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Galactosyl-diglyceride from Actinomyces viscosus
CHEMISTRY AND PHYSICS OF LIPIDS 12 (1974) 172-175. NORTH-HOLLAND PUBL. CO.
GALACTOSYL-DIGLYCERIDE
F R O M A C T I N O M Y C E S V I S C O S US
M. YRIBARREN and E. VILKAS Institut de Chimie des Substances Naturelles, CN.R.S., 91190. Gif sur Yvette, France and J. ROZANIS University of Western Ontario, London, Ontario, Canada
Received September 12, 1973 Accepted October 29, 1973 The glycolipids from Actinomyces viscosus were investigated. A 1-O-monogalactosyl-diglyceride was identified. Analysis of its fatty acids by mass spectrometry showed that they consisted largely of palmitic and stearic acids and their m onounsaturated horn ologues.
I. Introduction Although the glycosyl-diglycerides are widely distributed in Gram-positive bacteria [ 1], their presence in the order Actinomycetales was reported only once, in Streptomyces LA 7017 [2]. The present paper describes a monogalactosyl diglyceride isolated from Actinomyces viscosus (previously called Odontomyces vicosus) which has been implicated in root surface lesions of periodontally involved human teeth and in periodontal infection in animal-model experiments [3].
II. Materials and methods Actinomyces viscosus ATTCC 15987 was grown at 37°C in an anaerobic jar containing 5% CO2, 10% H2, 85% N2 washed three times with distilled water and freeze-dried. A. Extraction and purification o f lipids The freeze-dried cells were stirred with a mixture of ethanol-ether (1 : 1, v/v) for
M. Yribarren et al., Galactosyl-diglyceride from Actinomyces viscosus
173
12 hr at room temperature. This treatment was repeated three times and the combined extracts were evaporated to dryness in vacuo. The yield of lipid was 4% of the dry cell weight. The extraction was continued under the same conditions with chloroform-methanol (2 : 1, v/v) but only I% oflipids was obtained. The lipids were next treated with boiling acetone, the insoluble fraction contains mainly phospholipids. The soluble material was separated into neutral and glycolipid fractions by silicic acid chromatography [4]. The glycolipid was further purified by thin layer chromatography. B. Analytical methods
Hexoses were determined by the phenol-H2SO 4 method [5]. Paper chromatography of sugars, polyols and glycosides was carried out by descending technic in the solvent systems: (1) butan-l-ol-pyridine-water (6:4:3, v/v); (2) butan-l-ol-acetic acid-water (4:1:5, v/v). Compounds were detected by the aniline hydrogene phtalate for reducing sugars [6] ; the alkaline AgNO3 reagent for sugars and polyols [7], the periodate Schlff reagents for a-glycols [8]. Lipids were examined by thin layer chromatography in the systems: (3) chloroform-methanol-water (70:35:7, v/v); (4) chloroform-methanol-acetic acid-water (80 : 10 : 2 : 0, 75, v/v). Spots were detected with sulfuric acid 50%, an throne reagent [9], periodateSchiff reagents [8] Dittmer reagent specific for phosphorus compounds [10] and ninhydrine. Preparative thin layer chromatography were run on plates coated with silicic gel G (Merck) 1 mm thick. Samples of glycolipid and glycoside were hydrolysed by HCI 2 N, at 100°C for 3 hr [11 ]. The hydrolysate evaporated to dryness in vacuo was redissolved in a minimum quantity of water and examined by paper chromatography. Glycolipid was deacetylated with sodium methoxide according to Marinetti [12]. Fatty acids were analysed by mass spectrometry as their methyl esters. Mass spectrometry of glycosyl-diglyceride as its acetate derivative was performed with an AEI MS9 instrument operating at 70 eV and an ion temperature of 2 3 0 240°C (Institut de Chimie des Substances Naturelles, Gif-sur-Yvette). Peracetylation of the glycolipid was effected with acetic anhydride in pyridine at room temperature for 24 hr.
