Manipulation of the Phospholipid Composition of Tissue Culture Cells*

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1 Proc. Nat. Acad. Sci. USA Vol. 71, No. 10, pp , October 1974 Manipulation of the Phospholipid Composition of Tissue Culture Cells* (polar head group/fatty acid/lm cells/membrane) MICHAEL GLASERt, KAREN A. FERGUSONT, AND P. ROY VAGELOS Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, Missouri Contributed by P. Roy Vagelos, August 7, 1974 ABSTRACT Methods have been devised to signifiantly alter the phospholiopid composition of LM cells grown. in serum-free tissue culture medium. The polar head groups of the phospholipids, as well as th' acyl groups of these lipids, can be changeil. When LM cells were grown in medium containing choline analogues, either N-met'hylethanolaminb or NN-dimethylethanolamine, or the unnatural analogues, 1-2-amino-1-butanol or 3-amino-1- propanol, up to 50% of the cellular phospholipids contained the analogue supplied. When linoleate was added to the cells as a bovine serum albumin complex, up to 40% of the fatty acids of the phosplholipids were linoleate. Under the conditions discuss&l, the polar head group composition, the fatty acid compositions or both together could be varied in the membrane phospholipids. The composition of the fatty acids in the membrane phospholipids can be altered by supplementing the growth medium of a variety of microorganisms with fatty acids (1, 2). This has provided a valuable method for studying the role of fatty acids in many problems of membrane structure and function. There are, however, many problems unique to mammalian cells that have been difficult to approach experimentally. In addition, relatively little is known about the role of the polar head group of the phospholipids in biological membranes. Mutants of Neurospora crassa deficient in phosphatidyl choline (PC) have been isolated which have defects in the methylation pathway that converts phosphatidyl ethanolamine (PE) to PC by the step-wise addition of methyl groups (3). When N-methylethanolamine (MEA) or N,N-dimethylethanolamine (DMEA) was added to the growth medium, it was converted into the corresponding phospholipid, phosphatidyl methylethanolamine (PMEA) or phosphatidyl dimethylethanolamine (PIIMEA) (4). These metabolic intermediates, PMEA and PDMEA, are present in very small amounts in normal cells, but in these mutants they could ac- Abbreviations: Phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), phosphatidyl methylethanolamine (PMEA), phosphatidyl dimethylethanolamine (PDMEA), phosphatidyl butanolamine (PBA), and phosphatidyl propanolamine (PPA) designate phospholipids with head groups consisting of ethanolamine (E), choline (C), N-methylethanolamine (MEA), N,Ndimethylethanolamine (DMEA), 1-2-amino-1-butanol (BA), and 3-amino-1-propanol (PA), respectively. * A preliminary report of this work has been published [Glaser, M., Ferguson, K. A. & Bayer, W. H. (1974) Fed. Proc. 33, 1296]. t Present address: Department of Biochemistry, University of Illinois, Urbana, Ill t Present address: Department of Chemistry, Eastern Illinois University, Charleston, Ill cumulate to a considerable extent. Recently, a concerted effort has been made to change the fatty acid composition of animal cells growing in lipid-free tissue culture medium (5-7). In order to get optimal incorporation of fatty acids without deleterious effects on cell growth, it was necessary to add desthiobiotin to block endogenous fatty acid synthesis and fatty acids in the form of Tween esters. In this paper, methods are described that make it possible to manipulate the polar head groups of the phospholipids of LM cells grown in serum-free tissue culture medium. The polar head groups can be changed alone or in conjunction with the fatty acids of the phospholipids. MATERIALS AND METHODS Cells. A strain of mouse fibroblasts, LM cells, was obtained from the American Type Culture Collection. Culture Medium and Growth Conditions. Monolayer cultures were grown at 370 in Falcon 75-cm2 tissue culture flasks in 10 ml of Higuchi's medium (8) containing 20 mm N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffers ph 7.4. This medium is a chemically defined, lipid-free medium that supports growth of LM cells indefinitely in the absence of serum. The generation time was approximately 20 hr. Cells were harvested by scraping the flasks with perforated cellophane. Linoleate supplementation was carried out by adding the fatty acid complexed to bovine serum albumin (Pentex, fatty acid-free) to the medium. The method of Spector and Hoak (9) was used to complex the fatty acid to bovine serum albumin. Cells that were to be supplemented with choline analogues were harvested and centrifuged in an International PR-1 centrifuge for 5 min at 1000 rpm. The cells were resuspended in medium without choline and distributed into tissue culture flasks. The analogues were added at a concentration of 40,.Lg/ml (all analogues were obtained from Eastman Kodak Co., except 1-2-amino-1-butanol, which was obtained from Aldrich Chemical Co.). Lipid Composition. For determination of the lipid composition, flasks were rinsed twice and then harvested in 5 ml of phosphate-buffered saline [Dulbecco and Vogt's phosphatebuffered saline without calcium and magnesium (10)1. After centrifugation and resuspension in 0.8 ml of phosphatebuffered saline, the lipid was extracted by the method of Bligh and Dyer (11) as described by Ames (12). For quantitation of the phospholipid composition, cells were prelabeled for several generations (about 5 days) prior to ex- 4072

