Molecular and Biochemical Parasitology, 53 (1992) 71-78 C¢~ 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00
71
MOLBIO01741
Sulfate metabolism in Entamoeba histolytica Tilly B a k k e r - G r u n w a l d a n d B a r b a r a G e i l h o r n Department of Microbiology, University of Osnabru'ck, Osnabrffck, Germany (Received 20 September 1991; accepted 7 February 1992)
Sulfate fluxes and sulfate metabolites in Entamoeba histolytica were characterized employing [35S]sulfate as a marker. Sulfate was taken up both across the plasma membrane and by pinocytosis; in growth medium (sulfate concentration, 1.1 mM) total uptake was 1.5 /~mol h - l (5 x 10 7 cells) -I. The fate of sulfate within the cells was investigated by thin-layer chromatography. Major metabolites (together > 3/~mol (5 x l 0 7 cells)-l) were monoethyl sulfate and 3-cholesteryl sulfate; both these products were released into the growth medium. As minor components we identified the activated sulfate derivatives, adenosine-5'-phosphosulfate and 3'-phosphoadenosine-5'-phosphosulfate. In addition, up to 10% of the sulfate taken up was incorporated into high-molecular weight material (possibly proteoglycans). We propose that sulfurylation of cholesterol may play a role in controlling membrane sterol content. Key words: Sulphate transport; Sulphate ester; Cholesteryl sulphate; Entamoeba histolytica
Introduction
E. histolytica, the causative agent of amebiasis in man, exhibits several structural and biochemical features that have been interpreted as archaic [1-3]. In this context it seemed worthwhile to investigate sulfate metabolism in this organism. In higher eukaryotic cells, sulfate is activated to adenosine-5'-phosphosulfate (APS) and 3'-phosphoadenosine-5'phosphosulfate (PAPS). The sulfuryl group of PAPS may be transferred to a range of organic acceptors. Sulfurylation of low-molecular weight compounds (e.g., aromatic alcohols) often serves as a detoxification mechanism [4]. Cells also synthesize high-molecular weight sulfate esters, in particular proteoglycan sulfates; these are generally important components Correspondence address: Tilly Bakker-Grunwald, Universit~t Osnabriick, Abt. Mikrobiologie, Barbarastrasse I1, D-4500 Osnabriick, Germany. Fax: + + 49 541 969 2870. Abbreviations: APS, adenosine-5'-phosphosulfate; PAPS, 3'phosphoadenosine-5'-phosphosulfate; HPTLC, high-performance thin-layer chromatography; PEI, polyethylene imine.
of the intravesicular and extracellular matrix [5]. In the present work, we have identified the main products of sulfate metabolism in E. histolytica by labeling with [35S]sulfate. We found that activation apparently proceeded through APS and PAPS, as in higher eukaryotes. Most of the sulfate taken up was converted to either monoethyl sulfate or 3cholesteryl sulfate; a minor fraction was incorporated into high-molecular weight material. We present a quantitative overview of the fluxes of sulfate and these metabolites in cells maintained in growth medium.
Materials and Methods
Cell culture. E. histolytica HMI:IMSS was grown axenically at 36°C in TYI-S-33 (growth medium) [6,7] with 15% serum, supplemented with penicillin (100 U ml-~) and streptomycin sulfate (100 #g ml-l). To harvest the cells, cultures in late log growth were chilled and spun at 400 x g. The amebae were washed once in growth medium; all incubations were in growth medium at 36°C.
72
Uptake of[SSS]sulfate. Amebae were suspended at 3-4 × 10° cells ml -~ growth medium. At zero time, [35S]sulfate (Amersham) was added at 0.5 /tCi ml -~ At the indicated times, 0.2 ml-aliquots were mixed with 1 ml ice-cold PBS#8 (NaCI, 150 mM/K2HPO4, 20 mM/KH2PO4,7.5 mM, pH 7.1; ref. 7). The cells were spun down (1 s at 13000 × g). The supernatant was aspirated, and the ceils were washed twice more with 1 ml PBS#8. The final cell pellet was solubilized in 0.4 M NaOH and subjected to liquid scintillation counting (with quench correction based on channel ratio). Methanolic extraction of sulfate metabolites. Cells (2 × 106) were suspended in 60 /A growth medium. At the indicated times after addition of [35S]sulfate (2 #Ci), 10-/A aliquots were mixed with 1 ml ice-cold PBS#8 and spun (1 s at 13 000 × g). The cells were washed twice in PBS#8 and taken up in 0.5-1.0 ml ice-cold methanol. The mixture was kept at - 2 0 ° C for > 3 0 min. Denatured protein was spun down (1 min at 13 000 × g); the clear supernatant is denoted as methanolic cell extract. To investigate the release of 35S-containing metabolites, 2 × 106 cells were incubated for 1 h in 60 /~1 growth medium with 2 /~Ci [35S]sulfate. Subsequently, 1 mi ice-cold growth medium was added, and the cells were spun down and washed twice more with ! ml growth medium. They were then resuspended in 1 ml growth medium and reincubated at 36°C. At the indicated times, aliquots were centrifuged (1 s at 13000 × g). The supernatant was pipetted off and mixed with a 10fold volume of ice-cold methanol. Denatured protein was removed by centrifugation; the clear supernatant is denoted as methanolic medium extract. The cell pellet was washed once with PBS#8 and extracted with methanol as described above. Cell and medium extracts were subjected to liquid scintillation counting and high-performance thin-layer chromatography (HPTLC). For the latter, the extracts were concentrated up to 50-fold by rotary evaporation under vacuum (Speed Vac).
Determination of sulfate concentration.
The
sulfate concentration of TYI-S-33 was determined turbidimetricallv by precipitation with Ba 2+ [8]; we establishe~t a value of 1. I mM, the contributions of the medium components being 0.5 mM (broth), 0.2 mM (serum) and 0.4 mM (streptomycin sulfate), respectively. The sulfate content of the amebac was determined as Ba2"-precipitable radioactivity in cells grown on [SS]sulfate. ~'~ To this end, methanolic cell extract (see above) was acidified with trichloroacetic acid (final concentration, 50 g 1- l), and carrier sulfate and BaCI2 were added to final concentrations of 5 and 10 mM, respectively. Samples were frozen and left at - 2 0 ° C for > 30 min, and were then thawed and spun for 1 min at 13000 × g. Ba 2"precipitable radioactivity was calculated as the difference in radioactivity of the supernatant with that of a control without Ba 2.. Under the conditions described, stock [35S]sulfate was precipitated for >99.8% (not shown).
