Isolation and characterization of metallothioneins from cadmium-loaded mussel Mytilus edulis

Comp. Biochem. Physiol., Vol. 66C, pp. 179 to 182

0306-4492/80/0701-0179502.00/0

O Pergamon Press Ltd 1980. Printed in Great Britain

ISOLATION AND CHARACTERIZATION OF METALLOTHIONEINS FROM CADMIUM-LOADED MUSSEL M Y T I L U S EDULIS F. FRANKENNE*, F. NOF.L-LAMBOTand A. DISTECHE Laboratory of Oceanology, University of Li6ge, Institute of Chemistry, B-4000 Liege, Sart-Tilman, Belgium

(Received 23 October 1979) Abstract--1. Cadmium-binding proteins have been isolated from mussels kept in C d 2 + rich seawater. 2. Based upon their elution behavior in two chromatographic systems, Cd2+/SH ratio of about 3, paucity of both aromatic amino acids and absorbance at 280 nm, abundance of cysteinyl residues (>25%) and low molecular weight (< 11,000), those proteins meet the criteria for classification as metallothioneins. INTRODUCTION

Metallothioneins (MT) are low molecular weight proteins (6000-10,000) characterized by their high cystein content ( + 3 0 % of the residues) and their strong affinity for heavy metals (K~igi & Vallee, 1960, 1961; K~igi et al., 1974). Though their physiological function is not yet understood, little doubt remains they are implicated in a mechanism of detoxification against heavy metals poisoning (Piscator, 1964; Nordberg et al., 1971; Kimura et al., 1974; Yau & Mennear, 1977). These proteins have been extensively studied in mammals, where there are mainly found in liver and kidney (Kiigi & Vallee, 1960, 1961; Pulido et al., 1966; Btihler & K~igi, 1974; Olafson & Thompson, 1974; Syversen, 1975; Lee et al., 1977). The presence of metallothionein is also well established in birds (Weser et al., 1973; Webb & Daniel, 1975) and fishes (Olafson & Thompson, 1974; Bouquegneau et al., 1975; Marafante, 1976; Overnell et al., 1977; No~lLambot et al., 1978; Frankenne et aL, unpublished results). F r o m the papers of No61-Lambot (1976), NoS1Lambot et al. (1978), Howard & Nickless (1977), Brown et al. (1977) and Talbot & Magee (1978), it appears that invertebrates are able to synthesize Cdbinding, low molecular weight proteins in response to Cd intoxication, but till the recent work of Olafson et al. (1979), on crustacean, no protein isolated from intertebrate had been characterized as MT. The aim of this work was to isolate and characterize the Cd-binding, low molecular weight components detected earlier in the mussel Mytilus edulis by NoiflLambot (1976) in order to establish whether they belong or not to the MT's family. MATERIALS A N D M E T H O D S

Mussels are kept for 2 weeks in aerated seawater containing 0.5 ppm of Cd 2 + (CdCI2 form).

* Present address: Institut de Pathologie, C.H.U., Bat. B23, B-4000 Sart Tilman/Liege I, Belgium.

