ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 196, No. 1, August, pp. 186-191, 1979

Glucosphingolipid

Synthesis in the Cellular Slime Mold

Dictyostelium

Discoideum

l

EDMUND V. CREAN2 AND EDWARD F. ROSSOMANDO Department of Oral Biology,

University of Connecticut Health Center, Farmington,

Connecticut

060.92

Received December 19, 19’78;revised March 22, 1979 The transfer of glucose from UDP-[ WJglucose to endogenous acceptors has been examined disco&urn. In the presence of using a particulate fraction prepared from Dictyostelium EDTA, the sole product is a W-labeled glucolipid with the chemical and chromatographic properties of a monoglucosylceramide containing a hydroxy fatty acid. While the reaction does not require a divalent cation, various cations stimulate or inhibit the transferase activity. Other nucleotide sugars have little or no effect on the reaction. Preliminary studies suggest that glycosphingolipid metabolism is developmentally regulated in D. discoideum.

At the onset of starvation, the independent amoebae of the cellular slime mold Dictyostelium discoideum enter a developmental program during which the cells aggregate to construct fruiting bodies consisting of two distinct cell types (1). This system thus represents a useful model for examining the processes of cellular differentiation and morphogenesis. The presence of glycosubstances on cell surfaces has caused some speculation about the involvement of these substances in the regulation of cell growth, movement, recognition, and adhesion. Changes in lectin binding components (2-5), the appearance of a carbohydrate binding protein (6, 7), and developmental regulation of glycosidase (8, 9>, and glycosyltransferase (10, 11) activities are observed during D. discoideum development, raising the possibility that glycosubstances play a role in various aspects of the morphogenetic process. In addition, aggregation-defective mutants possessabnormal membrane glycolipids (12) and glycoproteins (13). While we have previously reported the synthesis of a mannosylphosphoryl polyprenol by D. discoideum (14), little else is known regarding glycolipid biosynthesis in this organism. In ’ This research was supported in part by Grant DE 03715 from the U. S. Public Health Service. 2 Address correspondence to this author. 0003-9861/79/090186-06$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.

this report we describe the transfer of glucose from UDP-glucose to endogenous acceptors by an activity present in a D. discoideum particulate fraction and present evidence to suggest that the principal product is a glucosyl sphingolipid. MATERIALS

AND METHODS

Cell culture. An axenic strain (AX-~) of D. discoideum derived from the parent stock NC-4 was used

for all experiments. Cells were grown on a gyratory shaker at 23°C in HL-5 medium (15) which contained 15 g Thiotone per liter, 7.5 g yeast extract per liter, 73 mM glucose, 3.6 mM N%HPO,, and 0.9 mM KH,PO, (final pH 6.1). Log phase cells were harvested at a titer of l-3 x lo6 cells/ml, while stationary phase cells were harvested at l-2 x 10’ cells/ml. To initiate development, log phase cells were harvested, resuspended a 1.5 x 10’ cells/ml in 17 mM potassium phosphate (pH 6.1) containing 2 mM MgSO, (KPM buffer),3 and placed on a gyratory shaker at 23°C. Under these conditions the amoebae became aggregation competent (16) within lo-12 h. Preparation of the total particulate fraction. Stationary phase amoebae were employed in all preparations except where otherwise noted, and in each case approximately 4 x 108cellswere used per preparation. Cells were harvested from growth medium by centrifu3 Abbreviations used: KPM, potassium phosphatemagnesium sulfate; PMSF, phenylmethylsulfonylfluoride; DTT, diothiothreitol; MeOH, methanol; SDS, sodium dodecyl sulfate; DOG, sodium deoxycholate; Glc, glucose; Gal, galactose; GlcNac, N-acetylglucosamine; Man, mannose. 186

