Báo cáo khoa học: Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches potx

Diversity of metallothioneins in the American oyster, Crassostrea virginica , revealed by transcriptomic and proteomic approaches Matthew J. Jenny 1 , Amy H. Ringwood 4 , Kevin Schey 2 , Gregory W. Warr 3 and Robert W. Chapman 4 1 Marine Biomedicine and Environmental Sciences Center, 2 Department of Cell and Molecular Pharmacology and 3 Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA; 4 Marine Resources Research Institute, South Carolina Department of Natural Resources, Charleston, SC, USA Metallothioneins are typically low relative molecular mass (6000–7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys- X n -Cys) and a prolate ellipsoid shape containing single a- and b-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan ab-domain structure. Oyster metallo- thioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of ab-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the a-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four a-domains and that lack a b-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evo- lutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family. Keywords: cadmium; gene expression; MALDI-TOF; met- allothionein; oyster. Metallothioneins (MTs) are a superfamily of ubiquitously expressed metal-binding proteins that can be upregulated by metal exposure, oxidative stress and immune challenge. Typical MTs are low relative molecular mass (M r ) (6000– 7000) proteins of high thiol content that lack histidine and aromatic amino acids [1,2]. While they are functionally diverse, they play major roles in metal homeostasis and detoxification. The defining characteristic of MTs is the high cysteine content ( 30%) and conserved Cys-X n -Cys motifs, where X can be any amino acid other than cysteine. The proteins typically have a one- or two-domain structure and bind multiple mono- and divalent metal ions. The structure of MTs, and the nature of their metal-binding, reveal extensive evolutionary diversification. While fungi and early diverged metazoans have small, single-domain MT proteins capable of binding up to eight monovalent metal ions [3–6], most MTs are comprised of two domains, designated a and b, which are capable of binding metals independently and are separated by a short linker region [7,8]. The a-domain typically contains 11 or 12 cysteines, binds four divalent metal cations, and is believed to convey structure and stability to the protein [9]. In contrast, the b-domain contains nine cysteines, binds three divalent metal cations and participates in metal exchange reactions invol- ving glutathione-shuttling with zinc- and copper-requiring apoproteins [10–12]. Some crustacean MTs deviate from this canonical structure, possessing two b-domains capable of binding six metal cations [13]. While the induction of MTs by various metals, partic- ularly cadmium, has been established in a variety of metazoan taxa [14–17], to date only one MT, a cadmium- inducible isoform, has been identified from Crassostrea virginica [18], although biochemical studies indicated the presence of two cadmium-binding proteins of 10 and 24 kDa [19,20]. Several metal-rich proteins, representing putative MTs, have been identified in control and metals- treated C. virginica larvae [21], and the presence of multiple MT isoforms has been demonstrated in other molluscan species [22–26], including bivalves (the blue mussel, Mytilus edulis [23] and the Pacific oyster, Crassostrea gigas [27]) and gastropods (the terrestrial snail, Helix pomatia [25,26]). It is clear that MT characteristics, especially amongst the invertebrates, are more varied than previously believed. Two metal-specific MTs, a copper-specific isoform isolated from mantle tissue and a cadmium-inducible isoform isolated from the midgut gland, have been found in the Correspondence to R. W. Chapman, Marine Resources Research Institute, South Carolina Department of Natural Resources, Hollings Marine Laboratory, 331 Fort Johnson Road, Charleston, SC 29412, USA. Fax: + 1 843 762 8737, Tel.: + 1 843 762 8860, E-mail: chapmanr@mrd.dnr.state.sc.us Abbreviations: Cv, Crassostrea virginica; IAA, iodoacetic acid; M r , relative molecular mass; MT, metallothionein. (Received 21 January 2004, accepted 4 March 2004) Eur. J. Biochem. 271, 1702–1712 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04071.x snail [25,26], and in the Pacific oyster a MT comprised of three metal-binding domains (abb), has been reported [27]. Here we report the results of a study, combining tran- scriptomic and proteomic approaches, designed to increase our knowledge of the structure and function of oyster MTs in the American oyster, C. virginica, and to shed light on the evolutionary diversification of this supergene family. Experimental procedures Collection of C. virginica Adult C. virginica were collected from Lighthouse Creek and Sweetgrass Creek (Charleston, SC, USA) or St. Pierre, ACE Basin (National Estuarine Reserve, SC, USA) and maintained in aerated natural seawater (25 ppt salinity, 1 lm filtered) at the Marine Resources Research Institute, South Carolina Department of Natural Resources (MRRI, SCDNR). Oysters