The soft part of the animals is then homogenized in 2 vols of ice-cold 0.5 M sucrose using an Ultra-Turrax (IKA) homogenizer. The homogenate is centrifuged at 37,000g for 1 hr at 4°C. The pellets are resuspended in 1 vol of 0.5 M sucrose and recentrifuged in the same conditions. To the pooled supernatants, acetone (Merck, reinst) at -30°C is added dropwise, under magnetic stirring, to a concentration of 45%. After 30 min, the mixture is centrifuged and the pellets discarded. The concentration of acetone of the supernatant is then raised to 80% in the same way. After 45 min, the supernatant is discarded. During this fractionation step, care is taken to maintain the temperature of the mixture under 4°C. The fraction 45-80% is dissolved in the minimum of 0.05M NH4HCO3 and immediately applied on a 5 x 50 cm column of Ultrogel AcA 54 (LKB), equilibrated with the same buffer. Fractions are monitored for absorbance at 215 and 250nm. The 250 nm absorbing fractions, corresponding to the elution characteristics of metallothioneins, are pooled, concentrated under N2 pressure using an Amicon UM2 membrane and equilibrated in 15 mM Tris-Cl (pH 8.5) on a 2 x 20 cm column of Sephadex G-25. The mixture is then applied on a 1.5 x 30cm column of DEAE cellulose (Whatmann DE 52) equilibrated against the same buffer. The column is further eluted with a linear gradient of 0-0.6 M NaCI produced by a Varigrad (Biichler) device. Those fractions corresponding to the 250nm peak are pooled, concentrated as above, desalted by filtration on a 2 x 20cm column of Sephadex G-25 equilibrated in 0.05 M NH4HCOa and concentrated again. Polyacrylamide gel slab electrophoresis have been achieved according to Perrie & Perry (1970), using a laboratory-designed apparatus, with T = 10.2, C = 2.6, in 20 mM Tris-glycine, 8 M urea (pH 8.6). Gels are stained with 0.6% Coomassie blue G-250 (Serva) in acetic acid: methanol:water (1:4.5:4.5), or sliced and dissolved by incubation of 48 hrs in 30% H202 at 50°C for analysis of metal content. For amino acid analysis, lyophilized samples are hydro* lysed at ll0°C for 24hr with constant boiling HC1 in sealed evacuated tubes. In order to estimate the cysteine content as cysteic acid, the samples are oxidized with performic acid before hydrolysis according to Hirs (1967). Analyses are performed with a modified Bechman Amino Acid Analyser, model 120. Cd, Zn and Cu contents are determined by atomic absorption spectrophotometry, using a model 370A Perkin-Elmer flame spectrophotometer. 179

180

F. FRANKENNE,F. NOi~L-LAMBOTand A. DISTECHE

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Fig. 1. Gel-filtration diagrams on Ultrogel AcA 54 of the 45-80~ acetone fraction of Mytilus edulis in 50mM NH4HCO3. 5 ml fractions are collected. MT designates the metailothionein containing fractions.

The u.v. spectrum is taken in 50 mM NH4HCOa using a model 124 Hitachi Perkin-Elmer spectrophotometer.

RESULTS The AcA 54 elution profile for the 45-80% acetonic fraction is shown in Fig. 1. Two main peaks of absorbance at 215nm are observed, one in the 6000-10,000 range and a second one corresponding to small molecules, i.e. small peptides, free amino acids and nucleic acids. The acetone fractionation eliminates most of the high molecular weight proteins with a resulting important decrease of the 215 nm absorbante in the void volume region. The first peak, associated with a high 250nm absorbance, which is characteristic of Cd-thioneins, is therefore designated as MT on the elution profile. By polyacrylamide gel electrophoresis analysis, the pooled MT fraction is resolved into several components (Fig. 2), the faster ones containing Cd. When the combined MT fraction is rechromatographied on DEAE cellulose, only one peak appears on the elution profile (Fig. 3), broad at 215nm, but sharpqr at 250 nm. In t~ose 250 nm absorbing fractions, no other components than those containing Cd are present as can be seen on the polyacrylamide gel electrophoresis of the pooled fractions (Fig. 4). Table 1 shows the mole percentage composition of the amino acids in the Cd-containing components (MT).

Cd 2+, Zn 2+ and Cu 2÷ are bound to the proteins in the proportion of 9.5, 0.25 and 0.25 gram-atoms, respectively, for 30 cysteinyl residues. The u.v. spectrum is shown in Fig. 5. DISCUSSION The cystein residue content of the Cd-containing fraction (> 25%) helps to establish these components as metallothioneins. Such large percentages of cystein are consistent with data derived for MT isolated from mammals, birds and fishes (Nordberg, 1972); Olafson & Thompson, 1974; Weser et al., 1973). Also in agreement with known characteristics of MTs is the almost complete lack of aromatic amino acids and the relative abundance of glycine and serine. The Cd-containing components are eluted from the Ultrogel AcA 54 at the same elution volume than mini m h o - -

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Fig. 3. Chromatography on DEAE-cellulose of pooled MT fractions from Ultrogel AcA 54 filtration (see Fig. 1). Buffered NaCI gradient 0--0.6M at pH 8.5. Column: 1.5 x 30 cm; flow rate: 20 ml/hr. Fractions of 4 ml.