GLUCOSYLTRANSFERASE

IN Dictyostelium

discoideum

187

gation for 5 min at 4,000g and washed once with 0.1 M tracted two additional times with 16 ml CHCl,:MeOH Tris- HCl buffer (pH 7.5).The cell pellet was then covered (2:l). All CHCl, extracts were combined and washed with a few milliliters of liquid N,. After the N, had twice with 10 ml HzO. The washed extract was dried boiled away, the pellet was suspended in 25 ml of a room down by rotary evaporation and the residue dissolved temperature solution of 4 mM Tris-HCI buffer (pH in 5 ml CHC&:MeOH (1:4) plus 0.5 ml of 1 N NaOH and 7.5) containing 4 mM NaF and 1 mM PMSF and vor- incubated at 37°C for 30 min. To this was added 0.5 ml texed. The lysate was cooled to 5°C and centrifuged for of 1 N HCl, 1 ml MeOH, 2 ml CHCl,, and 2 ml of H,O. 1 h at 100,OOOgand the pellet (total particulate frac- The CHCl, phase was removed and dried by rotary tion) was suspended at 5-6 mg/ml in a buffer contain- evaporation. The residue was dissolved in 5 ml of ing 10%(v/v) glycerol, 1 mM DTT, 1 mM PMSF, 0.5 mM CHCla:MeOH:H,O (10:10:3, v/v) and applied to a 0.5 ml NaF, and 0.5 mM Tris-HCl buffer (pH 7.5). Protein column of DEAE-cellulose acetate (19) equilibrated concentrations were determined by the procedure of with the same solvent and then eluted with 10 ml of Lowry et al. (17) using bovine serum albumin as solvent. The eluate was dried down, suspended in 5 ml standard. of CHCl,, and applied to a 2-ml column of Bio.Sil A. Glucosyltransferase assay. The standard assay mix- The column was washed with 10 ml of CHCl, and then ture contained the following components in a total the product was eluted in the glycosphingolipid fracvolume of 0.1 ml: 8% glycerol, 0.8 mM DTT, 1.25 mM tion (20) with 20 ml of acetone:MeOH (91). This fracEDTA, 4.4 ELM UDP-[Wlglucose (220 mCi/mmol), 50 tion was dried down and the residue suspended in 5 ml of CHCl, and applied to a 6 ml Bio’Sil A column. The mM Tris-HCI (pH 7.0), and O-400 pg total particulate protein. Incubations were performed at 30°C for O-2 column was washed with 30 ml CHCl, and then the h and terminated by adding 2 ml of ice-cold 10% (w/v) purified glycolipid product was eluted with 30 ml of trichloroacetic acid containing 0.1 M NaHZPO, and 0.1 CHCl,:MeOH (9:l). Chemical analysis of the glucolipid product. SamM NhP,O,. After cooling for 10 min on ice, the precipitates were collected on Whatman GF/C filters and the ples containing up to 0.3 pmol of product were heated radioactivity measured by liquid scintillation counting. overnight at 80°C in 1.5 ml of 0.75 N HCl in dry MeOH. To distinguish between lipid and nonlipid products, After cooling, this solution was extracted three times reactions were terminated by extraction with CHCl,: with 2 ml of hexane. The methanolic phase was then dried down and analyzed for long chain bases (21) using MeOH (2:1, v/v) as previously described (14). 1-tetradecylamine as the standard. The aqueous phases Chromatography and electrophoresis. Glycolipids were chromatographed on NEN-silica gel 0 plates in derived from the sphingosine assay were hydrolyzed for 3 h in 3 N HCl at 100°C to liberate free sugars CHCI,:MeOH:H,O (100:42:6, v/v), CHCl,:MeOH:H,O (60:35:8), or propanol:H,O (4:l). Glycolipids were de- which were measured enzymatically by the procedures tected with an orcinol spray (18) and by exposure to of Finch et al. (22). CrO, oxidation of the purified glucoIZ vapors. Radioactive products were located by scrap- lipid was performed essentially as described by Danishefsky et al. (23). ing off l-cm sections of the plate for liquid scintillation Radioactivity measurements. Radioactivity was counting. Sugars were chromatographed on Analtech MN300 cellulose plates (developed two to three times) measured using a Nuclear Chicago Isocap-300 liquid using 1-butanol:pyridine:H,O (6:4:3, v/v) or ethyl scintillation system. All filters, chromatography scrapacetate:pyridine:acetic acid:H20 (36:36:7:21)as solvents. ings, paper strips, and organic solvent extracts were Radioactive products were located as described above, dried in scintillation vials and counted in Liquifluor. Materials. Thiotone and yeast extract were purwhile others were detected with a periodic acid-AgNO, chased from BBL Laboratories. PMSF was obtained stain. Paper electrophoresis was performed on Whatman from Calbiochem, while 1-tetradecylamine was from 3MM paper strips at 350 V for l-2 h in 1% (w/v) sodium Aldrich. UDP-[W]Glc and UDP-[WIGal (200-300 tetraborate. Strips were cut into l-cm segments for mCi/mmol, uniformly labeled) and Liquifluor were liquid scintillation counting or were stained with periodic from New England Nuclear. CrO, was obtained from Eastman and glycolipid standards were from acid-AgNO, reagents. Large-scale preparation of the glucolipid product. A Miles Research Products. All other chemicals were large-scale reaction mixture containing 3 g of total purchased from Sigma. Bio.Sil A was obtained from particulate protein and 1 mM UDP-Glc suspended in Bio-Rad Laboratories. 10 ml of 10 mM MgCl,-50 mM Tris-HCl (pH 7.0) was incubated overnight at 30°C with a few drops of toluene RESULTS added to inhibit microbial growth. The reaction was stopped by adding 24 ml of CHCl,:MeOH (2:1, v/v). In Properties of the Glucosyltransferase some cases 200 ~1 of a radioactive glucosyltransferase Reaction incubation was added at this point as an internal standAs shown in Fig. 1, incubation of the staard for product recovery. The CHCl, phase was removed and the aqueous phase and interphase was ex- tionary phase total particulate fraction with