Ill. Results and discussion
The total lipid extract accounted for approximatively 5% dry weight of Actino-
174
M. Yribarrenet al., Galactosyl-diglyceridefrom Actinomyces viscosus
myces viscosus and consisted mainly of neutral lipids (60 to 65%), phospholipids (30%)* and glycolipids (5 to 10%). The glycolipid fraction eluted from a silicic acid column with chloroform-acetone (1 : 1, v/v) was shown by thin layer chromatography (solvent 3) to contain three components A: R f = 0.86, B: R f = 0.73 and C: R f = 0.53 which give a blue-purple color with anthrone and blue-gray color with periodate-Schiff reagents [ 13 ] typical of glycolipids. They were negative to reagents specific for phosphorus and aminocompounds. A small quantity of ninhydrine positive, phosphorus-negative material was detected in the area of R f between 0.15 and 0.4. Only a pure sample of component A was obtained by preparative TLC in sufficient quantity for further study. The total acid hydrolysis of compound A yielded fatty acids, galactose and glycerol. The ratio of galactose (17%) and of fatty acids (70%) suggested a monogalactosyl diglyceride structure. Deacylation yielded a water soluble non reducing glycoside Rgal (solvent 1) = 0.88 which gave a rapidly developing purple color with the periodate-Schiff reagents, indicative of a 1-substituted glycerol. The fatty acids of the glycolipid consisted largely of palmitic and stearic acids and their monounsaturated homologues. The structure of monogalactosyl diglyceride was confirmed by mass spectrometry. The spectrum shows that in fact the monogalactosyl diglyceride is a mixture of glycolipid homologues containing different fatty acids mainly C16, C16:1 , C 17, C18 and C18:1. The parent peaks are at m/e 898* (with two palmatic acids) 926,940 and 954 (the latter with two stearic acids); the peaks at m/e 896,938 and 952 correspond to the glycolipids containing one unsaturation. The peaks at m/e 237,239,253,265 and 267 are due to ions corresponding to acyl groups. A very strong peak at m/e 331 corresponds to the oxonium ion formed by cleavage of the glycosidic bond and ionization of te ring oxygen atom. Further fragmentation of the oxonium ion by loss of molecules of acetic acid and ketene gives peaks at m/e 289,229 and 169 [14]. The series of peaks at m/e 533,549, 551,577,579 and 605 is due to loss of carbohydrate moiety from the molecular ion. The peaks at m/e 313,339,341 and 367 are characteristic of an acid being attached directly to the glycerol skeleton [15 ]. The lack of material did not permit us to establish the stereochemical configuration, however the results obtained indicate that the compound A is a 1-O-monogalactosyl-2,3-diglyceride. * The phospholipids were tentatively identified according to their chromatographic behaviour and examination of their hydrolysis and deacylation products as eardiolipin, phosphatidylethanolamine and phosphatidyl inositol. * Galactosyl dipalmitoyl glyceride = M÷898.
M. Yribarren et al., Galactosyl-diglyceridefrom A ctinomyces viscosus
175
The glycolipid fraction ofActinomyces viscosus represents 5 to 10% of the total lipids and contains the monogalactosyl diglyceride as major component. Although the monogalactosyl diglyceride only was identified, the presence of diglycosyl diglyceride could not be excluded. The elution of two other bands (Rf = 0.73 and R f = 0.53) did not yields sufficient material for detailed studies but both contain galactose. The difficulty o f their hydrolysis may also be mentionned. According to Shaw [ 1] the monoglycosyl diglycerides are known to be the biosynthetic precursors of the diglycosyl diglycerides but they are not usually accumulated in significant amounts. A few organisms however: some species o f Arthrobacter [11] andMycoplasma mycotdes [16] and laidlawii [17] contain those glycolipids as principal component. So far as we know Actinomyces viscosus is the first representative of the genus of Actinomyces to be shown to contain such a glycolipid. Examination of their distribution among the other members of the same genus will perhaps enable certain taxonomic relationship to be established.
