Purification and properties of novel molluscan metallothioneins

ARCHIVESOFBIOCHEMISTRYAND BIOPHYSICS Vol. 2’73,No. 2, September, pp. 403-413,1989

Purification and Properties of Novel Molluscan Metallothioneins’ G. ROESIJADI,*r’ SANDRA KIELLAND,?

AND

PAUL KLERKS*

*University of Maryland, Centerfor Envircmmentnl and E&a&e Studies, Chesapeake Biological Laboratory, Box $8, Solomons, Maryland 20688,and ~Lkpartment of Bbchemistrg and Microbidogy, University of Victoria, Vi&aria, British Columbia, Canada Received January 27,1989, and in revised form May 8,1989

Two low-molecular-mass cadmium-induced, cadmium-, zinc-binding proteins were purified from the oyster Crassostrea virginica using procedures that included acetone precipitation, Sephadex gel chromatography, and anion-exchange and reverse-phase high-performance liquid chromatography. Although they could be cleanly separated from each other, they exhibited similar molecular weights, metal and amino acid compositions, and electrophoretic behavior. These proteins were glycine-rich, in addition to being cysteine-rich, and lacked methionine, histidine, arginine, and the aromatic amino acids phenylalanine and tyrosine. Determination of the NHz-terminal amino acid sequence of these molecules showed that they were identical in primary structure in this region and differed only in that one had a blocked NHz-terminal. This provided an explanation for the isolation of two proteins with otherwise identical characteristics. Serine was the NHz-terminal amino acid. The sequence was most similar to that of vertebrate metallothioneins when compared with other proteins, which included metallothioneins from other invertebrate phyla. All cysteines in the first 27 residues of the oyster metallothionein aligned with those in the mammalian forms. On this basis, these proteins were 0 1989 Academic Press. Inc. classified as class I metallothioneins.

Low-molecular-mass, metal-binding proteins classified as metallothioneins are ubiquitously distributed in the animal kingdom (1). While many from mammalian sources have been characterized in considerable detail (2), similar studies on metallothioneins from invertebrate species are relatively scarce despite the numerous studies on mechanisms for trace element regulation in this group. Notable exceptions are those of a crab (3~9, DrosopMu (6), and sea urchin (7). Studies on metallothioneins from nonmammalian sources indicate variance in structure from the mammalian form (2). These differences can reflect differences in ‘This study was supported in part by Grant DEFG0586ER60469 from the U.S. Department of Energy. ’ To whom correspondence should be addressed.

functional properties of the proteins as occurs with metallothioneins of the crab Scylla m-rata, whose metal cluster arrangement consists of two three-metal clusters, rather than the one three-metal and one four-metal cluster observed in mammalian species (4). The three-metal cluster is considered to possess lower metal affinity in comparison with the fourmetal cluster, a situation with obvious biochemical and physiological implications. Although numerous studies have been conducted on metal-binding proteins of molluscan species, those that have attempted to characterize the proteins are few and conflicting in their findings (8-14). The differences, undoubtedly, reflect difficulties encountered in purification of the proteins, as well as possible phenotypic diversity. The development of successful isolation techniques for molluscan metal-

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0003-9861/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

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lothioneins has eluded investigators and hindered progress in advancing our understanding of the biochemistry of metallothionein in this widespread group. Thus, the metal-binding protein of the oyster, Cmssostrea uirginica, which is the first invertebrate species for which a metallothionein-like protein was reported to occur (E), is yet to be identified as metallothionein. Previously unsuccessful purification of metallothioneins in this animal was attributed to technical problems associated with chromatographic and electrophoretic behavior during isolations (12). We have recently been able to purify two forms of a Cd-induced, metal-binding protein from C. GrgKca and present here a description of the purification procedure and some properties of the proteins. The sole difference between the two proteins appeared to be a blocked NHz-terminal in one. On the basis of the NHz-terminal amino acid sequence, they could be classified as class I metallothioneins since they exhibited a high degree of similarity with mammalian metallothionein. Of the known metallothioneins, those from the trout were shown to be most closely related to those described here for the oyster. MATERIALS

AND METHODS

Animals. Oysters of the species C virginioa were purchased from a local Waterman at Solomons, Maryland. They were rinsed, scrubbed to remove adhering organisms and placed in a tank with flowing water (17°C and 157~salinity) from Chesapeake Bay. Oysters were then exposed to 200 pg Cd.liter-’ Chesapeake Bay water for 21 days in static tanks at room temperature (-20°C). The water was replaced every second day. Following exposure, the soft tissues were removed from the shells and stored at -70°C. Isolation of km-molecular-mass, dmium-binding proteins Oysters were partially thawed, then homogenized in 2 vol of ice-cold buffer of 20 mM Tris-HCl, 275 mM NaCl, 5 mM DTP at pH 8.6. Phenylmethylsulfonyl fluoride was added to the sample at a final con-

3 Abbreviations used: DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; AA, atomic absorption; TPCK, L-ltosylamino-S-phenylethylchloromethylketone; CdBP, cadmium-binding protein; EDTA, ethylenediaminetetraacetate; PTH, phenylthiohydantoin.

