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Fruiting-inducing activity of cerebrosides observed with Schizophyllum commune
Biochimica et Biophysica Acta, 719 (1982) 612-618
612
Elsevier Biomedical Press BBA21286
FRUITING-INDUCING ACTIVITY OF CEREBROSIDES OBSERVED WITH S C H I Z O P H Y L L U M COMMUNE GENSHIRO
K A W A I a and Y O N O S U K E
IKEDA b
UNoda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken and b Nodai Research Institute, the Tokyo University of Agriculture, 1-1 Sakuragaoka, Setagaya-ku, Tokyo (Japan) (Received June 3rd, 1982)
Key words: Fruiting-inducing activity," Cerebroside; (S. commune)
Isolation and identification of substances having an activity to stimulate the fruiting body formation of Schizophyllum c o m m u n e were attempted. The active principles in its mycelia were divided into four fractions by sequential purification with silica gel column and reverse-phase HPLC column chromatography. By infrared spectra and thin-layer chromatography, the active substances in these four fractions were revealed as cerebrosides. About 0.1 ttg of the cerebroside fractions showed a discriminative stimulating activity on S. c o m m u n e when tested by the method these authors adopted. The active substance in the fraction II was N-2'-hydroxypalmitoyl-l-O-glucosyl-nonadecasphingadienine. The cerebrosides from pea seeds and Fusicoccure a m y g d a l i showed the similar activity on S. commune, but some commercial synthetic cerebrosides and cerebrosides from bovine and porcine brains exhibited no stimulating activity. Only definite cerebrosides with special structures seem to be able to induce the fruiting of S. commune.
Introduction
The phenomenon of fruiting body formation in Basidiomycetes is regarded as a model system for studies of differentiation and morphogenesis mechanisms of fungi. Among Basidiomycetes, Schizophyllum commune is most frequently used for these studies, because it grows well on simple solid media and completes its life cycle in as short as ten days. The organism is heterothallic; that is, a haploid homokaryotic mycelium derived from a single basidiospore is in principle self-sterile and a dikaryotic mycelium bred from two compatible homokarions is able to form fruiting bodies. Leonard and Dick [1] reported that some sorts of extracts prepared from several fungi were capable of inducing fruiting body formation when tested on homokaryotic mycelia of S. commune. Fruiting-inducing substance from Cladosporium cladosporioides was water-soluble, dialysable, and 0304-4165/82/0000-000/$02.75 © 1982 Elsevier Biomedical Press
stable to heating. Moreover, the substance was described to be resistant to acid and alkaline hydrolysis and several degradating enzymes. Rusmin and Leonard [2] purified another fruiting-inducing substance from cultivated mushroom (Agaricus bisporus) and described its chemical and physical properties without referring to its chemical structure. Independently, Uno and Ishikawa [3,4] reported that fruiting-inducing substances in homokaryotic mycelia of Coprinus macrorhizus were identical with cyclic AMP and some inhibitors of phosphodiesterase. In S. commune, however, Schwalb [5] wrote that he could not find any sign that cyclic nucleotides would act positively on the fruiting body formation. The present study was begun due to a casual observation of an unusual phenomenon that occurred between a strain of S. commune and a strain of Penicillium species. Abundant fruiting
613 bodies were observed on a contact line of two mycelial mats. An active principle able to induce fruiting of dikaryotyic and homokaryotic mycelia of S. commune was recovered in the acetone extract of the Penicillium mycelia. Since a similar principle was also recovered in the acetone extract of the S. commune itself, the principle chosen as the target of first-step investigation. As will be described in this paper, the principle has been identified as a certain sort of cerebroside by physical and chemical analyses. The principle in the acetone extract of the Penicillium mycelia will be reported elsewhere. Materials and Methods
Bioassay of fruiting-inducing activity. Basic assay methods were as follows. An appropriate amount of sample was dissolved in a suitable solvent. The solution (basically 20 ~1) was applied onto a paper disc (8 mm diameter, 0.7 mm thickness) and the solvent was removed under reduced pressure. A control disc was prepared in the same way by applying ethanol instead of the test solution. A dikaryotic strain of S. commune (IFO 6502) was spotted on the center of the malt-yeast agar medium [3] in a 9 cm Petri dish, and grown at 23-25°C. Three to four days after the inoculation, the test and control discs were placed on a margin of the plate. The plate was placed upside down and incubated for another two weeks under white fluorescent light (100-200 lux). Usually, more abundant fruiting bodies were observed around the test disc. All procedures were carried out aseptically. When the activity was to be indicated in term of units, the original solution was diluted serially by two-fold with ethanol and the least quantity which apparently gave a larger number of fruiting bodies than the control was defined as one unit. As the dilution was done by two-fold system, allowance of every unit is from x 0.5 to x 2.0. Thin-layer chromatography. Thin-layer chromatography was conducted either on precoated silica gel 60 plates (E. Merck, Darmstadt, F.R.G.) using (a) chloroform/methanol/water (65 : 25 : 4, v/v), (b) chloroform/methanol/85% formic acid (70: 18: 12, v/v), or on borate-impregnated silica gel plates prepared as described by Kean [6] using
