Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropod Littorina littorea
Institute of Biochemistry and Department of Biology, College of Natural Sciences, Carleton University, 1125 Colonel By Drive, Ottawa Ontario, Canada K1S 5B6
Author for correspondence (e-mail:
kenneth_storey{at}carleton.ca)
Accepted 22 April 2002
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Summary |
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Key words: environmental stress, gene expression, metallothionein, invertebrate, anaerobiosis, freeze tolerance, periwinkle, Littorina littorea
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Introduction |
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In recent studies, our laboratory has begun to document the gene-expression
responses involved in natural stress tolerance in L. littorea. Larade
et al. (2001) reported
transcriptional upregulation of the ribosomal protein L26 during anoxia, and
proposed that upregulation of the protein might function to stabilize the
existing mRNA pool during the anoxic period until normal oxygen levels are
resumed. More recently, Larade and Storey
(2002
) described a novel cDNA
that is both transcribed and translated during anoxia exposure, supporting a
role in anoxia survival. In the present study, we report the isolation of a
metallothionein (MT) cDNA that is upregulated in response to both anoxia and
freezing exposures in L. littorea.
MTs are a family of low molecular mass, cysteine-rich proteins that
characteristically bind both essential and non-essential metal ions in a
metal/sulfur complex. Although all MTs show similar functional properties and
are uniformly found in species ranging from microorganisms to mammals, they
are notoriously variable in terms of amino acid composition and sequence.
Despite this, there are enough similarities among primary structures to permit
a classification scheme comprising 15 families, as defined by Binz and Kagi
(1999) (see also
http://www.expasy.ch/cgi-bin/lists?metallo.txt).
MTs are commonly composed of 5875 amino acids, approximately 30% of
which are cysteine residues and few, if any, are aromatics.
Characteristically, the cysteines are arranged in repeating Cys-X-Cys motifs
throughout the sequence. Although a definitive picture of their cellular
function has yet to unfold, the metal-binding characteristic confers several
roles to MTs, including: (1) homeostasis of essential trace metals, (2)
segregation of toxic heavy metals (e.g. cadmium, mercury, lead), and (3) a
reservoir for copper and zinc, which can then be donated to other
metalloproteins such as transcription factors. Studies have also produced
several lines of evidence to indicate that MTs have antioxidant properties,
either directly, due to their many, readily oxidized cysteine residues, or
indirectly, by binding heavy metals ions (e.g. copper). This latter action
prevents metal ions from participating in the Fenton reaction, which is a
primary source of highly reactive hydroxyl radicals in cells
(Viarengo et al., 1999
).
MTs have been sequenced from several mollusc species
(Unger et al., 1991; Dallinger
et al., 1993
,
1997
;
Mackay et al., 1993
) and MT
protein levels in tissues of marine molluscs rise in response to heavy metal
challenge (especially cadmium) (Langston and Zhou,
1986
,
1987
;
Langston et al., 1989
;
Boutet et al., 2002
). However,
the present study is the first to clone and sequence the MT cDNA from a marine
gastropod and, more importantly, the first to show that MT expression is
induced in response to naturally occurring environmental stresses. Here we
report the upregulation of MT expression in L. littorea during anoxia
or freezing exposures, and propose several mechanisms by which MT may
contribute to stress tolerance.
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Materials and methods |
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cDNA cloning, screening and sequence analysis
Screening of the anoxic library
RNA isolations, cDNA library construction, screening and northern blot
analysis were all performed essentially as described previously
(Cai and Storey, 1996).
Poly(A)+ mRNA was isolated from the foot muscle of snails exposed
to 1, 12 or 24 h of anoxia. Equal amounts of mRNA from each time point were
pooled (totalling 5 µg) and then used to construct a cDNA library in the
LambdaZap vector, which was then subjected to two rounds of differential
screening. Plaques (
2.5x104 pfu 24 cm-2
plate) were grown on lawns of Escherichia coli (XL1 blue) and
transferred to duplicate nylon membranes (Hybond N, Amersham Biosciences, Baie
d'Urfe, Quebec, Canada). These were hybridized at 42°C for 16 h with
32P-labelled single-stranded cDNA probes synthesized from the
poly(A)+ RNA prepared from foot muscle of aerobic (control)
versus anoxic snails. The anoxic probe was also synthesized using a
composite pool of mRNA (equal amounts from 1, 12 and 24 h anoxic snails).