188

CREAN AND ROSSOMANDO

UDP-[14C]Glc in the presence of EDTA yielded only CHCl,:MeOH-soluble products. Although metal ions were not required for - 12 the reaction, the activity was sensitive to 2 various divalent metal ion concentrations g IO(Fig. 2). However, over 90% of the product cl was still CHCl,:MeOH-soluble even in the presence of 10 mM MgCl,. Paper electroE8 phoresis in borate buffer indicated that those % metals which were inhibitory did not pro3 mote UDP-Glc degradation. A pH optimum E of 7.0-7.1 was obtained in Tris-HCl buffer with O-10 lllM MgCl, and the initial rate of s4 the reaction was linear with respect to pro0 v tein at concentrations from 0 to 4 mg/ml. 3 2 The detergents SDS, DOC, and lysolecic3 thin stimulated the reaction up to twofold at Oconcentrations up to 0.01% (w/v>, but were I I I I 0 IO 20 30 40 inhibitory at higher concentrations. The nonionic detergents NP-40 and Triton X-100 [METAL ION] ( mM ) had no effect at low concentrations (O0.01%) but were inhibitory at higher FIG. 2. Effects of divalent metal ion concentrations concentrations. on the rate of glucose transfer. Reactions were run for The addition of 50 j.~uMUDP-GlcNac or 15 min at 30°C under the conditions of Fig. 1, except GDP-Man to the standard reaction mixture that various concentrations of MgCl, (O), MnCl, (O), had no effect on the activity, while 50 PM SrCl, (A), or CuCl, (A) were added in place of 1.25 mM EDTA (X). The data points represent transfer to UDP-Gal yielded a lo-15% inhibition. Standard reaction mixtures containing CHCl,:MeOH-soluble products only. UDP-[*4C]Gal in place of UDP-[14C]Glc revealed that some Gal was transferred to lipid products, but at only 5% of the extent that Glc was transferred under the same conditions. Characterization of the 14C-labeled Glucolipid Product