(c) chloroform/methanol/water/15 M NH4OH (280 : 70 : 6 : 1, v/v). Substances were located with iodine vapor, 50% aqueous sulfuric acid reagent or anthrone-sulfuric acid reagent. Hydrolysis of the cerebroside. The cerebroside (5 mg) was refluxed with 4 ml of 1 N HC1 in methanol at 70°C for 18 h according to Gaver and Sweeley [7]. After cooling, resulting long-chain base, fatty acid methyl ester, and methyl glycoside were fractionated by the method of Miyazawa et al. [8]. Isolation of the long-chain base was also attempted by the Ba(OH)2 hydrolysis method of Morrison and Hay [9], but the yield was very low. The cerebroside was decomposed also to ceramides following the procedure of HammarstriSm [10], and the ceramide was purified by thin-layer chromatography (adsorbent: silica gel, G.E. Merck; solvent: chloroform/methanol = 97 : 3, v/v). Gas-liquid chromatography. Appropriate amount (0.5-2 mg) of the fatty acid methyl ester, the long-chain base, the methyl glycoside, and the ceramide were mixed with a sufficient silanes reagent to make up a 1% solution. The silanes reagent, prepared according to Carter and Gaver [11], consisted of 1.6 ml trimethylchlorosilane, 2.6 ml hexamethyldisilazane, and 2.0 ml dry pyridine. The trimethylsilyl ether derivatives were analysed with a Shimazu 4CMPF Gaschromatograph (Shimazu Seisakusho Co., Ltd., Kyoto). The column was a glass tube of 0.3 x 200 cm packed with 2.5% OV-17 on 60-80 mesh Uniport HPS (Gasukuro Kogyo Co., Ltd., Tokyo), and the flow rate of nitrogen was 40 ml/min. The column was kept at 170, 200, 220, and 330°C, respectively, for the trimethylsilyl ether derivatives of methyl glycosides, fatty acid methyl esters, long-chain bases, and ceramides. Gas-liquid chromatography-mass spectrometry. GC-MS analysis was performed on an Hitachi RMU-7M instrument (Hitachi Seisakusho Co., Ltd., Tokyo). The column was a stainless steel tube of 0.3 x 200 cm packed with 2.5% OV-17 on 60-80 mesh Uniport HPS. The column was kept at 160, 200, 210, and 300°C, respectively, for the trimethylsilyl ether derivatives of methyl glycosides, fatty acid methyl esters, long-chain bases, and ceramides. Mass spectra were obtained under the following conditions: ion source temperature, 210°C; electron energy, 70 eV; ion accelerator,
614
voltage 3.2 kV; and ionizing current 51 /~A. Authentic cerebrosides. The authentic cerebrosides from bovine and porcine brains were purchased from Funakoshi Pharmaceutical Co., Ltd., T o k y o . N-Stearoyl-DL-dihydrogalactocerebroside and N-stearoyl-DL-dihydroglucocerebroside were purchased from Miles Laboratories, Inc., Elkhart, U.S.A. N-Palmitoyl-DL-dihydroglucocerebroside was purchased from Sigma Chemical Co., U.S.A. The cerebrosides from pea seeds were prepared according to the method of Miyazawa et al. [8]. Cerebroside from Fusicoccum amygdali was the generous gift of Professor A. Ballio [ 12]. Results
Purification of active substances S. commune IFO 6502 was cultivated aerobically at 25°C for 5 days in a 30-1 jar fermenter containing a 20-1 malt-yeast medium [3]. The mycelia (dry wt., 85.2 g) were harvested by filtration, washed several times with distilled water, suspended in 2 1 acetone, and stood at room temperature. After 3 days, the acetone suspension was filtered and the residue was re-extracted three times. The solvent was removed from the pooled extracts by vacuum evaporation and the resulting aqueous suspension was treated three times with ethyl acetate for extraction. The extract was dried over anhydrous sodium sulfate and then concentrated to dryness in vacuo, yielding 6.1 g of oily crude extract. The culture filtrate and the residual aqueous solution after the extraction with ethyl acetate showed slight or no inducing activity, but the ethyl acetate extract showed considerably high activity. The crude extract was subjected to silica gel column chromatography and eluted sequentially with chloroform, chloroform/acetone (1:2, v/v), acetone, and methanol. A greater part of the activity was recovered in the chloroform/acetone eluate. This fraction was evaporated to dryness. This active fraction was then loaded on a Lober column size A LiCroprep Si 60 (E. Merck) and eluted with chloroform/methanol (Fig. 1). The strong activity was found in fractions 75-110. These fractions were pooled and evaporated to dryness.
CHCLj/MeOH (vlv)l
I : 0 124 : I
9:1
I
I
4:1
I
3:2
I
2:3
I
03
02
i °. O(
0
100 Frocbon number
200
300
Fig. 1. Profile of the silica gel column re-chromatography. The chloroform/acetone fraction from the silica gel column chromatography was dissolved in 50 ml chloroform, loaded on Lober column size A LiCroprep Si 60 (E. Merck), and eluted with chloroform/methanol. Fractions (20 ml) were collected. The anthrone-sulfuric acid test [13] was carried out with dried material from 200 V,1 of each fraction.
Further purification was carried out by a reverse-phase high-performance liquid chromatography (Fig. 2). Inducing activity was found in four peaks (I, II, III, and IV) of the ultraviolet absorption (220 nm). These fractions were collected by the same chromatography repeatedly. The purification procedures are summarized in Table I. The most part of the initial activity is recovered in HPLC-I, II, III and IV (Fig. 3). The specific activities are nearly equal among the four,
E
tO
d
.< 0
10
20 Retention
30
40
time ( min )
Fig. 2. Profile of the high-performance liquid chromatography. Fractions 75-110 from the silica gel column re-chromatography were dissolved in 2 ml ethanol, and a portion (40 rtl) was loaded on a reverse-phase column (size: 250 x 7.2 mm; packing: TSK-GEL LS-410, Toyosodakogyo Co., Ltd., Tokyo). The elution was performed with methanol/water (92 : 8, v/v), and 1 min fractions (1.92 ml) were collected.
615 TABLE I SHIFT OF F R U I T I N G - I N D U C I N G ACTIVITY IN THE COURSE OF PURIFICATION As described in the text, the allowance of each unit is from ×0.5 to ×2, Fraction
Crude extract Silica gel column Lober column Si 60 HPLC I II III IV
Dry weight (rag)
Total activity (units)
Specific activity (units/mg)
6 100 296 92 2.1 64.1 3.3 12.2
800000 1000 000 1000 000 20000 700 000 30000 150000
130 3 500 11000 10000 11000 9000 12000
.
