Apparent deficiency of metallothionein in the liver of the Antarctic icefish Chionodraco hamatus. Identification and isolation of a zinc-containing protein unlike metallothionein

Comp. Biochem. Physiol.Vol. 103B,No. 1, pp. 201-207, 1992 Printed in Great Britain

0305-0491/92$5.00+ 0.00 © 1992PergamonPress Ltd

APPARENT DEFICIENCY OF METALLOTHIONEIN IN THE LIVER OF THE ANTARCTIC ICEFISH CHIONODRACO HAMATUS. IDENTIFICATION AND ISOLATION OF A ZINC-CONTAINING PROTEIN U N L I K E METALLOTHIONEIN ROSARIASCUDIERO,PIETROPAOLODE PRISCO, LAURACAMARDELLA,ROSSANAD'AvINO, GUIDO DI PRISCO and ELIO PARISI* CNR Institute of Protein Biochemistry and Enzymology, via Marconi 10, 80125 Napoli, Italy. Tel.: 081-725-7111; Fax: 081-636-123 (Received 28 January 1992; accepted 6 March 1992) Abstract--1. A zinc-bindingprotein has been isolated and purified from the liver of the icefish Chionodraco hamatus. 2. The icefish Zn-protein has characteristics distinct from those of metallothionein. 3. The amino acid composition shows a low content of cysteine and a high content of glutamate and aspartate. 4. No metallothionein has been detected in the extracts from icefish liver.

INTRODUCTION

Zinc is known to be an essential nutrient for all living systems. Usually zinc binds via metal thiolate bonds to cysteyl residues of proteins named metallothioneins (MT) present in most cells (Hamer, 1986; Kiigi and Nordberg, 1979). In order to be classified as MT, a protein must fulfil the following requirements: molecular weight of 6000-7000, high metal and cysteine contents, unique amino acid sequence including a fixed distribution of cysteyl residues, no aromatic amino acids, nor histidine (Kiigi and Kojima, 1987; K~igi and Schiiffer, 1988). Although metal-binding proteins from several organisms meet the required specifications for MT, evidence shows the existence in mammalian and non-mammalian species of metal-binding proteins with properties that exclude their classification as MT (Stone and Overnell, 1985). Metal-binding proteins lacking the features of MT have been detected in the gonads of mammals (Waalkes and Perantoni, 1986; Deagen and Whanger, 1985; Waalkes et al., 1988a, b and c) and in the ovaries of spotted sea trouts and Atlantic croakers (Baer and Thomas, 1991). These proteins have a low molecular weight and contain a low amount of cysteine and a high amount of glutamate. The physiological role of these metal-binding proteins is not known at the moment, nor is it known whether they are present in other vertebrate tissues as well. We have isolated a metal-binding protein from the liver of the Antarctic fish Chionodraco hamatus. This organism is one of the species of the family Channichthyidae (also known as "icefish"). The 16 species of this largely endemic family have remained *To whom correspondence should be addressed.

isolated south of the Antarctic Convergence. This oceanic frontal system, currently running between 50°S and 60°S, was developed approximately 25 million years ago, following the opening of the Drake Passage (Barker and BurreU, 1977; Kennett, 1977). The most striking peculiarity of icefish is the total lack of haemoglobin in the colourless blood (Ruud, 1954). The results of the present study show that the main low molecular weight metal-binding protein in C. hamatus liver is not an MT, but a component with characteristics similar to gonadal metal-binding proteins. MATERIALS AND METHODS Chemicals Dithiothreitol, phenylmethylsulfonykfluorideand standard proteins for PAGE were from Sigma Chemicals Co. (St Louis, MO); acrylamide, bis-acrylamide, sodium dodecylsulfate from Bio-Rad (Richmond, CA). All other reagents were of analytical grade. Animals and preparation of acetone powders Adults specimens of Chionodraco hamatus were collected by means of gill nets, set 100 m-deep on the sea floor in the proximity of Terra Nova Bay Station (Italy, 74° 42' S, 164° 07' E); fish were kept in aquaria supplied with running, aerated sea-water at approximately - 1°C. Adult samples of sea-ecl (Conger conger) were collected in the bay of Naples and kept in aquaria at a temperature of approx. 18°C. Livers were quickly removed and homogenized in 5 vol of acetone prechilled at -20°C. The homogenate was filtered on a Buchner funnel using a Whatman 3 MM paper filter. The resulting residue was extracted three times in the same way. The final residue was dried and stored at 4°C. All the following procedures were carried out at 0-4°C. One gram of acetone powder was extracted by homogenization with 10 ml of 50 mM Tris-HCl buffer pH 8.6 containing 2 mM dithiothreitol, 100#M phenylmethylsulfonylfluorideusing an Ultraturrax cell homogeniser (5 strokes of 2 min each). The extract was centrifuged at 10,000g for 30 min and the