Thin-layer chromatography. Analytical runs were performed on (i) HPTLC silica layers (Merck 60F254, height of plates 10 cm), solvent 1-butanol/acetic acid/H20, 6:2:2 (v/v), or (ii) 0.1 mm-polyethylene imine-(PEI-)cellulose F layers (Merck, height of plates 20 cm), solvent sat. (NH4)2504/1 M Na-acetate/2-propanol, 80:19:1 (v/v). In the former system, hydrophobic compounds move fastest; the latter system has anion-exchange properties [9]. After the solvent had ascended approx. 80% of the total height, plates were allowed to dry. For autoradiography we employed Kodak XO-Mat; films were exposed for periods ranging from overnight up to one month. Identification of low-molecular weight sulJate derivatives. APS and PAPS in methanolic amebal extract were identified by co-chromatography with the respective nonradioactive compounds (Sigma); the latter were visualized by fluorescence quenching under UV [9]. Also, 3-cholesteryl sulfate ('Y' in Fig. 2) was identified by co-chromatography with nonradioactive standard (Sigma); it was localized by staining with sulfuric acid in ethanol (10 g 1- '; ref. 9). As 3-cholesteryl sulfate is immobile on
73
the polyethylene imine layer (Fig. 2), colocalization was confirmed on a silica layer employing a different solvent (2-propanol/ chloroform/methanol/H20, 10:10:5:2 (v/v); results not shown). Monoethyl sulfate ('X' in Fig. 2) was isolated in9Pure form from the amebae. To this end, 10 cells were extracted with HCIO4 (5 M). The extract was neutralized with KOH, and KCIO4 was removed by centrifugation. To trace 'X', we added methanolic extract of cells incubated for 1 h with 355042- (see above). From this stage on, the degree of purification of 'X' was assessed by TLC followed by autoradiography and ninhydrin staining (most of the contaminants being ninhydrinpositive). The labeled mixture was freeze-dried and the residue was extracted with methanol (40 ml). The extract was kept overnight at - 2 0 ° C and the precipitate that had formed (mainly inorganic salt) was spun down and discarded. The supernatant was evaporated to dryness. The residue was dissolved in H20 (20 ml) and applied to an anion exchange column (AGIX8 from BioRad, 5 ml, in N O 3 - form). Upon elution with a linear gradient of KNO3 (0-1 M, 20 ml) one radioactive peak containing 'X' was obtained between 0.5 and 0.7 M KNO3. We further purified the material on a preparative silica layer (Merck 60F254s, eluted with l-butanol/acetic acid/H20, 6:2:2 (v/v)). The band containing 'X' was scraped off and extracted with methanol. The final yield of 'X' was approximately 3 mg. The compound was dissolved in 2H20 and subjected to IH-NMR spectroscopy. The spectrum turned out to be very simple, consisting of a triplet at 1.28 ppm and a quadruplet at 4.09 ppm with relative intensities of 1.60:1. From this, and the fact that 'X' was obviously a negatively charged sulfate derivative, we concluded that it must correspond to monoethyl sulfate. We confirmed this identification by chemical synthesis of monoethyl sulfate [10] followed by both ]H-NMR spectroscopy and co-chromatography with 'X' (results not shown).
h with [35S]sulfate (5 #Ci), washed twice with ice-cold PBS#8 and extracted with 1 ml buffer containing urea (7 M)/Triton X100 (5 g 1-i)/ NaCI (20 mM)/Tris-HCl (20 mM, pH 7.3) and iodoacetamide (2 mM). The extract was spun for 10 min at 40000 x g, and the supernatant was applied to an FPLC Superose-12 column (Pharmacia). Molecular weights were estimated by comparison with a standard protein mixture (Pharmacia).
Gel filtration of high-molecular weight sulfate derivatives. 3 x 10 cells were incubated for I
Fig. 1. Sulfate uptake by E. histolytica. O, Control; 0 , plus cytochalasin B (20 /~M). See Methods for details of experimental procedures.
Calculations. 5 × 107 cells correspond to 1 ml packed cell pellet, and contain 0.10 g protein. Extracellular space contributes 10% and the pinocytic vesicle compartment 40% to the packed cell volume [11]. Results
Sulfate uptake. [35S]Sulfate was taken up by the amebae in a time-dependent manner; in growth medium (sulfate concentration, 1.1 mM; see Methods) the initial rate of uptake was 1.5 /~mol h -1 (5 x 107 cells) -1 (Fig. 1). Generally, 2 routes contribute to uptake in E. histolytica: pinocytosis (indiscriminately for all solutes) and transport across the plasma I
i
1
1.0
~-
0.5
/ 1
I
I
20
40
60
time (rain)
74
membrane (for some solutes only) [12]. To discriminate between these two routes, we blocked pinocytosis with cytochalasin B [13]. This inhibited the initial rate of transport by about one-fourth (Fig. 1). We conclude that sulfate was taken up both by diffusion or transport across the plasma membrane and by pinocytosis, the former contributing about 3 times as much as the latter under these conditions.
Formation of low-molecular weight sulfate metabolites. We proceeded to investigate the intracellular fate of [35S]sulfate by HPTLC followed by autoradiography. Fig. 2 shows the pattern of radioactivity of a methanolic cell extract prepared 1 h after addition of [35S]sulfate to the cells. As analytical layers we employed both silica (left) and PEIcellulose (right). The patterns obtained were
A
B SO42"
approximately reciprocal: e.g., free sulfate, which remained at the origin in the left-hand system, moved with the front in the right-hand system. Strikingly, [35S]suifate as such was barely detectable in the extract: most of it had apparently been converted into 2 major and 2 or 3 minor derivatives. Two of the minor spots corresponded to APS and PAPS, respectively; these are the activated forms of sulfate normally found in eukaryotic cells. The most pronounced compound (marked 'X' in Fig. 2) was purified by anion exchange and preparative layer chromatography, and subsequently identified by its IH-NMR spectrum as monoethyl sulfate (see Methods for details). The second major spot, 'Y', was shown to correspond with 3-cholesteryl sulfate by co-chromatography with this compound (see Methods). One additional minor component ('Z') has not yet been identified.
