Three calpains and ecdysone receptor in the land crab Gecarcinus lateralis: sequences, expression and effects of elevated ecdysteroid induced by eyestalk ablation
1 Department of Biology, Colorado State University, Fort Collins, CO 80523,
USA
2 Bodega Marine Laboratory, University of California, Davis, Bodega Bay, CA
94923, USA
* Author for correspondence (e-mail: don{at}lamar.colostate.edu)
Accepted 15 June 2005
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Summary |
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Key words: Crustacea, Arthropoda, calpain, tissue distribution, steroid hormone, molting, ecdysone, gene expression, mRNA, ecdysone receptor, DNA sequence, amino acid sequence, cloning, cDNA, muscle atrophy, skeletal muscle
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Introduction |
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Calpain genes have been organized into two general categories, based on the
presence (`typical' or `EF-hand' calpains) or absence (`atypical' or
`non-EF-hand' calpains) of a calmodulin-like domain (dIV) in the C-terminus
(Goll et al., 2003;
Huang and Wang, 2001
;
Suzuki et al., 2004
). Domain
IV contains five EF-hand motifs, the first three of which (EF-1, EF-2 and
EF-3) bind Ca2+ with varying affinities
(Hosfield et al., 1999
). The
fifth EF-hand (EF-5) mediates dimerization with a 28-kDa regulatory subunit in
vertebrate heterodimeric calpains (Maki et
al., 2002
). Atypical calpains are either truncated, e.g. lobster
CalpM, nematode CPL-1, mammalian nCl-2' and Drosophila
Dm-CalpA' (Sorimachi et al.,
1993
; Theopold et al.,
1995
; Yu and Mykles,
2003
), or have the C-terminal region replaced by a different
sequence, e.g. the `T domain' in mammalian calpain 5 (Capn5) and nematode
TRA-3 (Barnes and Hodgkin,
1996
; Dear et al.,
1997
) or the `SOL domain' in mammalian SOLH and
Drosophila SOL or Calpain D
(Delaney et al., 1991
;
Friedrich et al., 2004
;
Kamei et al., 1998
). SOL and
SOLH have six Zn-finger motifs in the N-terminal region, suggesting that they
are DNA-binding proteins (Delaney et al.,
1991
; Kamei et al.,
1998
).
Calpains are involved in the selective degradation of myofibrillar proteins
during a molt-induced atrophy in the large claws of decapod crustaceans
(Mykles, 1992,
1998
). Over several weeks,
there is as much as a 78% reduction in muscle mass, which facilitates
withdrawal of the appendages at molt
(Mykles and Skinner, 1982a
;
Skinner, 1966
). Crustacean
calpains degrade myofibrillar proteins to acid-soluble products and show about
2-fold greater activity in atrophic muscle
(Mykles, 1990
; Mykles and
Skinner, 1982b
,
1983
). Lobster muscle contains
four calpain activities, which were initially termed Ca2+-dependent
proteinases (CDP I, CDP IIa, CDP IIb and CDP III; native masses 310, 125, 195
and 59 kDa,respectively; Mykles and
Skinner, 1986
). All require millimolar Ca2+ for full
activity in vitro and are inhibited by cysteine protease inhibitors
(Mykles and Skinner, 1983
,
1986
). CDP I has not been well
characterized and it is the least efficient of the four calpains in degrading
myofibrillar proteins (Mattson and Mykles,
1993
). CDP IIb is a homodimer of a 95-kDa subunit and is related
to Dm-CalpA, as determined by immunological analysis
(Beyette et al., 1993
;
Beyette and Mykles, 1997
). In
western blots, CDP IIa reacts with an antibody directed to a peptide sequence
in the active site of mammalian calpains but not with polyclonal antibodies
raised against mammalian µ- and m-calpains or Dm-CalpA
(Beyette et al., 1997
). These
results indicate that CDP IIa differs in structure from typical calpains. CDP
IIa and CDP IIb are the most effective in degrading myofibrillar proteins,
such as myosin heavy and light chains, actin, tropomyosin and troponin
(Mattson and Mykles, 1993
). A
cDNA encoding CDP III was cloned and characterized in lobster (Ha-CalpM;
Yu and Mykles, 2003
). Ha-CalpM
lacks the calmodulin-like domain in the C-terminus that is characteristic of
typical calpains. It is highly expressed in skeletal muscle, but its mRNA and
protein levels do not change significantly over the molting cycle. It may be
involved in restructuring and/or maintaining contractile structures in
crustacean skeletal muscle (Yu and Mykles,
2003
).