were allowed to depurate in the labor- atory for 24–96 h before use and were fed a phytoplankton suspension consisting of Chaetocerus gracilis Strain (Bacil- lariophyceae) and Isochrysis galbana Strain (Prymnesio- phyceae) every 48 h while maintained in the laboratory. cDNA library construction from C. virginica 24 h D-veliger Gametes were stripped, under sterile conditions, from four female and three male oysters and mixed to allow fertilization to occur. Fertilized eggs were diluted with sterile natural seawater to 50 embryos per mL and incubated for 24 h, under control conditions or conditions containing metal treatments – either copper (0.16 l M )or cadmium (0.18 l M ) – until the D-veliger developmental stage was reached. Three separate cDNA libraries were constructed from  200 000 D-veliger larvae from each treatment. RNA was isolated using the RNeasy Miniprep kits (Qiagen) and cloned using the PCR-based SMART cDNA Library Construction Kit (Clontech Laboratories, Inc.). Library construction has been described in detail previously [28]. Library screening Each library was differentially screened by plating 3 · 10 5 plaque-forming units, which were transferred to replicate nitrocellulose filters (Schleicher & Schuell BioScience, Inc., NH, USA). Filters were prehybridized at 65 °C in Church- Gilbert solution [29] and incubated overnight with probes generated from the cDNA of oyster cadmium-binding MT (generously provided by G. Roesijadi, Florida Atlantic University, Boca Raton, FL, USA) [18]. Probes were radiolabeled with random hexanucleotide primers, Klenow DNA polymerase, and 50 lCi of [ 32 P]dATP[aP] (Perkin- Elmer, Boston, MA, USA). MT cDNA positive plaques were purified through subsequent screenings. Plasmids were isolated from positive plaques using QiagenÒ Turboprep 96 kits on the QiagenÒ Biorobot 9600, according to the manufacturer’s instructions. All plasmid samples were sequenced in both directions by the Biotechnology Resource Laboratory, Medical University of South Carolina, using the ClontechÒ sequencing 5¢ (5¢-AGCTCCGAGATCTG GACGAGC-3¢)and3¢ (5¢-TAATACGACTCACTATA GGGC-3¢) primers for the pTriplEx2 plasmid. Experimental metal challenges for expression analysis Adult oysters were exposed to equimolar concentrations (0.25 l M ) of copper, cadmium, or zinc for a period of 96 h. Gill and hepatopancreas tissues were dissected for total RNA isolation using the methods previously described. For protein analysis, adult oysters were treated with 0.44 l M of cadmium for 96 h before hepatopancreas tissue was dissec- ted, flash frozen in liquid nitrogen and stored at )80 °C. Typically, the hepatopancreas tissues from two to three oysters were combined to increase the protein yield. RT-PCR Multiple cDNAs identified from library screenings were compared in order to design consensus primers for the concurrent amplification of both CvMT-I and -II isoforms; forward consensus primer (5¢-GCCGAYTGTAYCACAG ACAC-3¢) and reverse consensus primer (5¢-CTCTYATT RGTCGAGCGYTC-3¢). Total RNA was isolated with the RNeasy Miniprep kits (Qiagen). First-strand cDNA was synthesized from  1 lg of total RNA using an oligo (dT) primer and 200 U of M-MLV reverse transcriptase (Promega). Complementary isoforms were amplified with 25 cycles of PCR under the following conditions: denatur- ation at 94 °C for 30 s, annealing at 55 °Cfor60s,and extension at 72 °Cfor60s. TOPO TA cloning of RT-PCR products RT-PCR products from four separate reactions for each primer set (control, copper- and cadmium-treated C. vir- ginica larvae; and cadmium-treated adult hepatopancreas tissue) were cloned into the pCRÒ2.1-TOPO vector accord- ing to the manufacturer’s instructions (TOPO TA Cloning Kit; Invitrogen Corporation). The pCRÒ2.1-TOPO con- structs were transformed into chemically competent XL1 Blue MRF¢ Escherichia coli cells. Plasmids were isolated using QiagenÒ Turboprep 96 kits on the QiagenÒ Biorobot 9600, according to the manufacturer’s instructions. Plasmid samples were sequenced by SeqWright, Inc. (Houston, TX, USA) using M13 forward and reverse sequencing primers. Any unresolved nucleotides were confirmed with additional sequencing by the Biotechnology Resource Laboratory (Medical University of South Carolina), using the internal consensus primers previously described in the RT-PCR protocols as well as two additional internal consensus primers (5¢-CGCCTCTCATTGGTCGAGCGC-3¢)and (5¢-GARCGCTCGACYATTRAGAG-3¢). The sequences were deposited in the NCBI nonredundant database with sequential accession numbers AY331695 to AY331707. Genomic Southern blot analysis Genomic DNA was prepared from individual oysters using the total tissue remaining after removal of gonadal and hepatopancreatic tissue. The frozen tissue was ground with a mortar and pestle, transferred to lysis buffer (100 m M EDTA, 50 m M Tris/HCl pH 8.0, 1% SDS) containing Ó FEBS 2004 Metallothionein diversity in the American oyster (Eur. J. Biochem. 