AND KLERKS

centration of 0.1 mM immediately prior to homogenization. Homogenates were centrifuged at sO,OOOg for 90 min. The cytosolic fraction was then subjected to acetone precipitation at 45 and 80% final acetone concentration (v/v, assuming additive volume) as described previously (14). The pellet obtained after precipitation in 80% acetone was redissolved in 20 mM Tris, pH 8.6,5 mM DTT and separated by gel chromatography on Sephadex G-75 (5 X 80-cm column) with 20 mM Tris-HCl, pH 8.6, 2 mM DTT as the mobile phase. Absorbance at 254 nm was continuously monitored, and fractions were collected for cadmium analysis. Fractions corresponding to the low-molecularmass, cadmium-binding protein (apparent molecular mass = 13 kDa) were pooled and concentrated in an Amicon ultrafiltration cell with a YM2 membrane. Cadmium-binding proteins isolated in the previous step were next separated by anion-exchange HPLC with a TSK DEAE 5PW column (7.5 cm X 7.5 mm, Bio-Rad Laboratories, Richmond, CA). The starting buffer (A) was 20 mM Tris-HCl, pH 7.5, and the limit buffer (B) 400 mM Tris-HCl, pH 7.5. Eluents were monitored at 254 nm, and fractions were collected and analyzed for cadmium. After the positions of the cadmium-binding proteins were determined under analytical conditions, “semipreparative” separations were conducted on the same column by making serial injections under initial conditions, then eluting the proteins by starting the gradient program. Total injection volumes could be scaled up to 400X that of analytical separations by this method [i.e., from 5 pl in analytical scale to 4 X 500 rl(=2 ml) in semipreparative scale] without loss of resolution. In these runs, cadmium-binding proteins were collected manually, aliquoted, and stored at -70°C. The use of analytical separations to determine elution profiles and semipreparative isolations for collection of purified proteins was also followed in subsequent HPLC steps. These preparations were checked for purity by PAGE and analyzed for sulfhydryl and cadmium, copper, and zinc content. They were then concentrated by precipitation in 80% acetone at -2O”C, redissolved in 0.1% trifluoroacetic acid, and further purified on a C4 reverse-phase column (Synchropak RP-4 column, 25 cm X 4.1-mm analytical column and 5 cm X 4.1-mm guard column, Synchrom, Inc., Linden, IA). For this system, buffer A was 0.1% trifluoroacetic acid, buffer B was 0.1% trifluoroacetic acid containing 50% acetonitrile (v/v, assuming additive volume). Absorbance was monitored at 220 nm since the chromophore at 254 nm due to the cadmium-thiolate bond is abolished in the 0.1% trifluoroacetic acid (pH 2.1). Automatic background correction was employed in these runs. Our procedures for reverse-phase HPLC of metallothioneins are basically similar to those described previously (16). Gradients utilized for HPLC separations are described under Results. The HPLC system (Waters Chromatography Division, Millipore Corp.)