Release of low-molecular weight sulfate metabolites. After addition of [35S]sulfate to the medium, the radioactive metabolites appeared in the cells in a time-dependent manner (Fig. 3). Clearly, the intensity of the spots was still
"X" "Z"
ChoI-SO4"..~ APS ~ EtSO 4-
ny. ~PAPS
APS "Z", P A P S
..... APS
,,X,i
S0420 SO42-
__
,~
~
~
,~,
Fig. 2. Autoradiographic HPTLC patterns of a methanolic extract from cells labeled for 1 h with 135S]sulfate. A, silica system; B, PEl-cellulose system. For further experimental details, see Methods.
15
30
60
min
Fig. 3. Time dependence of the appearance of radioactive sulfate metabolites. At the indicated times after addition of laSS]sulfate to the growth medium, cells were extracted with methanol and samples analyzed by HPTLC on silica as described in Methods.
75 A
B
Chol-SO 4" ~ _ _ APS EtSO 4"
S042-
~!
~ 0
15
30
60
120
mln
0
15
30
60
120
min
Fig. 4. Disappearance of radioactive metabolites from the cells into the medium. Cells were loaded for I h with [35S]sulfate, washed, and resuspended in unlabeled growth medium. At the indicated times, aliquots of suspension were centrifuged. Cells and medium were extracted with methanol and samples analyzed by HPTLC on silica. For further details, see Methods. (A) Cell extracts; (B) medium extracts. increasing after 1 h, which indicates that equilibration o f the m e t a b o l i t e pools was a relatively slow process. This will be c o n f i r m e d by q u a n t i t a t i v e d a t a presented below. F o r the next e x p e r i m e n t (Fig. 4), cells were incubated for I h with [35S]suifate, washed, and resuspended in n o n r a d i o a c t i v e g r o w t h m e d i u m . D u r i n g the next 2 h, radioactivity d i s a p p e a r e d f r o m the cells (Fig. 4A); c o n c o m i t a n t l y , m o n o e t h y l and cholesteryl ester, s o m e free sulfate, a n d up to 2 additional c o m p o u n d s a p p e a r e d in the m e d i u m (Fig. 4B). Since there was very little free sulfate in the cells at a n y time (Fig. 4A), the sulfate in the m e d i u m m u s t have been due to b r e a k d o w n o f metabolites.
High-molecular weight sulfate derivatives. In p r e p a r i n g the cell extracts for the a b o v e experiments, we noticed that a few percent o f the radioactivity i n c o r p o r a t e d by the cells was insoluble in m e t h a n o l (see T a b l e I). This suggested the presence o f h i g h - m o l e c u l a r weight sulfate derivatives. We therefore solubilized a m e b a e that had been loaded for one h with [35S]sulfate in detergent and subjected the solubilizate to gel filtration (see M e t h o d s for details). This w a y we o b t a i n e d three 35Scontaining peaks, o f 110, 42 and 10 k D a (results not shown). Quantitation of sulfate metabolites.
T o deter-
TABLE I Contents of radioactive metabolites in cells grown on [35S]sulfate Components Total a Methanol-soluble Ba2+-precipitable h Methanol-insoluble c
Low-molecular weight Free sulfate High-molecular weight
Content (/~mol (5
x
10 7
cells) ~)
3.8 + 0.4 3.4 + 0.4 < 0.2 0.36 + 0.12
Values are means (+ SD) for three cell suspensions, including the one used for Fig. 5. " Sum of values for methanol-soluble and insoluble fractions. b Sulfate was precipitated by Ba2- as described in Methods. The methanol-insoluble residue was washed once with 1 ml methanol, digested in 0.4 M NaOH and subjected to scintillation counting.
76
A
B
present in the growth medium than in the cells.
Discussion
C h o l S O 4"
--
EtSO 4-
-"+, ".r'~ j.'."
S042-
--
Fig. 5. Patterns of radioactivity from a cell suspension grown on [35S]sulfate. 10 ml growth medium was inoculated with 5 × l 0 4 cells; [35S]sulfate was added at 0.5 ~Ci (10 ml)- i. After 72 h, total cell count had increa~d to approximately 3 × 10 6. Cells and medium were extracted with methanol and samples analyzed by HPTLC on silica as described in Methods. (A) Cell extract; (B) medium extract.