Acknowledgements The authors wish to thank Professor E. Lederer for his advice and Dr. B.C. Das for usefall, discussions. This work was supported, in part, by the "World Health Organization", by the "Ligue Nationale Fran~aise centre le Cancer" and the "Fondation pour la Recherche M6dicale Fran~aise".
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
N. Shaw, Baeteriol. Rev. 34 (1970) 365 L.D. Bergelson, S.G. Batrakov and T.V. Pilipenko, Chem. Phys. Lipids 4 (1970) 181 S. BeUack and H.V. Jordan, Arch. Oral. Biol. 17 (1972) 175 M.L. Vorbeck and G.V. Marinetti, J. Lipid Res. 6 (1965) 3 M. Dubois, R.A. GiUes, J.K. Hamilton, P.A. Rebers and P.F. Smith, Anal. Chem. 28 (1956) 350 S.M. Partridge, Nature 164 (1948) 443 W.E. Trevelyan, D.P. Procter and J.S. Harrison, Nature 166 (1950) 444 J. Baddfley, J.G. Buchanan, R.E. Handsehumacker and J.F. Prescott, J. Chem. Soc. (1956) 2818 T. Yamakhawa, Irie and M. Iwanaga, J. Biochem., 48 (1960) 490 J.C. Dittmer and R.L. Lester, J. Lipid Res. 5 (1964) 126 N. Shaw and D. Stead, J. Bacteriol. 107 (1971) 130 G.V. Marinetti, J. Lipid Res., 3 (1962) 1 N. Shaw, Biochim. Biophys. Acta, 164 (1968)435 K. Biemann, D.C. de Jongh and H.K. Schoes, J. Amer. Chem. Soc., 85 (1963) 1763 M. Barber and T.O. Merren, Tetrahedron Letters 18 (1964) 1063 P. Plackett, Biochemistry, 6 (1967) 2746 N. Shaw, P.F. Smith and W.L. Koostra, Biochem. J. 107 (1968) 329
GALACTOSYL-DIGLYCERIDE
F R O M A C T I N O M Y C E S V I S C O S US
M. YRIBARREN and E. VILKAS Institut de Chimie des Substances Naturelles, CN.R.S., 91190. Gif sur Yvette, France and J. ROZANIS University of Western Ontario, London, Ontario, Canada
Received September 12, 1973 Accepted October 29, 1973 The glycolipids from Actinomyces viscosus were investigated. A 1-O-monogalactosyl-diglyceride was identified. Analysis of its fatty acids by mass spectrometry showed that they consisted largely of palmitic and stearic acids and their m onounsaturated horn ologues.
I. Introduction Although the glycosyl-diglycerides are widely distributed in Gram-positive bacteria [ 1], their presence in the order Actinomycetales was reported only once, in Streptomyces LA 7017 [2]. The present paper describes a monogalactosyl diglyceride isolated from Actinomyces viscosus (previously called Odontomyces vicosus) which has been implicated in root surface lesions of periodontally involved human teeth and in periodontal infection in animal-model experiments [3].