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consisted of two Model M-45 pumps, a Model 680 gradient programmer, a Model 490 programmable variable wavelength detector, and a U6K injector. PAGE and SDS-PAGE. PAGE of cadmium-binding proteins obtained after the ion-exchange HPLC step used the Ornstein-Davis (1’7, 18) buffer system, and SDS-PAGE used the buffer system of Laemmli (19). Samples purified by reverse-phase HPLC were not successfully detected by electrophoresis. Gels in both buffer systems were made of 15% T/2.5% C (total acrylamide/proportion of total contributed by bisacrylamide) for the separation gel and 3% T/2.5% C for the stacking gel. Following electrophoresis, gels were fixed in 12.5% trichloracetic acid (w/v); equilibrated with 45% methanol, 10% acetic acid; then stained with Coomassie blue (Serva blue R). Cytosolic extracts of gills of Cd-exposed (50 pg Cd. liter-’ for 2 weeks) and unexposed oysters were prepared by homogenization in sample buffer and centrifugation at 80,000~ and also analyzed in the Ornstein-Davis system. Metal analysis. Fractions collected during Sephadex and ion-exchange HPLC isolations were analyzed for cadmium by graphite furnace AA using a PerkinElmer 503 AA spectrophotometer. Purified proteins obtained at the ion-exchange step were analyzed for cadmium, copper, and zinc following digestion in hot, concentrated nitric acid, evaporation of the acid, and redissolution in 0.4 N nitric acid. Digested samples were analyzed for cadmium and zinc using the flame unit of a Perkin-Elmer 5000 AA spectrophotometer. Copper was analyzed with the graphite furnace. Sulfydryl analysis. Sulfhydryl groups in proteins obtained after the ion-exchange step were analyzed using Ellman’s reagent [5,5’-dithiobis(2-nitrobenzoic acid)] (20). Amino acid analysis. Proteins samples isolated by reverse-phase HPLC were hydrolyzed in 6 N HCl for 24 h, then analyzed using a Beckman 119CL automated amino acid analyzer. Cysteine analysis was determined on performic acid oxidized samples (21). Values reported for each protein were based on replicate analyses. S-alkylation of proteins. Proteins purified by reverse-phase HPLC were modified by reaction with iodoacetamide. Proteins were dialyzed against 20 mM Tris-HCI, pH 8.6, 10 mM D’IT, 50 mM EDTA, mixed 1:l with the same buffer containing a suspension of guanidine equivalent to 12 M for a final concentration of 6 M guanidine, and incubated at 3’7°C for 20 min. Iodoacetamide was then added to 0.12 M final concentration. Samples were incubated in the dark for 45 min at room temperature, and the reaction was stopped by dialysis against 0.1% trifluoroacetic acid. In the case of one of the proteins (designated CdBPZ), the formation of an irregularly shaped crystal was observed during dialysis in 0.1% trifluoroacetic acid. The crystalized material was dissolved by dialysis

against 50 mM Tris-HCl, pH 8.6. Alkylated proteins were repurified by reverse-phase HPLC as described above. Molecular mass estimation. Molecular mass of the alkylated proteins was determined by gel permeation HPLC using a TSK SW3000 column (two 30 cm X 7.5mm columns in series with a 10 cm X 7.5-mm guard column, Altex, Berkeley, CA) and 0.1% trifluoroacetic acid as the mobile phase. Eluents were monitored at 220 nm. Myoglobin (17.2 kDa), cytochrome c (11.7 kDa), aprotinin (6.5 kDa), and rabbit metallothionein-2 (5.9 kDa) (all from Sigma Chemical Co. and alkylated with iodoacetamide) were used as standards. These were monitored at 254 and 280 nm. NH&erminal amino acid sequence Both alkylated and unalkylated proteins that had been purified by reverse-phase HPLC were analyzed for the NHc-terminal amino acid sequence by gas phase sequencing with use of an Applied Biosystems Model 470A sequenator with on-line analysis of the phenylthiohydantoin of the NH*-terminal amino acid. Standard procedures recommended by the manufacturer were used for the analyses. Peptide maps. Peptides prepared by tryptic proteolysis of S-alkylated proteins (TPCK-treated trypsin; Sigma Chemical Co.) were chromatographed on a reverse-phase HPLC system (pBondapak C-18 column, 3.9 mm X 15 cm; Waters Chromatography Division, Millipore Corp.) using the trifluoroacetic acid/acetonitrile solvent system described above. Approximately 60 pg of each protein was dissolved in 100 J 0.1 M NH4HC03. At intervals of 0, 3, and 15 h, 10 pl containing 0.6 fig trypsin (dissolved in 1 rnM HCl) was added to each protein. The digestions were conducted at 37°C and stopped at 18 h by drying under a Nz gas stream. Each sample was redissolved in 0.1% trifluoroacetic acid, then chromatographed. RESULTS

The low-molecular-mass, cadmium-binding proteins of I?. virginica eluted from Sephadex at a position equivalent to about 13 kDa apparent molecular mass (Fig. 1). Separation of this cadmium-binding fraction by anion-exchange HPLC resulted in the clear resolution of two cadmium-binding proteins designated CdBPl and CdBPZ (Fig. 2). These were collected in semipreparative isolations and used in further purification and characterization. Electrophoresis of the two proteins using the Ornstein-Davis method, which resulted in identical responses with a single major band with an Rf = 0.58 and a faint band of Rf = 0.31 (Fig. 3, lanes 1 and 2), was