mine the concentrations of intracellular sulfate and its metabolites, we equilibrated cells on [35S]sulfate by allowing them to grow in the presence of the isotope. From Fig. 5A it can be seen that also under these conditions monoethyl sulfate and cholesteryl sulfate were the major low-molecular weight sulfate derivatives. The ethyl derivative was clearly detectable in the growth medium (Fig. 5B); cholesteryl sulfate, which according to Fig. 4B should also be exported from the cells, may have been degraded by extracellular sulfatase activity. Table I summarizes the content of sulfate metabolites calculated from this and 2 other experiments. As judged from its precipitability by Ba 2~ less than 5% of cellular radioactivity was' associated with free sulfate; this confirms the qualitative picture supplied by the TLC patterns (Figs. 2-5). By the time the cells were harvested, approximately 6% of the amount of sulfate originally present in the medium had been converted into a Ba 2"soluble form (not shown) most of which should be monoethyl sulfate (Fig. 5B). At that stage, over 4 times more of this compound was
Overview of sulfate metabolism. Sulfate metabolism in E. histolytica appears to be relatively simple. As shown above (Fig. 1), sulfate was taken up both across the plasma membrane (about 75% ) and by pinocytosis (about 25%). At 1.5 /lmol h -~ (5 x 10 7 cells) 1 total sulfate uptake was relatively slow: two orders of magnitude slower than the uptake of glucose [14], but comparable to that of ieucine or methionine at an extracellular concentration of 1 mM (unpublished data). The content of free sulfate (0.4 l~mol (5 x 10 7 cells)- 1; ref. 12) if the vesicle compartment were impermeable: from this we conclude that most of the sulfate taken up by pinocytosis was subsequently transported into the cytoplasm, too. There, sulfate was transferred+ presumably after activation to APS and PAPS, to 2 major low-molecular weight acceptors, ethanol and cholesterol. The products, monoethyi sulfate and cholesteryl sulfate, reached relatively high (together >3 /~mol (5 x 10 7 cells)-~) steady-state concentrations. We have no indication as to the subcellular localization of these compounds, but we presume at least part was sequestered within the endocytic vesicles. Effiux of radioactivity was mainly in the form of monoethyl sulfate. At a doubling time of the cells of 12 h, some 20% of the sulfate taken up would be needed for the growth-associated expansion of the different sulfate ester pools; these include the 3 high-molecular weight components mentioned above. We should stress again that the data presented apply to cells incubated in growth medium. Amebae growing within the human host will probably contain the same set of sulfate metabolites, but their concentrations could well be very different. Function ~[+sulfate esters. Remarkably, over 90% of the sulfate taken up was converted to
77
either monoethyl sulfate or cholesteryl sulfate. Sulfate conjugation often plays an important role in the detoxication of noxious organic compounds [4]. We therefore considered the possibility that the formation of ethyl and cholesteryl sulfate in the amebae could serve a similar function. Entamoeba depends on externally-added cholesterol for growth [15]. In growth medium, this cholesterol is recruited from the cholesteryl esters present in serum lipoproteins. In this context we propose that cholesteryl sulfotransferase activity could have a function in removing any surplus membrane cholesterol; this way it could supplement other regulatory mechanisms such as the adjustment of the ratio of saturated to unsaturated fatty acids [ 16]. By contrast, a similar role for ethyl sulfurylation seems improbable. Amebae generate ethanol by fermentation [1-3]. In addition, growth medium contains about 10 mM ethanol (added as a solvent for lipid-soluble vitamins; refs. 6, 7). However, as the cells grow well on this mixture, this obviously does not excessively bother them. Moreover, if intended as a mechanism of detoxication, sulfurylation of ethanol would be extremely ineffective: less than 1% of the ethanol initially present was converted into the sulfate ester over a 3-day time course. Therefore, rather than looking for a function, we surmise that the sulfotransferase for cholesterol may accept ethanol as an alternative substrate; this would be in line with the range of specificities established for other sterol sulfotransferases [17]. If so, monoethyl sulfate would be an inherently wasteful product. We estimate, assumin8 a rate of uptake of glucose of/> 120 pmol h - " (5 x 107 cells) -1 [14], a yield of up to 7 ATP per glucose [1] and an investment of 3 ATP per ethyl sulfate synthesized, that cells maintained in growth medium continuously invest up to 0.5% of the energy they generate into its synthesis. Finally, a few more words on the highmolecular weight sulfate derivatives. We have proposed [12] that, similar to the situation in mast cells and natural killer cells [5], proteo-
glycan sulfates may serve in the retention of amebapain (and possibly other soluble digestive enzymes) upon exocytic fusion of intracellular vesicles with the plasma membrane. In this context it will be important to further characterize the high-molecular weight material and to assess its binding properties.
Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft (SFB 171 Project C2) and by the Fonds der Chemischen Industrie. We thank Dr. Helmut Rosemeyer for N M R spectroscopy, Dr. Willi Hoppe for invaluable advice, Mr. Andr6 Schlochtermeyer for help with the FPLC and Mrs. Dietlind Henrichs for excellent technical assistance.
References 1 Reeves, R.E. (1984) Metabolism of Entamoeba histolytica Schaudinn, 1903. Adv. Parasitol. 23, 105142. 2 McLaughlin, J. and Aley, S. (1985) The biochemistry and functional morphology of the Entamoeba. J. Protozool. 32, 221-240 3 Avron, B. and Chayen, A. (1988) Biochemistry of Entamoeba: a review. Cell Biochem. Funct. 6, 71-86. 4 Jakoby, W.B. and Ziegler, D.M. (1990) The enzymes of detoxication. J. Biol. Chem. 265, 20715-20718. 5 Fransson, L.-A. (1987) Structure and function of cellassociated proteoglycans. Trends Biochem. Sci. 12, 406 411. 6 Diamond, L.S., Harlow, D.R. and Cunnick, C.C. (1978) A new medium for axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72, 431-432. 7 Diamond, L.S. (1988) Cultivation of Entamoeba histolytica in vitro. In: Amebiasis. Human infection by Entamoeba histolytica (Ravdin, J.l., ed.), pp. 27-40. Wiley, New York. 8 Sorbo, B. (1987) Sulfate: turbidimetric and nephelometric methods. Methods Enzymol. 143, 3-6. 9 Stahl, E. (1969) Thin-Layer Chromatography. A Laboratory Handbook. Springer, Berlin. I0 Lloyd, A.G., Tudball, N. and Dodgson, K.S. (1961) Infrared studies on sulphate esters, III. O-Sulphate esters of alcohols, amino alcohols and hydroxylated amino alcohols. Biochim. Biophys. Acta 52, 413-419. II L6hden, U. and Bakker-Grunwald, T. (1989) Magnetic separation of pinocytic vesicles of defined age from Entamoeba histolytica. Anal. Biochem. 182, 77 83. 12 Bakker-Grunwald, T. (1991) Transport and compartmentation in Entamoeba histolytica. In: Biochemical
78 Protozoology as a Basis for Drug Design (Coombs. G.H. and North. M., eds), pp. 367 376. Taylor and Francis, London. 13 Bakker-Grunwald, T., L6hden, U. and Trissl, D. (1985) Effects of cytochalasin B on N a ' content and cell volume of Entamoeba histolytica. Biochim. Biophys. Acta815, 170 174. 14 Serrano, R. and Reeves, R.E. (1974) Glucose transport in Entamoeba histolytica. Biochem. J. 144. 43-48. 15 Latour, N.G., Reeves, R.E. and Guidry, M.A. (1965)
Steroid requirements of Fntamoeha histolyt&.a. Exp. Parasitol. 16, 18 22. 16 Van Vliet, H.H.D.M., Op den Kamp, J.A.F. and Van Deenen, L.I,.M. (1975) Lipid auxotrophy and the effect on lipid composition of E. invadens. J. Protozool. 22, 428 432. 17 Lyon. E.S. and Jakoby, W.B. (1980) The identity of alcohol sulfotransferases with hydroxysteroid sulfotransferases. Arch. Biochem. Biophys. 202, 474--481.