.
.
. . . . . . . . . . . . .
_ . . _
20
Fig. 4. Infrared spectrum of H P L C - I I substance. The infrared spectrum was taken on an infrared spectrophotometer (A-202
type, Japan Spectroscopic Co., Ltd., Tokyo), using 150 mg KBr pellets containing 1 mg HPLC-II substance.
Identification of the active fractions Each of four HPLC fractions (I-IV) gave a single spot on a thin-layer chromatogram when treated with aqueous sulfuric acid reagent and the spot fraction retained the inducing activity. These .
!'° Wavec,umber (cm-~)
and each stands for about 100-fold activity of that of the crude extract.
.
6O
~!!!?~
Fig. 3. Fruiting body-inducing activity of the isolated cerebroside (HPLC-II substance). From the top to the fight 100, 10, 1, and 0 p,g/disc. The test is S. c o m m u n e IFO 6502.
spots were negative to molybdenum blue reagent [14]. The R E values (0.62 on a silica gel plate with solvent system a, 0.58 with b, and 0.16 on borateimpregnated plate with c) were identical with each other and coincided with those of the glucocerebroside from F. amygdali [12]. The ultraviolet spectra of the HPLC fractions showed an equal end absorption. Their infrared spectra resembled each other and with those of the cerebrosides from bovine brain and F. amygdali. Fig. 4 shows the spectrum of HPLC-II substance, having peaks at diagnostic value of 3300-3400 (H-bonded OH), 1640 and 1530 (aliphatic secondary amide), 1000-1100 (alcoholic CO), and 890 m / e (13-glycopyranoside linkage). From above observations, the active substances in HPLC-I, II, and III, and IV were considered as cerebrosides.
The active substances in HPLC-H HPLC-II, the largest fraction from the HPLC, was analyzed further. When dried, it gave a white powder which melted at 196°C. The powder yielded prospective methyl glycoside, fatty acid methyl ester, and long-chain base on methanolysis [7]. Prospective ceramide was obtained by periodate opening of the glycoside ring, reduction with NaBHa and hydrolysis under a mild acid condition [9,15]. (a) Glycoside fraction. Trimethylsilyl ether derivatives of the methyl glycoside fraction yielded a single peak when tested on GLC. The retention
616 time of the peak was identical with that of a authentic preparation of trimethylsilyl ether derivative of a-methyl-D-glucoside, and its mass spectrum coincided with that of methyl 2,3,4,6-tetrakis-O-trimethylsilyl-c~-D-glucopyranoside [ 16]. (b) Fatty acid methyl ester fraction. Trimethylsilyl ether derivatives of the fatty acid methyl ester fraction yielded nearly a single peak (97% in the peaks) when tested on GLC. The mass spectrum of this peak exhibited ions of 358 (M÷), 343 (MCH3), 315 (M-CH3-CO), and 299 m / e (MCOOCH3). These patterns of ionic strength corresponded to those of 2-O-trimethylsilyl-palmitate
100 (M-a -IS)
~1
,4 ~1 tb.1
-> 25
o,~ ....... 100
(M-SOl2} (M-~-73)) 6(~ S31 (M-SO) (M.90.103) (M.103) 691 (M-,~(M 1
I
, n, ,1 .......... 200
300
I
~-0~,,~ b
,:o,,, ~, .... 400
500
600
~L .... 700
k~,, 800
role
Fig. 6. Mass spectrum of the ceramide (as trimethylsilylether derivative). The ceramide was prepared from HPLC-II substance as described in the text. The fragments are illustrated according to Hammarstr~m et al. [20].
[17]. (c) Long-chain base fraction. A gas-liquid chromatogram of the trimethylsilyl ether derivatives of the long-chain base fraction is shown in Fig. 5. The largest peak (No. 3) stands for 34% of the whole peak area and the mass spectrum of this peak exhibited ions of 455 (M+), 440 (M-CH3), 352 (M-CH2OSiMe3), 350 (M-HOSiMe3-CH3), and 323 m / e (M-NH2C2H3OSiMe3) specifying 1,3-bis-O- trimethylsilyl-nonadecasphingadienine [18]. The mass spectra of the other peaks did not exhibit the set of ions (M ÷ , M-CH 3, MCH2OSiMe3, M-NH2C2H3OSiMe3) specifying trimethylsilyl ether derivatives of long-chain bases [19]. Peaks 1, 2, 5, 6, and 7 were thought to be
artifacts since the long-chain base fraction obtained by Ba(OH)2 hydrolysis gave rise to only peaks 3 and 4 on GLC. (d) Ceramide fraction. The GLC of the trimethylsilyl ether derivatives of the ceramide fraction gave nearly a single peak (97% in the peaks) and the mass spectrum of the peaks showed ions corresponding to those of 1,3,2'-tris-O-trimethylsilyl-N2'-hydroxypalmitoyl-nonadecasphingadienine (Fig. 6). Above results indicate that the main constituent in HPLC-II is N-2'-hydroxypalmitoyl-l-O-glucosyl-nonadecasphingadienine.
Activity of other cerebrosides
'!
Samples (up to 0.5 mg) of some other known cerebrosides were applied onto paper discs and served for assay of fruiting-inducing activity. The two cerebrosides from bovine brain and porcine brain, and three synthetic cerebrosides (Nstearoyl-DL-dihydrogalactocerebroside, N-stearoylDL-dihydroglucocerebroside, and N-palmitoyl-DLdihydroglucocerebroside) exhibited no activity on S. commune mycelia, but the cerebrosides from F. amygdali and pea seeds showed activity equivalent with those of S. commune.
ill 0
g
5
Discussion
o
;o
2'o
I
Retention time (min)
Fig. 5. Gas-liquid chromatogram of the long-chain base fraction (as trimethylsilylether derivatives).The base fraction was prepared from aqueous HCI methanolysis of HPLC-II substance.