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OO

20

40

60

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FIG. 1. Sephadex G-75 chromatogram of the cadmium-exposed oyster cytosol previously subjected to acetone precipitation. The precipitate obtained after treatment with 80% acetone was redissolved in buffer and applied to the column. The column was equilibrated in 20 mM Tris-HCI, pH 8.6,2 mM DTT at 4°C. Other details of the procedure are described under Materials and Methods. Proteins later characterized as oyster metallothionein eluted with fraction 60 as the peak. This peak was pooled for further purification and analysis.

not sufficiently powerful to resolve proteins previously separated from each other by anion-exchange HPLC. The presence of

AND

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the major band in cytosolic extracts of cadmium-exposed tissues and its absence in nonexposed oyster-gill tissues verified the inducible nature of the former (Fig. 3, lanes 3 and 4). The minor band was not distinguishable from the general background staining of the cytosolic samples. Additionally, an unidentified, fast-migrating, protein that was heavily stained in the control cytosol was diminished in the cadmium-exposed cytosol. SDS-PAGE of the semipurified proteins confirmed the results above: i.e., identical responses for CdBPl and CdBP2 with a major and minor band in each (gel not shown). Apparent molecular masses were calculated as 15.5 kDa for the major band and 20.8 kDa for the minor band. Analyses for the metal composition of the two proteins and comparison with the respective sulfhydryl contents were made at this time since the next purification step entailed reverse-phase HPLC in 0.1% trifluoroacetic acid, which would result in removal of most metal ions from the proteins. The results showed that measured

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FIG. 2. Anion-exchange HPLC of the oyster metallothionein peak. (a) Absorbance profile at 254 nm (b) cadmium profile. Buffer A was 20 mM Tris-HCl, pH 7.5; buffer B was 400 mM Tris-HCI, pH 7.5. The proteins were injected with the system under initial conditions, i.e., 0% buffer B. The gradient was started 5 min after the injection and went from 0% buffer B to 50% B over 30 min. The oyster metallothioneins obtained after gel chromatography on Sephadex G-75 were separated by anion-exchange HPLC into two peaks designated CdBPl and CdBP2. The absorbance peaks associated with CdBPl and CdBP2 were coincident with the cadmium peaks, confirming their identity as cadmium-binding proteins.

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FIG. 3. PAGE of cadmium-binding proteins purified by anion-exchange HPLC and cytosolic extracts of gills from cadmium-exposed and control oysters. The buffer system was that of Ornstein-Davis. Lane 1, CdBPl; lane 2, CdBP2; lane 3, cytosol of control oyster gills; lane 4, cytosol of cadmium-exposed oyster gill. Each of the purified proteins separated into two bands, with the more heavily stained, faster migrating band corresponding with a cadmium-induced protein in the exposed cytosol.

values for sulfydryl groups in the protein solutions compared favorably with predicted values calculated from the metal analysis, assuming characteristic Cys: metal ratios of 3:l for both cadmium and zinc and 2:l for copper bound to metallothionein. Measured values were 1.57 and 1.42 SH/ml for CdBPl and CdBP2, respectively; predicted values were 1.57 and 1.37 SH/ml, respectively. Thus, the proteins were sufficiently pure at this stage to exhibit metal-binding properties consistent with the expected relationship between SH groups and metal for metallothionein. The relative amounts of metals on each protein were similar with about 75% cadmium, 25% zinc, and a trace of copper (Table I). The metal content of the CdBPs calculated with use of the metal, sulfydryl, and amino

acid compositional (see below) data indicated a total of about 7 g-atom metal/m01 protein for each protein (Table I). Reverse-phase HPLC of CdBPl and CdBP2 verified the singular nature of the proteins obtained in the previous ion-exchange step (Figs. 4a, 4b, and 4c) and effected further purification. Amino acid analyses of purified CdBPl and CdBP2 (Table II) indicated that the two proteins were virtually identical in composition with high amounts of cysteine (29%), glycine (16%), and lysine (13%) and an absence of methionine, leucine, tyrosine, phenylalanine, histidine, and arginine. Samples analyzed prior to the reverse-phase HPLC purification step exhibited lower levels of cysteine and the presence of small amounts of aromatic residues (data not shown). Molecular mass estimates of S-alkylated proteins by gel permeation HPLC resulted in values of 7.2 kDa for CdBPl and 6.5 kDa for CdBP2. These were in general agreement with the minimum molecular mass of 6.6 kDa calculated from the amino acid compositions of both proteins (Table II). The NHz-terminal amino acid sequence of CdBPl was determined through residue 27 (Table III). This region included serine as the NHz-terminal amino acid and three Cys-X-Cys sequences considered characteristic of metallothionein. Residue 17 was not identified and may be a modified amino acid. CdBP2, on the other hand, was blocked at the NHz-terminal by an undeTABLE

