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Study of the long chain bases of sphingomyelin of Entomophthora coronata
179
Biochimica et Biophyeica Acta, 486 (1977) 179-182 0 EIaevierlNorth-Holland Biomedical Press
BBA 66914
STUDY OF THE LONG CHAIN BASES OF SPHINGOMYELIN OF ENTOMOPHTHORA
CORONATA
C. DE BIEVRE and F. MARIAT
Service de Mycologic, Znstitut Pasteur, 25 rue du Dr. Roux, Paris 15e’me (France) (Received
May IOth, 1976)
Sphingomyelin was isolated from a strain of Entomophthom coronata, a fungus pathogenic for humans. After hydrolysis, the long chain bases were converted to their dinitrophenyl (N,pH) derivatives and the aldehydes prepared by oxidizing these compounds with periodic acid. The aldehydes were studied by gas chromatography. Twelve different aldehydes were identified, the chain distribution ranging from C 14 to C,,. The prominent chains were unsaturated. Straight and branched chains were found. The most abundant parent base which formed 52% of the total aldehyde was a 1,3dihydroxy-2-aminohexadecene.
Introduction In a previous commuflication [ 11, the presence of sphingomyelin in strains of Entomophthora coronata, a fungus pathogenic for humans, was reported. The long chain bases obtained were studied by thin layer chromatography as their N,pH derivatives. The bases were principally sphingenine homologues. Saturated bases (sphinganine homologues) which were found in very smalI quantity were sometimes difficult to detect on the chromatograms. Trihydroxy bases (hydroxysphinganine homologues) were absent or in too small amounts to be seen on the chromatogram. The presence of unsaturated bases as the major fraction was considered as a point of interest, because up to now the fungal bases were found to be either of the hydroxysphinganine type or of the sphinganine type. In the present paper, some data are given about the paraffm chain structure (length and methyl group) by studying the aldehydes produced by oxidation of the bases with periodic acid.
Nomencl&we according to IWAC-IUB
[21.
180
Materials and Methods E. coronatu strain No. 1047, obtained from our Institute and had been isolated from a human case of phycomycosis. Cultures were obtained on Sabouraud dextrose liquid medium at 25°C in stable Roux flasks. The mycelium was harvested at 7 days. A chemical standard of brain sphingomyelin was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. The sphingenine and the aldehydes of this sphingomyelin were prepared in the laboratory. Myristaldehyde and palmitaldehyde were obtained from Fluka, Switzerland.
Preparation of lung chain bases This was performed by hydrolys~g the sphingo~pids with HCl in aqueous methanol (HCI, 9.4 ml; water, 8.6 ml; made up to 100 ml with methanol) at 70°C for 13 h, and purification by chromatography [3]. Preparation
qf the NzpH derivatives of the long chain bases
The long chain bases (up to 5 mg) were converted to their correspondtig NzpH derivatives [ 41, by dissolving them in 1 ml of methanol containing 5 ~1 of l-fluoro-2,4dinitrobenzene, then 4 ml of 2 M potassium borate, pH 10.5, were slowly added. The mixture was allowed to stand 30 mm at 60°C. Purification of the N&H derivatives was achieved on a silicic column [4]. beak
tion of the aldehydes
The NzpH derivatives which are stable (in contrast to the free bases) could be easily oxidized by periodic acid in acetic acid according to the technique described by Karlsson et al. [ 51. Characterization of aldehydes
The aldehydes were studied directly by gas chromatography with a Hewlett Packard apparatus (5750) equipped with a flame ionization detector. A 1.8 m stainless steel column of 15% diethyleneglycol succinate on 120 mesh gas Chrom Q was operated at 150°C with nitrogen as carrier gas. The aldehydes were identified by using the following standards: ~mi~dehyde, myristaldehyde and those prepared from the standard sphin~mye~~, mostly 16 : 1 aldehyde and iso 16 : 1 aldehyde; and by plotting the logarithm of their retention time versus the chain length. For the branched chain aldehyde, the equivalent chain length was used. Results and Discussion The chromatogram (Fig. 1) shows at least 12 peaks. The aldehydes characterized and their probably parent bases are listed in Table I. The unsaturated bases constituted 93% of the total bases encountered. The major peak, number 6, corresponds to a 1,3~ihydroxy-2~~ohexade~ne representing 52+%of the total. The previous de~~~atiorl of sphingen~e homologue [l] was confirmed by the present study of aldehydes which were found to be mostly unsaturated. The saturated aldehydes were considered as produced by sphinganine base type
181
Ng.l. GM
chromatogrem of aldehydee obtained by oxidationof the longchainbaeaof rpblngomyeltn.
