Cadmium-thionein in Tetrahymena thermophila and Tetrahymena pyriformis

Europ.J.Protisto!. 26,1 76-1 81 (1990) October 19, 1990

Eu ropea n Journal of

PROTISTOLOGY

Cadmium-thionein in Tetrahymena thermophila and Tetrahymena pyriformis Ester Piccinni, Paola Irato and Laura Guidolin Dipartimento di 8iologia,

Univerut« di Padova, Padova, Italia

SUMMARY Th e treatm ent of Tetrahymena thermophila with cadmium causes a reduction in growth rate accord ing to dose ; almo st all the metal is accumulated in the cytosol where th e Zn content is also increased threefold . Bio-Gel and Water I 60 (HPLC) column chromato graph y show that Cd and Zn are bound to a protein with an ultra violet (UV) spectrum that appears to be similar to that of Cdmetalloth ioneins isolated by higher organisms, but its molecular weight is greater: about 28 000 D, comparable to that of metallothionein isolated from Tetrahym ena pyriformis. Further pur ification of these proteins by ion exchange chromatograph y revealed the presence of two peaks, con sidered as two isoforms of the metallothioneins present in both T. thermophila and T. pyriformis (MT 1 and MT 2). Their amino acid analyses confirm ed that they are different isometallothioneins, MT 1 and MT 2, with about 30 % cysteine, and aspa rtic acid, glycine and lysine as major amino acids. From our analyses we may conclud e th at Tetrahymena pyriformis MTs are similar to those present in invertebrates and vertebrates, while Tetrahymena thermophila MTs are peculiar in that they have cyclic amin o acid histidine in both MT 1 and MT 2; furthermore, aromatic amino acid phenylalanin e is also present in MT 2.

Introduction Metallothioneins (MTs) are a class of protein occurring in the animal kingdom , fungi and plants as well as in eukaryotic and prokaryotic micro-organisms [3]. MTs have been shown to be indu ced by heavy metals, mostly Cd, Zn and Cu, and to be respon sible for the accumulation of these metals in cells. Thu s, MTs are believed to play an important role in detoxification, although their general role in the metabolism of essential and non -essential metal s cannot be excluded [1,4]. Although the toxic effects of heavy metals on protozoa are well-documented, the occurrence of MT s or other binding molecules in these organisms is little known [7, 10, 11]. The effects of Cd on Tetrahym ena pyriformis have been reported in man y pape rs [8]. Furthermore, it has been demonstrated that Cd induces a chelating molecule with an unusually high molecular weight, about 24 000 D, with an amino acid compo sition very similar to that of MTs described for mamm als [11]. 0932 -4739/90/0026-01 76$3.50/0

With the aim of verifying if a Cd-linking protein with a similar molecular weight is common in the genus Tetrahymena, we chose another organism of the Tetrahymena complex, Tetrahym ena thermophila, and treated it with Cd to induce the formation of a Cd-bind ing protein. Data on the accumul ation of Cd and characteristics of the protein found in Tetrahym ena thermophila are described here and compared to tho se of Tetrahym ena pyriformis. Material and Methods

Cell Preparation Tetrahymena thermophila strain B VII and T etrahym ena pyriformis GL were grown axe nically in the same medium, as previously reported, at 32 °C and 25 °C, respectively [11]. Cd was added to the medium as CdCl2 x 2.5 H 20 at a final concentra tion of 2 ~g Cd/ml. Cell density was determined by counting the cells in a Biirker cha mber. © 1990 by Gustav Fischer Verlag, Stu ttgar t

Metallothionein in Tetrahymena Cells harvested by centrifugation on the second or third days of culture were rinsed in 50 mM Tris-HCl buffer, pH 7.5, then homogenized in the cold in a Polytron homogenizer in the same buffer with 5 mM ascorbic acid added. The homogenate was centrifuged at 48 000 g for 2 h at 5 °C and the pellet was washed with the same buffer.