All of the CHCl,:MeOH-soluble product obtained from a standard glycosyltransferase reaction migrated as a single radioactive component on silica gel plates in CHCl,: MeOH:H,O (100:42:6,v/v) and CHCl,:MeOH: H,O (60~358). In each system the product comigrated with a galactosylceramide standard which contained a hydroxy fatty acid. In addition, the product was stable to mild acid FIG. 1. Time course of glucose transfer from UDP- and base treatment (24), indicating that it [‘4C]glucose to endogenous acceptors. Each reaction was not a glucosyl phosphoryl polyprenol mixture contained 8% glycerol, 0.8 mM DTT, 1.25 mM or a glucosyl glycerolipid. Strong acid hyEDTA, 4.4 ELMUDP-[Ylglucose (340,000 cpminmol), drolysis (3 h at 100°C in 3 N HCl) yielded 50 mM Tris-HCl (pH 7.0), and 300 yg total particulate protein in a final volume of 0.1 ml. Incubations were [‘4C]Glc as the only radioactive product performed at 30°C and at the times indicated the detectable on thin-layer chromatography. amounts of CHCl,:MeOH-soluble (X) and insoluble (0) The 14C-labeledglycolipid was not bound by products were determined (14). a DEAE-cellulose column (acetate form)

GLUCOSYLTRANSFERASE

when equilibrated and eluted with CHCl,: MeOH:H,O (10:10:3, v/v), suggesting that it was not anionic. Finally when chromatographed on a silicic acid column as described by Esselman et al. (20), the 14C-labeled glucolipid was eluted in the acetone:MeOH fraction which normally contains glycosphingolipids. The properties of the 14C-labeled glycolipid were used to develop a scheme for large-scale purification of the product. To obtain suitable quantities of the product for chemical analysis, a large-scale incubation containing 3 g of total particulate fraction was extracted as described under Materials and Methods. Preparation in this manner yielded approximately 0.5 pmol of glucolipid from 3 g of total particulate material. The addition of 14C-labeledglucolipid as an internal standard at the beginning of the purification procedure indicated that the final yield of product was nearly 80% of the theoretical yield. The purified glucolipid yielded a single I,- and orcinol-positive spot on thin-layer chromatography in a variety of solvent systems and in each case the glucolipid comigrated with a galactocerebroside standard which contained a hydroxy fatty acid. When a sample of the purified glucolipid was subjected to acid methanolysis and analyzed for long chain base and sugar content as described under Materials and Methods, a long chain base:glucose molar ratio of 0.8: 1.0 was obtained, suggesting that the product was indeed a monoglucosylceramide. No other sugars were detectable by the enzymatic assays employed. The glucose of the purified glycolipid was totally susceptible to oxidation by CrO,, indicating a p-glycosidic linkage (23). These results identify the glucolipid as a p-glucosylceramide. Developmental Studies on Glucosphingolipid Biosynthesis

Total particulate fractions, prepared from cells at various early times of development up to the aggregation competent stage, were assayed for glucosyltransferase activity in an attempt to obtain evidence for quantitative or qualitative changes in glucolipid biosynthesis. As shown by the data in

IN Dictyostelium

189

discoideum

Table I, the measurement of initial rates of glucose transfer to endogenous acceptors yielded variable results in our hands. While the results obtained with cells derived from any individual suspension in KPM were very reproducible, the results obtained from separate suspensions prepared on different days were quite variable. For example, in Experiment 1 of Table I, the specific activity of the transferase decreased during this period of development, while in Experiment 2 a peak of activity was observed about 7 h after suspension in KPM. This variability prohibited the use of transfer to endogenous acceptors to evaluate developmental regulation of the activity. When the products of the transferase reactions from the experiments of Table I were examined by thin-layer chromatography the same glucosphingolipid as described above was the only product formed under standard assay conditions. Even when 10 lllM MgCl, was added to the reaction mixtures, 90-95% of the radioactivity incorporated was still accounted for as glucocerebroside in each case. In this case the remaining 5-10% of radioactive product was not extracted from the reaction mixture by CHC1,:MeOH (2:1, v/v) or CHCl,:MeOH TABLE

I

GLUCOSYLTRANSFERASE ACTIVITYIN DEVELOPING CELLS Activity

(pmol/min/mg protein)”

Time” (h)

Expt 1

Expt 2

0 1 3 5 7 10

2.3 1.2 1.1 0.9 0.9 0.9

2.4 2.5 2.1 1.7 2.4 2.2

a Vegetative phase cells were harvested and suspended in KPM buffer as described under Materials and Methods. At the indicated times, samples of cells were harvested for preparation of the total particulate fractions. b The initial rate of formation of TCA-precipitable products was measured over the first 10 min in standard reaction mixtures containing 10 mM MgCl,.