271) 1703 20 lgÆlL )1 proteinase K and incubated overnight at 55 °C. Genomic DNA was extracted with phenol/chloroform/ isoamyl alcohol (25 : 24 : 1) and precipitated with 70% ethanol. Separate restriction digests were performed on 7.5 lg of genomic DNA with one of three enzymes, EcoRI, AvaII, or BamHI (Gibco BRL). The resulting fragments were separated on 0.8% agarose gels and transferred to nitrocellulose membrane (Nytran; Schleider & Schuell), using an upward transfer technique, in 20 · NaCl/Cit (3 M NaCl, 0.3 M sodium citrate, pH 7.0). Hybridization was performed using [ 32 P]dATP[aP]-labelled CvMT-I probes. Because of the strong similarity in DNA sequence, the probes generated from CvMT-I will hybridize with all identified CvMT-II isoforms. Northern blot analysis Total RNA from adult oysters (5 lg) was electrophoresed in a 1.2% agarose, 0.6% formaldehyde gel. Denatured RNA was transferred to Nytran membrane, using an upward transfer technique, in 10 · SSPE (1.5 M NaCl, 100 m M NaH 2 PO 4 ,10m M EDTA, pH 7.4). Hybridization was performed in Denhardts reagent buffer [50% forma- mide, 1% SDS, 5 · SSPE (750 m M NaCl, 50 m M NaH 2 PO 4 ,5m M EDTA, pH 7.4], and 2 · Denhardts reagent [0.04% Ficoll 400, 0.04% poly(vinylpyrrolidone), 0.04% BSA)], overnight at 42 °C, with probes generated by random priming from the cDNA of CvMT-I. After autoradiography, membranes were stripped by boiling in 0.1% SDS and rehybridized with probes for b-actin (ACCN_ BG624786). Fractionation of metal-binding proteins by size-exclusion chromatography Hepatopancreatic tissue samples (1 : 2.5 ratio of tissue to buffer; g/mL) were partially thawed in buffer (30 m M NH 4 HCO 3 , pH 8.2) containing 1 m M dithiothreitol and 1m M phenylmethanesulfonyl fluoride. Samples were homo- genized under helium gas and centrifuged (32 000 g)for 60 min at 4 °C. Supernatant was removed and centrifuged (32 000 g) for an additional 30 min at 4 °C and filtered through a 0.45 l M membrane. Proteins were first separated by size-exclusion HPLC on a Superdex 75 PC 3.2/30 column (Pharmacia Biotech, Inc.) with 30 m M NH 4 HCO 3 contain- ing 1 m M dithiothreitol at a flow rate of 0.5 mLÆmin )1 . Fractions were collected every 30 s and monitored for cad- mium, zinc, and copper using a Perkin-Elmer AAnalyst Model 700 atomic absorption spectrophotometer. Commer- cially available rabbit MT (Sigma Chemical Co.) was used to approximate the elution time of comparable oyster MTs. Partial purification of metal-binding proteins by HPLC Anion-exchange HPLC was used to characterize the cadmium-rich pools isolated by size-exclusion chromato- graphy. Proteins were separated using an anion-exchange column (TSKgel DEAE-5PW) with a 35 min linear gradi- ent of 30–350 m M NH 4 HCO 3 containing 1 m M dithiothre- itol (pH 8.2). Proteins were eluted at a flow rate of 0.65 mLÆmin )1 and fractions were collected at 30 s intervals. A25lL aliquot was removed from each fraction, dried, reconstituted in 2% HNO 3 and analyzed for cadmium, zinc, and copper by atomic absorption spectrophotometry. The remaining fraction was frozen at )20 °C until analysis by MALDI-TOF. Determination of mass and cysteine content by MALDI-TOF MS Anion exchange-HPLC fractions, representing individual metal-rich peaks (molecular mass range of 6–22 kDa), were concentrated to 100 lL volumes using Centricon YM-3 filter devices. Samples were acidified with  15 lLof trifluoroacetic acid to a pH range of 2–3 and diluted to 1.5 mL with 2.5% trifluoroacetic acid. Samples were con- centrated to 100 lL by centrifuging with the YM-3 filter devices and demetallated by washing the concentrate with 1 mL of double distilled H 2 O through YM-3 filters until a final volume of  200 lL was achieved. These samples were lyophilized and reconstituted in 100 lLofdenaturing buffer (6 M guanidine/HCl, 0.5 M Tris/HCl, 4 m M EDTA; pH 8.0). A 20 lL sample was stored at )80 °Cformass determination of the native proteins. The remaining 80 lL was subjected to carboxymethylation with iodoacetic acid (IAA). Briefly, the sample was diluted into 920 lLof denaturing buffer deoxygenated with N 2 gas. The remaining steps were performed under N 2 gas with deoxygenated reagents. Sixty microlitres of 100 m M dithiothreitol was added to the sample, which was then incubated at 37 °Cfor 90 min, after which 120 lLof0.2 M IAA was added and incubated continued at 37 °Cfor120mininthedark. Samples suspected to contain MTs in the 6000–7000 M r range were concentrated using the YM-3 filter devices and washed with ddH 2 O. Samples believed to represent the high M r isoforms (> 15 000) were subjected to a buffer exchange by elution through a Superdex 75 PC column in 10 m M NH 4 HCO 3 , lyophilization with a speedvac and reconstitution in 25 lLofddH 2 O.Thenativeandcarb- oxymethylated proteins were desalted with ZipTip C18 pip- ette tips (Millipore) and eluted in 0.1% trifluoroacetic acid containing 50% acetonitrile. Samples were diluted in three parts sinapinic acid matrix (50 m M 3,5-dimethoxy- 4-hydroxycinnamic acid/70% acetonitrile/0.1% trifluoro- acetic acid) and the mass was determined by MALDI-TOF MS (Voyager-DE STR BioSpectrometry Workstation; Applied Biosystems). Cysteine content was determined by subtracting the mass of the native protein from the carboxymethylated protein and dividing by 58 Da (mass of the IAA derivative). The metal-rich fractions believed to correspond to the small M r MT ( 4000) isoforms were lyophilized and reconstituted in 60 lL of denaturing buffer (6 M guanidine/ HCl, 0.5 M Tris/HCl, 2 m M EDTA; pH 8.2). YM-3 filters or buffer exchange were likely to result in significant loss of sample, so the demetallation step was not performed. A10lL aliquot of sample was stored at )80 °Cformass determination of the native proteins. A modified method was used for carboxymethylation of the remaining 50 lL. Briefly, the sample was deoxygenated with N 2 gas and 2 lL of 100 m M dithiothreitol was added before the sample was incubated at 45 °C for 60 min, after which 6 lLof0.2 M IAA was added and incubated at 45 °C for 60 min in the dark. The native and carboxymethylated proteins were 1704 M. J. Jenny et al. (Eur. J. Biochem. 