II. Materials and methods Actinomyces viscosus ATTCC 15987 was grown at 37°C in an anaerobic jar containing 5% CO2, 10% H2, 85% N2 washed three times with distilled water and freeze-dried. A. Extraction and purification o f lipids The freeze-dried cells were stirred with a mixture of ethanol-ether (1 : 1, v/v) for
M. Yribarren et al., Galactosyl-diglyceride from Actinomyces viscosus
173
12 hr at room temperature. This treatment was repeated three times and the combined extracts were evaporated to dryness in vacuo. The yield of lipid was 4% of the dry cell weight. The extraction was continued under the same conditions with chloroform-methanol (2 : 1, v/v) but only I% oflipids was obtained. The lipids were next treated with boiling acetone, the insoluble fraction contains mainly phospholipids. The soluble material was separated into neutral and glycolipid fractions by silicic acid chromatography [4]. The glycolipid was further purified by thin layer chromatography. B. Analytical methods
Hexoses were determined by the phenol-H2SO 4 method [5]. Paper chromatography of sugars, polyols and glycosides was carried out by descending technic in the solvent systems: (1) butan-l-ol-pyridine-water (6:4:3, v/v); (2) butan-l-ol-acetic acid-water (4:1:5, v/v). Compounds were detected by the aniline hydrogene phtalate for reducing sugars [6] ; the alkaline AgNO3 reagent for sugars and polyols [7], the periodate Schlff reagents for a-glycols [8]. Lipids were examined by thin layer chromatography in the systems: (3) chloroform-methanol-water (70:35:7, v/v); (4) chloroform-methanol-acetic acid-water (80 : 10 : 2 : 0, 75, v/v). Spots were detected with sulfuric acid 50%, an throne reagent [9], periodateSchiff reagents [8] Dittmer reagent specific for phosphorus compounds [10] and ninhydrine. Preparative thin layer chromatography were run on plates coated with silicic gel G (Merck) 1 mm thick. Samples of glycolipid and glycoside were hydrolysed by HCI 2 N, at 100°C for 3 hr [11 ]. The hydrolysate evaporated to dryness in vacuo was redissolved in a minimum quantity of water and examined by paper chromatography. Glycolipid was deacetylated with sodium methoxide according to Marinetti [12]. Fatty acids were analysed by mass spectrometry as their methyl esters. Mass spectrometry of glycosyl-diglyceride as its acetate derivative was performed with an AEI MS9 instrument operating at 70 eV and an ion temperature of 2 3 0 240°C (Institut de Chimie des Substances Naturelles, Gif-sur-Yvette). Peracetylation of the glycolipid was effected with acetic anhydride in pyridine at room temperature for 24 hr.
Ill. Results and discussion
The total lipid extract accounted for approximatively 5% dry weight of Actino-
174
M. Yribarrenet al., Galactosyl-diglyceridefrom Actinomyces viscosus
myces viscosus and consisted mainly of neutral lipids (60 to 65%), phospholipids (30%)* and glycolipids (5 to 10%). The glycolipid fraction eluted from a silicic acid column with chloroform-acetone (1 : 1, v/v) was shown by thin layer chromatography (solvent 3) to contain three components A: R f = 0.86, B: R f = 0.73 and C: R f = 0.53 which give a blue-purple color with anthrone and blue-gray color with periodate-Schiff reagents [ 13 ] typical of glycolipids. They were negative to reagents specific for phosphorus and aminocompounds. A small quantity of ninhydrine positive, phosphorus-negative material was detected in the area of R f between 0.15 and 0.4. Only a pure sample of component A was obtained by preparative TLC in sufficient quantity for further study. The total acid hydrolysis of compound A yielded fatty acids, galactose and glycerol. The ratio of galactose (17%) and of fatty acids (70%) suggested a monogalactosyl diglyceride structure. Deacylation yielded a water soluble non reducing glycoside Rgal (solvent 1) = 0.88 which gave a rapidly developing purple color with the periodate-Schiff reagents, indicative of a 1-substituted glycerol. The fatty acids of the glycolipid consisted largely of palmitic and stearic acids and their monounsaturated homologues. The structure of monogalactosyl diglyceride was confirmed by mass spectrometry. The spectrum shows that in fact the monogalactosyl diglyceride is a mixture of glycolipid homologues containing different fatty acids mainly C16, C16:1 , C 17, C18 and C18:1. The parent peaks are at m/e 898* (with two palmatic acids) 926,940 and 954 (the latter with two stearic acids); the peaks at m/e 896,938 and 952 correspond to the glycolipids containing one unsaturation. The peaks at m/e 237,239,253,265 and 267 are due to ions corresponding to acyl groups. A very strong peak at m/e 331 corresponds to the oxonium ion formed by cleavage of the glycosidic bond and ionization of te ring oxygen atom. Further fragmentation of the oxonium ion by loss of molecules of acetic acid and ketene gives peaks at m/e 289,229 and 169 [14]. The series of peaks at m/e 533,549, 551,577,579 and 605 is due to loss of carbohydrate moiety from the molecular ion. The peaks at m/e 313,339,341 and 367 are characteristic of an acid being attached directly to the glycerol skeleton [15 ]. The lack of material did not permit us to establish the stereochemical configuration, however the results obtained indicate that the compound A is a 1-O-monogalactosyl-2,3-diglyceride. * The phospholipids were tentatively identified according to their chromatographic behaviour and examination of their hydrolysis and deacylation products as eardiolipin, phosphatidylethanolamine and phosphatidyl inositol. * Galactosyl dipalmitoyl glyceride = M÷898.
