Jun 16, 1987 - From the $Department of Biology, Faculty of Science, Ocharwmizu University, ... Bunkyo-ku, Tokyo 113, the 11 Department of Biophysics and.
Vol. 262, No. 34, Issue of December 5, pp. 16719-16723,1987 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
0 1987 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Poriferasterol Monoglucoside Associated with Differentiation of Physarum polycephalum* (Received for publication, June 16, 1987)
Kimiko Murakami-MurofushiSg, Kazuko Nakamura$, Jiro OhtaS, MinoruSuzukill, Akemi Suzukill, Hiromu MurofushiII, and Takao Yokota** From the $Department of Biology, Faculty of Science, Ocharwmizu University,Bunkyo-ku, Tokyo 112, the VDepartment of Metabolism, The Tokyo Metropolitan Institute of Medical Science,Bunkyo-ku, Tokyo 113, the 11 Department of Biophysics and Biochemistry, Faculty of Science, and the **Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan
A glycolipid which was expressed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum, has been examined. In the amoeboid stage, cellsdid not contain this glycolipid, but after conjugation of the haploid cells, thissubstance appeared and increased in its amount. From structural studies of thepurified glycolipid, it has been identified as poriferasterol monoglucoside.
In the life cycle of a true slime mold, Physarum polycephnlum, ithas haploid and diploid stages (1-3). Haploid myxoamoebae are germinated from spores under moist conditions, and theyconjugate in pairs to form diploid plasmodia. At the amoeboid stage, cells recognize different mating types and fuse to form zygotes. Zygotes grow and fuse each other and differentiate into young plasmodia. These steps are genetically regulated; zygote formation occurs only between amoebae carrying different alleles of a multiallelic locus (48 ) , and differentiation of the zygotes to plasmodia is also genetically determined (4,9, 10). Tiny young plasmodia grow up as multinuclear plasmodia, and they fuse each other without anyartificial treatments. During the course of differentiation from haploid amoebae to diploid plasmodia, the change of membrane characteristics might occur. We examined lipid composition of the membranes of the cells at both stages and found an expression of a novel glycolipid which wascorrelated with a process of differentiation. This glycolipid was purified and characterized and was identified as poriferasterol monoglucoside. EXPERIMENTAL PROCEDURES AND RESULTS’
Expression of a Glycolipid during the Differentiation of P. polycephulum from Haploid Amoebae to Diploid PlasmodiaTLC analysis of crude lipid fractions (see “Experimental
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed. Portions of this paper (including “Experimental Procedures,” part of “Results,” and Figs. 3-9) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of astandard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 87M-2032, cite the authors, and include a check or money order for $5.20 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
Procedures”) showed the patternof nonpolar glycoconjugates of haploid cells differed from that of diploid cells. Fig.1shows the TLC patternsof the nonpolar glycoconjugates from haploid amoebae, strain J, or diploid plasmodia of P. polycephalum. There was no difference between two different mating types of myxoamoebae, strains J and F. Four major carbohydrate-containing bands from haploid cells were designated as A to D according to their mobility. Five major sugar-containing bands were obtained in the extract from diploid cells: three of them were corresponding to A, C, and D from haploid cells, and other two bands designated as E and F were characteristics for diploid stage. Band D showed blue color and others showed purple colors. Among these nonpolar glycoconjugates, substance F was expressed after conjugation of haploid amoebae and increased in its amountduring the differentiation into diploid plasmodia (Fig. 2). A content of this substance was maintained constantly under different culture conditions either on rolled oats or in semidefined culture media. Substance E appeared after the differentiated plasmodia were transferred into culture media. When the crude membrane fraction was extracted, bands A and B were not detected, but other glycoconjugates were also extracted and almost the same results were obtained. Purification andstructural analysis of glycolipid F are presented in the MiniprintSupplement. From the evidence described in the Miniprint Supplement, the structureof substance F was designated as that shown in Fig. 10 (poriferasterol monoglucoside). DISCUSSION
In this work it is shown that a novel glycolipid was expressed during a differentiation of haploid amoebae of P. polycephalum into diploid plasmodia. The glycolipid was purified and identified as poriferasterol monoglucoside. Poriferasterol was a major sterol component in haploid cells of P. polycephalum (34) as well as in diploid cells (25). But in a haploid stage, the poriferasterol monoglucoside was completely absent, and during the differentiation into diploid plasmodia it appeared and increased in its amount. Other steryl-glucosides and their derivatives could not be detected in both stages. The steryl-monoglucosides and their 6”O-acyl derivatives are known as common constituents of higher plants (27, 28), andtheir biological functions have been suggested to be metabolically active components of plant membrane structure (291, intercellular transporters of sterols (30), or glucose carriers through cell membranes (31, 32). Amoeboid cells are uni-nuclear and behave like protozoan soil-amoebae on solid substrata or amoebo-flagellate in non-
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Poriferasterol Monoglucoside: Differentiation of Physarum
FIG. 1. Thin layer chromatogram of nonpolar glycoconjugates.Plate a, developed in solvent I;plate b, developed in solvent 11. Lane 1, mixture of glycolipids from rat brain (SCMH, galactosylsulfatide; CDH, lactosylceramide; Gm, GM,, gangliosides having different length of sugar chains); lane 2, crude extract from myxoamoebae; lane 3, crude extract from plasmodia. Bands were visualized byspraying with orcinolH2SO4.