Plaque lifts were washed at room temperature with low stringency (2x
SSC, 0.2% w/v sodium dodecyl sulfate) to high stringency (0.2x SSC, 0.2%
w/v sodium dodecyl sulfate) washes (SSC, 150 mmol l-1 NaCl, 15 mmol
l-1 sodium citrate). Plaques showing a higher signal with the
anoxic versus aerobic probe were purified with a second round of
plaque hybridization. Duplicate lifts were concurrently hybridized, washed and
exposed to film. The purified clones in pBluescript plasmid vectors were
rescued by in vivo excision using ExAssist helper phage (Stratagene,
La Jolla, CA, USA). Plasmids were digested with EcoRI and
XhoI to release the cDNA insert.
Screening of the freeze library
A cDNA library was prepared from foot muscle of freeze-exposed snails in
the same manner as for the anoxic library but using equal amounts of
poly(A)+ mRNA isolated from snails frozen for 1, 12 and 24 h.
Screening was conducted with 32P-labelled probes made from mRNA
isolated from control (aerobic, unfrozen) versus frozen snails
(synthesized from a composite pool of mRNA from 1, 12 and 24 h frozen
snails).
Northern blot analysis
Total RNA was isolated from organs of control, anoxic or frozen snails
using TRIzolTM (Gibco Life Technologies, Carlsbad, CA, USA), and
separated on formaldehydeagarose gels using 16 µg of total RNA per
lane. After transfer to Hybond N membrane by capillary action, the quality of
total RNA was assessed by the identification of a well-defined ribosomal band
(stained with ethidium bromide). The LLMET probe was synthesized by
random primer labelling of the clone insert
(Sambrook et al., 1989). Blots
were hybridized overnight with labelled probe (107 c.p.m.
ml-1 hybridization solution) at 42°C, followed by low (2x
SSC) and high (0.2x SSC) stringency washes until there was a strong
contrast between the signal and background, and then exposed to film.
Transcript levels were quantified by scanning the X-ray autoradiogram using a
Scan Jet3C scanner (Hewlett Packard, Mississauga, Ontario, Canada). The image
was acquired using DeskScan (v2.2) software in conjunction with Imagequant
V3.22 to quantify pixel density (Amersham Biosciences). The ribosomal bands
were quantified and these values were used to evaluate any differences in
loading between lanes. RNA transcript sizes were estimated based on comparison
with the migration of standards in an RNA ladder (Gibco Life
Technologies).
DNA sequencing and analysis
The clone was sequenced in both directions by Canadian Molecular Research
Services (Ottawa, Ontario) and Bio S&T (Lachine, Quebec, Canada), using an
automated sequencing procedure. Clone identification was attempted
via sequence homology searches as determined with the NCBI blastn and
blastx programs. Sequence analyses (alignments, comparisons, translations)
were completed using DNAman vI (Lynnon BioSoft, Vaudreuil, Quebec, Canada).
Percentage identities between L. littorea MT and other mollusc MT
sequences were calculated from individual pairwise alignments as described in
DNAman. A homology tree of invertebrate MT sequences was generated with
sequences obtained from GenBank using the protein alignment matrix PAM
(Dayhoff et al., 1978) as
employed in DNAman.
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Results |
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At 100 amino acids in length and a predicted molecular mass of 10 kDa, the
protein encoded by LLMET (Ll-MT;
Fig. 1) is significantly larger
than most other MTs, regardless of species. Ll-MT also contained a
correspondingly higher number of cysteine residues (27), 20 of which fall into
the Cys-X-Cys motif pattern that is characteristic of all MTs. As with other
mollusc MTs, Ll-MT lacks the additional Cys-Cys pattern found in crustacean,
vertebrate, nematode and echinoderm MTs.
Fig. 2A shows a comparison of
the Ll-MT amino acid sequence with those of MTs from three other mollusc
species: the copper-binding (Dallinger et
al., 1997) and cadmium-binding
(Dallinger et al., 1993
) MTs
from the land snail Helix pomatia; isoform MT20-Ib from the blue
mussel Mytilus edulis (Mackay et
al., 1993
) and MT from the eastern oyster Crassostrea
virginica (Unger et al.,
1991
). Although these sequences are quite variable, the alignment
of the cysteine residues is conserved. All 18 of the cysteine residues of the
land snail MTs (both the copper- and cadmium-binding isoforms) were aligned
with those of the marine snail L. littorea, albeit with the insertion
of selected spaces. The oyster and blue mussel sequences have more cysteine
residues (21 and 23, respectively), but 18 of them also align with those
shared by the L. littorea and H. pomatia sequences.