Proc. Nat. Acad. Sci. USA 71 (1974) posure to choline analogues by adding approximately 50 /LCi of [32P]phosphate (New England Nuclear Corp., carrierfree) to each flask.

2 Proc. Nat. Acad. Sci. USA 71 (1974) posure to choline analogues by adding approximately 50 /LCi of [32P]phosphate (New England Nuclear Corp., carrierfree) to each flask. The cells were exposed to the same concentration of [32P]phosphate throughout the experiment. After extractions, the phospholipids were separated by two-dimensional thin-layer chromatography on Silica Gel G (250 Om thick, Analtech), made visible by autoradiography (12), scraped, and counted in 3a70 scintillation fluid (Research Products International Corp.). Solvent system I consisted of chloroform-methanol-water (65:25:4) in the first dimension and n-butanol-glacial acetic acid-water (6:2:2) in the second dimension. Solvent system II consisted of chloroformmethanol-28% aqueous ammonia (65:35:5) in the first dimension and chloroform-acetone-methanol-glacial acetic acid-water (5:2:1:1:0.5) in the second dimension (13). For fatty acid determinations, the phospholipids were first separated from the neutral lipids on a short silicic acid column, and the fatty acids were determined as their methyl esters by gas-liquid chromatography. Desmosterol content was determined by the method of Sokoloff and Rothblat (14). Synthesis of Phospholipid Standards. Dioleyl derivatives of PMEA, PDMEA, PBA, and PPA were synthesized from dioleyl-pc (generously provided by Drs. Frederick A. Dombrose and Craig M. Jackson) and the appropriate amino alcohol by the reaction catalyzed by phospholipase D (Sigma) (15), using the conditions of Yang et al. (16). The product was purified by preparative one-dimensional thin-layer. chromatography on Silica Gel G with chloroform-methanol-water (65:25:4). The plate was exposed to iodine vapor to make the phospholipids visible. The portion of the plate containing the product was scraped and the phospholipid was eluted with chloroform-methanol-water (50:100:5). Protein Determination. After the cells had been washed, harvested, and resuspended in phosphate-buffered saline, an aliquot was removed, diluted with 0.5 M KOH, and used for the protein determination. Alternatively, if the entire flask was to be used for a protein determination, the flasks were rinsed twice with 5 ml of phosphate-buffered saline and 1 ml of 0.5 M KOH was added. After 5 min the cells had dissolved. Samples were heated at 600 for 10 min to reduce the viscosity, and the protein was determined by a microbiuret method (17). Deacylation and Determination of the Polar Head Groups of Phospholipids. A mild alkaline hydrolysis to deacylate phospholipids was carried out by the procedure of Dittmer and Wells (18). The products (glycerolphosphate esters) were examined by thin-layer chromatography on cellulose plates (Polygram CEL 300, Brinkmann Instruments, Inc.) in isopropanol-28% aqueous ammonia-water (7:1: 2), which separates intact and deacylated phospholipids (19). The polar head group was identified by further hydrolyzing the deacylated phospholipids in 6 M HCl for 3 hr at This liberates the free amino alcohol, which can be identified by gas-liquid chromatography. The procedure of Lester and White (20), which involves drying down the amine hydrochloride and dissolving it in alkaline methanol, was used. The free amine was chromatographed on a column of 25% K. A. Ferguson, M. Glaser, W. H. Bayer, and P. R. Vagelos, manuscript submitted. Altering the Phospholipid Composition of LM Cells 4073 DAYS Growth of LM cells in medium containing choline or FIG. 1. different choline analogues. At day zero, cells were harvested and distributed into flasks containing 40 Ag/ml of choline (0), DMEA (A), MEA (X<), PA (0), BA (0), or no analogue (0). Flasks were harvested at different times, and the protein contents were determined. Triton X-100 with 2.5% NaOH on Chromosorb P 80/100 mesh (Supelco). The column was run at 950 with a flow rate of 100 ml/min. DMEA, MEA, E, BA, and PA had retention times of 9.0, 21.2, 27.5, 50.5, and 56.3 min, respectively. RESULTS A strain of mouse fibroblast L cells, LM cells, was chosen for this study because it can be grown and subcultured indefinitely in a lipid-free, chemically defined medium. Most growth media contain choline, and many cells, including L cells, require choline for growth (21). This is also true for LM ce]ls growing in monolayer culture (Fig. 1). After about 2 days in medium without choline, the cells became rounded and detached from the flask. A pumber of choline analogues, which were substituted for choline in the medium, allowed growth for several days. DMEA, which closely resembles choline, permitted growth of the cells, and cells have been successfully propagated with DMEA for several months. MEA, PA, and BA permitted growth for several days at a nearly normal growth rate with little change in morphology. After that time the cells became rounded and detached from the flask. LM cells under the normal growth conditions used for the experiments contained about 22.4% PE, 51.8% PC, 5.6% sphingomyelin, 13.6% phosphatidyl inositol plus phosphatidyl serine, 3.9% cardiolipin, and the remaining 2.7% in several minor phospholipid species. The composition was determined by labeling the cells with [32P]phosphate, extracting the phospholipids, separating them on two-dimensional thin-layer chromatography, and visualizing the spots by autoradiography. When the cells were grown on choline analogues, new spots appeared on the autoradiogram due to the phospholipids with the new polar head groups. After three days' growth on MEA, DMEA, BA, or PA, the corresponding phospholipids comprised 45.1%, 50.6%, 35.4%, and 45.0% of the total phospholipids, respectively (Table 1). The presence of the new phospholipids was generally accompanied by a decrease in the percent of PC. There was a slight drop in the percent of PE, depending on which analogue was present, and there were