271) Ó FEBS 2004 purified with ZipTip C18 pipette tips and eluted in 0.1% tri- fluoroacetic acid containing 50% acetonitrile. Samples were diluted in three parts a-cyano matrix (50 m M a-cyano-4- hydroxycinnamic acid/70% acetonitrile/0.1% trifluoroace- tic acid) and the mass was determined by MALDI-TOF MS. Results Diversity of oyster MTs at the level of the transcriptome cDNA libraries constructed from control, cadmium-, and copper-treated larvae were screened with a probe represent- ing the oyster MT originally reported [18]. From these respective libraries, 20, 25, and 38 clones were plaque purified, and 10 distinct isoforms were identified and com- pletely sequenced (NCBI accession numbers AY331695 to AY331707; also viewable at http://www.marinegenomics. org). The nucleotide sequences obtained all showed strong similarity (> 85%) to the known oyster MT that was used as the probe and which is designated as CvMT-IA. Although this strong sequence conservation indicates that all of the sequenced MTs belong to the same family, the 10 sequences could be divided into two separate subfamilies (CvMT-I and CvMT-II), based on their conceptual trans- lation and the inferred domain structure of the encoded MTs (Fig. 1A,B). Two clones were of the CvMT-I sub- family and eight clones were of the CvMT-II subfamily. The designation CvMT-I is used to represent the traditional class of molluscan MT proteins with a- and b-domains and 21 conserved cysteines [30]. In addition to CvMT-IA, a novel isoform of the same subfamily (CvMT-IB)wasidentified from the control (nonmetals challenged) larval cDNA library (Fig. 1B), and showed five amino acid substitutions in the a-domain, one in the b-domain and conservation of all 21 cysteines. The CvMT-II subfamily is distinguished by the presence of only a-domains in its conceptual translation. This structure arises from the presence of a mutation (AfiT) which converts a lysine codon (AAG) in the linker region separating the a- and b-domains into a stop codon (TAG). The CvMT-II subfamily is exemplified by two related isoforms (CvMT-IIA and -IIB) which, in conceptual translation, are single a-domain peptides of inferred M r of  4100. The CvMT-II subfamily contains additional mem- bers (designated CvMT-IIC through CvMT-IIH)inwhich two, three and four a-domains are encoded (Fig. 1A), and have inferred M r values of  9200, 14 600 and 20 200, respectively. Inferred exon structure of the CvMT-IA gene The sequencing of MT cDNAs revealed several partially spliced CvMT-IA transcripts which, taken together, permit- ted deduction of the intron/exon structure of the CvMT-IA gene, as shown in Fig. 2A. This deduced gene structure was Fig. 1. Diversity of metallothionein (MT) isoforms from Crassostrea virginica. (A) Schematic representation of diversity in the CvMTI/II family, demonstrating the characteristic domain structures of the isoforms characterized as cDNA. The cysteine-rich domains are classified as either a or b, based on the number and configuration of the cysteine motifs. The Ônoncoding exonÕ region represents the b-domain region of the transcript that has been truncated by the introduction of a stop codon in the linker region. (B) Amino acid sequences of representative isoforms from the CvMT-I/II family, as deduced from cDNA sequences. Conserved residues are designated by (d) and the linker region (KVK) between the two domains is underlined. The alignment demonstrates the presence of the three a-domains present in CvMT-IIC,witheachdomainbeingpresentedasaseparate, rather than contiguous, sequence. Ó FEBS 2004 Metallothionein diversity in the American oyster (Eur. J. Biochem. 271) 1705 compared with the structures reported for the C. virginica MTA gene (NCBI accession number AF506977) and the C. gigas MT1 gene [22], as shown in Fig. 2B. The CvMT-I gene was found to have the same intron phase (1,1) as the CvMTA gene, whereas the C. gigas MT1 gene had a (1,2) intron phase. The three genes differed remarkably in their intron lengths. The first introns in the two CvMT-I genes were the only ones of similar length. The second intron was much shorter in CvMT-IA than in CvMTA; however, it shared 86% identity with the last 92 nucleotides of the second intron of CvMTA. The second intron of CvMT-IA was found to contain a noncanonical donor splice site (AG/tT, Fig. 2A), which has been reported as a rare splice site variant in mammalian genes [31]. Taken together with the cDNA cloning data, these results strongly suggest that in C. virginica