We have presented evidences that the fruitinginducing substances in mycelia of S. commune are a family of cerebrosides and that at least one of them is identical with N-2'-hydroxypalmitoyl-1O-glucosyl-nonadecasphingadienine. The possibil-
617
ity that the activity resides in a contaminating substance will be ruled out from the following observations: (1) the total activity in the HPLC fractions was nearly equal to the activity in the crude extract, (2) the specific activities of the four HPLC fractions were intrinsically identical, (3) the activity was recovered in a single spot on a thinlayer plate, (4) the cerebrosides from pea seeds and from F. amygdali (crystalline homogeneous cerebroside [12]) have been proved as stimulating as the cerebrosides from S. commune. Meanwhile, the case dose not imply that all members of the cerebroside family hold the similar activity as described later. Chemically, cerebrosides are members of glycosphingolipids and are known to be the constituents of cell membranes [21]. They have been described as being able to stimulate activity of the rat brain cholesterol ester hydrolase in myelin sheath [22], activity of the membrane-bound acid protease of Aspergillus oryzae [23] and aggregation of human thrombocytes and erythrocytes [24], although these stimulating activities are generally weaker than those of other lipids such as phospholipids and gangliosides. The stimulating effect on the fruiting body formation of S. commune reported in this paper is the most dramatic in this sense. The three cerebrosides, that is, the cerebrosides from S. commune (major constituent; N-2'-hydroxypalmitoyl- 1- O-glucosyl-nonadecasphingadienine), the cerebroside from F. amygdali (N-2'hydroxy- 3'-trans-octadecenoyl- 1- O-fl-D-glucosyl9-methyl-cis-4,X-8-sphingadienine [12]), and the cerebrosides from pea seeds (major constituent [8]; N-2'-hydroxytricosanoyl- 1-O-glucopyranosyl-dihydrosphingosine), were able to induce the fruiting body formation of S. commune, but the cerebrosides from bovine brain (major constituents [9]; N-cerebronoyl-l-O-galactosyl-sphingosine, and Nlignoceroyl-l-O-galactosyl-sphingosine), the cerebrosides from porcine brain, and some synthetic cerebrosides (N-stearoyl-l-O-fl-D-galactosyl-DL-dihydrosphingosine, N-stearoyl- l-O-fl-Dglucosyl-DL-dihydrosphingosine, and N-palmitoyl1-O-fl-D-glucosyl-DL-dihydrosphingosine) were unable to induce the fruiting body formation of the same strain. Therefore, it must be stated that only a limited number of cerebrosides hold the inducing activity.
The long-chain base of the cerebroside in HPLC-II (nonadecasphingadienine) has not yet been characterized with regard to its fine structure, but its molecular weight is identical with that of the cerebroside from F. amygdali. Judging from the data in the present study, the active cerebrosides seem to involve, at least, a glucose and a 2-hydroxy fatty acid moiety in their structures. As described above, the cerebrosides have been isolated from mycelia of S. commune IFO 6502 and showed effectiveness on the fruiting body formation of the same strain. Therefore, it may be surmised that the substances play an important role in morphogenesis of the fungus. But it does not always imply that the cerebrosides are the sole substance(s) which stimulate the fruiting body formation. An experiment carried out expounding on this view will be reported elsewhere. The effect of the isolated and the authentic cerebrosides on the fruiting body formation of Basidiomycetes other than S. commune has also been studied. Some of the tested cerebrosides have exhibited a similar effect on certain strains. But, because different criteria must be used for judgement, these studies are not refered to in this paper.
Acknowledgements The authors wish to express their hearty thanks to Drs. M. Nagasawa and N. Saito, Noda Institute for Scientific Research, for the valuable suggestions and discussions. The authors are also indebted to Drs. M. Kikuchi and T. Aishima, Noda Institute for Scientific Research, Dr. H. Yamane, University of Tokyo, and Dr. M. Sasaki and Mr. N. Nunomura, Kikkoman Central Research Laboratories, in the chemical and physical analyses. The authors' thanks are also extended to Miss T. Kurokawa and Miss M. Yamazaki who served as technical assistants.