+(CH3)Z 22.4 51.8 25.8 - - Methylethanolamine HOCH2CH2NHCH3 9.8 15.2 26.7 45.1 3. X Dimethylethanolamine HOCH2CH2N(CH3)2 17.3 8.9 22.4 0.7 50.6 NH2 1-2-Aminobutanol HOCH2CHCH2CH3 13.9 11.1 39.6 35.

3 4074 Biochemistry: Glaser et al. Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 1. Phospholipid composition of LM cells grown in medium containing different analogues of choline Phospholipid composition (%) Supplement Structure PE PC Other PMEA PDMEA PBA PPA Choline HOCH2CH2N +(CH3)Z Methylethanolamine HOCH2CH2NHCH X Dimethylethanolamine HOCH2CH2N(CH3) NH2 1-2-Aminobutanol HOCH2CHCH2CH Aminopropanol HOCH2CH2CH2NH Choline + linoleate Methylethanolamine + linoleate Dimethylethanolamine + linoleate Aminobutanol + linoleate Cells were incubated for 3 days in medium containing choline or different analogues (40 Mg/ml). In some samples, linoleate (20.ug/ml) was added as the bovine serum albumin complex 16 hr before cells were harvested. Then they were harvested and their phospholipid composition determined as described in Matorials and Methods. -, none detectable. no significant differences in the amounts of the minor phospholipids. In LM cells the methylation pathway that converts PE to PC must function at a very low level and cannot be the major pathway for the synthesis of PC. The PDMEA (3.1%) that was present when cells were supplemented with MEA could be accounted for by a small amount of DMEA in the MEA solution. The new [32P]phospholipids synthesized by the LM cells were identified by several methods. They chromatographed with synthetic standard phospholipids (synthesized from PC and the appropriate amino alcohol by phospholipase D) on two-dimensional thin-layer chromatography in two solvent systems. Solvent system I separated all the phospholipids '(Fig. 2) with the exception of PPA which was not separated from PE, 'although sometimes two spots could be distinguished. In solvent system II, PPA ran slightly slower than PE in the first dimension and could, therefore, be separated from it.' We further characterized the phospholipids synthesized by the LM cells by eluting the spots from the two-dimensional thin-layer plate and showing that they contained fatty acids. Also, isolated [32P]phospholipids were mixed with the'corresponding standard phospholipids and subjected to mild alkaline hydrolysis. The unlabeled and 32P-'labeled watersoluble products (glycerolphosphate esters) cochromatographed on thin-layer cellulose plates. Finally, the phospholipids isolated from the cells by two-dimensional chromatography were deacylated, and the glyce'rolphosphate esters were subjected to acid hydrolysis to liberate the polar head groups (amino alcohols). Gas-liquid chromatography of each polar head group gave the same retention time as the appropriate standard. The choline analogues were efficiently converted into phospholipids by the cells, and by 3 days the amount incorporated reached a plateau (Fig. 3). Fatty acids were taken up by LM cells and converted into phospholipids. Sixteen hours after the addition of linoleate (20 Mg/ml) as the bovine serum albumin complex to the medium, 32.6% of the fatty acids in the phospholipids were linoleate (Table 2), and no toxic effects were apparent. When cells were grown on choline analogues for 2 days prior to the addition of linoleate, the amount of linoleate incorporated went up slightly (35.3% linoleate with DMEA and 37.5% linoleate with MEA). Small changes were seen in the amounts of other fatty acids. The fatty acid compositions showed no significant differences when the cells were grown on MEA, DMEA, or BA. Conversely, there was a slight but reproducible increase in the' incorporation of the choline analogues when linoleate was added to the medium'(table 1). LM cells grown under the conditions of the' experiment in unsupplemented medium contained 15.4,ug of desmosterol per mg of protein. This is different from the value of 7.5 ug of desmosterol per mg of protein for L cells grown on sterol-free medium (14, 22), but it may simply reflect the straip of cells Qr the growth conditions. In two experiments when LM cells were grown on DMEA, MEA, PA, or BA for 3 days, the average sterol content was 11.5, 12.2, 11.6, and 17.9 ug of desmosterol per mg of protein, respectively. PE *PBA *PMEA J#PDM\EA Origin. FIG. 2. Two-dimensional thin-layer chromatogram of phospholipids. About 0.04 Mmole of each phospholipid was spotted at the origin and developed in solvent system I. The origin was at the lower right-hand corner. The first dimension was towards the top of the plate, and the second dimension was from right to left. The phospholipids were made visible by the phosphate spray of Vaskovsky and Kostetsky (23). PC

