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

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

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

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

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

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-

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

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

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

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-

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

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

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-

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

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)

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

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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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