Fig. 2A also shows the presence
of the conserved C-terminal sequence pattern,
Cys-X-Cys-X(3)-Cys-Thr-Gly-X(3)-Cys-X-Cys-X(3)-Cys-X-Cys-Lys
(Binz and Kagi, 1999
). This
motif is characteristic of mollusc MTs and its presence in Ll-MT confirms that
the L. littorea protein is a family 2 (mollusc) metallothionein.
Overall, Ll-MT showed the strongest sequence identity with the proteins from
H. pomatia (56% identical to the cadmium-induced isoform, 45%
identical to the copper-specific) as well as 47% and 43% identity with oyster
and blue mussel MTs.
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Metallothionein homology relationship
To further support the identity of the L. littorea clone as
encoding a metallothionein, a homology tree was generated from a multiple
alignment of Ll-MT and eight other invertebrate metallothionein sequences
(Fig. 2B). Ll-MT is most
similar to the other gastropod sequences, sharing 51% identity with the H.
pomatia sequences, and sequences from gastropod molluscs are distinctly
different from those of bivalve MTs. The crustacean sequences share the
highest percentage identity (74%) but are distinct from the mollusc MTs. Both
nematode and sea urchin MTs show very little sequence similarity with the
molluscan proteins.
Northern blot analysis
The expression of LLMET during freezing or anoxia exposure of
L. littorea was assessed by northern blot hybridization using
radiolabeled probe synthesized from the digested insert of the
LLMET plasmid. LLMET expression in both foot muscle and
hepatopancreas was analyzed in control snails at 5°C, snails subjected to
1, 12 or 24 h of stress exposure (either anoxia under an N2 gas
atmosphere at 5°C or freezing at -8°C), and snails given 24 h of
recovery at 5°C after 6 days of stress. Although LLMET was
isolated from cDNA libraries synthesized from foot muscle mRNA, northern blot
hybridization revealed that the transcript was also present in the
hepatopancreas, an expression pattern that is consistent with the broad
distribution of MTs among the tissues of these and other molluscs. For
example, MT protein has been documented in the gill, kidney and digestive
gland of L. littorea (Bebianno and
Langston, 1998) as well as the foot and adductor muscles of the
mussel Mytilus galloprovincialis
(Carpene et al., 1983
).
The LLMET probe hybridized with a 1.2 kb band and the pattern of stress-induced transcript accumulation was similar under both freezing and anoxia stresses. In foot muscle (Fig. 3A), LLMET transcripts were present in low amounts in control snails but, during anoxia, transcript levels increased significantly (P<0.05) by twofold after only 1 h of anoxia exposure and further rose to a maximum of 2.8-fold higher than control values after 12 h. LLMET transcripts declined to near-control values by 24 h of anoxia exposure, but recovery under normoxic conditions induced another significant increase to 1.7-fold that of control animals. The response to freezing in the foot muscle was a significant 2.6-fold increase in transcript levels within 1 h of freezing at -8°C with a continuing increase to reach a 3.3-fold maximum level after 24 h of freezing. After thawing for 24 h, transcript levels had decreased slightly but were still 2.2-fold greater than control levels. The effects of anoxia and freezing exposure on LLMET transcript levels in hepatopancreas are shown in Fig. 3B. Transcript levels did not rise as quickly as in foot muscle (after 1 h levels were not significantly increased) but after 12 h of anoxia transcript levels peaked at a value that was much greater than in muscle, 5.2-fold higher than control values. As for foot muscle, this level of expression declined after 24 h to a value threefold higher than control levels, followed by a slight increase to 4.7-fold higher than controls after 24 h of normoxic recovery. The response to freezing was somewhat faster in hepatopancreas; transcript levels rose significantly by 3.3-fold within 1 h and reached a maximum expression level of sixfold greater than control values by 12 h. Transcripts fell sharply after freezing at -8°C for 24 h and, in contrast to the response during recovery from anoxia, there was no secondary increase in transcript levels during thawing recovery.
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Discussion |
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Since the 1970s, the concentrations of heavy metals in the tissues of
marine molluscs have been used as a bioindicator of the level of pollution in
the marine environment (Phillips,
1977). More recently, researchers have focused on MT protein
levels as an indicator of heavy metal content in tissues because exposure to
metals such as Cd, Zn and Hg seems to be a universal inducer of MT
(Hamza-Chaffai et al., 2000
;
Boutet et al., 2002
;
Ceratto et al., 2002
). This
correlation has led several laboratories to pursue the cloning of molluscan MT
gene sequences in order to elucidate the controls on MT gene expression and
understand how MT protein levels are regulated in vivo
(Tanguy et al., 2001
;
Ceratto et al., 2002
). As a
result, the MT gene family is relatively well described in marine
invertebrates. In this study, we report the isolation of a metallothionein
cDNA, denoted LLMET, from the foot muscle of marine periwinkles
L. littorea, analyze the Ll-MT protein sequence encoded by it, and
illustrate the pattern of gene expression in response to anoxia and freezing
stresses.