The peak numbers refer to Table I.
because the thin layer chromatography studies showed that the hydroxysphinganine was absent or present in negligible amount l . Only four saturated bases were indicated: branched (n-2) (ho),-16 : 0, (ho),-16 : 0, (ho)z-17 : 0 and (ho),-20 : 0 (abbreviations: see footnote Table I). Others could have been overlooked on the chromatogram due to small amounts. Although free bases could be studied and transformed directly into aldehydes, the NlpH derivatives were used because they were more easily purified than the free bases. They were stable and oxidized by periodic acid. Other workers have studied aldehydes [4,5]. The use of Ccl,, as solvent and storage at -20°C have prevented degradation over long periods, thus facilitating their study. The use of a 15% diethyleneglycolsuccinate column gave separation of
TABLE I DISTRIBUTION
OF LONG CHAIN BASES IN SPHINGOMYELIN
Peak no.
Aldehyde
Parent base
(n -2)14:0 * 14:o 12:l l&O (n -33)13:1 l 13:l 14:l (n -2) 16:l *
(n -2)(ho)2-16:O * )(ho)2-16:O (ho)2-14:l (hoj2-17:O (n -3)(ho)2-16:l (ho)2-16:l (ho)2-16:1 (n -2)(ho)2-17:l * (ho)z-17:l (ho)2-20:O (n - 2)(hoj2-13:l* (bo)y13:l (n - 2)(ho)2-19:l*
OF E. CORONATA Percent of parent base
-
-1 2 3 4 6 6 7
a
l&l 18:O
9
10
(n-2)16:1
*
11 12
16:l (n--2)17:1
l
0.6 1.4 4.4 1.0 9.7 62.0 3.9 6.7 4.3 6.1 2.9 3.4
*(n-2) meane a methylbrench in wmition 2 from the metbylend (eeeref.6);(ho)2 mane d?OXY.
~~~
brcdd&:
brenched(n-2),(ho)3-17 : O.(ho)3-18 : O.@'+-21
;O.
dihy-
182
saturated from unsaturated aldehydes and also of straight from branched chains. The occurrence of sphingomyelin and sphingenine homologue bases in a fungus is important. The presence of sphingomyelin in fungi is probably rare in contrast to its wide distribution in animal organisms. Its long chain base composition would also require more investigation. It would be useful to confirm the present result by combined gas chromatography-mass spectrometry because saturated, unsaturated or branched aldehydes are often difficult to distinguish by using only gas chromatography. In previous papers about fungal sphingolipids, it was reported that the bases belonged to the saturated series [6-111. Weiss and Stiller [6] in their detailed study on the sphingolipids of Fusurium lini and Phycomyces blukesleanus found hydroxysphinganine and sphinganine homologues and no sphingenine type base. Sometimes, traces (about 1%) of sphingenine were discovered [ 8,9]. In E. coronata, the situation is exactly the reverse. As the strain studied contained unusual major long chain bases, it would be interesting to extend this investigation to other pathogenic fungi and determine if the distribution of their bases can be related to the host-parasite interaction. References 1 2 3 4 5 6 7 8 9 10
de Bievm, C. (1974) Ann. Microbial. (Inst. Pasteur) 125 B. 471-487 I.U.P.A.C.-I.U.B. (1970) Eur. J. Biocbem. 12.1-2 Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil. Chem. Sot. 42,294-298 Karlseon. K.A. (1970) Chem. Phys. Lipid 5.6-43 Karlsson, K.A.. SamueLmon. B.E. and Steen, G.O. (1973) Biochim. Biophys. Acta 3161336-362 Weiss. B. and Stiller. R.L. (1972) Biochemistry 11,4552-4557 Weiss. B. and Stiller, R.L. (1973) Lipid 8 (1). 25-30 Stanacev, NE. and K&es. M. (1963) Can. J. Biochem. Physiol. 41.1330-1335 Tyorinoja, K.. N urminen, T. and Suomalainen. H. (1974) Biochem. J. 141.133-139 Stodola, F.H. and Wickerham. L.J. (1960) J. Biol. Chem. 235.2584-2535
Biochimica et Biophyeica Acta, 486 (1977) 179-182 0 EIaevierlNorth-Holland Biomedical Press
BBA 66914
STUDY OF THE LONG CHAIN BASES OF SPHINGOMYELIN OF ENTOMOPHTHORA
CORONATA
C. DE BIEVRE and F. MARIAT
Service de Mycologic, Znstitut Pasteur, 25 rue du Dr. Roux, Paris 15e’me (France) (Received
May IOth, 1976)
Sphingomyelin was isolated from a strain of Entomophthom coronata, a fungus pathogenic for humans. After hydrolysis, the long chain bases were converted to their dinitrophenyl (N,pH) derivatives and the aldehydes prepared by oxidizing these compounds with periodic acid. The aldehydes were studied by gas chromatography. Twelve different aldehydes were identified, the chain distribution ranging from C 14 to C,,. The prominent chains were unsaturated. Straight and branched chains were found. The most abundant parent base which formed 52% of the total aldehyde was a 1,3dihydroxy-2-aminohexadecene.