F -E
J
CDH, SCMH.
-D -C
GM3-
GMI -
-F -E -D
-B CDH \ SCMH
GDI~
-A 1
2
3
-C JB -A
GM3.
1 2
3
A C D H L X M H-
FIG.2. A, thin layer chromatogram of crude lipids from Physarum at various developing stages. Lane I, mixture of glycolipids from rat brain (see Fig. 1); lane 2,O days after matingof the haploid cells; lane 3, 2 days after mating; lane 4, 4 days after mating; lane 5, 6 days after mating. Plate a, developed in solvent I; plate b, developed in solvent 11. Bands were visualizedby spraying with orcinolH2SO4. B , content of substance F during differentiation of Physarum from haploid amoebae into diploid plasmodia. The content of substance F was expressed as the amount of lipid-bound glucose. o " 0 , percent of conjugated cells; 0 - 0 , content of glycolipid F.
-F
-D
F
PhysarumDifferentiation of Poriferasterol Monoglucoside:
'
OH
FIG. 10. Structure of the glycolipid F.
nutrient liquid. Diploid cells grow as multinuclear plasmodia in which an intranuclear mitosis occurs, and they fuse each other very easily without any artificial treatments. The cell membrane of plasmodia seems to have higher fluidity than that of haploid amoebae, and when the cell membrane has been injured the repair of it is completed immediately. Plasmodia are capable of growth in liquid or on agar media, but amoebae, except for rare mutant strains (33), can be cultured only on bacterial lawns. Amoebae may not be able to utilize glucose and other small molecules, but plasmodia maybe capable of utilizing them as nutrients. Poriferasterol monoglucoside may have someactive functionsinmembranes showing such interesting properties. It seems that the role of poriferasterol monoglucoside inthe plasmodial membrane must be examined with purified membrane components and compared to propertiesof the amoeboid membrane. It is also important to examine regulation the of the enzyme, UDP-g1ucose:poriferasterol glucosyltransferase,which synthesizes substance F, during the process of differentiation of P. polycephalum. REFERENCES 1. Alexopoulos, C. J. (1982) in Cell Biology of Physarum and Didymium (Aldrich, H. C., and Daniel, J. W., eds) Vol. I, pp. 3-23, Academic Press, New York 2. Wick, R. J., and Sauer,H. W. (1982) in Cell Biology of Physarum and Didymium (Aldrich, H. C., and Daniel, J. W., eds) Vol. 11, pp. 3-20, Academic Press, New York 3. Raub, T. J., and Aldrich, H. C. (1982) in Cell Biology of Physarum and Didymium (Aldrich, H. C., and Daniel, J. W., eds) Vol. 11, pp. 21-75, Academic Press, New York 4. Dee, J. (1982) in Cell Biology of Physarum and Didymium (Ald-
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rich, H. C., and Daniel, J. W., eds) Vol. 11, pp. 211-251, Academic Press, New York 5. Dee, J. (1978) Genet. Res. 31,85-92 6. Kirouac-Brunet, J., Masson, S., and Pallotta, D. (1980) Can. J. Genet. Cytol. 23,9-16 7. Youngman, P. J., Pallotta, D. J., Hosler, B., Struhl, G., and Holt, C. E. (1979) Genetics 91, 683-693 8. Youngman, P. J., Anderson, R. W., and Holt, C. E. (1981) Genetics 97,513-530 9. Collins, 0. R. (1961) Am. J. Bot. 4 8 , 674-683 10. Dee, J. (1966) J. Protozool. 13, 610-616 11. Taniguchi, M., Yamazaki, K., and Ohta, J. (1978) Cell Struct. F ~ n c t 3, . 181-190 12. Murakami-Murofushi, K., Hiratsuka, A., and Ohta, J. (1984) Cell Struct. Funct. 9, 311-315 13. Daniel, J. W., and Rusch, H. P. (1961) J. Gen. Microbiol. 25,4759 14. Camp, W. G. (1936) Bull. Torrey Bot. Club 63, 205-210 15. Murakami-Murofushi, K., Minowa, Y., Yamada, R., and Ohta, J. (1986) Cell Struct. Funct. 11, 219-225 16. Barden, A., Lemieux G., and Pallotta,D. (1983) Biochim. Biophys. Acta 730,25-31 17. Nagai, Y., and Iwamori, M. (1980) Adu. Exp. Med. Biol. 125,1321 18. Svennerholm, L. (1957) Biochim. Biophys. Acta 24,604-611 19. Murakami-Murofushi, K., Nakamura, K., Ishizuka, I., and Ohta, J. (1985) Anal. Biochem. 