71
MOLBIO01741
Sulfate metabolism in Entamoeba histolytica Tilly B a k k e r - G r u n w a l d a n d B a r b a r a G e i l h o r n Department of Microbiology, University of Osnabru'ck, Osnabrffck, Germany (Received 20 September 1991; accepted 7 February 1992)
Sulfate fluxes and sulfate metabolites in Entamoeba histolytica were characterized employing [35S]sulfate as a marker. Sulfate was taken up both across the plasma membrane and by pinocytosis; in growth medium (sulfate concentration, 1.1 mM) total uptake was 1.5 /~mol h - l (5 x 10 7 cells) -I. The fate of sulfate within the cells was investigated by thin-layer chromatography. Major metabolites (together > 3/~mol (5 x l 0 7 cells)-l) were monoethyl sulfate and 3-cholesteryl sulfate; both these products were released into the growth medium. As minor components we identified the activated sulfate derivatives, adenosine-5'-phosphosulfate and 3'-phosphoadenosine-5'-phosphosulfate. In addition, up to 10% of the sulfate taken up was incorporated into high-molecular weight material (possibly proteoglycans). We propose that sulfurylation of cholesterol may play a role in controlling membrane sterol content. Key words: Sulphate transport; Sulphate ester; Cholesteryl sulphate; Entamoeba histolytica
Introduction
E. histolytica, the causative agent of amebiasis in man, exhibits several structural and biochemical features that have been interpreted as archaic [1-3]. In this context it seemed worthwhile to investigate sulfate metabolism in this organism. In higher eukaryotic cells, sulfate is activated to adenosine-5'-phosphosulfate (APS) and 3'-phosphoadenosine-5'phosphosulfate (PAPS). The sulfuryl group of PAPS may be transferred to a range of organic acceptors. Sulfurylation of low-molecular weight compounds (e.g., aromatic alcohols) often serves as a detoxification mechanism [4]. Cells also synthesize high-molecular weight sulfate esters, in particular proteoglycan sulfates; these are generally important components Correspondence address: Tilly Bakker-Grunwald, Universit~t Osnabriick, Abt. Mikrobiologie, Barbarastrasse I1, D-4500 Osnabriick, Germany. Fax: + + 49 541 969 2870. Abbreviations: APS, adenosine-5'-phosphosulfate; PAPS, 3'phosphoadenosine-5'-phosphosulfate; HPTLC, high-performance thin-layer chromatography; PEI, polyethylene imine.
of the intravesicular and extracellular matrix [5]. In the present work, we have identified the main products of sulfate metabolism in E. histolytica by labeling with [35S]sulfate. We found that activation apparently proceeded through APS and PAPS, as in higher eukaryotes. Most of the sulfate taken up was converted to either monoethyl sulfate or 3cholesteryl sulfate; a minor fraction was incorporated into high-molecular weight material. We present a quantitative overview of the fluxes of sulfate and these metabolites in cells maintained in growth medium.
Materials and Methods
Cell culture. E. histolytica HMI:IMSS was grown axenically at 36°C in TYI-S-33 (growth medium) [6,7] with 15% serum, supplemented with penicillin (100 U ml-~) and streptomycin sulfate (100 #g ml-l). To harvest the cells, cultures in late log growth were chilled and spun at 400 x g. The amebae were washed once in growth medium; all incubations were in growth medium at 36°C.
72
Uptake of[SSS]sulfate. Amebae were suspended at 3-4 × 10° cells ml -~ growth medium. At zero time, [35S]sulfate (Amersham) was added at 0.5 /tCi ml -~ At the indicated times, 0.2 ml-aliquots were mixed with 1 ml ice-cold PBS#8 (NaCI, 150 mM/K2HPO4, 20 mM/KH2PO4,7.5 mM, pH 7.1; ref. 7). The cells were spun down (1 s at 13000 × g). The supernatant was aspirated, and the ceils were washed twice more with 1 ml PBS#8. The final cell pellet was solubilized in 0.4 M NaOH and subjected to liquid scintillation counting (with quench correction based on channel ratio). Methanolic extraction of sulfate metabolites. Cells (2 × 106) were suspended in 60 /A growth medium. At the indicated times after addition of [35S]sulfate (2 #Ci), 10-/A aliquots were mixed with 1 ml ice-cold PBS#8 and spun (1 s at 13 000 × g). The cells were washed twice in PBS#8 and taken up in 0.5-1.0 ml ice-cold methanol. The mixture was kept at - 2 0 ° C for > 3 0 min. Denatured protein was spun down (1 min at 13 000 × g); the clear supernatant is denoted as methanolic cell extract. To investigate the release of 35S-containing metabolites, 2 × 106 cells were incubated for 1 h in 60 /~1 growth medium with 2 /~Ci [35S]sulfate. Subsequently, 1 mi ice-cold growth medium was added, and the cells were spun down and washed twice more with ! ml growth medium. They were then resuspended in 1 ml growth medium and reincubated at 36°C. At the indicated times, aliquots were centrifuged (1 s at 13000 × g). The supernatant was pipetted off and mixed with a 10fold volume of ice-cold methanol. Denatured protein was removed by centrifugation; the clear supernatant is denoted as methanolic medium extract. The cell pellet was washed once with PBS#8 and extracted with methanol as described above. Cell and medium extracts were subjected to liquid scintillation counting and high-performance thin-layer chromatography (HPTLC). For the latter, the extracts were concentrated up to 50-fold by rotary evaporation under vacuum (Speed Vac).
Determination of sulfate concentration.
The
sulfate concentration of TYI-S-33 was determined turbidimetricallv by precipitation with Ba 2+ [8]; we establishe~t a value of 1. I mM, the contributions of the medium components being 0.5 mM (broth), 0.2 mM (serum) and 0.4 mM (streptomycin sulfate), respectively. The sulfate content of the amebac was determined as Ba2"-precipitable radioactivity in cells grown on [SS]sulfate. ~'~ To this end, methanolic cell extract (see above) was acidified with trichloroacetic acid (final concentration, 50 g 1- l), and carrier sulfate and BaCI2 were added to final concentrations of 5 and 10 mM, respectively. Samples were frozen and left at - 2 0 ° C for > 30 min, and were then thawed and spun for 1 min at 13000 × g. Ba 2"precipitable radioactivity was calculated as the difference in radioactivity of the supernatant with that of a control without Ba 2.. Under the conditions described, stock [35S]sulfate was precipitated for >99.8% (not shown).