Introduction In a previous commuflication [ 11, the presence of sphingomyelin in strains of Entomophthora coronata, a fungus pathogenic for humans, was reported. The long chain bases obtained were studied by thin layer chromatography as their N,pH derivatives. The bases were principally sphingenine homologues. Saturated bases (sphinganine homologues) which were found in very smalI quantity were sometimes difficult to detect on the chromatograms. Trihydroxy bases (hydroxysphinganine homologues) were absent or in too small amounts to be seen on the chromatogram. The presence of unsaturated bases as the major fraction was considered as a point of interest, because up to now the fungal bases were found to be either of the hydroxysphinganine type or of the sphinganine type. In the present paper, some data are given about the paraffm chain structure (length and methyl group) by studying the aldehydes produced by oxidation of the bases with periodic acid.
Nomencl&we according to IWAC-IUB
[21.
180
Materials and Methods E. coronatu strain No. 1047, obtained from our Institute and had been isolated from a human case of phycomycosis. Cultures were obtained on Sabouraud dextrose liquid medium at 25°C in stable Roux flasks. The mycelium was harvested at 7 days. A chemical standard of brain sphingomyelin was obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. The sphingenine and the aldehydes of this sphingomyelin were prepared in the laboratory. Myristaldehyde and palmitaldehyde were obtained from Fluka, Switzerland.
Preparation of lung chain bases This was performed by hydrolys~g the sphingo~pids with HCl in aqueous methanol (HCI, 9.4 ml; water, 8.6 ml; made up to 100 ml with methanol) at 70°C for 13 h, and purification by chromatography [3]. Preparation
qf the NzpH derivatives of the long chain bases
The long chain bases (up to 5 mg) were converted to their correspondtig NzpH derivatives [ 41, by dissolving them in 1 ml of methanol containing 5 ~1 of l-fluoro-2,4dinitrobenzene, then 4 ml of 2 M potassium borate, pH 10.5, were slowly added. The mixture was allowed to stand 30 mm at 60°C. Purification of the N&H derivatives was achieved on a silicic column [4]. beak
tion of the aldehydes
The NzpH derivatives which are stable (in contrast to the free bases) could be easily oxidized by periodic acid in acetic acid according to the technique described by Karlsson et al. [ 51. Characterization of aldehydes
The aldehydes were studied directly by gas chromatography with a Hewlett Packard apparatus (5750) equipped with a flame ionization detector. A 1.8 m stainless steel column of 15% diethyleneglycol succinate on 120 mesh gas Chrom Q was operated at 150°C with nitrogen as carrier gas. The aldehydes were identified by using the following standards: ~mi~dehyde, myristaldehyde and those prepared from the standard sphin~mye~~, mostly 16 : 1 aldehyde and iso 16 : 1 aldehyde; and by plotting the logarithm of their retention time versus the chain length. For the branched chain aldehyde, the equivalent chain length was used. Results and Discussion The chromatogram (Fig. 1) shows at least 12 peaks. The aldehydes characterized and their probably parent bases are listed in Table I. The unsaturated bases constituted 93% of the total bases encountered. The major peak, number 6, corresponds to a 1,3~ihydroxy-2~~ohexade~ne representing 52+%of the total. The previous de~~~atiorl of sphingen~e homologue [l] was confirmed by the present study of aldehydes which were found to be mostly unsaturated. The saturated aldehydes were considered as produced by sphinganine base type
181
Ng.l. GM
chromatogrem of aldehydee obtained by oxidationof the longchainbaeaof rpblngomyeltn.