M. Yribarren et al., Galactosyl-diglyceridefrom A ctinomyces viscosus
175
The glycolipid fraction ofActinomyces viscosus represents 5 to 10% of the total lipids and contains the monogalactosyl diglyceride as major component. Although the monogalactosyl diglyceride only was identified, the presence of diglycosyl diglyceride could not be excluded. The elution of two other bands (Rf = 0.73 and R f = 0.53) did not yields sufficient material for detailed studies but both contain galactose. The difficulty o f their hydrolysis may also be mentionned. According to Shaw [ 1] the monoglycosyl diglycerides are known to be the biosynthetic precursors of the diglycosyl diglycerides but they are not usually accumulated in significant amounts. A few organisms however: some species o f Arthrobacter [11] andMycoplasma mycotdes [16] and laidlawii [17] contain those glycolipids as principal component. So far as we know Actinomyces viscosus is the first representative of the genus of Actinomyces to be shown to contain such a glycolipid. Examination of their distribution among the other members of the same genus will perhaps enable certain taxonomic relationship to be established.
Acknowledgements The authors wish to thank Professor E. Lederer for his advice and Dr. B.C. Das for usefall, discussions. This work was supported, in part, by the "World Health Organization", by the "Ligue Nationale Fran~aise centre le Cancer" and the "Fondation pour la Recherche M6dicale Fran~aise".
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]
N. Shaw, Baeteriol. Rev. 34 (1970) 365 L.D. Bergelson, S.G. Batrakov and T.V. Pilipenko, Chem. Phys. Lipids 4 (1970) 181 S. BeUack and H.V. Jordan, Arch. Oral. Biol. 17 (1972) 175 M.L. Vorbeck and G.V. Marinetti, J. Lipid Res. 6 (1965) 3 M. Dubois, R.A. GiUes, J.K. Hamilton, P.A. Rebers and P.F. Smith, Anal. Chem. 28 (1956) 350 S.M. Partridge, Nature 164 (1948) 443 W.E. Trevelyan, D.P. Procter and J.S. Harrison, Nature 166 (1950) 444 J. Baddfley, J.G. Buchanan, R.E. Handsehumacker and J.F. Prescott, J. Chem. Soc. (1956) 2818 T. Yamakhawa, Irie and M. Iwanaga, J. Biochem., 48 (1960) 490 J.C. Dittmer and R.L. Lester, J. Lipid Res. 5 (1964) 126 N. Shaw and D. Stead, J. Bacteriol. 107 (1971) 130 G.V. Marinetti, J. Lipid Res., 3 (1962) 1 N. Shaw, Biochim. Biophys. Acta, 164 (1968)435 K. Biemann, D.C. de Jongh and H.K. Schoes, J. Amer. Chem. Soc., 85 (1963) 1763 M. Barber and T.O. Merren, Tetrahedron Letters 18 (1964) 1063 P. Plackett, Biochemistry, 6 (1967) 2746 N. Shaw, P.F. Smith and W.L. Koostra, Biochem. J. 107 (1968) 329