Thin-layer chromatography. Analytical runs were performed on (i) HPTLC silica layers (Merck 60F254, height of plates 10 cm), solvent 1-butanol/acetic acid/H20, 6:2:2 (v/v), or (ii) 0.1 mm-polyethylene imine-(PEI-)cellulose F layers (Merck, height of plates 20 cm), solvent sat. (NH4)2504/1 M Na-acetate/2-propanol, 80:19:1 (v/v). In the former system, hydrophobic compounds move fastest; the latter system has anion-exchange properties [9]. After the solvent had ascended approx. 80% of the total height, plates were allowed to dry. For autoradiography we employed Kodak XO-Mat; films were exposed for periods ranging from overnight up to one month. Identification of low-molecular weight sulJate derivatives. APS and PAPS in methanolic amebal extract were identified by co-chromatography with the respective nonradioactive compounds (Sigma); the latter were visualized by fluorescence quenching under UV [9]. Also, 3-cholesteryl sulfate ('Y' in Fig. 2) was identified by co-chromatography with nonradioactive standard (Sigma); it was localized by staining with sulfuric acid in ethanol (10 g 1- '; ref. 9). As 3-cholesteryl sulfate is immobile on
73
the polyethylene imine layer (Fig. 2), colocalization was confirmed on a silica layer employing a different solvent (2-propanol/ chloroform/methanol/H20, 10:10:5:2 (v/v); results not shown). Monoethyl sulfate ('X' in Fig. 2) was isolated in9Pure form from the amebae. To this end, 10 cells were extracted with HCIO4 (5 M). The extract was neutralized with KOH, and KCIO4 was removed by centrifugation. To trace 'X', we added methanolic extract of cells incubated for 1 h with 355042- (see above). From this stage on, the degree of purification of 'X' was assessed by TLC followed by autoradiography and ninhydrin staining (most of the contaminants being ninhydrinpositive). The labeled mixture was freeze-dried and the residue was extracted with methanol (40 ml). The extract was kept overnight at - 2 0 ° C and the precipitate that had formed (mainly inorganic salt) was spun down and discarded. The supernatant was evaporated to dryness. The residue was dissolved in H20 (20 ml) and applied to an anion exchange column (AGIX8 from BioRad, 5 ml, in N O 3 - form). Upon elution with a linear gradient of KNO3 (0-1 M, 20 ml) one radioactive peak containing 'X' was obtained between 0.5 and 0.7 M KNO3. We further purified the material on a preparative silica layer (Merck 60F254s, eluted with l-butanol/acetic acid/H20, 6:2:2 (v/v)). The band containing 'X' was scraped off and extracted with methanol. The final yield of 'X' was approximately 3 mg. The compound was dissolved in 2H20 and subjected to IH-NMR spectroscopy. The spectrum turned out to be very simple, consisting of a triplet at 1.28 ppm and a quadruplet at 4.09 ppm with relative intensities of 1.60:1. From this, and the fact that 'X' was obviously a negatively charged sulfate derivative, we concluded that it must correspond to monoethyl sulfate. We confirmed this identification by chemical synthesis of monoethyl sulfate [10] followed by both ]H-NMR spectroscopy and co-chromatography with 'X' (results not shown).
h with [35S]sulfate (5 #Ci), washed twice with ice-cold PBS#8 and extracted with 1 ml buffer containing urea (7 M)/Triton X100 (5 g 1-i)/ NaCI (20 mM)/Tris-HCl (20 mM, pH 7.3) and iodoacetamide (2 mM). The extract was spun for 10 min at 40000 x g, and the supernatant was applied to an FPLC Superose-12 column (Pharmacia). Molecular weights were estimated by comparison with a standard protein mixture (Pharmacia).
Gel filtration of high-molecular weight sulfate derivatives. 3 x 10 cells were incubated for I
Fig. 1. Sulfate uptake by E. histolytica. O, Control; 0 , plus cytochalasin B (20 /~M). See Methods for details of experimental procedures.
Calculations. 5 × 107 cells correspond to 1 ml packed cell pellet, and contain 0.10 g protein. Extracellular space contributes 10% and the pinocytic vesicle compartment 40% to the packed cell volume [11]. Results
Sulfate uptake. [35S]Sulfate was taken up by the amebae in a time-dependent manner; in growth medium (sulfate concentration, 1.1 mM; see Methods) the initial rate of uptake was 1.5 /~mol h -1 (5 x 107 cells) -1 (Fig. 1). Generally, 2 routes contribute to uptake in E. histolytica: pinocytosis (indiscriminately for all solutes) and transport across the plasma I
i
1
1.0
~-
0.5
/ 1
I
I
20
40
60
time (rain)
74
membrane (for some solutes only) [12]. To discriminate between these two routes, we blocked pinocytosis with cytochalasin B [13]. This inhibited the initial rate of transport by about one-fourth (Fig. 1). We conclude that sulfate was taken up both by diffusion or transport across the plasma membrane and by pinocytosis, the former contributing about 3 times as much as the latter under these conditions.
Formation of low-molecular weight sulfate metabolites. We proceeded to investigate the intracellular fate of [35S]sulfate by HPTLC followed by autoradiography. Fig. 2 shows the pattern of radioactivity of a methanolic cell extract prepared 1 h after addition of [35S]sulfate to the cells. As analytical layers we employed both silica (left) and PEIcellulose (right). The patterns obtained were
A
B SO42"
approximately reciprocal: e.g., free sulfate, which remained at the origin in the left-hand system, moved with the front in the right-hand system. Strikingly, [35S]suifate as such was barely detectable in the extract: most of it had apparently been converted into 2 major and 2 or 3 minor derivatives. Two of the minor spots corresponded to APS and PAPS, respectively; these are the activated forms of sulfate normally found in eukaryotic cells. The most pronounced compound (marked 'X' in Fig. 2) was purified by anion exchange and preparative layer chromatography, and subsequently identified by its IH-NMR spectrum as monoethyl sulfate (see Methods for details). The second major spot, 'Y', was shown to correspond with 3-cholesteryl sulfate by co-chromatography with this compound (see Methods). One additional minor component ('Z') has not yet been identified.
