. and Pieringer, R. A. ( 1971) J. Biol. Chem. 246.4216-4223 Brown, D. M., Hatl. G. E. and Higson. H. M. (1958) J. Chem. Sot. London. i 36~I)% Baer, E. and Kates, M. (1948) J. Biol. Chem. 175, 79-88 Burger, M. M. and Glascr, L. (1966) J. Biol. Chem. 241, 494-506 Bailly, M. C. (1942) Bull. Sot. Chim. 9. 314-350 Olsen, R. W. and Ballou. C. E. (1971) J. Biol. Chem. 246, 3305-3313 Shaw. N.. Smith, P. F. and Verhey, H. M. (1972) Biochem. J. $29, 167. 173 Watson, M. J., Tyler, J. M., Buchanan, J. G. and Baddiley, J. (1972) Biochem. J. 130.45-54 Yoshida, K., line, N.. Kamata, T. and Kato, K. (1969) Chem. Pharm. Bull. 17, I I 23--t 127 Plackett, P. (1967) Biochemistry 6, 2746-2754 Reeves, R. E., Latour, N. G. and Loustean. R. J. (1964) Biochemistry 3, I r48--1249 Garner, E. F.. Goldstein, I. J., Montgomery, R. and Smith. F. (1958) J. Am. Chem. SOC. 80, I 206--I 208
387 32 33 34 35 36 37 38 39 40 41 42
Kates, M., Yengoyan, L. S. and Sastry, P. S. (1965) Biochim. Biophys. Acta 98, 252-268 Joo, C. N. and Kates, M. (1968) Biochim. Biophys. Acta 152, 800-803 Hancock, A. J. and Kates, M. (1972) Chem. Phys. Lipids 8, 87-90 Oshima, M. and Yamakawa, T. (1972) Biochem. Biophys. Res. Commun. 49, 185-191 Wilkinson, S. G. and Bell, M. E. (1971) Biochim. Biophys. Acta 248, 293-299 Fischer, W., Landgraf, H. R. and Herrmann, J. (1973) Biochim. Biophys. Acta 306, 353-367 Smith, P. F. (1972) Biochim. Biophys. Acta 280, 375-382 Pieringer, R. A. (1968) J. Biol. Chem. 243, 4894-4903 Pieringer, R. A. (1972) Biochem. Biophys. Res. Commun. 49, 502-507 Lands, W. E. M. and Hart, P. (1965) J. Biol. Chem. 240, 1905-1911 Van Golde, L. M. G., Scherphof, G. L. and Van Deenen, L. L. M. (1969) Biochim. Biophys. Acta 176, 635-637 43 Kanoh, H. (1969) Biochim. Biophys. Acta 176, 756-763
et Bioplrysicrr Ar,frr. 348 ( I 974) 370.-387 Scientific Publishing Company. Amsterdam
- Printed
in The Netherlands
BBA 56450
BIOCHEMICAL
CHANGES
PENNSYLVANICUS VII. STRUCTURE
(Received
December
AFTER
1N B~F~~~BACTERiU~ CELL WALL
B~Fi~~~~~~ VAR
1NHlBlTlON
OF THE PHOSPHOGALACTOLIPIDS
14th.
1973)
SUMMARY
The structures of two major phosphogaiactoiipids from BiJdohacreritml bifidum var. pennsylvanicus have been established by chemical analysis and chemical and enzymatical degradation procedures. The compounds were identified as 3-U-f6’-(snglycero-1 -phosphoryl)-/3-D-galactofuranosyI)-snI ,2-diglyceride and its lysoderivative. A third minor phosphogalactolipid was a diacyl derivative of the former compound. About 10-200,~ of these compounds appeared to be gala~topyranosyi derivatives. Phosphorylgalactosylglycerol and galactose phosphate were identified as one of the products of alkaline and acid hydrolysis, respectively. Marked differences were present in the fatty acid composition of monogala~tosyldigly~eride, phosp~latidylglycerol and the two main phosphogalactolipids.
INTRODUCTION
TWO unknown phosphate-containing lipids, earlier [I] designated as Compounds I 5 and I 7, have been detected in B#idobacterium hzji’dwu var. penns!.lvanic’lts. These compounds constituted a considerable amount of the total lipid radioactivity of cell wall synthesis by lack of human after incorporation of 32P. After inhibition milk, which contains essential growth factors [2] the galactose content of the total lipids, the ratio of digalactosylto monogaia~tosyldigiycerides and the percentages of these unknown phosphate-containing lipids were considerably decreased [i, 41. These unknown lipids were also detected in 14 B@dobacterium strains of various origin, but were not present in 9 ~ac~obucii~~~ strains [5]. Originally [I] the compounds were characterized as polyglycerof phosphohpids, because glycerophosphorylglycerol was detected as one of the deacylation products. However, after extensive purification of the unknown lipids, this compound proved to be absent after deacylation. Because further investigations led to an identification of galactose as a component, the unknown lipids could belong to the class of phosphoglycolipids [6, 71. The structure of the six major galactosyldiglycerides in 3. h13dt4m var. ~e~ln~~~7~~a~liclis was reported
371
earlier [S]. In this paper we report the elucidation of the structure of the two major phosphogalactolipids and give also some information about the structure of a third phosphogalactolipid, which was earlier [I] designated as Compound 6. The investigations were hampered by the fact that the compounds were present in both galactofuranoside and galactopyranoside forms which could not be separated. The composition of the intact lipids and a mild alkaline deacylation product was analyzed. Their structure was established by identification of the products obtained by chemical and enzymatical degradation procedures. MATERIALS
AND
METHODS