The peak numbers refer to Table I.
because the thin layer chromatography studies showed that the hydroxysphinganine was absent or present in negligible amount l . Only four saturated bases were indicated: branched (n-2) (ho),-16 : 0, (ho),-16 : 0, (ho)z-17 : 0 and (ho),-20 : 0 (abbreviations: see footnote Table I). Others could have been overlooked on the chromatogram due to small amounts. Although free bases could be studied and transformed directly into aldehydes, the NlpH derivatives were used because they were more easily purified than the free bases. They were stable and oxidized by periodic acid. Other workers have studied aldehydes [4,5]. The use of Ccl,, as solvent and storage at -20°C have prevented degradation over long periods, thus facilitating their study. The use of a 15% diethyleneglycolsuccinate column gave separation of
TABLE I DISTRIBUTION
OF LONG CHAIN BASES IN SPHINGOMYELIN
Peak no.
Aldehyde
Parent base
(n -2)14:0 * 14:o 12:l l&O (n -33)13:1 l 13:l 14:l (n -2) 16:l *
(n -2)(ho)2-16:O * )(ho)2-16:O (ho)2-14:l (hoj2-17:O (n -3)(ho)2-16:l (ho)2-16:l (ho)2-16:1 (n -2)(ho)2-17:l * (ho)z-17:l (ho)2-20:O (n - 2)(hoj2-13:l* (bo)y13:l (n - 2)(ho)2-19:l*
OF E. CORONATA Percent of parent base
-
-1 2 3 4 6 6 7
a
l&l 18:O
9
10
(n-2)16:1
*
11 12
16:l (n--2)17:1
l
0.6 1.4 4.4 1.0 9.7 62.0 3.9 6.7 4.3 6.1 2.9 3.4
*(n-2) meane a methylbrench in wmition 2 from the metbylend (eeeref.6);(ho)2 mane d?OXY.
~~~
brcdd&:
brenched(n-2),(ho)3-17 : O.(ho)3-18 : O.@'+-21
;O.
dihy-
182
saturated from unsaturated aldehydes and also of straight from branched chains. The occurrence of sphingomyelin and sphingenine homologue bases in a fungus is important. The presence of sphingomyelin in fungi is probably rare in contrast to its wide distribution in animal organisms. Its long chain base composition would also require more investigation. It would be useful to confirm the present result by combined gas chromatography-mass spectrometry because saturated, unsaturated or branched aldehydes are often difficult to distinguish by using only gas chromatography. In previous papers about fungal sphingolipids, it was reported that the bases belonged to the saturated series [6-111. Weiss and Stiller [6] in their detailed study on the sphingolipids of Fusurium lini and Phycomyces blukesleanus found hydroxysphinganine and sphinganine homologues and no sphingenine type base. Sometimes, traces (about 1%) of sphingenine were discovered [ 8,9]. In E. coronata, the situation is exactly the reverse. As the strain studied contained unusual major long chain bases, it would be interesting to extend this investigation to other pathogenic fungi and determine if the distribution of their bases can be related to the host-parasite interaction. References 1 2 3 4 5 6 7 8 9 10
de Bievm, C. (1974) Ann. Microbial. (Inst. Pasteur) 125 B. 471-487 I.U.P.A.C.-I.U.B. (1970) Eur. J. Biocbem. 12.1-2 Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil. Chem. Sot. 42,294-298 Karlseon. K.A. (1970) Chem. Phys. Lipid 5.6-43 Karlsson, K.A.. SamueLmon. B.E. and Steen, G.O. (1973) Biochim. Biophys. Acta 3161336-362 Weiss. B. and Stiller. R.L. (1972) Biochemistry 11,4552-4557 Weiss. B. and Stiller, R.L. (1973) Lipid 8 (1). 25-30 Stanacev, NE. and K&es. M. (1963) Can. J. Biochem. Physiol. 41.1330-1335 Tyorinoja, K.. N urminen, T. and Suomalainen. H. (1974) Biochem. J. 141.133-139 Stodola, F.H. and Wickerham. L.J. (1960) J. Biol. Chem. 235.2584-2535