References 1 Leonard, T.J. and Dick, S. (1968) Proc. Natl. Acad. Sci. U.S.A. 59, 745-751 2 Rusmin, S. and Leonard, T.J. (1978) Plant Physiol. 61, 538-543 3 Uno, I. and Ishikawa, T. (1973) J. Bacteriol. 113, 1240-1248 4 Uno, I. and Ishikawa, T. (1973)J. Bacteriol. 113, 1249-1255 5 Schwalb, M.N. (1974) Arch. Microbiol. 96, 17-20
618 6 Kean, E.L. (1966) J. Lipid Res. 7, 449-452 7 Gaver, R.C. and Sweeley, C.C. (1965) J. Am. Oil Chem. Soc. 42, 294-298 8 Miyazawa, T., Ito, S. and Fujino, Y. (1974) Agric. Biol. Chem. 38, 1387-1391 9 Morrison, W.R. and Hay, J.D. (1970) Biochim. Biophys. Acta 202, 460-467 10 HammarstriSm, S. (1970) Eur. J. Biochim. 15, 581-591 11 Carter, H.E. and Gaver, R.C. (1967) J. Lipid Res. 8, 391-395 12 Ballio, A., Casinovi, C.G., Framondino, M., Marino, G., Nota, G. and Santurbano, B. (1979) Biochim. Biophys. Acta 573, 51-60 13 Horikoshi, K. (1958) Kagakunoryoiki Zokan 34, 36-39 14 Dittmer, J.C. and Lester, R.L. (1964) J. Lipid Res. 5, 126-127 15 Carter, H.E., Rothfus, J.A. and Gigg, R. (1961) J. Lipid Res. 2, 228-234
16 DeJongh, D.C., Radford, T., Hribar, J.D., Hanessian, S., Bieber, M., Dawson, G. and Sweeley, C.C. (1969) J. Am. Chem. Soc. 91, 1728-1740 17 Capella, P., Galli, C. and Fumagalli, R. (1968) Lipids 3, 431-438 18 Fujino, Y. and Ohnishi, M. (1977) Biochim. Biophys. Acta 486, 161-171 19 Karlsson, K. (1965) Acta Chem. Scand. 19, 2425-2427 20 Hammarstr6m, S., Samuelsson, B. and Samuelsson, K. (1970) J. Lipid Res. 11, 150-157 21 O'Brien, J.S., Fillerup, D.L. and Mead, J.F. (1964) J. Lipid. Res. 5, 109-116 22 Igarashi, M. and Suzuki, K. (1976) J. Neurochem. 27, 859-866 23 Tsujita, Y. and Endo, A. (1978) Eur. J. Biochem. 84, 347-353 24 Mkheyan, E.E., Akopov, S.E. and Sotskii, O.P. (1981) Zh, Eksp. Klin. Med. 21, 20-24 (Chem. Abs. (1981) 95, 94645a)
612
Elsevier Biomedical Press BBA21286
FRUITING-INDUCING ACTIVITY OF CEREBROSIDES OBSERVED WITH S C H I Z O P H Y L L U M COMMUNE GENSHIRO
K A W A I a and Y O N O S U K E
IKEDA b
UNoda Institute for Scientific Research, 399 Noda, Noda-shi, Chiba-ken and b Nodai Research Institute, the Tokyo University of Agriculture, 1-1 Sakuragaoka, Setagaya-ku, Tokyo (Japan) (Received June 3rd, 1982)
Key words: Fruiting-inducing activity," Cerebroside; (S. commune)
Isolation and identification of substances having an activity to stimulate the fruiting body formation of Schizophyllum c o m m u n e were attempted. The active principles in its mycelia were divided into four fractions by sequential purification with silica gel column and reverse-phase HPLC column chromatography. By infrared spectra and thin-layer chromatography, the active substances in these four fractions were revealed as cerebrosides. About 0.1 ttg of the cerebroside fractions showed a discriminative stimulating activity on S. c o m m u n e when tested by the method these authors adopted. The active substance in the fraction II was N-2'-hydroxypalmitoyl-l-O-glucosyl-nonadecasphingadienine. The cerebrosides from pea seeds and Fusicoccure a m y g d a l i showed the similar activity on S. commune, but some commercial synthetic cerebrosides and cerebrosides from bovine and porcine brains exhibited no stimulating activity. Only definite cerebrosides with special structures seem to be able to induce the fruiting of S. commune.
Introduction
The phenomenon of fruiting body formation in Basidiomycetes is regarded as a model system for studies of differentiation and morphogenesis mechanisms of fungi. Among Basidiomycetes, Schizophyllum commune is most frequently used for these studies, because it grows well on simple solid media and completes its life cycle in as short as ten days. The organism is heterothallic; that is, a haploid homokaryotic mycelium derived from a single basidiospore is in principle self-sterile and a dikaryotic mycelium bred from two compatible homokarions is able to form fruiting bodies. Leonard and Dick [1] reported that some sorts of extracts prepared from several fungi were capable of inducing fruiting body formation when tested on homokaryotic mycelia of S. commune. Fruiting-inducing substance from Cladosporium cladosporioides was water-soluble, dialysable, and 0304-4165/82/0000-000/$02.75 © 1982 Elsevier Biomedical Press
stable to heating. Moreover, the substance was described to be resistant to acid and alkaline hydrolysis and several degradating enzymes. Rusmin and Leonard [2] purified another fruiting-inducing substance from cultivated mushroom (Agaricus bisporus) and described its chemical and physical properties without referring to its chemical structure. Independently, Uno and Ishikawa [3,4] reported that fruiting-inducing substances in homokaryotic mycelia of Coprinus macrorhizus were identical with cyclic AMP and some inhibitors of phosphodiesterase. In S. commune, however, Schwalb [5] wrote that he could not find any sign that cyclic nucleotides would act positively on the fruiting body formation. The present study was begun due to a casual observation of an unusual phenomenon that occurred between a strain of S. commune and a strain of Penicillium species. Abundant fruiting
613 bodies were observed on a contact line of two mycelial mats. An active principle able to induce fruiting of dikaryotyic and homokaryotic mycelia of S. commune was recovered in the acetone extract of the Penicillium mycelia. Since a similar principle was also recovered in the acetone extract of the S. commune itself, the principle chosen as the target of first-step investigation. As will be described in this paper, the principle has been identified as a certain sort of cerebroside by physical and chemical analyses. The principle in the acetone extract of the Penicillium mycelia will be reported elsewhere. Materials and Methods