Cultivation of the organism in 8-1 cultures for I 6 h and extraction of lipids were performed as described previously [I]. Cells were washed twice with 0.2 M sodium acetate buffer (pH 5.0). Incorporation of 32Pi was made in 8oo-ml cultures by addition of I mCi of Na,H3’P04 and cultivation for 16 h. The lipids from these cultures were mixed with the non-radioactive lipids. Phospholipids were obtained by silicic acid fractionation of total lipids [I]. They were subfractionated on silicic acid columns with chloroform-methanol mixtures [I]. The purification of the two unknown compounds, phosphatidylglycerol and diphosphatidylglycerol was achieved by preparative thin-layer chromatography on silicagel G (Merck, Darmstadt, Germany) in the Solvent Systems I, chloroform-methanol-7 M ammonia (60:35: 5, by vol.) and 2, chloroform-methanol-acetic acid-water (125 : 37 : 9.5 : I .5, by vol.). For a fina. step silicagel HR with addition of 3 % magnesium silicate was used with Solvent System 3, chloroform-methanol-water (65 : 25 : 4, by vol.) as developer. After detection by autoradiography or scanning, the compounds were eluted from the silicagel with chloroform-methanol-water (IO : IO : I, by voi.). Lipids were stored under benzene-methanol (4:1, by vol.) at -20 “C. Hydrolysis procedures and isolation qf hydrolysis products Deacylation products of the phosphate-containing lipids were prepared according to a modified procedure of Dawson [9]. Before addition of the reagents the lipids were dissolved in 0.2 ml chloroform. The hydrolysis was repeated once. Paper electrophoresis for 2 h at 30 V/cm in Solvent System 4, pyridine-acetic acid-water (I :IO: 89, by vol.) (pH 3.5) was used for separation of the deacylation products. After detection by autoradiography they were eluted with water and purified by repeated electrophoresis or paper chromatography in the Solvent System 5, r-propanol-conc. ammonia-water (6 : 3 : I, by vol.). Acid hydrolysis of the deacylation products was performed by heating for 2 h at IOO “C in 2 M HCl. Alkaline hydrolysis was carried out by heating for 3 h at IOO “C in I M NaOH. Hydrolysis products were isolated by preparative paper electrophoresis in Solvent System 4. The on the origin remaining galactosylglycerol compounds were eluted and separated by paper chromatography in Solvent System 6, butanol-pyridine-water (6:4: 3, by vol.). Detection was carried out by scanning of radioactivity, autoradiography or staining with one of the reagents described previously [I, 81. 32P. a-glycerophosphate, glycerophosphorylglycerol, galactose 6-phosphate, glycerol, galactose and /I-galactosylglycerol compounds prepared from the monogalactosyldiglycerides [8] were used as reference substances for the qualitative identification of
372
the hydrolysis products by paper efectrophoresis in System 4 and paper chromatography in the Systems 5,6 and 7, ethylacetate-pyridine-water (10:4:3, by vol.). Preparations used for analytical determinatiolls, mass spectrometry and proton magnetic resonance spectroscopy were purified additionally by chromatography on acid washed Schfeicher and Schiifl paper No. 2045~ in Solvent System 6. Enzymatic hydrol.vsis procedures Enzymes were obtained from Boehringer and Soehne, Mannheim, unless otherwise indicated. Hydrolysis by phospholipase A, (EC 3. I. 1.4). An amount of I .o jlmole (30.000 cpm) [3zP]phosphogafactolipid, [32P]phosphatidyfgfycerof or egg lecithin was dispersed in I .5 ml of diethyf ether and shaken with 0.2 ml of o. I M Tris-mafeate buffer (pH 7.0) containing 0.5 hlmofe CaCf, and 6-10 units phosphofipase A, (Crofalus adamaffte~s, Sigma Chemical Co., St. Louis MO. or Crotalus terr. fur) for 2-4 hat 37 “C. Incubation of phosphogalactol~pids was also performed in 5 ml of 0.1 M Tris-HCI buffer (pH 8.0) containing 2 mg sodium deoxychotate, 12.5 /lmofes CaCf, and 6 units phosphofipase A,, the activity of which was checked using egg yolk lipoprotein [IO]. If_ydrolysis by phospholipase C (EC 3.1.4.3). Incubation of I jtmofe (30000 cpm) [32P]phosphogafactofipid, [32P]phosphatidyfgfycerof or egg lecithin was carried out with 0.3 ml of 0.2 M Tris-mafeate buffer (pH 7.4), containing 4 Itmoles CaCfz and 2 units of phospholipase C from Bacillus cereus for 4 h at 37 ‘C. The phosphofipase C preparation was obtained from Dr R. Zwaaf, Department of Riochemistry, University of Utrecht. The activity of the enzyme was measured by continuous titration of the acid produced by hydrolysis of egg yolk lipoprotein [I I 1. Hydrolysis by p~~~sp~?o~~~ase D (EC 3.1.4.4). Amounts of I /[mole [“2P]phosphate containiilg lipids were dispersed in 2 ml diethyf ether and shaken with 0.8 ml of 0.05 M sodium acetate buffer (pH 5.6) containing 4.5 pmofes CaCf, and 2.8 mg (I .4 units) phospholipase D from cabbage. Incubation was performed for 4-16 h at 37 “C.