Release of low-molecular weight sulfate metabolites. After addition of [35S]sulfate to the medium, the radioactive metabolites appeared in the cells in a time-dependent manner (Fig. 3). Clearly, the intensity of the spots was still
"X" "Z"
ChoI-SO4"..~ APS ~ EtSO 4-
ny. ~PAPS
APS "Z", P A P S
..... APS
,,X,i
S0420 SO42-
__
,~
~
~
,~,
Fig. 2. Autoradiographic HPTLC patterns of a methanolic extract from cells labeled for 1 h with 135S]sulfate. A, silica system; B, PEl-cellulose system. For further experimental details, see Methods.
15
30
60
min
Fig. 3. Time dependence of the appearance of radioactive sulfate metabolites. At the indicated times after addition of laSS]sulfate to the growth medium, cells were extracted with methanol and samples analyzed by HPTLC on silica as described in Methods.
75 A
B
Chol-SO 4" ~ _ _ APS EtSO 4"
S042-
~!
~ 0
15
30
60
120
mln
0
15
30
60
120
min
Fig. 4. Disappearance of radioactive metabolites from the cells into the medium. Cells were loaded for I h with [35S]sulfate, washed, and resuspended in unlabeled growth medium. At the indicated times, aliquots of suspension were centrifuged. Cells and medium were extracted with methanol and samples analyzed by HPTLC on silica. For further details, see Methods. (A) Cell extracts; (B) medium extracts. increasing after 1 h, which indicates that equilibration o f the m e t a b o l i t e pools was a relatively slow process. This will be c o n f i r m e d by q u a n t i t a t i v e d a t a presented below. F o r the next e x p e r i m e n t (Fig. 4), cells were incubated for I h with [35S]suifate, washed, and resuspended in n o n r a d i o a c t i v e g r o w t h m e d i u m . D u r i n g the next 2 h, radioactivity d i s a p p e a r e d f r o m the cells (Fig. 4A); c o n c o m i t a n t l y , m o n o e t h y l and cholesteryl ester, s o m e free sulfate, a n d up to 2 additional c o m p o u n d s a p p e a r e d in the m e d i u m (Fig. 4B). Since there was very little free sulfate in the cells at a n y time (Fig. 4A), the sulfate in the m e d i u m m u s t have been due to b r e a k d o w n o f metabolites.
High-molecular weight sulfate derivatives. In p r e p a r i n g the cell extracts for the a b o v e experiments, we noticed that a few percent o f the radioactivity i n c o r p o r a t e d by the cells was insoluble in m e t h a n o l (see T a b l e I). This suggested the presence o f h i g h - m o l e c u l a r weight sulfate derivatives. We therefore solubilized a m e b a e that had been loaded for one h with [35S]sulfate in detergent and subjected the solubilizate to gel filtration (see M e t h o d s for details). This w a y we o b t a i n e d three 35Scontaining peaks, o f 110, 42 and 10 k D a (results not shown). Quantitation of sulfate metabolites.
T o deter-
TABLE I Contents of radioactive metabolites in cells grown on [35S]sulfate Components Total a Methanol-soluble Ba2+-precipitable h Methanol-insoluble c
Low-molecular weight Free sulfate High-molecular weight
Content (/~mol (5
x
10 7
cells) ~)
3.8 + 0.4 3.4 + 0.4 < 0.2 0.36 + 0.12
Values are means (+ SD) for three cell suspensions, including the one used for Fig. 5. " Sum of values for methanol-soluble and insoluble fractions. b Sulfate was precipitated by Ba2- as described in Methods. The methanol-insoluble residue was washed once with 1 ml methanol, digested in 0.4 M NaOH and subjected to scintillation counting.
76
A
B
present in the growth medium than in the cells.
Discussion
C h o l S O 4"
--
EtSO 4-
-"+, ".r'~ j.'."
S042-
--
Fig. 5. Patterns of radioactivity from a cell suspension grown on [35S]sulfate. 10 ml growth medium was inoculated with 5 × l 0 4 cells; [35S]sulfate was added at 0.5 ~Ci (10 ml)- i. After 72 h, total cell count had increa~d to approximately 3 × 10 6. Cells and medium were extracted with methanol and samples analyzed by HPTLC on silica as described in Methods. (A) Cell extract; (B) medium extract.
mine the concentrations of intracellular sulfate and its metabolites, we equilibrated cells on [35S]sulfate by allowing them to grow in the presence of the isotope. From Fig. 5A it can be seen that also under these conditions monoethyl sulfate and cholesteryl sulfate were the major low-molecular weight sulfate derivatives. The ethyl derivative was clearly detectable in the growth medium (Fig. 5B); cholesteryl sulfate, which according to Fig. 4B should also be exported from the cells, may have been degraded by extracellular sulfatase activity. Table I summarizes the content of sulfate metabolites calculated from this and 2 other experiments. As judged from its precipitability by Ba 2~ less than 5% of cellular radioactivity was' associated with free sulfate; this confirms the qualitative picture supplied by the TLC patterns (Figs. 2-5). By the time the cells were harvested, approximately 6% of the amount of sulfate originally present in the medium had been converted into a Ba 2"soluble form (not shown) most of which should be monoethyl sulfate (Fig. 5B). At that stage, over 4 times more of this compound was
Overview of sulfate metabolism. Sulfate metabolism in E. histolytica appears to be relatively simple. As shown above (Fig. 1), sulfate was taken up both across the plasma membrane (about 75% ) and by pinocytosis (about 25%). At 1.5 /lmol h -~ (5 x 10 7 cells) 1 total sulfate uptake was relatively slow: two orders of magnitude slower than the uptake of glucose [14], but comparable to that of ieucine or methionine at an extracellular concentration of 1 mM (unpublished data). The content of free sulfate (0.4 l~mol (5 x 10 7 cells)- 1; ref. 12) if the vesicle compartment were impermeable: from this we conclude that most of the sulfate taken up by pinocytosis was subsequently transported into the cytoplasm, too. There, sulfate was transferred+ presumably after activation to APS and PAPS, to 2 major low-molecular weight acceptors, ethanol and cholesterol. The products, monoethyi sulfate and cholesteryl sulfate, reached relatively high (together >3 /~mol (5 x 10 7 cells)-~) steady-state concentrations. We have no indication as to the subcellular localization of these compounds, but we presume at least part was sequestered within the endocytic vesicles. Effiux of radioactivity was mainly in the form of monoethyl sulfate. At a doubling time of the cells of 12 h, some 20% of the sulfate taken up would be needed for the growth-associated expansion of the different sulfate ester pools; these include the 3 high-molecular weight components mentioned above. We should stress again that the data presented apply to cells incubated in growth medium. Amebae growing within the human host will probably contain the same set of sulfate metabolites, but their concentrations could well be very different. Function ~[+sulfate esters. Remarkably, over 90% of the sulfate taken up was converted to