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resulting supernatant was centrifuged again at 100,000g for 1 hr. Gel filtration chromatography

Five millilitres o f the 100,000g supernatant, containing approximately 70 mg protein, were loaded on a Sephadex G-75 column ( l . 8 x 3 6 c m ) equilibrated with 10mM Tris-HC1 buffer pH 8.6 containing 2 m M dithiothreitol and 100gM phenylmethylsulfonylfluoride. The column was eluted with the same buffer at a flow rate of 1 ml min-L Fractions of 1 ml were analysed for zinc and protein content. The elution distribution coefficient K D was determined according to the equation K o = ( V e - V o ) / ( V t - Vo), where Ve is the elution volume, V0 the void volume, and Vt the column volume.

Ion-exchange chromatograph),

The fractions collected from gel filtration were pooled and directly loaded on a column of DEAE-Sephadex A-25 (I x 10cm) equilibrated with the same buffer used in gel filtration. The column was washed with three volumes of equilibration buffer and developed with a linear gradient from 0 to 400 m M NaCI in equilibration buffer at a flow rate of 1 ml min i. The eluate was monitored for the absorbance at 280 nm and each fraction was analysed for zinc content. Anion-exchange F P L C

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desalted sample was loaded on a Mono-Q HR-5/5 FPLC column 0aharmacia, Uppsala, Sweden) equilibrated with 10mM Tris-HCl pH 8.6. The column was washed with equilibration buffer and developed with a linear gradient from 0 to 500 mM NaC1 at a flow rate of I ml min- i. The eluate was monitored at 280 nm. Fractions of 1 ml were analysed for zinc content.

with an extract prepared from sea-eel liver showed a peak of zinc and thiol groups at the position of MT (Fig. 2). The C. hamatus Zn-containing protein, eluted in the low molecular weight region of the Sephadex column, was chromatographed on a DEAE-Sephadex A-25 column giving a single symmetrical peak of Zn-bound component which eluted at 0.2 M NaCI (Fig. 3). The protein recovery based on the amount of zinc was between 40 and 60%. The pooled peak from DEAE-Sephadex was dialysed, and further purified by ion-exchange FPLC using a Mono-Q column. The elution profile obtained (Fig. 4) showed several zinc-containing fractions, suggesting microhetcrogencity similar to that reported for MT (Hunziker and K/igi, 1985). The major metalcontaining protein fraction was concentrated and used for further characterisation. SDS--PAGE of this protein showed a single band. The molecular weight was calculated to be 11,000 (Fig. 5). The amino acid composition of the protein isolated by ion-exchange FPLC confirmed the results obtained with the thiol-group reagent. The C. hamatus zinccontaining protein has a low cysteine content, with glycine, aspartate and glutamate as the predominant amino acids. Table 1 reports the amino acid composition of the icefish protein together with that of zincbinding proteins from other sources. The results of cluster analysis on these data (Fig. 6) show that the icefish protein, the gonadal zinc-binding proteins and the selenium-binding protein from ovine heart are together, apart from MT. Within this cluster it is possible to identify various subclusters. In general, proteins from the same source show a tendency to group together in the same subcluster.