I

METALCOMPOSITIONOFCADMIUM-BINDINGPROTEINS OFTHE OYSTERCrassostrea wirginica

Cd” Zn” CU” C Cd, Zn, Cu” Cd:Zn:Cu ratio

CdBPl

CdBP2

5.0 1.7 0.1 6.8 3.O:l.O:O.l

4.7 1.6 0.1 6.4 3.0:1.0:0.1

“Gram-atoms metal per molecule protein; calculated from metal and SH concentrations in protein samples and amino acid compositional data in Table II.

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b

FIG. 4. Reverse-phase HPLC of CdBPl and CdBPB Proteins used in this procedure were previously isolated by anion-exchange HPLC as in Fig. 2. (a, b) CdBPl and CdBP2 chromatographed individually. (c) CdBPl and CdBP2 recombined and chromatographed. Buffer A: 0.1% trifluoroacetic acid, buffer B: A containing 50% acetonitrile. Proteins were injected under initial conditions, i.e., 0% buffer B. The gradient was started after 5 min at 0% B and went from 0 to 70% B in 30 min. Each protein sample eluted as a single major peak with one or two very minor peaks at the base (a and b). When the two proteins were recombined then chromatographed in the reverse-phase system (c), the major peaks separated from each other with near baseline resolution at the expected positions, verifying the independent nature of the two proteins. The minor peaks associated with each of the proteins were removed when the proteins were collected in “semipreparative” runs.

termined moiety, and initial attempts to analyze the unalkylated and alkylated forms were unsuccessful. Since CdBPl and CdBP2 were similar in electrophoretic behavior, metal content, and amino acid composition, it was assumed that the sequences were also similar, and an experiment that was expected to provide a partial sequence in the NHe-terminal region was conducted in the sequenator. The basis for this experiment was the expectation of an acid-labile, Asp-Pro bond at residues 2 and 3, which was to be cleaved by acid treatment prior to sequencing. Cleavage of this bond was expected to result in two peptides: a blocked Ser-Asp dipeptide that would not be detected and the remainder of the molecule starting with Pro-3. We planned to determine the NHz-terminal sequence of the latter peptide for comparison with CdBPl. To conduct this experiment, CdBP2 was applied wet, subjected to gaseous trifluoroacetic acid, and allowed to sit for 10 min. The sample was then blown dry

and wetted again, and the previous steps were repeated. Following the second cycle of acidification and drying, the normal sequencing run was initiated. This procedure resulted in the unexpected removal of the block and detection of serine, as well as proline, as NHa-terminal amino acids. Three peptides could have been generated by unblocking a peptide identical in other respects to CdBPl and cleaving it between Asp-2 and Pro-3: (i) the dipeptide Ser-Asp, (ii) the remainder of the molecule beginning with Pro-3, and (iii) the intact peptide beginning with Ser-1, if the block was removed and only a portion of the molecules was cleaved at the Asp-2-Pro-3 bond. Serine would represent the NHz-terminal of both the dipeptide and the intact peptide, and proline would represent the NHa-terminal of the remaining peptide beginning with Pro-3. The result would be the apparent detection of two peptides, as we found. Subsequent Edman degradation cycles also resulted in the generation of paired

MOLLUSCAN METALLOTHIONEINS TABLE II AMINO ACID COMPOSITION OFCADMIUM-BINDING PROTEINSISOLATEDFROM THEOYSTER Crasmstrea

virginim

Residues/ molecule Amino acid Asx Thr Ser Glx Pro GUY Ala cys/2 Val Met Ile Leu 5r Phe His LYS Arg Total

Nearest integer”

CdBPl

CdBP2

CdBPl and CdBP2

4.8 5.0 5.2 3.3 3.6 10.6 3.6 19.8 0.7 0 0.6 0 0 0 0 9.0 0

5.0 5.4 5.7 3.2 3.4 10.9 3.6 19.5 0.9 0 0.6 0 0 0 0 9.0 0

5 5 5 3 4 11 4 20 1 0 1 0 0 0 0 9 0 68b

a Calculated on the basis of nine Lys per molecule deduced from the 10 tryptic peptides generated during hydrolysis prior to peptide mapping; CdBPl and CdBP2 are considered to be identical in amino acid composition. *Minimum metal-free molecular mass: CdBPl and CdBPL = 6.6 kDa based on nearest integer values.