77
either monoethyl sulfate or cholesteryl sulfate. Sulfate conjugation often plays an important role in the detoxication of noxious organic compounds [4]. We therefore considered the possibility that the formation of ethyl and cholesteryl sulfate in the amebae could serve a similar function. Entamoeba depends on externally-added cholesterol for growth [15]. In growth medium, this cholesterol is recruited from the cholesteryl esters present in serum lipoproteins. In this context we propose that cholesteryl sulfotransferase activity could have a function in removing any surplus membrane cholesterol; this way it could supplement other regulatory mechanisms such as the adjustment of the ratio of saturated to unsaturated fatty acids [ 16]. By contrast, a similar role for ethyl sulfurylation seems improbable. Amebae generate ethanol by fermentation [1-3]. In addition, growth medium contains about 10 mM ethanol (added as a solvent for lipid-soluble vitamins; refs. 6, 7). However, as the cells grow well on this mixture, this obviously does not excessively bother them. Moreover, if intended as a mechanism of detoxication, sulfurylation of ethanol would be extremely ineffective: less than 1% of the ethanol initially present was converted into the sulfate ester over a 3-day time course. Therefore, rather than looking for a function, we surmise that the sulfotransferase for cholesterol may accept ethanol as an alternative substrate; this would be in line with the range of specificities established for other sterol sulfotransferases [17]. If so, monoethyl sulfate would be an inherently wasteful product. We estimate, assumin8 a rate of uptake of glucose of/> 120 pmol h - " (5 x 107 cells) -1 [14], a yield of up to 7 ATP per glucose [1] and an investment of 3 ATP per ethyl sulfate synthesized, that cells maintained in growth medium continuously invest up to 0.5% of the energy they generate into its synthesis. Finally, a few more words on the highmolecular weight sulfate derivatives. We have proposed [12] that, similar to the situation in mast cells and natural killer cells [5], proteo-
glycan sulfates may serve in the retention of amebapain (and possibly other soluble digestive enzymes) upon exocytic fusion of intracellular vesicles with the plasma membrane. In this context it will be important to further characterize the high-molecular weight material and to assess its binding properties.
Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft (SFB 171 Project C2) and by the Fonds der Chemischen Industrie. We thank Dr. Helmut Rosemeyer for N M R spectroscopy, Dr. Willi Hoppe for invaluable advice, Mr. Andr6 Schlochtermeyer for help with the FPLC and Mrs. Dietlind Henrichs for excellent technical assistance.
References 1 Reeves, R.E. (1984) Metabolism of Entamoeba histolytica Schaudinn, 1903. Adv. Parasitol. 23, 105142. 2 McLaughlin, J. and Aley, S. (1985) The biochemistry and functional morphology of the Entamoeba. J. Protozool. 32, 221-240 3 Avron, B. and Chayen, A. (1988) Biochemistry of Entamoeba: a review. Cell Biochem. Funct. 6, 71-86. 4 Jakoby, W.B. and Ziegler, D.M. (1990) The enzymes of detoxication. J. Biol. Chem. 265, 20715-20718. 5 Fransson, L.-A. (1987) Structure and function of cellassociated proteoglycans. Trends Biochem. Sci. 12, 406 411. 6 Diamond, L.S., Harlow, D.R. and Cunnick, C.C. (1978) A new medium for axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans. R. Soc. Trop. Med. Hyg. 72, 431-432. 7 Diamond, L.S. (1988) Cultivation of Entamoeba histolytica in vitro. In: Amebiasis. Human infection by Entamoeba histolytica (Ravdin, J.l., ed.), pp. 27-40. Wiley, New York. 8 Sorbo, B. (1987) Sulfate: turbidimetric and nephelometric methods. Methods Enzymol. 143, 3-6. 9 Stahl, E. (1969) Thin-Layer Chromatography. A Laboratory Handbook. Springer, Berlin. I0 Lloyd, A.G., Tudball, N. and Dodgson, K.S. (1961) Infrared studies on sulphate esters, III. O-Sulphate esters of alcohols, amino alcohols and hydroxylated amino alcohols. Biochim. Biophys. Acta 52, 413-419. II L6hden, U. and Bakker-Grunwald, T. (1989) Magnetic separation of pinocytic vesicles of defined age from Entamoeba histolytica. Anal. Biochem. 182, 77 83. 12 Bakker-Grunwald, T. (1991) Transport and compartmentation in Entamoeba histolytica. In: Biochemical
78 Protozoology as a Basis for Drug Design (Coombs. G.H. and North. M., eds), pp. 367 376. Taylor and Francis, London. 13 Bakker-Grunwald, T., L6hden, U. and Trissl, D. (1985) Effects of cytochalasin B on N a ' content and cell volume of Entamoeba histolytica. Biochim. Biophys. Acta815, 170 174. 14 Serrano, R. and Reeves, R.E. (1974) Glucose transport in Entamoeba histolytica. Biochem. J. 144. 43-48. 15 Latour, N.G., Reeves, R.E. and Guidry, M.A. (1965)
Steroid requirements of Fntamoeha histolyt&.a. Exp. Parasitol. 16, 18 22. 16 Van Vliet, H.H.D.M., Op den Kamp, J.A.F. and Van Deenen, L.I,.M. (1975) Lipid auxotrophy and the effect on lipid composition of E. invadens. J. Protozool. 22, 428 432. 17 Lyon. E.S. and Jakoby, W.B. (1980) The identity of alcohol sulfotransferases with hydroxysteroid sulfotransferases. Arch. Biochem. Biophys. 202, 474--481.