Hydrolysis b.v pancreatic lipase (EC 3. I. 1.3). E3’PIPhosphate-containing lipids were sonicated with a Bransson sonifier Model Br2 for 30 s in an amount of I jtmole with 5 ml of 0.05 M Tris-HCl buffer (pH 8.0) containing 2 mg sodium deoxycholate and 12.5 [rmofes CaCI,. 1.2 ml of these emulsions were incubated with 25 /moles CaCf, and 50 pg pork pancreatic fipase for 2 h at 23 “C. The pure lipase preparation was obtained from Dr R. Verger, Department of Biochemistry, University of Utrecht. The activity of the preparation was checked using tributyrine as substrate [12]. ~~~dro~?tsisby p~?os~~l~odiesteras~ (EC 3.1.4.1). Incubation of water-soluble phosphodiesters was performed in 0.1 M Tris-HCI buffer (pH 8.9) for 3 h at 37 ‘C with IO units phosphodiesterase from Russel’s viper venom (B grade, Calbiochem). When the phosphogalactofipids were used as substrate, an equal volume diethyl ether was added and the incubation mixture shaken violently for 4 h at 30 C. NydroEysis by phosphomonoesterases. Incubation with alkaline phosphatase (EC 3.1.3. I) from calf intestine was performed in a 0.1 M Tris-HCI buffer (pH 8.2), from potatoes was used in a O. I ?W citrate containing 1 mM Mg” +. Acid phosphatase buffer (pH 5.6). Incubations were performed for 4 h at 37 “C. Hydrolysis by gaiac’tosidases. The water-soluble hydrolysis products were incubated with cc-galactosidase from coffee beans and with P-galactosidase from
373
Escherichiu coli for 3-16 h according previously described procedures [8]. The same check procedures were followed. After incubation with lipolytic enzymes or phosphodiesterase the procedure of Bligh and Dyer [13] was followed to separate the lipid-soluble and the water-soluble fractions. The amount and the identity of the (radioactive) products were determined as described below. Periodate oxidation
Periodate oxidation was always performed at 22 “C. Lipids were dissolved in chloroform-ethanol (I : 3, by vol.) and treated with 0.25 vol. IO mM NaIO, at pH 5.0 for 24 h. The isolated deacylation and hydrolysis products were oxidized in 10-20 mM NaIO, at pH 6.0. Mild periodate oxidation was performed in 5 mM NaTO, for 15 min. Smith degradation of the phosphogalactolipid was performed in chloroformethanol (I : 3, by vol.) after addition of 0.25 vol. 0.05 M NaIO, for 72 h at 23 “C. The oxidation mixture was treated with ethyleneglycol and KBH, [8]. Thereafter the lipid was deacylated in 0.1 M methanolic NaOH. The inorganic ions were separated and the oxidation product hydrolyzed in 2 M HCI for 2 h at IOO “C. For Smith degradation of water-soluble compounds 0.05 M periodate (pH 6.0) was used for 16 h. After addition of ethyleneglycol the oxidation products were reduced with KBH, for at least 16 h at 4 “C. The residual KBH, was destroyed with acetic acid and K+ and borate ions eliminated [8]. The products were partially used for studies by chromatography or electrophoresis. Another part was hydrolyzed for 2 h in 2 M HCI at IOO“C or for 72 h in 6 M HCI at 120 “C. The thus obtained products were analyzed by paper electrophoresis and chromatography or by gas-liquid chromatography together with reference compounds. Treatment with HF
Degradation of lipids and their deacylation products was performed with 60 % HF according to the procedure of Toon et al. [14] for 16-24 h at 4 “C. The solutions were neutralized with a LiOH solution and freeze dried. Lipids were extracted with chloroform-methanol (2:1, by vol). The detection of glycerol was performed as described by Fischer et al. [I 51. Analytical procedures
The purity of the isolated phospholipids and phosphogalactolipids were checked in at least three systems by thin-layer chromatography in Solvent Systems I, 2, 3 or 8, chloroform-methanolconc. ammonia (70: 20: 2, by vol.) or by chromatography on silicagel impregnated paper (Whatman S G 81) in System 9, chloroformmethanoldiisobutylketone-acetic acid-water (45 : I 5 :30 : 20: 4, by vol.) [I]. They were also checked on their deacylation products. The purity of the isolated deacylation products and the hydrolysis products was checked by paper electrophoresis in System 4 and by paper chromatography in the Systems 5, 6 and 7. The identity of the hydrolysis products was established by analysis and comparison with reference compounds in the same systems. Staining procedures have been given previously [I, 81. Analytical methods for galactose, glycerol, formaldehyde, periodate, ester
374
groups, phosphorus and total hexose have been described previously [I, X]. strGlycerol j-phosphate was determined with glycerol-j-phosphate dehydrogenase (EC 1.1.1.8) [r6]. Gas chromatography of trimethyl-silylated degradation products from lipids and their deacylation products was performed on the same columns and under the same conditions as previously used [8]. Preparation and gas chromatography of fatty acid methyl esters were also described before [t7]. Nonadecanoic acid methyl ester was used as an internal standard for quantitative analysis. The radioactivity of an aliquot of the organic phase after Bligh and Dyer [I 31 extraction was measured after evaporation of the solvent in IO ml toluene, containing 4 g Omnifluor (NEN Chemicals GmbH, Dreieichenhain, W. Germany) per 1. An aliquot of 0.5--1.0 ml of aqueous solutions was counted in to ml Aquasol (Merck, Darmstadt). Radioactive compounds on thin-layer plates or chromatograms were located by scanning or by exposure for 24-96 h of Kodak X-ray film. The radioactivity of the spots was measured by liquid-scintillation counting [I]. Optical rotations were measured with a Perkin-Elmer Model 141 polarimeter. Glycosides were dissolved in water. Pertrimethylsilylation of the galactosylglycerol compounds followed by mass spectrometry and PMR spectroscopy were performed as described by Kamerling and co-workers [I 8, I 91. RESULTS