181

Cadmium metallothioneins in Mytilus e l....1

Table 1. Amino acid composition of the Cd2+-binding proteins isolated from Cd 2+ loaded mussels Amino acid

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Fig. 4. Polyacrylamide gel electrophoresis of the MT fraction obtained by filtration on Ultrogel AcA 54 and chromatography on DEAE-celIulose (see Fig. 3). For experimental conditions, see Fig. 2.

% Total residues

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Ser Glu Pro Gly Ala 1/2 Cys* Val Met Ile Leu Tyr Phe

7.9 4.5 6.8 15.8 5.2 25.5 3.8 <0.5 4 1.3 <0.5 1.3

parvalbumins, a 11,000-12,000 globular proteins family (Bhushana et al., 1973). Although the molecu* Determined as cysteic acid. lar weight of MT seems to lie in the 6500 range (K~igi et al., 1974), this is consistent with the elution volume obtained by gel-filtration of other MTs. The large This finding helps to establish the extended ubiStoke's radius (Kojima et al., 1976) of the MTs quity of those proteins and the universality of the explains this apparent discrepancy. U.v. spectrum (Fig. 5), with no absorbance peak at mechanism of detoxification against heavy metals in 280 nm and a shoulder at 250 nm is also very charac- which they are involved. teristic of Cd-thioneins (K/igi & Vallee, 1961). REFERENCES The high binding capacity for Cd by mussel's MTs and the stoechiometry of metal to cysteinyl metal BHUSHANA RAO K. S. P. & GERDAY CH. (1973) LOW binding sites (about three) is consistent with other molecular weight proteins of Pike (Esox lucius) white MTs too. muscles--II. Chemical and physical properties. Comp. Metallothioneins are known to occur in several Biochem. Physiol. 44B, 1113-1125. polymorphic forms in many species (e.g. Kojima et al., BOUQUEONEAUJ. M., GERDAYCH. & DISTECHEA. 0975) Fish Hg-binding thionein related to adaptation mechan1976), indicating that their synthesis is coded by mulisms. FEBS Lett. 55, 173-177. tiple gene loci. The presence of several Cd-containing components in the MT fraction from mussel's tissues BROWN D. A., BAWDENC. A., CHATELK. W. & PARSONS T. R. (1977) The wildlife community of Iona Island Jetty, is probably due to the existence of such variants. Vancouver, B.C., and heavy-metal pollution effects. This report is the first at our knowledge to conclusEnvir. Conserv. 4, 213-216. ively demonstrate, by chromatography, amino acid BLIHLERR. H. O. & Kgot J. H. R. (1974) Human hepatic analysis and other properties, that the low molecular metaliothionein. FEBS Lett. 39, 229-234. weight Cd-binding components synthesized in a mol- Hms C. H. W. (1967) Performic acid oxidation. In Methods lusc in response to Cd poisoning, are metallothioin Enzymology, Vol. XI, pp. 197-199. HOWARD A. G. & NICKLES$G. (1977) Heavy metal comneins. plexation in polluted molluscs--I. Limpets (Patella tmloata and Patella interraedia). Chem.-biol. Interact. 16, 107-114. A K~Ol J. H. R. & V^LL~E B. L. (1960) Metallothionein: a Cd- and Zn-containing protein from equine renal cortex. J. biol. Chem. 235, 3460-3465. K~.ol J. H. R. & VALLEEB. L. (1961) Metallothionein: a Cd- and Zn-containing protein from equine renal cortex--II. Physicochemical properties. J. biol. Chem. 236, 2435-2442. K~GI J. H. R., HIMMELHOCH S., WHANGER P. D., BETHUNE 0.5

0 200

J. L. 8£ VALLEE B. L.

250

300

Wavelength (rim)

Fig. 5: Ultraviolet absorption spectrum of the Cd-binding low molecular weight, components from Mytilus edulis in 50 mM NH4HCOs.

(1974) Equine hepatic and

renal

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