Isolation of Proteins The supernatants thus obtained were lyophilized and the MTs were purified by a combination of gel filtration, ion-exchange chromatography. Columns of Bio-Gel P-60 (2.6 em X 95 ern) and Bio-Gel P-30 (1.6 ern x 95 em), previously equilibrated with 0.1 M Tris-HCl buffer, pH 8, were used. Elution fractions were collected in 6.1 or 3.5 ml samples, respectively, with an LKB fraction collector. During gel chromatography the elution buffer was continuously bubbled with N 2 • Proteins were monitored either using an LKE 2138 Uvicord S or a Zeiss PMQ spectrophotometer at 280 or 254 nm. Folin-phenol reagent [6] was also used. The Bio-Gel P-30 metal-linking fractions were applied on a Pharmacia FPLC mod. LCC 500 equipped with a Mono-Q ion-exchange column in 10 mM Tris-HCl, pH 7.5, and 10 mM Tris-HCl plus 400 mM NaCl, pH 7.5, by using stepwise gradients. The flow rate was 1 ml/min. For determination of the apparent molecular weight, aliquots of 250 ul of metal-linking fractions from Bio-Gel P-30 were processed on a Beckman HPLC mod. 341 equipped with a Waters Protein PI-60 column. The elution buffer was 10 mM Tris-HCl, 0.2 M NaCl, pH 7.5, at a flow rate of 0.5 ml/min. Calibration runs were carried out with ovoalbumin (mol. wt 44000 D), myoglobin (mol. wt 17800 D), ribonuclease A (mol. wt 13 700 D) and bacitracin (mol. wt 1450 D). UV spectral analyses were performed with a Kontron mod. Uvikon 860 spectrophotometer in 10 mM Tris-HCl at pH 7.5.

>

177

40 min, 15 h 36 min in the presence of 2 ftg and 3 ug of Cd/ml, respectively (Fig. 1). Treatment with 2 ug Cd/ml for 2 days was chosen as the experimental condition for other analyses. Table 1 shows that Tetrahymena accumulates Cd; it is almost totally present in the soluble fraction, which contains more than 90% of the total metal in the homogenate. Purification and Metal Binding Proteins Cell-free extracts from treated cultures were subjected to chromatography on Bio-Gel P-60 and Bio-Gel P-30. Bio-Gel P-60 gives two peaks, one eluting near void volume (Vo); the second, eluting between 180 and 200 mls at VeNo 1.4, shows a higher absorbance at 254 nm

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Metal Analysis Cd and Zn were measured by atomic absorption spectroscopy with a Perkin-Elmer mod. 4000 spectrophotometer. The metals of homogenates and sediments were measured after wet ashing in concentrated HN0 3, (AristaR grade 60%) in a teflon bomb. All measurements were corrected for background absorption by using reagent blanks.

Amino Acid Analysis Amino acid analyses were performed on dialysed and hydrolysed samples, after performic acid oxidation, with 6 M HCl for 24 h, according to Hirs [2]. Samples were assayed on an LKB model 4101 analyser and a Carlo Erba mod. 3A30 analyser. The most likely set of residues and the range of molecular weights of the chain were obtained from comparison of four amino acid analyses. The data were handled by an Olivetti M 20 computer, using statistical procedures.

Results Growth Curve and Accumulation The growth curve of Tetrahymena thermophila shows that Cd reduces growth rate according to concentration. Reduplication time during the logarithmic phase is at about 3 h 36 min in the controls. It is enhanced up to 8 h

E

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5

3

2

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days

Fig. 1. Growth curves of Tetrahymena thermophila cells. • - control; 6. - 2 Itg Cdlml; 0 - 3 ug Cd/ml. Table 1. Metal content in control and treated cells after two days of culture (ltg!g dry wt) Cd Homogenate Sediment Control 2 Itg Cdlml 460

45

Zn Homogenate Sediment 70 200

200 150

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mls Fig. 2. Bio-Gel P-60 chromatography of cell-free extract of Cd-treated cells of T. thermophila. Column eluted with 0.1 M Tris-H Cl buffer, pH 8. Vo = void volume; Ve = elution volume. Cd is linked to peaks eluting at VeNo 1.4. Fractions between bars were collected.