SDS-PAGE

SDS-PAGE was performed according to Laemmli (1970). Slab gels contained a linear gradient from 9 to 12% acrylamidc in 8 M urea. The gels were run for 5 hr at 35 mA and stained with silver stain (Otsuka et al., 1988). CNBr myoglobin fragments were used as molecular weight standards. Data analysis

Cluster analysis was applied to amino acid composition data using the single linkage method with Pearson correlation coefficient as distance metric (Wilkinson, 1989). Other methods

Zinc content was determined by either Plasma Emission Spectrometry (Beckman) or Atomic Absorption Spectrometry (Perkin-Elmer). Thiol group determination was carried out according to Ellman (1959). Protein was determined according to Lowry et al. (1951). Amino acid analysis was performed either on a Carlo Erba model 3A29 automatic analyzer or on an Applied Biosystems model 420 derivatizer equipped with an automatic hydrolysis station; cysteine was determined as cysteic acid after performic acid oxidation (Hirs, 1967). RESULTS

The elution profile for the icefish liver extract run through a Sephadex G-75 column is shown in Fig. 1. Two peaks of zinc were eluted: the first with the void volume, the second at a KD value of 0.74. Horse kidney MT used as standard eluted at a KD value of 0.31, which is typical for MT (Vasak et al., 1984). The analysis by Eliman reagent on the low molecular weight fractions elutexi from the column did not detect thiol groups at the position where MT was expected to elute. In comparison, the elution profile obtained

DISCUSSION

The results reported in the present paper show that the characteristics of the major low molecular weight

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zinc-binding protein extracted from icefish liver are different from those of MT. The apparent deficiency of MT in C. hamatus liver seems to be a peculiarity of this species, since liver is usually considered a major site of MT synthesis (McCornic et al., 1981; Sato et al., 1984). In addition, metal-binding proteins

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related to mammalian MT have been identified in the liver of many groups of fishes (Overnell and Coombs, 1979; Kito et al., 1982; Hidalgo and Flos, 1986). It is possible that in C. hamatus the MT gene is either absent or expressed at a very low level.

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Fig. 5. (a) SDS PAGE of the icefish zinc-containing protein. The electrophoresis was carried out for 5 hr at 35 mA. Lane I: C. hamatus Zn-binding protein (DEAE-Sephadex A-25 step); lane II: CNBr myoglobin fragments; lane III: lysozyme (mol. wt 14,400)+ horse kidney MT (mol. wt 7000); lane IV: C. hamatus extract (low molecular weight fractions from the Sephadex G-75 step). (b) Molecular weight of the zinc-containing protein estimated on the basis of SDS-PAGE shown in (a). (S) Sample; (1) Myoglobin (mol. wt 16,950); (2) Fragment I + II (tool. wt 14,400); (3) Fragment I (mol. wt 8160); (4) Fragment II (mol. wt 6210); (5) Fragment III (mol. wt 2510).

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Amino acid Asp Glu Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys

Ieefish (tool%) 10.92 17.57 5.98 11.99 1.94 1.89 5.43 6.94 8.00 1.98 5.68 1.63 4.75 4.12 5.10 2.05 3.88

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Table 1. Aminoacidcompositionof zinc-bindingproteinsfrom ieefishand other sources Hamster Sea trout Croaker Croaker Ovine Rat Rat Rat ovary* ovary03* ovaryCI* Ovary C3" Heart? TestesI~ TestesII:~ MT I:~ (tool%) (mol%) (tool%) (tool%) (tool%) (mol%) (mol%) (tool%) 9.67 17.83 10.17 5.33 0.00 1.33 6.17 6.67 5.67 0.00 1.53 1.67 8.67 3.00 3.17 1.67 14.83

10.63 16.09 I 1.56 14.38 2.97 3.59 7.19 6.72 1.56 0.00 3.28 2.19 7.50 3.28 6.87 3.44 1.72

9.34 14.59 9.02 6.88 2.13 4.59 4.59 7.05 5.41 1.97 7.54 1.47 4.59 3.28 8.69 3.11 4.75

10.54 14.36 7.64 8.18 0.91 1.64 4.91 4,54 3.45 1.45 5.45 0.91 3.82 4.73 7.82 4.91 8.18

10.80 10.70 6.40 9,30 3.90 4.60 4.90 6.20 5.10 2.40 7.20 2.50 0.30 3.50 7.70 5.40 9.10

6.98 10.56 8.12 12,10 2.56 3.98 5.58 8.77 4.38 1.49 6.05 2.56 7.30 4.67 6.84 2.39 7.65

