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:
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 inegenomics.

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. In addition, we would like to thank the staff of the
ACE Basin National Estuarine Reserve and members of the South
Carolina Department of Natural Resources, Marine Resources
Research Institute, for their assistance with oyster collection.
Additional thanks to the MUSC Mass Spectrometry Facility. This

paper is the Charleston, SC Marine Genomics Group contribution
#4, #01-04 of the Cooperative Institute of Fisheries Molecular
Biology and #537 of the South Carolina Department of Natural
Resources. Research was supported by the National Oceanic and
Atmospheric Administration, National Marine Fisheries Service
(NA07FL0498) and National Science Foundation (EPS0083102).
Part of the research was conducted under an award from the
Estuarine Reserves Division, Office of Ocean and Coastal Resource
Management, National Ocean Service, National Oceanic and Atmo-
spheric Administration.
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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. (A) Diagram of CvMT-I and -II
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) 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).
1712 M. J. Jenny et al. (Eur. J. Biochem. 271) Ó FEBS 2004