The purification of the unknown phosphate-containing lipids in B. h$k/um var. pennsvl~~akx.tsproved to be rather complicated. Silicic acid column fractionation and thin-layer chromatography in 3 solvent systems were necessary to separate them from all contaminating lipids. Their Iocation on a two-dimensional thin-layer chromatogram is given in Fig. I. The unknown phosphate-containing lipids designated as 6, 15, 17 and 18 turned out to belong to the phosphogalactolipids. Results on the structure of the more apolar Lipid 6 will be described at the end of this section. Lipid 18 proved to be another salt form of Lipid 17, because it did only separate after extraction on the first thin-layer chromatogram. After elution it migrated together with Lipid 17. The compounds did not stain with ninhydrin. The presence of sulphate in the lipids could be excluded by lack of [3”S]sulphate incorporation into the lipids. Acid hydrolysis of the pure compounds in 2 M HCI at IOO “C for 2 h yielded glycerol, glycerol phosphate, galactose and galactose phosphate as constituents. The identity of these compounds was established by paper chromatography in 3 solvent systems (5, 6 and 7), that of glycerol and galactose also by gas chromatography. The results on the structure of galactose phosphate will be given in a separate paragraph. Quantitative analyses of the pure Lipids I 5 and I 7 demonstrated a molar ratio for hexose : glycerol: phosphate of about 1: 2 : I (Table I). Both lipid compounds were rapidly periodate-Schiff positive and yielded I mole formaldehyde per mole phosphate, indicating the presence of one 1,2-glycol group. The results of the fatty acid ester assays by the alkaline hydroxamate method and by gas chromatography showed both the presence of 2 moles fatty acids per mole phosphate in Lipid 15 and one in Lipid 17. A survey of the main chemical and enzymatical degradation procedures is
375
Fig. I. Autoradiogram of a two-dimensional thin-layer chromatogram on silicagel G of a lipidextract from cells grown with 32Pi. The chromatogram was developed in Direction I with chloroformmethanol-7 M ammonia (60: 35:5, by vol.) and in Direction II with chloroform-methanol-acetic acid-water (125: 37:9.5:1.5, by vol.). Identity of spots: I, diphosphatidylglycerol; z, acylphosphatidylglycerol; 4, triacyl-bis-(glycerophosphoryl)glycerol; 6, phosphogalactolipid together with phosphatidylglycerol, salt form; 7 and 8, phosphatidylglycerol; 9, alanylphosphatidylglycerol; IO, diacylbis-(glycerophosphoryl)glycerol; I I, monoacyl-bis-(glycerophosphoryl)glycerol ; I 3. not identified ; I 5, phosphogalactolipid; 17 and 18, phosphogalactolipid; 19, not identified. The spot at the upper right side of the autoradiogram originates from a marker. TABLE
I
ANALYTICAL Molar values
DATA
ON LIPIDS
15 AND
17 AND
THEIR
DEACYLATION
PRODUCT
ratios to phosphate are given as the mean of duplicate analyses on at least 2 preparations. of periodate rapidly used reflect the amounts used within 5 min. N.D., not determined. Compound
Phosphate Hexose Glycerol Galactose Formaldehyde Periodate rapidly used Periodate total used Acyl groups
I5
Compound
1.00 0.88 2.05
I .oo 0.69
0.54 0.76 0.90
0.51 0.74
1.02
1.88
I.95 N.D. 1.23 1.15
17
Deacylation
The
product
I .oo I .05 1.90 0.60 2.07
1.87 3.30
given in Fig. 2. Autoradiograms of an electropherogram and a paper chromatogram of 32P-containing degradation products are reproduced in Figs 3 and 4, respectively. The common mild alkaline hydrolysis product of the phosphogalactolipids (MPi 0.52 and RF in Solvent System 5 0.44) contained nearly all the lipid phosphorus, hexose
376
.“M .
c
: ‘i
ri
,A i.
Tt
.4 u
and glycerol, which indicated that these lipids contained only ester bonds. The deacylation product showed the same hexose: glycerol: phosphate ratio as the intact lipids, but an additional r,z-glycol group (Table I). The amounts of sugar estimated by the total hexose assay (anthron method) and the specific galactose determination in an acid hydrolyzate showed a ratio of about two. However another hexose did not appear to be present in the lipids. The
377
Fig. 3. Autoradiogram of an electropherogram of 32P-containing degradation products of Lipid 15 and phosphatidylglycerol. Water-soluble phosphate esters were separated by paper electrophoresis for zh at 30 V/cm in a pyridine-acetic acid-water buffer (I :IO: 89, by vol.) (pH 3.5). Identity of spots: I, Pi; 2, the mild alkaline deacylation product; 3. the deacylation product after periodate oxidation and reduction; 4. the same after addjtjonal acid hydrolysis; 5, the products after acid hydrolysis of the deacylation product; 6, the products after strong alkaline hydrolysis of the deacylation product; 7, glycerophosphorylglycerob The spots on the start line are only markers.
Fig. 4. Autoradiogram of a paper chromatogram of 32P-containing degradation products of Lipid 15, phospbatidylgIycero1 and diphosphatidylgIycero1. The chromatogram was developed with n-propanolcont. ammonia-water (6: 3:1, by vol.). The start line is marked by two dark spots. The following fractions were separated: I, the deacylation product of Lipid 15; 2, the deacylation product after oxidation and reduction; 3, the same after additional acid hydrolysis; 4, the products after acid hydrolysis of the deacylation product; 5, the products after strong alkaline hydrolysis of the deacylation product; 6, glycerophosphorylglycerol; 7, bis-(glycerophosphoryl)glycerol.
ratio became one, when galactose was assayed in an acid hydrolyzate, treated with alkaline phosphatase. These results suggested that a binding of phosphate to D-galactose was still present after acid hydrolysis. The detection of galactose phosphate as a component of the phosphogalactolipids excluded the possibility that the unknown lipids were glycerophosphorylglycerol derivatives. The earlier [I] reported observation of glycerophosphorylglycerol as one of the deacylation products could be explained by the presence of a small amount of lysophosphatidylglycerol in the preparations of the isolated phosphogalactolipid. The glycerophosphorylglycerol structure as a base for a phosphatidylglycerylgalactose was also disproven by the following observation. No glycerol could be detected after periodate oxidation of the deacylation product of the phosphogalactolipid, when the oxidation was only followed by addition of arsenite to destroy the excess of periodate, and acid hydrolysis. The same procedure applied on bis-(glycerophosphoryl)glycerol as a check yielded glycerol. The unknown lipids and their common deacylation product were not affected by treatment with phosphomonoesterases, suggesting a phosphodiester linkage. Phosphodiesterase from Russell’s viper venom did not split this linkage. This does not exclude a phosphodiester linkage in a phosphoglycolipid, because diglycosylphosphorylglycerol was also inert to this enzyme [20]. Alkalir~ehydrolysis c~fthe deacylatiotl product Strong alkaline hydrolysis (I M NaOH at IOO ‘C for 3 h) of the deacylation product yielded besides glycerol and glycerophosphate a polyol and a polyolphosphate MP, 0.58, (Fig. 3). The polyol contained equimolar amounts of glycerol and galactose, the polyolphosphate glycerol, galactose and phosphate in the same proportions. The relative amounts of glycerophosphate and the polyolphosphate agree with the amounts of galactosyiglycerol and glycerol, respectively (Table 11). Both phosphates were hydrolyzed by alkaline phosphatase, indicating that two monoesters were formed by alkaline hydrolysis from a diester. The polyol showed in the Solvent Systems 5 and 6 two spots, with R, values identical to the furanose and the pyranose TABLE