9.78 11.94 5.83 11.63 2.76 3.87 4.74 6.44 7.09 2.35 7.39 1.44 5.57 2.74 9.33 2.70 7.96

Rat MT IU/ (tool%)

8.57 2.44 18.02 12.32 0.32 0.24 0.63 6.24 4.19 0.14 4.35 2.59 26.65 0.32 0.62 0.19 12.19

7.52 6.52 10.94 7.94 0.24 0.36 4.13 8.84 3.70 0.27 2.52 2.00 28.06 1.36 1.14 0.38 13.09

*Adaptedfrom Baerand Thomas(1991). tFrom Whangeret al. (1987). :l:Adaptedfrom Waalkeset al. (1984). Compared to a typical MT, the C. hamatus protein has a higher molecular weight and a different amino acid composition. Among several differences, that of cysteine content is the most remarkable: the icefish metal-binding protein has a low amount of cysteine compared to 30% cysteine content of MT. In addition, at variance with MT, the C. hamatus protein has tyrosine and phenylalanine in appreciable amounts, together with an unusually high content of aspartate, glutamate and glycine. Low molecular weight metalloproteins that lack identity with MT have been described in a number of species (Stone and Overnell, 1985). Cadmium-binding proteins have been found in rat testes (Waalkes et al., 1984; Klaassen and Waalkes, 1987), in oyster (Ridlington and Fowler, 1979) and in mammalian (Waalkes et al., 1988c) and fish (Baer and Thomas, 1991) ovaries. All these proteins are characterised by a high content of acidic amino acids residues and a low cysteine content, similar to the C. hamatus protein. Interestingly, the amino acid composition of the latter is also similar to that of a selenium-containing protein isolated from ovine heart (Whanger et al., 1987).

Since it is known that zinc may interact with amino acids containing carboxyl groups (Jacobson and Turner, 1980), glutamate and aspartate in the C. hamatus protein may substitute for the limited availability of cysteyl residues needed in metal complex formation. However, it has been reported that zinc is capable of interacting with carboxyl groups more weakly than with cysteine (Jacobson and Turner, 1980). This means that a zinc-binding protein carrying acidic amino groups would require a higher concentration of free ions for metal sequestration, but would also release the bound metal more easily than MT, thus providing a more rapidly mobilised source of zinc. The physiological role of the C. hamatus zinccontaining protein is not clear at present. In general, MT and MT-like proteins are thought to be involved in heavy metal detoxication (Suda et al., 1974; Cherian and Goyer, 1978; Layton and Ferm, 1980; Roberts and Schnell, 1982), and in the storage (Garvey and Chang, 1981; Webb and Cain, 1982), transport and homeostasis (Cousins, 1985) of d ~° metals. It is possible that in C. hamatus many, if not all, functions ascribed to MT are carried out by the zinc-containing

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Fig. 6. Cluster analysis of the data in Table 1. Distance metric is l--Pearson correlation coefficient. Legend: (1) icefish liver; (2) hamster ovary; (3) sea trout ovary O-3; (4) croaker ovary C-3; (5) croaker ovary C-l; (6) rat testes---I; (7) rat testes--II; (8) rat MT--I; (9) rat MT--II; (10) ovine heart.

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protein described in this paper. Probably, the deficiency o f M T observed in C. hamatus is c o m p e n s a t e d for by the presence of this zinc-binding protein a n d by d e v e l o p m e n t of adaptive changes in the m e c h a n isms o f zinc homeostasis. It has been reported that n o n - M T metal-binding proteins are uninducible (Waalkes et al., 1988a); hence tissue c o n c e n t r a t i o n of these proteins is expected to be m u c h less a d a p t a b l e to a change in the intracellular level of metal. O n the other h a n d , the lack of M T does not necessarily have a negative effect on Z n a n d Cu homeostasis, as d e m o n s t r a t e d by the fact t h a t certain cultured t u m o u r cells lacking M T can grow almost n o r m a l l y ( C o m p e r e a n d Palmiter, 1981; C r a w f o r d et al., 1985). Acknowledgements--This research is in the framework of the Italian National Programme for Antarctic Research and was supported by the CNR Special Project Bioactive Peptides. We are particularly grateful to the CNR Research Area of Naples for the use of the Atomic Absorption Spectrometer.

REFERENCES

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