sulted in profiles that contained 10 peaks eluting between 12.5 and 30% acetonitrile (Table V) (chromatograms not shown). The first nine peaks in each profile, Tl to T9, eluted at the same relative positions in the two samples indicating that they were identical in the two proteins. TlOa and TlOb, which were the highest and most hydrophobic peaks in the respective profiles, differed slightly from each other in elution position. They also did not elute at positions of the intact proteins indicating that they were not unlysed parent compounds. They most likely were the NHz-terminal fragments remaining after lysis at the Lys-24 position. These would comprise roughly one-third of the respective molecules and be expected to dominate the profiles. These findings provided support for the possibility that the NH,-terminal block in CdBP2 is probably the single feature that distinguishes the two proteins from each other. TABLE III AMINO TERMINAL PRIMARYSTRUCTURE OFOYSTER CdBPl AND CdBP2 AND COMPARISON WITH

TROUTMT-AANDMOUSEMT~ Protein CDBPl CDBPZ Trout MT-A

residues, and a sequence was deduced using the CdBPl NHz-terminal sequence for comparison (Table IV). The deduced sequence indicated that the serine and proline residues generated in the first Edman cycle corresponded to Ser-1 and Pro-3, respectively, of CdBPl (Table III). CdBP2 was identical to CdBPl through residue 22, the last identifiable amino acid in CdBP2. Thus, we were able to fortuitiously remove the block and facilitate determination of the NHz-terminal sequence of CdBP2. We are not aware of other instances in which the NHz-terminal sequence of a blocked protein has been successfully analyzed in a sequenator. Reverse-phase HPLC of the tryptic peptides of the two Cd-binding proteins re-

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Mouse MT1

NHz-Terminal amino acid sequence SDPCNCIETGTCACSDXCPATGCKCGP--a XSDPCNCIETGTCACSDXCPATG------.. .. .. . . z::.:.:.. : ..: :.. MDPCECSKTGSCNCGGSCKCSNCACTS--"e :.: ..:.: :.. :. ..:::.. MDPNCSCSTGGSCTCTSSCACKNCKCTS--""

a X indicates presence of unidentified NHa-terminal block and unidentified residue at position 17. The latter is probably a modified amino acid. b Colon (:) indicates identical residues; period (.) indicates conservative replacement; comparisons are made with oyster CdBP. ’ Trout MT-A exhibited the highest score when the oyster NHa-terminal amino acid sequence was compared against a protein library using FASTP [Ref. (25)] and is shown here for reference. All mammalian MTs scored high as well, and mouse MT-l was the most closely matched of that group. Crab, sea urchin, and Drosuphilu MTs showed significant similarities with those of the oyster but did not score as high as the vertebrate proteins [see Ref. (2) and citations therein and Ref. (26) for entire amino acid sequences for these species].

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TABLE IV NH2-T~~?d1~~~ AMINO ACID SEQUENCE ANALYSIS OF TRIFLUOROACETIC ACID-CLEAVED CdBP2”

PTH amino acid (aa) Cycle no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

aal

aa

Ser Asp Pro /” CYS Asn

/’

CYS

Be Glu Thr GUY Thr CYS

Ala CYS Ser Asp ? Pro Ala Thr Gly/’

/ Pro Asn CYS Ile Glu Thr GUY Thr CYS Ala CYS Ser Asp ? CYS

Pro Ala Thr /

/ GUY Cys LYS

“Paired residues were generated at each Edman cycle (see text). Regions of apparent identity are connected by dashed lines. DISCUSSION

Metal and sulfhydryl content, amino acid composition, and NHz-terminal sequence analysis showed that the oyster, C. virginica, possessed two cadmium-inducible, low-molecular-mass, cadmium-, zincbinding proteins that could be classified as class I metallothioneins according to recently proposed nomenclature (22). Designated CdBPl and CdBP2, these two forms of metallothionein were separable by ionexchange and reverse-phase HPLC but exhibited similar electrophoretic behavior and metal and amino acid compositions. They appeared to be differentiated only by a blocked NHa-terminal in CdBPB Since such blocked proteins may not be easily detectable in the presence of similar, but un-