149,480-483 20. Toennies, G . , and Kolb, J. J. (1951) Anal. Chem. 23, 823-826 21. Dittmer, J. C., and Lester, R. L. (1964) J. Lipid Res. 5, 126-127 22. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 1 9 3 , 265-275 23. Radin, N. S.,Lavin, F. B., and Brown, J. R. (1955) J. Biol. Chem. 2 17,789-796 24. Momose, T., Ueda, Y., Yamamoto, K., Masumura T.,and Ohta K. (1963) Anal. Chem. 3 5 , 1751-1753 25. Bullock, E., and Dawson, C. J. (1976) J . Lipid Res. 17, 565-571 26. Rubinstein, J., Goad, L. J., Clague, A. D. H., and Mulheirn, L. J. (1976) Phytochemistry 15, 195-200 27. Lepage, M. (1964) J. Lipid Res. 5,587-592 28. Bush, P. B., and Grunwald, C. (1972) Plant Physiol. 50, 69-72 29. Grunwald, C. (1971) Plant Physiol. 48,653-655 30. Evans, F.J. (1972) J . Pharm. Pharmacol. 24,645-650 31. Smith, P. F. (1969) Lipids 4,331-336 32. Wojciechowski, Z.A., Zimowski, J., and Zielenska, M. (1976) Phytochemistry 15, 1681-1683 33. McCullough, C. H. R., Dee, J., and Foxon, J. L. (1978) J. Gen. Microbiol. 106, 297-306 34. Murakami-Murofushi, K., Nakamura, K., Ohta, J., and Yokota, T. (1987) Cell Struct. Funct. 12, in press
Poriferasterol Monoglucoside: Differentiation of Physarum
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Gas llauld chromatoqraohy (GLCI- For the detectlo" of conponentsofthe qlycollpld, homoqeneous substance was methanolyzed ~n 5% IlClImethanol, and the resultlnq methyl g l u c o s ~ d eand sterol were trimthylsllylatedand analyzed by G L C on a column of 38 O V - 1 0 1 .
Gas llould ChromatoqraDhy-mass spectrometry 1GClMSI- GClMS was carrled Out on J E O L (Japan1 DX-303 110n1zat10n uoltaqe, 70 e V I . GC COnditlOnS for free Sterols were a 5 follows: Column. J 8 W megabore colmm 08-1; flow rate of He carrier gas. 20 mllrnln; column temperature, 1OO'C to 296-C 132"CIrn~nl. For steryi acetates, GCIMS was performed under the followlnq condltlons: Column, J 6 W capillary column 08-1; He pressure, 0.7 kqlcrn); columntemperature, 230'C IiSOCratlCI.
D e t e r m l n a t m n o f N M RSDectI-a- The 4 0 0 MHz NMR Spectra were measured on J M N G X 400 IJCOL, Jdpanl. For a n Intact qlycollpld probe temperature was 27'C and the COrnPOUnd was dissolved i n DMSO. The acetylated sterol moiety o f t h e g l v c o 1Lpld and standard Sterols were dissolved Ln ?DCl I and NMR spectrduere rneaiuked using TMS as a n lnternal standard. Acetylation of Sterols- The Sterol was dissolved i n acetic anhydride contalnl n g 4 mq13.2 m i p-toluensulfonlc acid. The mixture was allowed to stand at room temperature overnlqht, ethyl acetate was added and washed wlth aqueous Sodium bicarbonate. Then, the ethyl a c e t a t e soIut10n was dried o v e r anhydrous sodlum sulfate and evaporated toqive whlte Crystals. Enzymatic hydrolysis- 8-Glucosldase treatment was done a s follows. T h e g l y c o Ilpld preparation 1400 Yql was dissolved i n 430 u l of 50 mM sodium Cltrate
buffer, pH 5.0, and 0.6 unlt of " - q l u c o s ~ d a ~~n e 20 u l of t h e same buffer and 450 u q Of sodium taurocholate were added. The mixture was xncubated at 37'C for 6 hrs and*addltxonal 0.6 unlt of the enzyme ( I n 20 u l of the buffer1 was added and lncubatlon was conttnued for additional 1 8 hrs. The reaction was stopped by the addltlon Of 2.0 mi Of n-hexane and centrifuged. The lower phase was washed three tlrnes With water, concentrated, dissolved l n 0.2 m l o f n-hexane and analyzed by TLC developed i n chloroform and GLC. o - G l ~ c o s l d a ~ e treatment was also done essentially as descrlbed above, but potasslum phosphate buffer. pH 6.8. was used lnstead of sodium c ~ r r d t ebuffer.