Bioassay of fruiting-inducing activity. Basic assay methods were as follows. An appropriate amount of sample was dissolved in a suitable solvent. The solution (basically 20 ~1) was applied onto a paper disc (8 mm diameter, 0.7 mm thickness) and the solvent was removed under reduced pressure. A control disc was prepared in the same way by applying ethanol instead of the test solution. A dikaryotic strain of S. commune (IFO 6502) was spotted on the center of the malt-yeast agar medium [3] in a 9 cm Petri dish, and grown at 23-25°C. Three to four days after the inoculation, the test and control discs were placed on a margin of the plate. The plate was placed upside down and incubated for another two weeks under white fluorescent light (100-200 lux). Usually, more abundant fruiting bodies were observed around the test disc. All procedures were carried out aseptically. When the activity was to be indicated in term of units, the original solution was diluted serially by two-fold with ethanol and the least quantity which apparently gave a larger number of fruiting bodies than the control was defined as one unit. As the dilution was done by two-fold system, allowance of every unit is from x 0.5 to x 2.0. Thin-layer chromatography. Thin-layer chromatography was conducted either on precoated silica gel 60 plates (E. Merck, Darmstadt, F.R.G.) using (a) chloroform/methanol/water (65 : 25 : 4, v/v), (b) chloroform/methanol/85% formic acid (70: 18: 12, v/v), or on borate-impregnated silica gel plates prepared as described by Kean [6] using
(c) chloroform/methanol/water/15 M NH4OH (280 : 70 : 6 : 1, v/v). Substances were located with iodine vapor, 50% aqueous sulfuric acid reagent or anthrone-sulfuric acid reagent. Hydrolysis of the cerebroside. The cerebroside (5 mg) was refluxed with 4 ml of 1 N HC1 in methanol at 70°C for 18 h according to Gaver and Sweeley [7]. After cooling, resulting long-chain base, fatty acid methyl ester, and methyl glycoside were fractionated by the method of Miyazawa et al. [8]. Isolation of the long-chain base was also attempted by the Ba(OH)2 hydrolysis method of Morrison and Hay [9], but the yield was very low. The cerebroside was decomposed also to ceramides following the procedure of HammarstriSm [10], and the ceramide was purified by thin-layer chromatography (adsorbent: silica gel, G.E. Merck; solvent: chloroform/methanol = 97 : 3, v/v). Gas-liquid chromatography. Appropriate amount (0.5-2 mg) of the fatty acid methyl ester, the long-chain base, the methyl glycoside, and the ceramide were mixed with a sufficient silanes reagent to make up a 1% solution. The silanes reagent, prepared according to Carter and Gaver [11], consisted of 1.6 ml trimethylchlorosilane, 2.6 ml hexamethyldisilazane, and 2.0 ml dry pyridine. The trimethylsilyl ether derivatives were analysed with a Shimazu 4CMPF Gaschromatograph (Shimazu Seisakusho Co., Ltd., Kyoto). The column was a glass tube of 0.3 x 200 cm packed with 2.5% OV-17 on 60-80 mesh Uniport HPS (Gasukuro Kogyo Co., Ltd., Tokyo), and the flow rate of nitrogen was 40 ml/min. The column was kept at 170, 200, 220, and 330°C, respectively, for the trimethylsilyl ether derivatives of methyl glycosides, fatty acid methyl esters, long-chain bases, and ceramides. Gas-liquid chromatography-mass spectrometry. GC-MS analysis was performed on an Hitachi RMU-7M instrument (Hitachi Seisakusho Co., Ltd., Tokyo). The column was a stainless steel tube of 0.3 x 200 cm packed with 2.5% OV-17 on 60-80 mesh Uniport HPS. The column was kept at 160, 200, 210, and 300°C, respectively, for the trimethylsilyl ether derivatives of methyl glycosides, fatty acid methyl esters, long-chain bases, and ceramides. Mass spectra were obtained under the following conditions: ion source temperature, 210°C; electron energy, 70 eV; ion accelerator,
614
voltage 3.2 kV; and ionizing current 51 /~A. Authentic cerebrosides. The authentic cerebrosides from bovine and porcine brains were purchased from Funakoshi Pharmaceutical Co., Ltd., T o k y o . N-Stearoyl-DL-dihydrogalactocerebroside and N-stearoyl-DL-dihydroglucocerebroside were purchased from Miles Laboratories, Inc., Elkhart, U.S.A. N-Palmitoyl-DL-dihydroglucocerebroside was purchased from Sigma Chemical Co., U.S.A. The cerebrosides from pea seeds were prepared according to the method of Miyazawa et al. [8]. Cerebroside from Fusicoccum amygdali was the generous gift of Professor A. Ballio [ 12]. Results
Purification of active substances S. commune IFO 6502 was cultivated aerobically at 25°C for 5 days in a 30-1 jar fermenter containing a 20-1 malt-yeast medium [3]. The mycelia (dry wt., 85.2 g) were harvested by filtration, washed several times with distilled water, suspended in 2 1 acetone, and stood at room temperature. After 3 days, the acetone suspension was filtered and the residue was re-extracted three times. The solvent was removed from the pooled extracts by vacuum evaporation and the resulting aqueous suspension was treated three times with ethyl acetate for extraction. The extract was dried over anhydrous sodium sulfate and then concentrated to dryness in vacuo, yielding 6.1 g of oily crude extract. The culture filtrate and the residual aqueous solution after the extraction with ethyl acetate showed slight or no inducing activity, but the ethyl acetate extract showed considerably high activity. The crude extract was subjected to silica gel column chromatography and eluted sequentially with chloroform, chloroform/acetone (1:2, v/v), acetone, and methanol. A greater part of the activity was recovered in the chloroform/acetone eluate. This fraction was evaporated to dryness. This active fraction was then loaded on a Lober column size A LiCroprep Si 60 (E. Merck) and eluted with chloroform/methanol (Fig. 1). The strong activity was found in fractions 75-110. These fractions were pooled and evaporated to dryness.
CHCLj/MeOH (vlv)l
I : 0 124 : I
9:1
I
I
4:1
I
3:2
I
2:3
I
03
02
i °. O(
0
100 Frocbon number
200
300
Fig. 1. Profile of the silica gel column re-chromatography. The chloroform/acetone fraction from the silica gel column chromatography was dissolved in 50 ml chloroform, loaded on Lober column size A LiCroprep Si 60 (E. Merck), and eluted with chloroform/methanol. Fractions (20 ml) were collected. The anthrone-sulfuric acid test [13] was carried out with dried material from 200 V,1 of each fraction.