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blocked, molecules, the possibility exists that the same situation may exist in metallothioneins of other species. The functional significance of an individual organism possessing metallothioneins that differ in only a modification at the NHz-terminal is not known, although possibilities may be receptor-recognition for transport, intracellular compartmentalization, or compartmentalization resulting from the metabolism of different cell types. These possibilities are currently being explored. From the results of reverse-phase HPLC separations of the intact proteins and tryptic peptides, it appears that the NHzterminal blocking moiety is most likely a small organic constitutent that is acid-la-

TABLE V RESULTS OF PEPTIDE MAPPING OF CdBPl AND CdRP2”

Relative peak heightb Peptide Tl T2 T3

T4 T5 T6 T7 T8 T9 TlOa TlOb

CdBPl

CdBP2

5.2

6.0

7.1

6.9

6.6 24.1 24.8 16.7 45.0 22.2 10.6 100

7.2 20.8 24.9 14.3 42.3 20.3 8.2 100

o Created by reverse-phase HPLC of tryptic digests of CdBPl and CdBP2; peptides were eluted by a linear gradient of 0 to 40% acetonitrile in 0.1% trifluoroacetic acid on a PBondapak C-18 column (3.9 mm X 15 cm) with a flow rate of 1 ml/min. Peptides were detected by absorbance at 220 nm. Ten peptide peaks designated Tl to TlO eluted between 12.5 and 30% acetonitrile and are arranged in order of increasing hydrophobicity. TlO was the highest and most hydrophobic of the peaks and was the only one which differed in the relative elution position in the two proteins. TlOa and TlOb most likely represent the NHzterminals of the respective proteins. bPeak heights were normalized to that of TlOa or TlOb, which were arbitrarily assigned values of 100.

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bile and contributes to the increased hydrophobicity of CdBP2 in comparison with that of CdBPl. The resolution of two protein peaks during ion-exchange separations and the slight difference in elution positions for the two during molecular mass estimations using a gel permeation column may have been facilitated by uncharacterized hydrophobic components of the two columns, rather than by the stated principles of separation. In each case, CdBP2 lagged slightly behind CdBPl in elution position. The molecular mass of CdBP2 estimated by gel permeation chromatography would, thus, be expected to be slightly smaller than that of CdBPl as was observed. The behavior of CdBP2 is reminiscent of proteins modified by NH,-terminal acylation (23). Highly elevated glycine levels such as that reported here are unusual for metallothioneins and may represent a molluscan variant of the molecule. The absence of methionine is consistent with other nonmammalian metallothioneins [e.g., in Ref. (2)] and indicated that metallothioneins in these types of organisms are subject to NH, - terminal modifications following initial synthesis that does not occur in the mammalian metallothioneins. Recent findings with yeast metallothionein (24) have shown that the first eight residues predicted from the nucleotide sequence are not present in the final form of the protein and not required for structural integrity or metal-binding properties. This region of the protein seems labile when considered from a comparative perspective since the NH,-terminals of various nonvertebrate metallothioneins exhibit departures from the mammalian form. In other studies with the oyster (12) and mussel (11, 13), two size variants of metallothionein with apparent molecular masses of 10 to 13 and 20 to 25 kDa were observed following the initial Sephadex G75 separation of the cytosol. Our results agree with those of Frazier (25), who also reported only a single peak at the lo-kDa position. We obtained similar results when cytosol was prepared and chromatographed in the absence of reducing agents

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and without prior acetone precipitation. Discrepancies between these findings and those obtained with mussels can be explained on the basis of diversity among different species of animals. Such an argument cannot be invoked to explain the differences observed by the different investigators who have examined metallothioneins from C virginicu since initial isolation procedures through gel chromatography have been similar. These differences may reflect phenotypic variability in this species. Use of a computerized search for amino acid sequence similarities (FASTP) (26) for comparison of the oyster metallothionein NHz-terminal sequence against sequences in a protein library resulted in the highest scores with the metallothioneins of vertebrate species. x values, which represent measures of statistical significance for such comparisons (26), were calculated for comparisons against selected animal metallothioneins (mouse metallothionein 1, green monkey metallothionein 2, horse metallothionein 2A, rainbow trout metallothioneins A and B, crab metallothionein 1, sea urchin metallothionein, and Drosophila metallothionein [(2) and citations therein; (27)]). In all cases, x values were >4 for initial scores and >lO for optimized scores, which allow for gaps. z values > 4 are believed to have biological significance, and those >lO are considered highly significant (26). With the exception of comparisons with sea urchin and Drosophila metallothioneins, the best alignments occurred with the NHz-terminal regions of the various metallothioneins (see Table III for alignment of oyster, trout, and mouse metallothionein NHz-terminal sequences). The rank order of FASTP scores, which indicates the degree of similarity to the oyster, was trout > mouse > crab > sea urchin > Drosophila On the basis of our data, the oyster metallothionein is structurally more closely related to vertebrate metallothioneins than it is to those of other invertebrate species studied thus far. The positions of cysteinyl residues in this region of the oyster and vertebrate metallothioneins were highly conserved, with all of the