The catalogue of characterized MTs covers a range of species including
vertebrates, molluscs, crustaceans, echinoderms and nematodes. All the
proteins are similar in terms of structure and function but, apart from the
conserved patterns of cysteine residues (C-X-C), show significant diversity in
nucleotide and amino acid sequences. Vertebrate MTs are uniformly
characterized by a low molecular mass (67 kDa), with 6068 amino
acids, 20 of which are cysteine residues and none are aromatic. By contrast,
MTs from marine invertebrates also have a high cysteine content, but may also
contain a few aromatic residues. Invertebrate MTs also display a broader range
of molecular mass, but with an unusually long N-terminal sequence, Ll-MT is
still 2538 residues longer than nearly all other invertebrate MTs
reported to date. A recent study by Park et al.
(2002), however, described a
MT containing 103 amino acids in the Asian periwinkle Littorina
brevicula, so a MT sequence of
100 amino acids may be characteristic
of this group of gastropods. Unfortunately, the study by Park et al.
(2002
) presented only a short
C-terminal sequence (21 residues) covering the highly conserved
mollusc-specific motif (boxed region in
Fig. 2A), so no comparison of
the two species can be made with respect to the novel N terminus that was
found in Ll-Mt. One unusual feature of MT in both littorine species, however,
was the presence of an aromatic phenylalanine residue that is not found in
other molluscs. Interestingly, Langston and Zhou
(1986
) reported the presence
of a 10 kDa cadmium-binding protein in L. littorea. They showed that
cadmium in L. littorea from unpolluted sites was primarily complexed
with a metal-binding protein of approximately 20 kDa, but during exposure to
high cadmium, a second binding protein of 10 kDa was induced, predominantly in
the digestive gland. Based on the equivalent molecular masses, the currently
identified Ll-MT protein may be the 10 kDa protein reported in this earlier
work. Together with the current information, these data suggest that
Littorina sp. may harbour a novel subclass of mollusc MTs.
Several amino acid motifs characterize the metallothionein family, the most
prevalent being the presence of a large number of cysteine residues found in
repeats of Cys-X-Cys. Although the MT sequences aligned in
Fig. 2A are all from molluscs,
the percentage identities are quite low. Despite this, 18 of the cysteine
residues align perfectly, with 14 in the predicted pattern. The defining
characteristic of the mollusc MT family is the presence of a conserved
C-terminal sequence that is unique to this group
[Cys-X-Cys-X(3)-Cys-Thr-Gly-X(3)-Cys-X-Cys-X(3)-Cys-X-Cys-Lys;
Binz and Kagi, 1999]. As
illustrated in Fig. 2A, this
motif was found in Ll-MT, confirming it is a metallothionein of the mollusc
class.
A unique biological feature of MT genes is their promiscuous expression in
response to a variety of agents and conditions. Depending on the system,
de novo synthesis of the protein can be induced by exposure to
selected metals, cytokines, tumour promoters, hormones and growth factors
(reviewed in Coyle et al.,
2002) as well as by environmental stresses, including hypoxia
(Murphy et al., 1999
). To our
knowledge, this paper is the only study to date to show enhanced MT gene
expression in response to natural environmental stresses in a stress-tolerant
animal. Northern blot analysis showed a three- to sixfold increase in MT mRNA
levels in response to both anoxia and freezing. The induction response was
essentially the same for both stresses, rising quickly and remaining high
through at least 12 h of stress exposure. Such a pattern indicates that a
common mechanism or trigger is responsible for activating the transcription of
the MT gene, most likely low oxygen tension. Anoxia is, by definition, a lack
of oxygen, whereas freezing creates an ischemic state where oxygen delivery to
tissues is interrupted due to hemolymph freezing. It is understandable,
therefore, that genes that are anoxia-induced could also respond to freezing
stress.
Possible functions of MT during anoxia and freezing
Despite their initial discovery as metal-binding proteins, MTs are clearly
multifunctional, and their roles may vary, not only in a species-specific but
stress-specific manner. A particularly appealing function is one of an
antioxidant, for which there is ample supportive evidence. Campagne et al.
(1999) demonstrated that
transgenic mice overexpressing the MT-1 isoform were protected against focal
cerebral ischemia and reperfusion injury. In their study, the mRNA levels for
MT1 increased 7.5-fold over baseline values upon application of ischemia, and
even though tissues were still damaged, the sizes of the affected regions were
approximately 40% smaller. In a reverse study, Lazo et al.