II
RELATIVE
AMOUNTS
OF DEGRADATION
PRODUCTS
OFTHE
DEACYLATtD
The deacylation product was hydrolyzed for z h in 2 M HCI or for 3 h Degradation products were separated by paper electrophoresis. The j*P containing products was measured and given in percentages of total 32P experiments. The ratio of glycerol and galactosylglycerol was determined and after acid hydrolysis of the neutral products after alkaline hydrolysis. the glycerophosphate isomers were assayed as described in the text. Product
Acid hydrolysis
Galactose phosphate Glycerophosphate Phosphorylgalactosylglycerol Glycerol Galactosylglycerol sn-Glycero I -phosphate s,Kilycero j-phosphate Glycero z-phosphate
33 63
Alkaline hydrolysis
s4 46 45 55
‘I 1 80 20 ~._~_~__
149 5’ ~_~
45 4
LIPID
15
in I M NaOH at loo C. activity of the phosphatractivity as the mean of z by a glycerol assay before The relative amount? of
379
isomers of galactosylglycerol, prepared from the monogalactosyl lipids of B. b$dum var. ~e~nsylva~icus [8]. The ratio of the furanose form (Iz, 0.43) and the pyranose form (RF 0.27) was determined after separation in Solvent System 6. Galactose assay after elution and acid hydrolysis resulted in a ratio of 81 :rg. Gas chromatographic analysis of the trimethylsilyl derivatives of the polyol gave also two compounds with retention times identical on two columns to those of the monogalactosylglycerol compounds derived from the gaiactosyldiglycerides. The main one (86%) proved to cochromatograph with 3-U-~-D-gaIactofuranosyl-~~-glycero1 from B. bl~d~rn var. ~e~~sy~vanic~s, the other (14 %) with the 3-~-~-D-galactopyranosyl-so-glycerol from spinach and B. bl$dum var. pennsylvanicus. The polyolphosphate appeared to be a mixture of two compounds with R, values of 0.3 I and 0.25, respectively in Solvent System 5. After phosphatase action on the polyolphosphate the same two forms of galactosylglycerol were detected as above. Thus the polyolphosphate appeared to be phosphory~galactosyig~ycerol, present in both furanose form (RF 0.31) and pyranose form (RF 0.~5). The appearance of phosphoryIgalactosylglycero1 at alkaline hydrolysis of the deacyiation product can be explained by the fact that cyclic phosphodiesters can be formed on the galactose molecule, in the case of the furanose form between carbon atoms 5 and 6, in the case of the pyranose form between carbon atoms 4 and 6. Only these carbon atoms are mentioned, because the phosphate group is linked to carbon atom 6 in the intact deacylation product, as will be shown in the next paragraph. The formation of phosphorylgaIactosylglycero1 is remarkable, because phosphoglucohpids do not yield any detectable product in which the phosphate-glucose linkage is retained, like phosphoryldiglucosylglycerol (7, 151. Phosphoglucolipids can not easily form a cyclic diester on the glucose molecule at alkaline hydrolysis, because the glycerophosphate is linked to carbon atom 6 of a gIucopyranoside with a trans hydroxyIgroup at Position 4. The cis- or trans-orientation of the neighbouring hydroxyl groups inff uences markedly the products of alkaline hydrolysis, as was established by Brown et al. [21 J. Acid hydrolysis
qf the deacylation product
Acid hydrolysis (2 M HCl, IOO“C, 2 h) of the deacylation product gave as products glycerophosphate and galactose phosphate (Table II), together with free glycerol and galactose. The identification of the galactose phosphate was accomplished by quantitative analysis, by paper chromatography and by periodate oxidation studies. Quantitative analysis showed after phosphatase action a galactose phosphate ratio of 1.0: 1.2. The galactose phosphate showed the same RF value as galactose 6phosphate in 2 solvent systems (5 and 6) and stained with ani~inephtaIate reagent. After treatment with KBHS and phosphatase no galactose was found with the gafactose dehydrogenase assay. Only galactitof could be detected by paper chromatography in this case. These observations and the relative good acid stability of the phosphate excluded the existence of a galactose r-phosphate structure. Results of periodate oxidation of the deacylation product excluded a location of phosphate on carbon atoms 2 and 3 (Table I). In the intact deacylation product ga~actopyranosylgIycero1 coufd be bound to glycerolphosphate at carbon atom 4 or 6 of galactose, galactofuranosylglycerol at atom 5 or 6. Acid hydrolysis of the pyranose isomer of the deacylation product could yield both galactose 4-phosphate and 6-phosphate, because formation of a cyclic
380