there is a large family of CvMT-I/II genes. In order to gain further insight into this gene family, genomic Southern blot analysis of two oysters was performed, using a probe that detects members of both the CvMT-I and CvMT-II subfamilies. The results (Fig. 3) are clearly compatible with the presence of multiple copies of these sequences in the oyster genome, and the variability of intensity between the hybridizing bands suggests that there may be multiple, closely linked CvMT-I/II sequences. The differences in restriction fragment length between the two oysters also indicated substantial allelic polymorphism in this gene family. CvMT-I and CvMT-II gene expression is induced by cadmium The expression of CvMT-I/II isoforms in gill and hepato- pancreas, and their upregulation by exposure to copper, zinc and cadmium, were examined by Northern blot analysis. The CvMT-I and CvMT-II messages were readily distinguishable by their relative electrophoretic mobility, as indicated in Fig. 4. While the results suggested that hepatopancreas has a higher basal expression level of CvMT-I/II isoforms than does gill tissue, it is clear that cadmium exposure strongly upregulated CvMT-I/II expres- sion in both tissues. However, there were no significant changes in the levels of CvMT-I/II expression following exposure to copper or zinc at the same concentration as cadmium. Fig. 2. A comparison of metallothionein (MT) gene structure in oysters. (A) Diagrammatic representation of two partially spliced transcripts of CvMT-IA. Exons are represented by boxes. Introns are represented by straight lines. The angled lines represent the intron spliced from the partially processed transcript. The second intron contains a nonca- nonical splice donor site (tTgAG). (B) Comparison of the proposed exon/intron structure of CvMT-IA with that of two characterized MT genes from Crassostrea virginica and C. gigas ( a AF506977, b AJ242657). Exons are designated by boxes with the number of nucleotides in each open reading frame. Introns are designated by lines, with the length (number of nucleotides) shown in parentheses. Fig. 3. Southern blot analysis of the CvMT-I/II gene family in Cras- sostrea virginica. Genomic DNA from two individual oysters was digested with EcoRI, AvaII and BamHI, and analyzed, after electro- phoresis and blot transfer, with a probe for the CvMT-IA cDNA. Each lane contains 7.5 lgofgenomicDNA. Fig. 4. Northern blot analysis of control and metal-treated tissues from adult Crassostre a virginica. Adult oysters were exposed to copper, cadmium and zinc for 96 h, and 5 lg of total RNA from gill and hepatopancreas tissues was analyzed by Northern blot using a probe for the CvMT-IA cDNA. 1706 M. J. Jenny et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Diversity of oyster MTs at the level of the proteome Studies of oyster MT proteins were undertaken, first, to confirm that the diversity of CvMT-I/II sequences seen at the cDNA level was reflected at the level of the expressed proteins and, second, to test the possibility that different isoforms of the CvMT-I subfamily preferentially associate with cadmium. In the initial characterization of oyster MTs, extracts of hepatopancreas from control and cadmium- exposed oysters were separated by gel filtration chromato- graphy. The eluted proteins were monitored at three wavelengths. The relative absorption at 220/280 nm allowed the detection of proteins (such as MTs) that are deficient in aromatic amino acids, while cadmium/thiol interactions yielded an increased absorbance at 254 nm. Comparison of the elution profiles identified three protein peaks in the cadmium-treated samples that showed specific increases of absorption at 220 and 254 nm, but not at 280 nm (Fig. 5A, right panel). These three fractions also corresponded to peaks of cadmium in the elution profile (Fig. 5B). The elution profile of the three cadmium-rich pools (A, B, C; Fig. 5B) was consistent with the predicted diverse M r values of the multiple CvMT-I/II isoforms detected at the cDNA level, but to determine the exact nature of these proteins, further analysis was undertaken to determine experiment- ally their M r and cysteine content. Each of the three pools was fractionated by anion-exchange HPLC, and the metals elution profile for the high and low M r pools were measured for cadmium, while the classic M r pool was also measured for copper and zinc (Fig. 6). The metals elution profiles for the high and low M r pools were determined from single representative samples of cadmium-exposed oysters. The metals elution profile of the classic M r pool was a composite of control and cadmium-treated oysters. The proteins in the fractions identified by the metals elution profiles were subjected to MALDI-MS analysis before and after deriva- tization with IAA. Typical MALDI-MS traces are shown, in Fig. 7, for the analysis of three of the fractions before and after derivatization. Overall, 10 proteins were identified, by MALDI-MS, whose M r and calculated cysteine content were consistent with their identification as members of the CvMT-I and CvMT-II subfamilies. Of these, three MTs (peaks e, f and g, Fig. 6B), of approximate M r 7242–7375, were characterized