(Fig. 2). Almost tot al Cd is associated with this peak, which was subsequently applied on a Bio-Gel P-30 column. The elution pattern is shown in Fig. 3. The metal-linking prot ein elutes as a single peak containing abo ut 22 ug Cd/mg pro tein and 2.7 ug Zn/m g protein. Mo lecular weight was also estimated by HPLC using a Water I 60 column . A single peak, correspo nding to an apparent molecular weight of 28000 D, was obtained (Fig. 4). The UV absorption spectrum of this prot ein is shown in Fig. 5. It shows low absorbance at 280 nm due to a very low content of aromatic amino acids, and a bro ad shoulder at 254 nm due to Cd-rhiolate coordination. Further purification analyses were performed on this fraction by FPLC using a Mono Q ion-exchange column (Fig. 6). The pooled peaks from Bio-Gel P-30 were resolved by this method into two main peak s. Most of the Cd (90%) is linked to fraction s 1 and 2 eluting at 15 and 17% NaCl respectively. Their UV absorbance spectrum is typical of classic metallothi oneins, and these two peaks were thus considered as isothioneins MT 1 and MT 2 of T. th ermophila. The fractions between the bars, each conta ining 25 ~g Cdlmg protein, were submitted to amino acid analysis. The data show n in

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retent ion t ime (m in s)

Fig. 4. HPLC chromatography of Tetrahymena thermoph ila Cd, Zn-thionein. Column (Water 1 60) eluted with 10 mM Tris-HCl buffer, pH 7.5, containing 0.2 M Na Cl. Single peak eluted with retention time of 28 000 D molecular weight.

Metallothionein in Tetrahymena . 179 1.0

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Table 2 give high values of cysteine, the absence of tyrosine but the presence of histidine for both MTs, and small amounts of phenylalanine in MT 1. Converting the analyses to the near est integer for each amino acid residue gives a minimum molecular weight of between 28 900 D and 26 800 D for MT 1 and 25700 D and 25 000 D for MT 2.

Tetrahymena pyriformis GL Similar analyses were performed on the metallothionein previously isolated from Tetrahym ena pyriformis [11]. The peak obtained from Bio-Gel P-30 was chromatographed by ion exchange Mono Q column (FPLC). The elution profil (Fig. 7) indicates the presence of two main peaks, eluting at approximately 16 and 20 % NaCl, containing about 70% Cd. Both peaks show UV absorbance spectra similar to those of classical MTs and link 23 and 33 f!g Cd/mg protein. The y may thu s be cons idered as two Tetrahymena pyriformis Cd-isothioneins MT 1 and MT 2. Preparative isolation of these two peaks and subsequent amino acid anal yses (Table 3) confirmed that they are different proteins, as shown by comparing the respective leucine and isoleucine values. The minimum molecular weights are calculated between 24 700 D and 22600 D for both proteins.

Fig. 6. Ion-exchange chromatography of T. thermophi/a Cd, Zn-thionein. Bio-Gel P-30 fraction was applied to a Mono Q column (FPLC) and eluted with a salt gradient: 10 mM Tris-HCl plus 400 mM NaC!, pH 7.5. Flow rate: 1 ml/rnin, Fractions between bars were collected and submitted to amino acid analysis.