4 Proc. Nat. Acad. Sci. USA 71 (1974) TABLE 2. Altering the Phospholipid Composition of LM Cells 4075 Fatty acid composition of the phospholipids in LM cells grown in medium containing different choline analogues and linoleate Fatty acid composition (%) Supplement 14:0 16:0 16:1 18:0 18:1 18:2 Choline Dimethylethanolamine Methylethanolamine l-2-aminobutanol Choline + linoleate Dimethylethanolamine + linoleate Methylethanolamine + linoleate Cells were grown as in the experiments of Table 1. Fatty acid composition was measured as described in Materials and Methods.-, none detectable. DAYS FIG. 3. Incorporation of choline analogues into phospholipids. At day zero, cells were harvested and distributed into flasks containing DMEA (A), MEA (A), PA (0), or BA (0). Flasks were harvested at different times, and the phospholipid compositions were determined by one-dimensional thin-layer chromatography: chloroform-methanol-water (65:100:4) was used for DMEA; n-butanol-glacial acetic acid-water (6:2:2) for MEA; chloroform-methanol-28% aqueous ammonia (65:35:5) for BA; and chloroform-methanol-28% aqueous ammonia (65:60:5) for PA. DISCUSSION Methods have been devised to dramatically alter the lipid composition of LM cells grown on serum-free and lipid-free tissue culture medium. The phospholipids can be altered with respect to the fatty acyl group alone, the polar head group alone, or both simultaneously. These changes in the lipid composition should have large effects on the structure and function of the cellular membranes. In recent years considerable effort has been directed towards understanding the role of the fatty acids in membrane phospholipids. By altering the fatty acid composition, the "fluidity" of the membrane and the activity of many membranous enzymes are affected (1, 2). The manipulations of the fatty acid composition in LM cells will be discussed in more detail elsewhere. The polar head groups of the phospholipids are also important. For example, there is more than a 30 difference in the transition temperatures from the gel-like to liquid crystalline phase for pure PC and PE containing the same acyl groups (in excess water) (24). It is possible that the cells have a regulatory mechanism that would compensate for the changes in the polar head group by altering, for example, the sterol content, and thus conferring the same overall physical properties on the membrane lipids. The data indicate that the desmosterol content was reduced when the cells were grown on some choline analogues. Although changes in the fatty acid composition of a phospholipid could also compensate for a change in the polar head group, no significant changes in the total fatty acid compositions were observed when cells were grown on different