in zinc-rich fractions, with peak g identified from a control oyster. All the other MTs were found in cadmium-rich fractions after 96 h of exposure of oysters to 0.44 l M of cadmium. As summarized in Table 1, putative MTs of the ab-domain structure (CvMT-I) and with one, three and four a-domains (CvMT-II), could readily be identified. Sequence diversity (of unknown extent) within the CvMT-I/II family, and uncertainties over post- translational modifications of MTs (such as N-acetylation [18]), probably contribute to the small divergence seen between the conceptual (cDNA translation) and experi- mentally observed M r values. Although the examination of MT representation in the oyster proteome was not exhaustive, it is clear from the data presented in Table 1 that there is a substantial complexity of the CvMT-I/II family. Discussion The data presented in this study were obtained by an initial transcriptomic and proteomic study, and reveal a diversity of oyster MTs that has implications for our understanding of the evolution of this gene family and for interpreting structure/function relationships in molluscan MTs. Diversity of oyster MTs at the transcriptomic level It is known from previous studies [22–26] that molluscan MTs show a diversity of structure that encompasses not only the canonical ab-domain structure, but also molecular forms in which this structure has been modified, e.g. as in the abb MT seen in the Pacific oyster, C. gigas [27]. The data reported here reveal a structural and functional diversity within the MT family of the American oyster (C. virginica) that, while proposed by prior studies at the protein level [19–21], has not previously been documented. Fig. 5. Gel filtration profile of cadmium- exposed hepatopancreas tissue from adult Crassostrea virginica. Extracts of hepatopan- creas tissue from control and cadmium- exposed adult oysters were subjected to gel filtration chromatography with a Superdex 75 PC 3.2/30 column. (A) Chromatograms clearly demonstrate three strong peaks (21, 25.5, and 31.5 min), detectable at 220 nm but not at 280 nm, in extracts of cadmium-treated tissues. The corresponding absorbance at 254 nm is consistent with cadmium–thiol interactions expected of metallothionein pro- teins. (B) Cadmium elution profile of the same samples demonstrates the presence of three cadmium-rich pools (A, B and C) corres- ponding to the 220 nm/254 nm absorbance. The rabbit MT (rMT) standard eluted at 26 min. Ó FEBS 2004 Metallothionein diversity in the American oyster (Eur. J. Biochem. 271) 1707 In particular, it is clear that in this species of oyster, the family of canonical ab-domain-containing MTs (the CvMT-I subfamily) has undergone substantial expansion to include MTs that solely express a-domains (the CvMT-II subfamily). While cDNA cloning showed the presence of CvMT-II transcripts encoding MTs with one to four a-domains, analysis at the protein level (discussed below) identified putative expressed molecules corresponding to three of these MTs: those with one, three and four a-domains. Analysis of cDNA sequences of CvMT-I/II clones, along with the intronic sequence of the CvMT-IA gene, permitted deduction of the series of events that probably led to the generation of genes encoding CvMT-II family members. Initially, the mutation of a lysine codon to a stop codon in the linker region would have truncated the MT protein after the a-domain. Subsequent tandem duplications of the a-encoding sequence (the first two exons) would then have readily generated the multiple CvMT-II genes identified in this study. While the data reported here confirmed that the CvMT- IA gene had the same pattern of three exons/two introns previously reported for a C. virginica MT gene (CvMTA, ACCN_AF506977) and for a C. gigas MT gene [22], the variations seen in intron length suggest that molluscan MT genes, while conforming to a basic exon structure, probably show, as predicted, substantial variations in their introns. The presence of a rare noncanonical donor splice site in the CvMT-IA gene (Fig. 2) suggests that this variation in intronic sequences may have implications for the expression of the oyster MT genes. While Southern blot analysis confirmed CvMT-I/II as a multigene family, it was unable to distinguish the total or relative genomic representation of the CvMT-I and CvMT-II subfamilies of genes. The expression of oyster MT genes in response to metals exposure was measured by Northern blot, and showed that there was apparent global upregulation of CvMT-I/II transcripts induced by cadmium, but not by comparable concentrations of copper or zinc. Variable baseline expres- sion of MTs was observed in unchallenged oysters (Fig. 4), but it is not known if this pattern of expression reflects prior exposure to metals or other stresses that may induce MT expression, or is representative of basal expression associ- ated with normal metals homeostasis. Diversity of oyster MTs at the proteomic level Characterization of oyster MTs was undertaken at the protein level in order to confirm and extend the results obtained from cDNA analysis. Gel filtration and anion- exchange chromatography, combined with MALDI-MS analysis, resulted in the