ester between atoms 4 and 6 of galactopyranose is possible, like Burger and Glaser [22] suggested at acid hydrolysis of polygalac~osy~glycerophosphate. The phosphodiester linkage of the furanose compound may be cleaved by forming a cyclic ester between carbon atoms 5 and 6 of galactose. In this way- phosphate migration can take place. Another possibility is that the glycosidic linkage is split earlier than the phosphodiester. In this case galactose 5- and 6-phosphate can also be expected as products of acid hydrolysis and phosphate migration. Galactose [32P]phosphate obtained by acid hydrolysis of the deacylation product was subjected to periodate oxidation and reduction with KBH,. The products were separated by paper electrophoresis and by paper chromatography in solvent system 5.49 “/, of the radioactivity was found as ethyleneglycol phosphate (MP, 0.93 and RF 0.29), 45 y0 as glycerophosphate. The formation of these two products can be explained by phosphate migration during the acid hydrolysis of the deacylation product, resulting in a mixture of galactose 5- and &phosphates. Galactose [32P]phosphate prepared from phosphorylgalactosylgly~ero~ by acid hydrolysis was also subjected to periodate oxidation. After reduction and electrophoresis the ratto of the 32Pactivities of glycol phosphate and glycerol phosphate was 1.5, which indicates a prevalence of galactose 6-phosphate. The linkage of the phosphate to galactose in the deacylation product could not definitively be established by these procedures. When the deacylation product was hydrolyzed under mild acid conditions another diester with an MP, value of 0.62 and a R, of 0.22 (Solvent System 5) could be isolated. Analysis gave for this compound a ratio phosphate: galactose: glycerol: r,2-glycoi of I .o: I .03:1.19:1.30 with an use of 5.02 moles periodate per mole phosphate. Only ethyleneglycol [32P]phosphate was observed as a phosphate-containing product after periodate oxidation, reduction and acid hydrolysis. These results give evidence for the structure of this diester as 6-~-gly~erophosphorylgalactose and establish thus the linkage of the phosphate to the carbon atom 6 of galactose in the deacylation product. From the acid hydrolysis experiments w e may conclude that the acid labile galactofuranosidic linkage is split first. The resulting dies&r glycerylphosphorylgalactose yields at further hydrolysis a mixture of both glycerol phosphate and galactose phosphate, The deacytated core of the phosphoglucolipids contains gly~opyranosidic linkages, which appear more acid-stable than the phosphodiester. The formation of a cyclic diester on the glucose moiety does not appear possible when the phosphate is linked at the 6-position and the glycosidic linkage is intact. Therefore phosphoglucolipids do not give any glucose phosphate at acid hydrolysis [7, 151. Though it seems possible, that the galactopyranoside isomer of our phosphogalactolipids yields also galactose phosphate at acid hydrolysis, we have no experimental evidence for this. We could not separately hydrolyze the furanose and pyranose isomers of the deacylation product, because they could not be isolated as pure compounds. Conjiguration qf’ the glyccrophosphate moiety The stereochemical configuration was determined on the glycerophosphate isolated after alkaline hydrolysis of the deacyIation product. The ratio of glycerol 3tl)-phosphate ~~-glycerophosphate) and glycerol 2-phosphate (~-glycerophosphate~ obtained after hydrolysis was investigated by periodate oxidation, followed by NaBH, reduction and paper electrophoretic separation of the resulting glycol phoswas phate and the residual glycerophosphate. The 32P activity of these compounds
381
determined. The ratios of both a-glycerophosphate and P-glycerophosphate obtained after acid or alkaline hydrolysis of the deacylation product (Table II) agree with those in the literature on hydrolysis of other glycerophosphodiesters [r5, 22-261. As the glycerophosphate moiety is originally present as a-isomer, which will be shown below, the isomerization observed indicates again a phosphodiester, which is known to be split via a cyclic intermediate. With stereospecific glycerophosphate dehydrogenase [I 61 we could identify after alkaline hydrolysis only 4.2 y< of the total glycerophosphate as sn-glycerol 3phosphate. This results in a value for st-glycerol I-phosphate of 45%. Under the same conditions glycerophosphorylglycerol gave after alkaline hydrolysis, as expected, 56 % glycerol 2-phosphate, 24 % sn-glycerol f-phosphate and 20 % sn-glycerol 3-phosphate. After alkaline hydrolysis a-glycerophosphate is mainly found in its original stereochemical configuration, like Baer and Kates [22] showed. Therefore these results give evidence that the deacylation product of the phosphogalactolipids contains the sn-glycerol I-phosphate. Periodate oxidation of‘ the deacylation product The deacylation product was readily attacked by periodate with a total consumption of about 3 moles per mole phosphate (Table I). The yield of 2 moles formaldehyde and the rapid use of 2 moles periodate indicated that the phosphate and galactose moieties are linked to a C-atoms of the glycerol molecules. The perlodate oxidation had to be carried out at pH 6.0, because of the acid lability of the glycosidic linkage. Periodate oxidation of the deacylation product at pH 4.0 gave after reduction and acid hydrolysis only ethyleneglycol phosphate as a phosphate-containing product. This established again the location of phosphate to carbon atom 6 of galactose, because otherwise glycerophosphate would also be detected as a product. After periodate oxidation of the deacylation product at pH 6.0 and NaBH, reduction a compound was obtained, which showed a higher electrophoretic mobility (MP, 0.68) and a higher R, value in Solvent System 5 (RF 0.49 in stead of 0.44). Acid hydrolysis resulted in two main phosphate compounds. One had the electropholetic and chromatographic properties of ethyleneglycol phosphate. This confirmed again the attachment of the phosphodiester linkage to a r C-atom of the glycerol moiety. The second phosphate showed a electrophoretic mobility (MP, 0.72) and RF value in Solvent System 5 (0.20) between glycetophosphate and galactose 6-phosphate, comparable with threitolphosphate [27]. Phosphatase treatment of this compound liberated a polyol, which showed at gas chromatography retention times identical on two columns with threitol. Besides threitol a small amount of glycerol was then also observed. Threitolphosphate could be formed by oxidation of a galactofuranose unit at carbon atoms 2 and 3. This confirms that the deacylation product is mainly present as a galactofuranoside. The glycerol observed originates from the galactopyranoside isomer. Configuration qf the galactose moiety The acid lability of the deacylation product, paper and gas chromatography of galactosylglycerol preparations isolated and the products after periodate oxidation of the deacylation product gave already strong evidence that the galactose moiety is mainly present in the furanose form. Studies on the configuration of the glycosidic linkage were performed on the deacylation product and the isolated galactosylglycerol.