Discussion The dose of 2 f!g Cd/ml, which induce s some inhibition of the growth rate of T. thermophila, is rather low when compared with the doses tolerated by T. pyriformis. In fact, we demonstrate that the latt er ciliate can be cultured without damage in the presence of 10 ug Cd/ml and that some inhibition of growth accompanied by ultrastructural alteration of mitochondria and nucleoli occurs at of 15 ug Cd/ml [5]. Although the growth of T. thermophila is faster than that of T. pyriformis (reduplication times 3 h 36 min and 7 h, respectively), the dose tolerated by T. pyriformis (10 f!g Cd/ml) is five times higher than that inducing some inhibition of growth in T. therm ophila. Despite the higher sensitivity of fast-growing cells to external factor s, the reported differences of the respon se of the two strains seems to indicate a lower tolerance of T. thermophila in our experimental conditions. However, onl y more specific experiments can confirm this observation.

180 . Piccinni et al. Table 2. Amino acid composition of Tetrahymena thermophila isometallothioneins 1.0

Amino acid Cys"

Asx Thr Ser Glx Pro Gly Ala Val Met He Leu Tyr Phe His Lys Try Arg

MT2

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III

...0

.0 III

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«

Total mol. wt HPLC mol. wt

MT-1 Nearest integer

Mol%

MT-2 Nearest integer

Mol%

63-59 29-28 21-20 20-17 21-18 9-8 21 14-12 5-4

25.33 11.85 8.38 7.66 8.24 3.7 8.81 5.33 2.01

67-61 26-25 14 15-14 13 10 18-17 9-8 4-3

30.42 12.29 6.67 6.76 6.25 4.76 8.23 4.15 1.63

4-3 3

1.45 1.35

5-4 10 24-23

1.83 4.11 9.94

249-230 28979-26782 28000

1 3

0.64 1.38

7 29-27

3.39 13.43

216-203 25685-24092 28000

* Calculated as cysteic acid. 4

8

12

16 mins

20

24

28

Fig. 7. Ion-exchange chromatography of T. pyriformis Cd, Zn-thionein. Fraction from Bio-Gel P-30 applied to Mono Q column (FPLC). All conditions as given in Fig. 6.

Table 3. Amino acid composition of Tetrahymena pyriformis isometallothioneins Amino acid

The same dose of 2 ug Cd/ml is sufficient to induce accumulation accompanied by an increase of Zn in the cells of T. thermophila. This synergistic effect is welldocumented for many organisms and may be explained by the enhancement of binding sites in the cells.This dose is in fact sufficient to induce the formation of Cd-Zn chelating molecules with about 30% cysteine and UV profiles similar to those of typical MTs, but the molecular weight of this protein is very high and unusual for MTs isolated from other invertebrates and vertebrates. We have already isolated a high molecular weight MT from another ciliate of the Tetrahymena complex, T. pyriformis: the probability that the native MTs in these protozoa are polymeric forms of 6000 D has been discussed [11]. Further purification of these compounds by ionexchange chromatography indicates the presence of two isoproteins in both T. pyriformis and T. thermophila. Different isoproteins are reported in pluricellular organisms. Their presence is related to the different tissues of each individual animal, or to heterogeneity of animals containing different protein patterns. The presence of isometallothioneins in Tetrahymena thermophila B VII

Cys* Asx Thr Ser

Glx Pro Gly Ala Val Met He Leu Tyr Phe His Lys Try Arg Total mol. wt HPLC mol. wt

MT-1 Nearest integer

Mol%

MT-2 Nearest integer

Mol%

58-53 29-27 17-16 18-17 15-14 7-6 23-21 9-8 5-4

27.09 13.56 8.24 8.36 7.05 3.09 10.70 4.01 2.27

62-57 29-28 18-17 16-15 18-17 7-6 25-23 9-8 4

29.14 13.88 8.69 7.61 8.45 3.31 11.75 4.23 2.06

2 3-2

0.80 1.19

29-27

13.64

23-22

10.89

215-197 24741-22686 24000

* Calculated as cysteic acid.