identification of 10 MTs in extracts of control and cadmium-treated oysters. On the basis of size and cysteine content, these could be identified, with confidence, as members of the CvMT-I/II family (Table 1). Because of the probable size of the CvMT-I/II gene family, uncertainties exist concerning the full range of sequences of the encoded MTs, as well as the potential post-translational modification of MTs. These uncertainties make difficult any attempt to correlate the observed oyster MT proteins with the isoforms inferred from the cDNA sequences. However, based on cadmium-binding, M r and cysteine content characteristics, it is highly likely that CvMT-IA, the prototypical oyster metallothionein [32], has been identified (Fig. 6B; peak d). In addition, we identified three MTs whose M r values ( 7200) were not consistent with the predicted characteristics of CvMT-IA or -IB. These three MTs were the only ones identified in this study in copper/ Fig. 6. Anion exchange HPLC of the cadmium-rich pools. The three cadmium-rich pools identified from gel filtration chromatography (asteriskedinFig.5A)weresubjectedtoanionexchangechromato- graphy. Metal elution profiles (cadmium and/or copper and zinc) were determined by spectrometry (PerkinElmer AAnalyst Model 700 atomic absorption spectrophotometer), and eluted proteins were analyzed by MALDI-MS to determine the M r and cysteine content (Fig. 7, Table 1). All peaks labeled with a lowercase letter (a–i) had a cysteine content consistent with their identification as metallothionein (MT). (A) Anion-exchange chromatography of the high molecular mass pool from gel filtration (Fig. 5) identified three cadmium-rich peaks (a–c) containing MTs. (B) Anion-exchange chromatography of the intermediate molecular mass pool from gel filtration (Fig. 5) identified four peaks (d–g) containing MTs. Only one strong cadmium- rich MT-containing peak (d) was recovered from this pool, but three MT-containing peaks (e–g) were identified as copper/zinc rich. (C) Anion-exchange chromatography of the low molecular mass pool from gel filtration (Fig. 5) identified two cadmium-rich peaks (h,i) containing MTs. 1708 M. J. Jenny et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Fig. 7. Identification of CvMTI/II family pro- teins by MALDI-TOF MS. The mass of the native and iodoacetic acid (IAA)-derivatized proteins from the metals-rich peaks (b,d,h; Fig. 6) identified by anion-exchange HPLC was determined by MALDI-TOF MS. Three representative trace spectra clearly illustrate the identification of metallothioneins (MTs) from each of the three M r pools based on gel filtration analysis (Fig. 5). Table 1. Characteristics of CvMT-I/II family members. Predicted, conceptual translation of nucleotide sequence; Observed, measured by MALDI- MS; Length, number of amino acid residues; M r , relative molecular mass calculated from the amino acid sequence; M r acetylated, relative molecular mass calculated assuming N-acetylation of the MT; Fraction, as designated in Figs 6 and 7; M r native, MALDI-MS of underivatized proteins; M r IAA, MALDI-MS of iodoacetic acid (IAA) derivatized proteins; Cys residues, number of cysteine residues calculated from the mass difference between the native and IAA-derivatized proteins measured by MALDI-MS; Subfamily, the exact assignment of isoform was not attempted. Isoform Predicted Observed Length M r M r acetylated Domain structure Cys residues Fraction M r native M r IAA Cys residues Subfamily CvMT-IA 74 7214 7256 a/b 21 d 7251.2 8471.1 21.1 CvMT-I CvMT-IB 74 7224 7266 a/b 21 e 7242.4 8469.4 21.1 CvMT-I f 7250.4 8470.6 21.0 CvMT-I g 7375.9 8390.4 17.5 CvMT-I CvMT-IIA 42 4097 4139 a 12 h 4106.4 4802.2 12.0 CvMT-II CvMT-IIB 42 4122 4164 a 12 h, i 4234.8 4933.2 12.0 CvMT-II CvMT-IIC 93 9250 9292 (a) 2 25 CvMT-II CvMT-IID 148 14 758 14 800 (a) 3 38 a 14 638.9 16 879.9 38.6 CvMT-II CvMT-IIE 148 14 592 14 634 (a) 3 38 b 14 640.7 16 856.4 38.2 CvMT-II CvMT-IIF 144 14 276 14 318 (a) 3 38 CvMT-II CvMT-IIG 203 20 202 20 244 (a) 4 51 b 20 478.9 23 310.9 48.8 CvMT-II CvMT-IH 200 19 777 19 819 (a) 4 51 c 20 461.2 23 342.1 49.7 CvMT-II Ó FEBS 2004 Metallothionein diversity in the American oyster (Eur. J. Biochem. 271) 1709 zinc-rich and cadmium-poor fractions, despite the fact that two were isolated from cadmium-treated oysters. Thus, they may represent novel CvMT-I isoforms with highly prefer- ential binding for copper/zinc. Observations presented here support the hypothesis that the a-domain of molluscan MTs has characteristics similar to those of other vertebrate and invertebrate species. The analysis of proteins induced by cadmium exposure (Figs 5–7) identified multiple CvMT-II isoforms, containing one, three and four a-domains, all associated with cad- mium-rich fractions. Although the data support the expres- sion and cadmium-inducibility of CvMT-II proteins, the mass accuracy of the analysis was not adequate to confirm the identity of specific CvMT-II isoforms identified as cDNA. Evolution of MT structure The MT b-domain has been proposed as the ancestral MT domain with a primary role in the homoeostasis of physiologically relevant metals, such as copper and zinc. It has been suggested that duplication of the