Further purification was carried out by a reverse-phase high-performance liquid chromatography (Fig. 2). Inducing activity was found in four peaks (I, II, III, and IV) of the ultraviolet absorption (220 nm). These fractions were collected by the same chromatography repeatedly. The purification procedures are summarized in Table I. The most part of the initial activity is recovered in HPLC-I, II, III and IV (Fig. 3). The specific activities are nearly equal among the four,
E
tO
d
.< 0
10
20 Retention
30
40
time ( min )
Fig. 2. Profile of the high-performance liquid chromatography. Fractions 75-110 from the silica gel column re-chromatography were dissolved in 2 ml ethanol, and a portion (40 rtl) was loaded on a reverse-phase column (size: 250 x 7.2 mm; packing: TSK-GEL LS-410, Toyosodakogyo Co., Ltd., Tokyo). The elution was performed with methanol/water (92 : 8, v/v), and 1 min fractions (1.92 ml) were collected.
615 TABLE I SHIFT OF F R U I T I N G - I N D U C I N G ACTIVITY IN THE COURSE OF PURIFICATION As described in the text, the allowance of each unit is from ×0.5 to ×2, Fraction
Crude extract Silica gel column Lober column Si 60 HPLC I II III IV
Dry weight (rag)
Total activity (units)
Specific activity (units/mg)
6 100 296 92 2.1 64.1 3.3 12.2
800000 1000 000 1000 000 20000 700 000 30000 150000
130 3 500 11000 10000 11000 9000 12000
.
.
.
. . . . . . . . . . . . .
_ . . _
20
Fig. 4. Infrared spectrum of H P L C - I I substance. The infrared spectrum was taken on an infrared spectrophotometer (A-202
type, Japan Spectroscopic Co., Ltd., Tokyo), using 150 mg KBr pellets containing 1 mg HPLC-II substance.
Identification of the active fractions Each of four HPLC fractions (I-IV) gave a single spot on a thin-layer chromatogram when treated with aqueous sulfuric acid reagent and the spot fraction retained the inducing activity. These .
!'° Wavec,umber (cm-~)
and each stands for about 100-fold activity of that of the crude extract.
.
6O
~!!!?~
Fig. 3. Fruiting body-inducing activity of the isolated cerebroside (HPLC-II substance). From the top to the fight 100, 10, 1, and 0 p,g/disc. The test is S. c o m m u n e IFO 6502.
spots were negative to molybdenum blue reagent [14]. The R E values (0.62 on a silica gel plate with solvent system a, 0.58 with b, and 0.16 on borateimpregnated plate with c) were identical with each other and coincided with those of the glucocerebroside from F. amygdali [12]. The ultraviolet spectra of the HPLC fractions showed an equal end absorption. Their infrared spectra resembled each other and with those of the cerebrosides from bovine brain and F. amygdali. Fig. 4 shows the spectrum of HPLC-II substance, having peaks at diagnostic value of 3300-3400 (H-bonded OH), 1640 and 1530 (aliphatic secondary amide), 1000-1100 (alcoholic CO), and 890 m / e (13-glycopyranoside linkage). From above observations, the active substances in HPLC-I, II, and III, and IV were considered as cerebrosides.
The active substances in HPLC-H HPLC-II, the largest fraction from the HPLC, was analyzed further. When dried, it gave a white powder which melted at 196°C. The powder yielded prospective methyl glycoside, fatty acid methyl ester, and long-chain base on methanolysis [7]. Prospective ceramide was obtained by periodate opening of the glycoside ring, reduction with NaBHa and hydrolysis under a mild acid condition [9,15]. (a) Glycoside fraction. Trimethylsilyl ether derivatives of the methyl glycoside fraction yielded a single peak when tested on GLC. The retention
616 time of the peak was identical with that of a authentic preparation of trimethylsilyl ether derivative of a-methyl-D-glucoside, and its mass spectrum coincided with that of methyl 2,3,4,6-tetrakis-O-trimethylsilyl-c~-D-glucopyranoside [ 16]. (b) Fatty acid methyl ester fraction. Trimethylsilyl ether derivatives of the fatty acid methyl ester fraction yielded nearly a single peak (97% in the peaks) when tested on GLC. The mass spectrum of this peak exhibited ions of 358 (M÷), 343 (MCH3), 315 (M-CH3-CO), and 299 m / e (MCOOCH3). These patterns of ionic strength corresponded to those of 2-O-trimethylsilyl-palmitate
100 (M-a -IS)
~1
,4 ~1 tb.1
-> 25
o,~ ....... 100
(M-SOl2} (M-~-73)) 6(~ S31 (M-SO) (M.90.103) (M.103) 691 (M-,~(M 1
I
, n, ,1 .......... 200
300
I
~-0~,,~ b
,:o,,, ~, .... 400
500
600
~L .... 700
k~,, 800
role
Fig. 6. Mass spectrum of the ceramide (as trimethylsilylether derivative). The ceramide was prepared from HPLC-II substance as described in the text. The fragments are illustrated according to Hammarstr~m et al. [20].