The deacylation product was stable against M- and /I-galactosidase. The deacylation products from other phosphoglycolipids were also not or only partially attacked by glycosidases [r5, 20, 261. The isolated galactosylglycerol was stable against a-galactosidase from coffee beans and only for 17% hydrolyzed by &galactosidase from Escherickia coli. This percentage agrees with the 14 and r9;:‘, fi-galactopyranosylglycerol detected by gas and paper chromatography, respectively. The stability of the /I-galactofuranosides to E. coli P-galactosidase was recently demonstrated [2X]. These results again indicate that the galactose residue is mainly present in the furanose form. The optical rotation values gave obvious evidence for a fi-furanose form. Two different preparations of the deacylation product showed a [a]n of -45.4 and -47.3 , respectively. The deacylation product was a mixture of both furanose and pyranose form, The purified galactofuranosylglycerol had a [u],, of - 102 “. Plackett [29] reported for a ~-~-galactofuranosylglycerol preparation a [~]u value of -80 ” f 13 , Reeves et al. [30] a value of -73 O. In analogy to that from the galactosyldigIycerides [8] the galactofuranosylglycerol preparation from the phosphogalactolipids of B. biju’lml var. permsvlwnicus is likely 3-0-/?-D-galactofuranosyl-sn_Rlycerol. The galactopyranosylglycerol had a [&I,, of -4. I ‘. Reported values for a 3-0-/?-D-galactopyranosyl-sr7-glycerol vary between -I- 7.5 and - 8.1 ” f8J. The mass spectrum of the trimethyIsilyfderivative of the isolated galactofurallosylglycero~ established its furanose form by the high ratios of the peaks nzje 217 and nlje 204 (217/204 = 19.8) and n?jr 205 and m/e 204 (205/204 = 1.8). Taking into account the mass spectral data, the PMR spectrum gave additional evidence for a fl configuration, because a coupling constant Jr .2 of o Hz was found (Kamerling, J. P., personal communications). Structure of’ the deacy1c;tio.n product From the analytical results on the intact compound and its degradation products we can conclude that the structure of the main deacylation product of Lipids 15 and 17 is: 3-0-[6’-sn-glycero-l-phosphoryl)-B_D-galactofuranosyl]-~~-glycerol. A minor amount of the galactopyranosyl isomer was also established. Location and compositim qf the fbrty ncids The intact phosphogalactolipids were completely resistant to the action of the phospholipases A, and C (Table 111) and II in contrast to phosphatidylglycerol and lecithin. Both Lipid Compounds 15 and 17 were attacked by pork pancreatic lipase, though Lipid 17 and the second ester bond of Lipid 15 were more slowly hydrolyzed like monoglycerides. These enzymatic hydrolysis experiments suggest that Compound 15 contains no phosphatidyl group, but a diglyceride unit linked to galactose. A free glycerophosphate molecule will be linked to galactose at the 6-position, because only this glycerol can contain the 1,2-glycol group, which is proven by the results of periodate oxydation. Both phosphogalactolipids gave a rapidly developing purple colour like phosphatidy~glycerol on periodate-Schiff staining at chromatograms and yielded one mole formaldehyde per mole phosphate (Table I). The presence of a non-acylated gfycerol molecule could also be established by its identification as a product after treatment of Lipid I 5 with 60 “/, HF. Other products of HF degradation were glycerophosphate, galactose and mono- and diglyceride. A lipid containing galactose could
383
TABLE
111
ACTION
OF PHOSPHOLIPASES
Extraction were
AND
15 and 17 were incubated
Lipids
according
separated
LIPASE
with lipolytic
enzymes
by thin-layer
activity over the fractions _. _ ._
chromatography
in Solvent
soluble
15 15
Lipid
I5 $
Lipid
15
Lipid Lipid
17
I
phospholipase lipase
Lipid t7llipasc _ -. _ -.-
and Methods.
distribution
of the 32P
_
soluble -.. 74
Lipid
73
78
21
28
72
18
79
6
94
9
91
5
95
64
36
-
-
-
-
of Lipid 15 -. ._ ._ 9 8
90
C
_
I. The
27 IO
A,
I 5T phospholipasc
System
.91 92
16
Lipid Lipid
Materials
Lipid 32P activity -. -. 15 Lyso-derivatives
Total 32P activity -.-_ -. -._ WaterLipid-
15
under
and lipid-soluble fractions. The lipids
is given in y,Lof the total activity as the mean of at least 2 experiments. -.-.-.
System
Lipid
as described
to Bligh and Dyer [13] gave water-soluble
_
._ _ -----
not be isolated. Presumably the furanosyl linkage is too labile in 60 % HF. This degradation method was not adequate therefore in this case to localize the fatty acids either both linked to glycerol or one to glycerol and one to galactose. The linkage of one fatty acid molecule of Lipid I 5 to galactose could also not definitively be excluded by periodate experiments, but periodate consumption of both Lipids 15 and 17 amounted only I mole per mole phosphate (Table I). Also higher concentrations up to 0.05 M did not result in a degradation of the galactose molecule. This could be proven by gas-chromatographic analysis after reduction with NaBH, and acid hydrolysis. However the organic solvent system and the steric inhibition 1311 by the diglyceride unit at C-I and the glycerophosphate group at C-6 may render the frans-glycol group of C-2 and C-3 inert to attack to periodate. The fatty acid composition of Lipid 15 showed marked differences with that of Lipid 17, monogalactosyldiglyceride and phosphatidylglycerol (Table IV). Lipid 17 can not be a I : I mixture of the two lyso-derivatives of Lipid 15. The markedly decreased content of octadecenoic and lactobacillic acid suggests a prevalence of the 2-acyl compound. These acids showed in phosphatidylglycerol from B. biJdunz var. TABLE
IV
FATTY
ACID
COMPOSITION
PHATIDYLGLYCEROLAND The percentage
OF
THE
PHOSPHOGALACTOLIPIDS
15 AND
of each fatty acid is given as the mean of duplicate
of each lipid. The fatty acid methyl esters arc designated
analyses of at least two preparations
by the number
of carbon atoms, followed
the number of double bonds. Fatty acid
14:o 16:o 16:1 18:o 18:1 Cycle- I9 : 0 -- .- .-
Monogalactosyl-
Phosphatidyl-
diglyccride --_-..___
glycerol
2. I 14.7
3.0 26.5
3.’ 25.7
3.8 23.0
45.6 6. I -. --
17, PHOS-
MONOGALACTOSYLDIGLYCERIDE
--
_..
36.5 8.7 _._ _._._..__
Compound -
_
__
5.1 33.0 5.4 20.5 28.6 5.7
I5
Compound
___._
_ I.5 55. ’ 3.0 28.0 II.9 _
_
I7
by
384
pennsylvanicus
degradation.