211-196 24340-22613 24000

Metallothionein in Tetrahymena . 181

and Tetrahymena pyriformis GL must be considered as an intrinsic characteristic of the strains. The main difference between the two isoproteins from Tetrahymena pyriformis regards the presence of longchain aliphatics in MT 1 (isoleucine and leucine), residues rarely found in other MTs. The amino acid compositions of MTs from T. pyriformis referred to a molecular weight of 6000 D are very similar to those of MTs of invertebrate and vertebrate organisms [10]. The composition of MTs from T. thermophila is unique. Beside isoleucine and leucine residues - at higher % in MT 1 - they both have some histidine; in addition, MT 1 also has phenylalanine, residues not found in other eukaryotic organisms. The amino acid composition of this protein seems to be comparable to that of cyanobacterial MT isolated from Synechococcus [9]. Reverse-phase methodology applied on these isothioneins are in progress to permit further studies on these particular thioneins. In conclusion, our present data indicate that, in the ciliate Tetrahymena, Cd induces peculiar chelating proteins, similar to the MTs of other organisms but having high molecular weight and some aliphatic residues. In addition, the MTs isolated from T. thermophila are unique, since they also have aromatic and cyclic residues which make these proteins comparable to those of prokaryotic organisms.

Acknowledgements We thank Dr. Hellung-Larsen, Department of Biochemistry B, Panum Institute, University of Copenhagen, for providing the strain of Tetrahymena thermophila B VII. This work was financed by a grant from the Ministero Pubblica Istruzione and Contr. C.N.R. 87.02953.04.

References 1 Albergoni V. and Piccinni E. (1983): Biological response to trace metals and their biochemical effects. In: Leppard G. G. (ed.): Trace element speciation in surface waters and its ecological implications, pp. 159-175. Plenum Publishing Corporation. 2 Hirs C. H. W. (1967): Determination of cystine as cysteic acid. In: Hirs C. H. W. (ed.): Methods in enzymology, vo!' 11, pp. 59-62. Academic Press, New York. 3 KagiJ. H. R. and Kojima Y. (1987): Chemistry and biochemistry of metallothionein. In: Kagi J. H. R. and Kojima Y. (eds.): Metallothionein II, pp. 25-61. Birkhauser Verlag, Basel-Boston. 4 Kagi J. H. R. and Schaffer A. (1988): Biochemistry of metallothionein. Biochemistry, 27, 8509-8515. 5 Irato P. and Albergoni V. (1989): Response of Tetrahymena pyriformis to high doses of Cd. J. Protozoo!., 36, 39. 6 Lowry O. H., Rosebrough N.]., Farr A. L. and Randall R. J. (1951): Protein measurements with the Folin-phenol reagent. J. Bio!' Chem., 193, 265-275. 7 Nagano T., Miwa M., Suketa Y. and Okada S. (1984): Isolation, physicochemical properties, and amino acid composition of a cadmium-binding protein from cadmiumtreated Chlorella ellipsoidea. J. Inorg. Biochem., 21, 61-71. 8 Nilsson J. R. (1989): Tetrahymena in cytotoxicology, with special reference to effects of heavy metals and selected drugs. Europ. J. Protisto!', 25, 2-25. 9 Olafson R. W. (1986): Physiologicaland chemical characterization of cyanobacterial metallothioneins. Environ. Health Perspect., 65, 71-75. 10 PiccinniE., Coppellotti O. and Guidolin L. (1985): Chelatins in Euglena gracilis and Ochromonas danica. Compo Biochem. Physio!', 82C, 29-36. 11 Piccinni E., Irato P., Coppellotti O. and Guidolin L. (1987): Biochemical and ultrastructural data on Tetrahymena pyriformis treated with copper and cadmium. J. Cell Sci., 88, 283-293.

Key words: Tetrahymena thermophila B VII - Tetrahymena pyriformis GL - Metallothionein - Cadmium Detoxification Ester Piccinni, Dipartimento di Biologia, Universita di Padova, Via Trieste, 75, 35121 Padova, Italia