190

CREAN

AND ROSSOMANDO

(12). Furthermore, the addition of 50 pM GDP-Man, UDP-GlcNac, and UDP-Gal to reaction mixtures containing UDP-[‘4C]Glc and 10 mM MgClz had no effect on the nature of the product formed at any stage of early development. Over 90% of the radioactive product was still present as the glucocerebroside. Thus, these in vitro experiments provided no evidence for qualitative changes in the products formed from UDP-[‘4ClGlc under these assay conditions. In addition, borate paper electrophoresis of the reaction products revealed that there was negligible breakdown of UDP-[14C]Glc to glucose l-phosphate or free glucose under the assay conditions employed, indicating that substrate degradation was not interfering with our attempts to detect changes in the glucosyltransferase activity or its products. We did obtain some evidence for developmental regulation of glucosphingolipid biosynthesis in the following experiments. Log phase amoebae were harvested and suspended in KPM buffer to initiate development as described under Materials and Methods. At 2-h intervals after suspension, samples containing 1 x lo9 cells were harvested, extracted, and chromatographed on a silicic acid column to isolate the glycosphingolipid fraction (20). These extracts were chromatographed on silica gel plates in CHCl,:MeOH:H,O (100:42:6, v/v) and sprayed with orcinol reagent to detect glycolipids (not shown). While no glucolipid was detectable in cells harvested after 0 and 2 h in KPM buffer, the glucolipid spot appeared at 4-6 h and remained present throughout 12 subsequent h of suspension in KPM buffer. The Rf of this spot was identical to that of the glucocerebroside product described above and was the only glycolipid which moved away from the origin of the chromatogram. While the biochemical basis for the appearance of the glucolipid remains to be elucidated, the results suggest that regulation of glycosphingolipid metabolism occurs during the early stages of D. discoideum development. DISCUSSION

The present report provides evidence for the synthesis of a glucocerebroside in vitro

by total particulate fractions prepared from While this manuscript was being prepared, the results of Rossler et al. (25), which describe glycolipid synthesis during the differentiation of D. discoideum, were published. We feel that our results complement and extend many of their observations on glucosphingolipid synthesis, as well as providing conclusive evidence that the product is a monoglucosylceramide. In contrast to the results of Rossler et al. (25), we have not observed appreciable transfer of Glc to acceptors which were not extractable with CHCI,:MeOH. Since our respective assay procedures are quite similar, the reason for this discrepancy is not evident. While in some experiments we did observe quantitative changes in glucosyltranstbrase activity similar to those described by Rossler et al. (25), the variability in our results and the problems attendant with studies of transfer to a limited quantity of endogenous acceptor made it difficult for us to conclude that this activity is developmentally regulated. However, the chromatographic evidence we obtained by extraction of glycolipids from developing cells clearly suggests that glycosphingolipid metabolism is developmentally regulated in D. discoideum. To our knowledge, the only similar observation was the appearance of a similar cerebroside spot in extracts of a mutant of D. discoideum which was reported by Hoffman and McMahon (13). It is interesting to note that Glc is transferred almost exclusively to a sphingolipid acceptor under these assay conditions while previous work has shown that Man in transferred from GDP-Man predominantly to polyprenol acceptors (14, 25) and GlcNac is transferred from UDP-GlcNac exclusively to endogenous protein acceptors (Crean, Lagerstedt, and Rossomando, submitted for publication). Each of these reactions represents an initial step in various pathways of glycolipid and glycoprotein biosynthesis and clearly raises the possibility of using D. discoideum to study these biosynthetic pathways and their possible roles in the developmental process. However, to our knowledge no one has yet reported the transfer of sugars to oligosaccharide-containing glycolipids or glycoproteins in D.