[17]. (c) Long-chain base fraction. A gas-liquid chromatogram of the trimethylsilyl ether derivatives of the long-chain base fraction is shown in Fig. 5. The largest peak (No. 3) stands for 34% of the whole peak area and the mass spectrum of this peak exhibited ions of 455 (M+), 440 (M-CH3), 352 (M-CH2OSiMe3), 350 (M-HOSiMe3-CH3), and 323 m / e (M-NH2C2H3OSiMe3) specifying 1,3-bis-O- trimethylsilyl-nonadecasphingadienine [18]. The mass spectra of the other peaks did not exhibit the set of ions (M ÷ , M-CH 3, MCH2OSiMe3, M-NH2C2H3OSiMe3) specifying trimethylsilyl ether derivatives of long-chain bases [19]. Peaks 1, 2, 5, 6, and 7 were thought to be
artifacts since the long-chain base fraction obtained by Ba(OH)2 hydrolysis gave rise to only peaks 3 and 4 on GLC. (d) Ceramide fraction. The GLC of the trimethylsilyl ether derivatives of the ceramide fraction gave nearly a single peak (97% in the peaks) and the mass spectrum of the peaks showed ions corresponding to those of 1,3,2'-tris-O-trimethylsilyl-N2'-hydroxypalmitoyl-nonadecasphingadienine (Fig. 6). Above results indicate that the main constituent in HPLC-II is N-2'-hydroxypalmitoyl-l-O-glucosyl-nonadecasphingadienine.
Activity of other cerebrosides
'!
Samples (up to 0.5 mg) of some other known cerebrosides were applied onto paper discs and served for assay of fruiting-inducing activity. The two cerebrosides from bovine brain and porcine brain, and three synthetic cerebrosides (Nstearoyl-DL-dihydrogalactocerebroside, N-stearoylDL-dihydroglucocerebroside, and N-palmitoyl-DLdihydroglucocerebroside) exhibited no activity on S. commune mycelia, but the cerebrosides from F. amygdali and pea seeds showed activity equivalent with those of S. commune.
ill 0
g
5
Discussion
o
;o
2'o
I
Retention time (min)
Fig. 5. Gas-liquid chromatogram of the long-chain base fraction (as trimethylsilylether derivatives).The base fraction was prepared from aqueous HCI methanolysis of HPLC-II substance.
We have presented evidences that the fruitinginducing substances in mycelia of S. commune are a family of cerebrosides and that at least one of them is identical with N-2'-hydroxypalmitoyl-1O-glucosyl-nonadecasphingadienine. The possibil-
617
ity that the activity resides in a contaminating substance will be ruled out from the following observations: (1) the total activity in the HPLC fractions was nearly equal to the activity in the crude extract, (2) the specific activities of the four HPLC fractions were intrinsically identical, (3) the activity was recovered in a single spot on a thinlayer plate, (4) the cerebrosides from pea seeds and from F. amygdali (crystalline homogeneous cerebroside [12]) have been proved as stimulating as the cerebrosides from S. commune. Meanwhile, the case dose not imply that all members of the cerebroside family hold the similar activity as described later. Chemically, cerebrosides are members of glycosphingolipids and are known to be the constituents of cell membranes [21]. They have been described as being able to stimulate activity of the rat brain cholesterol ester hydrolase in myelin sheath [22], activity of the membrane-bound acid protease of Aspergillus oryzae [23] and aggregation of human thrombocytes and erythrocytes [24], although these stimulating activities are generally weaker than those of other lipids such as phospholipids and gangliosides. The stimulating effect on the fruiting body formation of S. commune reported in this paper is the most dramatic in this sense. The three cerebrosides, that is, the cerebrosides from S. commune (major constituent; N-2'-hydroxypalmitoyl- 1- O-glucosyl-nonadecasphingadienine), the cerebroside from F. amygdali (N-2'hydroxy- 3'-trans-octadecenoyl- 1- O-fl-D-glucosyl9-methyl-cis-4,X-8-sphingadienine [12]), and the cerebrosides from pea seeds (major constituent [8]; N-2'-hydroxytricosanoyl- 1-O-glucopyranosyl-dihydrosphingosine), were able to induce the fruiting body formation of S. commune, but the cerebrosides from bovine brain (major constituents [9]; N-cerebronoyl-l-O-galactosyl-sphingosine, and Nlignoceroyl-l-O-galactosyl-sphingosine), the cerebrosides from porcine brain, and some synthetic cerebrosides (N-stearoyl-l-O-fl-D-galactosyl-DL-dihydrosphingosine, N-stearoyl- l-O-fl-Dglucosyl-DL-dihydrosphingosine, and N-palmitoyl1-O-fl-D-glucosyl-DL-dihydrosphingosine) were unable to induce the fruiting body formation of the same strain. Therefore, it must be stated that only a limited number of cerebrosides hold the inducing activity.
The long-chain base of the cerebroside in HPLC-II (nonadecasphingadienine) has not yet been characterized with regard to its fine structure, but its molecular weight is identical with that of the cerebroside from F. amygdali. Judging from the data in the present study, the active cerebrosides seem to involve, at least, a glucose and a 2-hydroxy fatty acid moiety in their structures. As described above, the cerebrosides have been isolated from mycelia of S. commune IFO 6502 and showed effectiveness on the fruiting body formation of the same strain. Therefore, it may be surmised that the substances play an important role in morphogenesis of the fungus. But it does not always imply that the cerebrosides are the sole substance(s) which stimulate the fruiting body formation. An experiment carried out expounding on this view will be reported elsewhere. The effect of the isolated and the authentic cerebrosides on the fruiting body formation of Basidiomycetes other than S. commune has also been studied. Some of the tested cerebrosides have exhibited a similar effect on certain strains. But, because different criteria must be used for judgement, these studies are not refered to in this paper.
Acknowledgements The authors wish to express their hearty thanks to Drs. M. Nagasawa and N. Saito, Noda Institute for Scientific Research, for the valuable suggestions and discussions. The authors are also indebted to Drs. M. Kikuchi and T. Aishima, Noda Institute for Scientific Research, Dr. H. Yamane, University of Tokyo, and Dr. M. Sasaki and Mr. N. Nunomura, Kikkoman Central Research Laboratories, in the chemical and physical analyses. The authors' thanks are also extended to Miss T. Kurokawa and Miss M. Yamazaki who served as technical assistants.
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