a strong preference for Position I, as was shown by phospholipase A In two strains of Streptococcus.faecalis the same preference was observed
[151. Structure
qf the more apolar phosphogalactolipid
Lipid 6 could not be isolated in sufficient amounts for analysis, because it amounts less than I y;‘, of total lipid phosphorus and is considerably contaminated with phosphatidylglycerol. Its deacylation product showed the same chromatographic and electrophoretic properties as the deacylation product of Compound 15. Analysis of the pure deacylation product gave a glycerol galactose phosphate ratio of 2: I : I. Alkaline hydrolysis gave the same products glycerol, galactosylglycerol, glycerophosphate and phosphorylgalactosylglycerol. The isolated glycerophosphate amounted 74 %, glycerol a-phosphate, 2 I “/;; sn-glycerol I -phosphate and 5 Y,,,al-glycerol J-phosphate. The configuration of the glycerophosphate unit is the same as in Lipids I 5 and I 7. Galactosylglycerol appeared to be about 90 “/I of the furanose form and IO )‘<:of the pyranose form. The mobility of Compound 6 on the thin-layer chromatograms points to the presence of four fatty acids. Between Lipids 6 and 15 another lipid was detected sometimes, which contained the same deacylation product and probably three fatty acids. The location of the fatty acids in Lipid 6 could not be established. A phosphatidy1 galactosyldiglyceride structure does not appear very likely, because the st7-Iphosphatidyl structure has been detected rarely in nature. Kates and coworkers [32-341 reported that the phytanyl ether analogs of phosphatidylglycerol and phosphatidylglycerophosphate and the sulphate ester of phosphatidylglycerol in Halobacterium cutirubum contained this unusual structure. The location of one or two acyl groups on the galactose molecule appears probable, because mono- and diacyl monogalactosyldiglycerides with a free carbon atom on Position 6 of galactose are present in B. b$dum var. pennsylvanicus [8]. Another argument for this location of the fatty acids is given in the next section. DISCUSSION
The unknown Lipids 6, 15 and J7 of B. b@dum var. pennsylvanicus proved to be phosphogalactolipids. The studies on their structure were hampered by the coexistence of furanose and pyranose forms and the acid lability of the galactofuranosyl linkage. The data obtained are compatible with the structure of Compound 15 (Fig. 5) as: 3-O-[6’-(sn-glycero-1-phosphoryl)-B-D-galactofuranosyl]-sn-I ,2-diglyceride. Lipid 17 has the same structure except one fatty acyl group. Lipid 6 is probably a diacyl derivative
Fig. 5. Structure
of the main phosphogalactolipid
of B. hifid~rm var. pennsqlwnicrrs
(Lipid
15).
385
of Compound 15, though its structure could not be completely established. About IO-2o;d of these compounds appeared to be present as galactopyranosyl compounds. Glycerylphosphoryl derivatives of galactosyldiglycerides have not been described earlier. The furanosyl structure of the sugar moiety is heretofore unique in bacterial phosphoglycolipids. We detected this form already in the acylated galactosyldiglycerides of B. bifidum var. pennsylvanicus [8]. It was also detected in the lipids of Mycoplasma mycoides [29], Bacteroides symbiosus [30] and Flavobacterium thermophi/urn [35]. Glycerylphosphoryldiglucosyl diglycerides have been isolated from a wide variety of bacteria [6, 7, I 5, 20, 261. A phosphatidyl derivative of monoglucosyldiglyceride was present in Pseudomonas diminuta [36]. Leuconostoc mesenteroides and Listeria monocytogenes contain glycerophosphorylgalactosylglucosyldiglycerides 171. Their structure has not yet been published. Phosphatidyldiglucosyldiglycerides have been shown to be present in Streptococci [IS, 371 and Acholeplasma laidlawii B [38]. The Phosphogalactolipids 6, I 5 and 17 from B. bQ?dum var. pennsylvanicus contain the unusual sn-glycerol r-phosphate like the glycerylphosphoryldiglucosyldiglyceride of Streptococci [15]. The enantiomorphous sn-glycerol 3-phosphate was present in the glycerylphosphoryldiglucosyldiglyceride of Acholeplasma laidlawii B [26] and the phosphatidylglucolipids of Srreprococci [IS, 371, A. laidlawii B [38] and Pseudomonas diminuta [36]. Phosphatidylglycerol and (acylated) monogalactosyldiglycerides can function as the precursors for the biosynthesis of the phosphogalactolipids of B. btjidum var. pennsylvanicus. Pieringer and co-worker gave evidence for his earlier suggestion [20,39) that a phosphoglucolipid in Streptococcus faecalis could be synthesized from diglucosyldiglyceride and phosphatidylglycerol and/or diphosphatidylglycerol [40]. Preliminary results with a particulate fraction from B. b$dum var. pennsylvanicus demonstrated that 32P activity from [32P]phosphatidylglycerol was incorporated into the Phosphogalactolipid Compound 6 and diphosphatidylglycerol. The incorporation into the phosphogalactolipid was increased by addition of diacyl monogalactosyldiglyceride. Radioactivity from [32P]diphosphatidylglycerol was not incorporated into Compound 6. The difference between phosphatidylglycerol and diphosphatidylglycerol in this system suggests that the srr-I-glycerophosphate unit is transferred in contrast to the phosphatidyi transfer in the S. faecufis system (4oJ. This agrees with a free sn-r-glycerophosphate group in Compound 6. Incorporation into Phosphogalactolipids 15 and 17 appeared to be absent in the used particulate system. A metabolic conversion from Lipid 6 by deacylation to Lipids 15 and 17 appears a real possibility in vivo because of the analogous stereoconfiguration of the glycerophosphate. Chase experiments with [32P]phosphate with intact cells sustain the precursor role of phosphatidylglycerol also for thesecompounds. The specific activities of phosphatidylglycerol and Lipids 15 and 17 changed with time in such a way that a real precursor-product relationship appears possible. Lipid 6 was present in a concentration too low for an investigation of its specific activity changes with time. The possible precursor role of the (acylated) monogalactosyldiglycerides will form an important point of further investigation. The differences in fatty acid composition between Lipid 15 and monogalactosyldiglyceride suggest that variations in fatty acid composition may play a role in the use of monogalactosyldiglyceride species for the biosynthesis of phosphogalactolipids. Contrary to our results the phosphoglucolipids. diglucosyldiglyceride and the other phospholipids from
Streptococci [37] showed the same fatty acid composition. Besides de novo synthesis a deacylation-reacylation cycle [4r] with lysoderivatives as intermediates may contribute to the final assessment of the fatty acid composition of our phosphogalactolipids like earlier was reported for the phospholipids in rat liver [42, 431. ACKNOWLEDGEMENTS
The authors are indebted to Mr E. Keizer for excellent technical assistance. Mass spectrometry and PMR spectroscopy were performed by Dr J. P. Kamerling, Department of Organic Chemistry, State University, Utrecht. Material support by the Netherlands Organization for the Advancement of Pure Research (ZWO-SON) is also gratefully acknowledged. REFERENCES I z 3 4
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