I
0
lb
20 3b l i m o ( min. )
lo
Fiq.5. Gas liquid chromatoqram of 0-trinethylsilyl derivatives of methyl lvcosides from IAI standard su ars and 81 1 colloid F. Peaks correspond to ?he follovlnq sugars : fucoserql-3; g a l ~ c t o ~4-6; e ~ qlucose, 7, 8 ; mannitol, 9; N-acetyl galactosamine, 1 0 , 1 1 ; N-acetyl neuramlnlc acid, 12. GLC analysis was done on a column o f 3% OV-101 at 15O-25O0c.
Other methods- Protein deternlnatlonwas done by the method of Lowry el nl.1221, hexose measurement was performed accordin9 to Lad," el el.1231, and sterol d c termlnatlon wag carrled out by the procedure descrlbed by Momose P C al. 1241. Meltlnq polnts were measured accordlnq to Bullock and Dawson 1251.
RESULTS
L
F ,
'E
D-
Thln laver chromatoarm of purifled non-volar qlvcoconluqates. Plate was developed I" solvent I. Lane 1 , crude llplds from myxoamoebae; 2, crude llplds from plasmodia; 3 , purifled A; 4, purlfled E; 5, purlfled C; 6, pur'lfied 0 ; 7. purlfled E; 8, purifred F. Bands were vlsuallred by Spraylnq vlth o r c ~ n o l - H ~ S O ~ . F~q.3.
CBA1 2 3 4 5 6 7 8
The sterol moiety of the substance F was subiected to GCIMS, and a molecular 10" at mle 412 and fragmentation 10" at mle 394 1M-H:O) was detected together Wlth number Of Other ionsIFig.781. The mass pattern Of the sterol of substance F was q u t e similar to that of standard St1qmasterol IFlq.7A1. The sterol rnolety was acetylated and also subiected to GCIMS lFlq.8CI. Acetylated o m qave no molecular lo", but, instead, a n abundant >on at ole394,' aolecularron. whlch wrrespondlng to the loss of acetlc acld from the expected dominated the spectra. Thls mass pattern is also qulte simllar to that ObServed for stigmasterol acetate IFlg.BA1. and that of poriferasterol acetate IFlq.88).
7?2
rnl
Flq.4. Fast atom bombardment mass Spectra Of substance F. FABIMS was carrled out on JEOL DX-303 mas5 spectrometer. When the SubstanceF was methandyred and trirnethylsilyl derivatives of methyl glycoside and sterol were analyzed by GLC. Fiq.5A shows GX patterns of trirnethylszlyl ITMSI derivathves o f some known sugars: fucose, galactose, glucose, N-acetyl galactosanlne, N-acetyl neuramlnlc acrd. The patternof TMSsugar from substance F corresponded to the qluCOSeas Shown l n Flq.58. NO other suqars were detected in the glycolipid F. Flq.6A shows the GLC patterns o f T H S derivatives Of Standard sterols andFig.68 is the pattern of TMS derlvatlve of Sterol molety Of the qlycollpld F. From these GLC patterns. the sterol molety Of the glycolipid F was consldered to be a StiqmaSterOl. NO other components were detected by GLC analysls undersome different conditions with some different c01ums.
m/e
Poriferasterol Monoglucoside: PhysarumDifferentiation of A.
'2al". -
I
C. IPY
7---
/ i
mle
16723
From above results, the sterol molety could not be ldentrtied a s stlgrnasterol or porlferasterol, latter one 1s a C-24 eplmer O f former one. The 4 0 0 MHz N M R spectra Of the sterol molety of the Substance F showed rt n o t to be a stlgmasterO1 but a Porlferasterol, ludglng from the methyl group chemlcal shifts I F q . 9 1 whlch were reported by Rublnsteln ef a 1 . ( 2 6 1 before. The melti n g p o m t of It was 1 5 6 - C and thls v a l u e also conflrmed that the sterol was not a s t l g r n a s t e r o l (m.p., 1 7 0 ' C l but a porlferasteral 1m.p.. 1 5 6 - C J as described by Bullock and Dawson ( 2 5 1 .