b-domain gave rise to a two-domain MT, and the subsequent divergence of the two domains eventually gave rise to the canonical a/b structure of the MTs [33]. The b-domain has a binding preference for copper [34], whereas the a-domain has a preference for cadmium and zinc [35]. This suggests that selective pressures may have led to the evolution of two domain MTs with specific functions carried out by the two domains, with the b-domain more important for metal homeostasis and the a-domain more important for metal storage and detoxification. This hypothesis is supported by the presence of single b-domain, copper-thionein systems present in Drosophila [4] and fungi [6,36] and the existence of the crustacean MTs, comprising two b-domains, that function in copper homeostasis related to the synthesis and degradation of hemocyanin [13,37]. Of interest is the single-domain MT peptide (containing 41 amino acids and capable of binding four cadmium ions) that has been identified in a terrestrial worm, Eisenia foetida [38]. This is a cadmium-inducible MT derived from a two-domain mole- cule by post-translational cleavage. The four-metal-cluster binding stoichiometry of this MT would suggest functional analogy to a single a-domain MT. This theory of domain duplication is further supported by the widespread occur- rence of the ab- and ba-domain structures of many invertebrate and vertebrate MTs and their roles in zinc homeostasis and cadmium detoxification. This notion can also explain the presence of the high molecular mass MT proteins, which may enhance metals-resistance in benthic and terrestrial organisms experiencing a greater exposure to metals owing to their ecological niche [27,39,40]. It should be acknowledged that the theory of gene duplication experiences some difficulties when invertebrate and verteb- rate MT gene structures are compared: in many inverte- brates, the a-domain is N-terminally encoded, whereas in vertebrates the reverse is the case, with the b-domain being N-terminally encoded [41]. Thus, true homology between the a- and b-domains of invertebrate and vertebrate domains would require an inversion within the MT gene of the a and b encoding segments, an event of which there is no obvious record in the genes. The presence of the three-domain abb MT protein from C. gigas [27] is an interesting contrast to the multiple a-domains of the CvMT-II isoforms. While both species of oyster appear to have adopted similar strategies for survival in environments that can be metals-rich, selective pressures and novel genetic mutations in C. virginica appear to have resulted in the unusual structure of the CvMT-II isoforms. Thus, under the general hypothesis discussed above, the differences in domain structure between CvMT-I and -II may represent an example of evolutionarily divergent domain functions. While the stability and metal-binding affinity of the CvMT-II proteins are not yet known, the cadmium-inducibility and in vivo cadmium-binding proper- ties of these proteins suggests similar roles in metals- detoxification and metals-resistance, as proposed for high molecular mass isoforms of MTs and other cysteine-rich proteins present in other species [27,40]. Acknowledgements The authors would like to thank Drs Paul Gross and Mats Lundquist, Darlene Middleton, and members of the Marine Genom- ics Program for support and advice with this study. We would also like to thank Dr G. Roesijadi for the generous donation of the cDNA for CvMT-IA. 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(A) Diagram of CvMT-I and -II Ó FEBS 2004 isoform structural diversity, as represented by cDNA (B) Conceptual translation of the two domains of CvMT-IA and the corresponding aligned sequences of CvMT-IIB and -IID The conceptual translation includes the residues ÔencodedÕ after the stop codon (*) present in the linker region between the a- and b-domains of CvMT-IIB and -IID (highlighted by a box) (C)... Wilkinson, D.G., Travaglini, E.C., Sternberg, E.J & Butt, T.R (1985) Sea urchin metallothionein sequence: key to an evolutionary diversity Proc Natl Acad Sci USA 82, 4992–4994 Supplementary material The following material is available from http:// blackwellpublishing.com/products/journals/suppmat/EJB/ EJB4071/EJB4071sm.htm Fig S1 The mutation of a lysine codon introduces a stop codon in CvMT-II isoforms... codon (*) present in the linker region between the a- and b-domains of CvMT-IIB and -IID (highlighted by a box) (C) Nucleotide sequence alignment showing the stop codon introduced by mutation of a lysine codon in the linker region of CvMT-IA (highlighted by a box) . Diversity of metallothioneins in the American oyster, Crassostrea virginica , revealed by transcriptomic and proteomic approaches Matthew J. Jenny 1 , Amy H. Ringwood 4 , Kevin Schey 2 ,. MT. This theory of domain duplication is further supported by the widespread occur- rence of the ab- and ba-domain structures of many invertebrate and vertebrate MTs and their roles in zinc homeostasis. between the a- and b-domains of invertebrate and vertebrate domains would require an inversion within the MT gene of the a and b encoding segments, an event of which there is no obvious record in the genes. The