D. discoideum.

GLUCOSYLTRANSFERASE

discoideum during the earliest stages of development. Our own preliminary attempts to use various lipids and proteins as exogenous acceptors in transferase assays with D. discoideurn have proven unsuccessful with the exception of using dolichol monophosphate as an exogenous acceptor in mannosyltransferase assays (14). Yet D. discoideum is known to contain glycolipids and glycoproteins which carry oligosaccharide moieties of substantial size (12). We are now in the process of isolating the structurally characterizing glycopeptides and glycolipids from growing and aggregation competent D. discoideum amoebae. It is anticipated that this will not only provide a more direct assessment of whether these structures are changing during the early stages of D. discoideum development but will also generate suitable materials for use as exogenous acceptors in studies on glycosyltransferase activities in this organism. REFERENCES 1. LOOMIS, W. R. (1975) Dictyostelium Discoideum: A Developmental System, Academic Press, New York. 2. WEEKS, G. (1975)J. Biol. Chem. 250,6706-6710. 3. REITHERMAN, R. W., ROSEN, S. A., FRAZIER, W. A., AND BARONDES, S. H. (1975)Proc. Nat. Acad. Sci. USA 72, 3541-3545. 4. GELTOSKY, J. E., SIV, C.-H., AND LERNER, R. A. (1976) Cell 8, 391-396. 5. CREAN, E. V., AND ROSSOMANDO, E. F. (1977) Biochem. Biophys. Res. Commun. 75,488-495. 6. ROSEN, S. D., KAFKA, J. A., SIMPSON, D. L., AND BARONDES, S. H. (1973) Proc. Nat. Acad. Sci. USA 70, 2554-2557. 7. SIU, C.-H., LERNER, R. A., MA, G., FIRTEL, R. A., AND LOOMIS, W. F. (1976) J. Mol. Biol. 100, 157-178. 8. LOOMIS, W. F., JR. (1969) J. Bacterial. 97, 1149-1154.

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9. LOOMIS, W. F., JR. (1970) J. Bacterial. 103, 375-381. 10. BAUER, R., RATH, M., ANDRISSE, H. (1971)Eur. J. Biochem. 21, 179-190. 11. ROGGE, H., NEISES, M., AND RISSE, H. (1977) Biochim. Biophys. Acta. 499, 273-277. 12. WILHELMS, O., LUDERITZ, O., WESTPHAL, O., AND GERISCH, G. (1974) Eur. J. Biochem. 48, 89-101. 13. HOFFMAN, S., AND MCMAHON, D. (1978) J. Biol. Chem. 253, 278-287. 14. CREAN, E. V., AND ROSSOMANM), E. F. (1977) Biochim. Biophys. Acta 498, 439-441. 15. WATTS, D. J., AND ASHWORTH, J. M. (1970) Biothem. J. 119, 171-174. 16. GERISCH, G. (1968) in Cm-rent Topics in Developmental Biology (Moscona, A. A., and Monroy, A., eds.), Vol. 3, pp. 157-197, Academic Press, New York. 17. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 18. LAINE, R. A., STELLNER, K., AND HAKOMORI, S. (1974) in Methods in Membrane Biology (Kern, E. D., ed.), Vol. 2, pp. 205-244, Plenum, New York. 19. ROUSER, G., KRICHEVSKY, G., YAMAMOTO, A., SIMON, G., GALLI, C., AND BAUMAN, A. J. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 14, pp. 272-317, Academic Press, New York. 20. ESSELMAN, W. J., LAINE, R. A., AND SWEELEY, C. C. (1972) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 28, pp. 140-156, Academic Press, New York. 21. LAUTER, K. J., AND TRAMS, E. G. (1962) J. Lipid Res. 3, 136-138. 22. FINCH, P. R., YUEN, R., SCHACHTER, H., AND MOSCARELLO, M. A. (1969) Anal. Biochem. 31, 296-305. 23. DANISHEFSKY, I., ZWEBEN, A., AND SLOMIANY, B. L. (1978) J. Biol. Chem. 253, 32-37. 24. RICHARDS, J. B., AND HEMMING, F. W. (1972) Biochem. J. 130, 77-93. 25. R~SSLER, H., PEUCKERT, W., RISSE, H.J., AND EIBL, H. J. (1978) Mol. Cell. Biochem. 20, 3-15.