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ROESIJADI,

KIELLAND,

cysteines in the oyster metallothionein aligning with those in the vertebrate metallothioneins (although the reverse was not true due to an additional cysteine in this portion of the vertebrate metallothioneins). When compared with the trout, the oyster metallothionein exhibited 42.3% identical residues and 84.6% corresponding residues (=identical residues and conservative replacements) over a 26 residue overlap (Table III). The higher affinity with the trout metallothionein, in comparison with that of the mouse, reflects the lower taxonomic position of fish relative to mammals in the vertebrate subphylum. Interestingly, the sea urchin metallothionein does not show significant similarity with mammalian metallothioneins (7) although both are from deuterostomes, while the similarity is significant when the comparison is made with the NHz-terminal segment described here for the oyster, a protostome. It was noted (7) that the sea urchin metallothionein is reversed in the placement of the NHz- and COOH-terminal halves of the molecule when compared with mammalian metallothioneins and has a COOH-terminal that is similar to the amino-terminal half of mammalian metallothionein, if the former is read in reverse polarity. We found with use of FASTP that the NHz-terminal halves of mammalian metallothioneins exhibited significant similarities with the COOH-terminal half of the sea urchin sequence read in reverse polarity. The central segments (7) of the respective molecules, which are contained in these halves, aligned exactly, in the area with the best overlap. Similar comparison of the oyster NHB-terminal sequence and the sea urchin metallothionein sequence did not result in significant similarities. The sea urchin metallothionein, therefore, exhibits greater similarity with mammalian metallothioneins than with oyster metallothioneins when analyzed in this manner. The overall findings were consistent with recent proposals for a molecular phylogeny based on 18 S ribosomal RNA sequences which aligns molluscs more closely with chordates in comparison with echinoderms (28); although, in this scheme, arthropods are more distantly re-

AND

KLERKS

lated to molluscs than either the chordates or echinoderms. The NHz-terminal sequence data also allowed us to infer characteristics related to the possible existence of metal clusters, which in mammalian metallothioneins occur as two forms within a single molecule (2). Cluster B is a three-metal-containing structure with metal atoms coordinated to the cysteines in the NHz-terminal residues 1 to 30, while cluster A is a four-metal-containing structure formed by residues 31 to 61 in the carboxyl-terminal region. The close alignment of the cysteinyl residues in the oyster metallothioneins’ NH,-terminal sequence with that in cluster B of mammalian metallothioneins supports the presence of a similar metal cluster in the structure of the oyster molecules. The crab metallothioneins, to which the oyster is less closely related, possess such a cluster in the NHz-terminal region (5); thus, providing further support for the presence of such a cluster in the oyster. Additionally, the metal analysis of oyster metallothionein indicating ‘7 g-atoms metal per protein molecule, primarily of cadmium and zinc, supported the possibility that the remainder of the molecule may be coordinated to four metal atoms in the cluster A arrangement. In summary, we have been able to purify low-molecular-mass cadmium-, zinc-binding proteins of a mollusc to a level of purity not previously achieved for this group by other investigators. These proteins can be classified as class I metallothioneins on the basis of the amino acid compositions and the NH2-terminal amino acid sequences. Thus, molluscs can now be included with arthropods as nonvertebrate animal phyla whose representatives possess Class I metallothionein. The molluscan metallothioneins lacked methionine as the NHz-terminal amino acid and contained high levels of glycine. They occurred as two forms, whose only difference, at this time, appears be the presence of a blocked NHz-terminal in one of them. ACKNOWLEDGMENTS We thank Drs. J. B. Blair, D. B. Bonar, and T. T. Chen for useful discussions and B. A. Fowler and

MOLLUSCAN

METALLOTHIONEINS

R. W. Olafson for reviewing an earlier version of the manuscript. Dr. Bonar provided the oysters. L. Matteson exposed oysters to cadmium.

413

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