(1995
) showed an enhanced
sensitivity to oxidative stress in cultured embryonic cells that lacked genes
for MT-I and II. A cold-induced MT response in the whole animal has also been
reported in the literature, although not in a stress-tolerant species; Beattie
et al. (1996
) found that MT was
induced in response to cold in rats that were transferred from 26°C to
6°C. Following an increase in mRNA content, the MT protein levels
increased 1.4- to 3-fold in kidney and liver, whereas the thermogenic organ,
brown adipose tissue, showed a 16-fold increase in MT protein. Thermogenesis
is a process that requires high oxygen consumption and is also accompanied by
a sharp rise in reactive oxygen species (ROS) generation. This led the
researchers to conclude that MT was produced to provide antioxidant
defense.
In invertebrates, additional supporting evidence for an antioxidant role
for MTs comes from studies of the effects of iron and cadmium on the
intertidal mussel Mytilus galloprovincialis. Because of its role in
the Fenton reaction, exposure to high levels of iron stimulates ROS production
whereas cadmium exposure does not. However, cadmium exposure did induce de
novo synthesis of MT protein and Viarengo et al.
(1999) showed that
pre-exposure to cadmium greatly increased the survival rate of mussels that
were subsequently exposed to iron in an anoxic environment. The protective
effect was attributed to the cadmium-dependent induction of MT, thus
supporting a role for MT in antioxidant defense. Interestingly, this
antioxidant effect of MT does not appear to due to iron binding, as MTs do not
bind iron particularly well. Rather, there is evidence that MT may be an
inherent antioxidant, scavenging ROS via thiolate oxidation of the
cysteine residues. Using cell-free systems, Thornalley and Vâsàk
(1985
) demonstrated that MT
could quench hydroxyl radicals, whereas Irato et al.
(2001
) showed the same result
could be achieved with living cells challenged with superoxide. Therefore, it
is interesting to note that Ll-MT contains 27 cysteine residues, 47
more than MTs from previously described species, and these additional
thiol-containing cysteine residues may allow for a stronger antioxidant
activity in this stress-tolerant species.
Injury caused by reactive oxygen species (ROS) is a major source of
cellular damage for organisms exposed to environmental stress. L.
littorea can be viewed as a model for reperfusion injury because long
bouts of anoxia or freezing are followed by a sudden reintroduction of oxygen.
Such rapid changes in oxygen availability can cause a burst of ROS production
sufficient to overwhelm the existing capacity of antioxidant defenses, thereby
leading to oxidative damage. Studies with land snails have shown that
antioxidant defenses are elevated when animals enter a dormant state
(aestivation), even though this state is associated with reduced oxygen
consumption (Hermes-Lima et al.,
1998). It was hypothesized that the rise in antioxidant defenses
during aestivation was a preparatory measure for combating oxidative stress
when metabolic rate rose rapidly during arousal. A similar response might
occur in L. littorea, antioxidant defenses being upgraded as a
response to anoxia or freezing stress in order to limit the potential for
damage from a burst of ROS production occurring as soon as oxygen is
reintroduced. The rapid upregulation of MT gene expression documented here
would support this idea as well as provide a way to sequester ions, such as
copper, that can also catalyze the Fenton reaction. Interestingly, anoxia
exposure of L. littorea also induces a rapid increase in transcript
and protein levels of ferritin, an iron-binding protein (K. Larade and K. B.
Storey, manuscript submitted for publication) as well as changes in the
activities of some antioxidant enzymes and a strong increase in reduced
glutathione content (Pannunzio and Storey,
1998
). Hence, MT expression in response to anoxia or freezing
stresses may be part of a concerted elevation of antioxidant defenses under
these conditions.
Conclusion
Whole animal exposure to freezing and anoxia induced transcription of
metallothionein in the tissues of Littorina littorea. The ability of
MT to function as an antioxidant and as a reservoir of essential metals could
contribute to survival under these stresses. The present results, in
demonstrating MT induction in response to anoxia and freezing stresses, also
suggest the need for caution in the use of MT protein levels as an index of
the level of heavy metal pollution in marine ecosystem. Clearly, MT is
inducible by multiple environmental stresses (anoxia, freezing, heavy metals),
so the use of MT as a marker for heavy metal pollution would only be valid if
`marker-species' are not simultaneously exposed to other stresses. This could
create a problem for the interpretation of long-term (year-round) studies,
because seasonal changes in exposure to environmental low oxygen and low
temperature could confound a correlation between MT and heavy metal
levels.
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Acknowledgments |
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Footnotes |
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References |
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