choline analogues. When LM cells were supplemented with choline analogues as well as linoleate, however, linoleate incorporation was slightly increased in the phospholipids. The addition of linoleate also increased slightly the incorporation of the choline analogues into the phospholipids. A more rigorous interpretation of these changes in terms of the role of lipids in membrane structure and function will have to await the isolation and characterization of the subcellular membrane fractions. We thank Mrs. Sze Mei Lau for excellent technical assistance. We also thank Drs. Frederick A. Dembrose and Craig M. Jackson for the advice and help in the synthesis of the phospholipids. K.A.F. is a National Institutes of Health Postdoctoral Fellow (AM-54171). This investigation was supported in part by NSF Grant GB-38676X and NIH Grant HL Machtiger, N. A. & Fox, C. F. (1973) Annu. Rev. Biochem. 42, Cronan, J. E., Jr. & Vagelos, P. R. (1972) Biochim. Biophys. Acta 265, Scarborough, G. A. & Nyc, J. G. (1967) J. Biol. Chem. 242, Crocken, B. J. & Nyc, J. G. (1964) J. Biol. Chem. 239, Wisnieski, B. J., Williams, R. E. & Fox, C. F. (1973) Proc. Nat. Acad. Sci. USA 70, Williams, R. E., Wisnieski, B. J., Rittenhouse, H. G. & Fox, C. F. (1974) Biochemistry 13, Rittenhouse, H. G. & Fox, C. F. (1974) Biochem. Biophys. Res. Commun. 57, Higuchi, K. (1970) J. Cell. Physiol. 75, Spector, A. A. & Hoak, J. C. (1969) Anal. Biochem. 32, Dulbecco, R. & Vogt, M. (1954) J. Exp. Med. 99, Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, Ames, G. F. (1968) J. Bacteriol. 95, Rouser, G., Kritchevsky, G. & Yamamoto, A. (1967) in Lipid Chromatographic Analysis, ed. Marinetti, G. V. (Marcel Dekker, Inc., New York), Vol. I, pp Sokoloff, L. & Rothblat, G. H. (1972) Biochim. Biophys. Acta 280, Dawson, R. M. C. (1967) Biochem. J. 102,

5 4076 Biochemistry: Glaser et al. 16. Yang, S. F., Freer, S. & Benson, A. A. (1967) J. Biol. Chem. 242, Munkres, K. D. & Richards, F. M. (1965) Arch. Biochem. Biophys. 109, Dittmer, J. C. & Wells, M. A. (1969) in Methods in Enzymology, ed. Lowenstein, J. M. (Academic Press, New York), XIV, pp Wells, M. A. (1972) Biochemistry 11, Proc. Nat. Acad. Sci. USA 71 (1974) 20. Lester, R. L. & White, D. C. (1967) J. Lipid Res. 8, Nagle, S. C., Jr. (1969) Appl. Microbiol. 17, Rothblat, G. H., Burns, C. H., Conner, R. L. & Landrey. J. R. (1970) Science 169, Vaskovsky, V. E. & Kostetsky, E. Y. (1968) J. Lipid Res. 9, Ladbrooke, B. D. & Chapman, D. (1969) Chem. Phys. Lipids 3,

Phospholipase D Activity of Gram-Negative Bacteria

JOURNAL OF BACTERIOLOGY, Dec. 1975, p. 1148-1152 Copyright 1975 American Society for Microbiology Vol. 124, No. 3 Printed in U.S.A. Phospholipase D Activity of Gram-Negative Bacteria R. COLE AND P. PROULX*