Molt-induced muscle atrophy is coincident with increasing concentrations of
ecdysteroids (ecdysone, 20-hydroxyecdysone and related compounds) in the
hemolymph (Skinner, 1985). In
the large claw muscles of fiddler crabs, ecdysone receptor (Up-EcR) mRNA level
is increased significantly in the premolt stage
(Chung et al., 1998b
). (We use
the term `ecdysone receptor' as per common usage; however, we recognize that
it is actually an ecdysteroid receptor.) This suggests that the molting
hormone, 20-hydroxyecdysone, initiates and sustains the elevated myofibrillar
protein degradation that results in the reduction of muscle mass. Ecdysteroids
are steroid hormones that regulate growth, development, reproduction and
molting in arthropods (Chang,
1993
; Kozlova and Thummel,
2000
). Ecdysteroids regulate gene transcription after binding to a
nuclear ecdysone receptor (EcR). In insects, EcR heterodimerizes with
ultraspiracle (USP), a vertebrate RXR ortholog, to form a functional hormone
receptor (Yao et al., 1993
);
it binds to a specific response element to induce expression of early response
genes, such as E74 and E75
(Thummel, 1996
). The products
of these early response genes are usually transcription factors that initiate
an ecdysteroid cascade reaction (Huet et
al., 1995
).
Here, we report the cloning of cDNAs encoding three calpain genes (Gl-CalpM, Gl-CalpB and Gl-CalpT) and ecdysone receptor (Gl-EcR) isolated from the tropical land crab Gecarcinus lateralis. The deduced sequences of the three calpains were compared with those of calpains from other species. The expression of the three calpains in nine tissues was quantified by real-time RT-PCR. The effects of elevated ecdysteroid, induced by eyestalk ablation, on calpain and EcR mRNA levels in claw and thoracic muscles were determined. The results suggest that Gl-CalpT is involved with the initiation of myofibrillar proteolysis induced by elevated ecdysteroid.
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Materials and methods |
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Cloning of calpains and ecdysone receptor (EcR) cDNAs
Partial calpain cDNAs were initially obtained by nested RT-PCR using
degenerate primers directed to highly conserved sequences in the protease
domain of a wide variety of calpains in the GenBank database
(http://www.ncbi.nlm.nih.gov),
including those from nematode (GenBank accession #NP502751), fruit fly
(#NP477047 and #AAD04331), rat (#NP058813) and human (#AAH08751). Conserved
sequences were identified by aligning proteins using the ClustalW program
(http://www.ebi.ac.uk/clustalw/index.html).
Two sets of degenerate primers were designed to anneal to DNA sequences
encoding LG(N/D/E)CW(L/F), EKA(Y/F)AK or (G/R)HAY(T/S)(V/I) in the catalytic
domain: CPN F1, (G/A)II (C/T)T(A/G/T/C) GG(A/G/T/C) (G/A)A(A/G/T/C) TG(C/T)
TGG; CPN F2, GA(G/A) AA(G/A) GC(A/G/T/C) (C/T)A(C/T) GC(A/G/T/C) AA(G/A); CPN
R1, IA(C/T) (A/G/T/C)(G/C)(A/T) (G/A)TA (A/G/T/C)GC (G/A)TG (IC); CPN R2,
(C/T)TT (A/G/T/C)GC (G/A)T(G/A) (A/G/T/C)GC (C/T)TT (C/T)TC. Partial sequences
encoding EcR were initially obtained by nested RT-PCR using degenerate primers
directed to highly conserved sequences in the DNA-binding (domain C; MMRKCQ
and CRL(K/R)KC) and ligand-binding (domain E; EYALL(T/A)A and DQI(A/T)LLK)
domains of EcR cDNAs in the GenBank database, as described above: EcR F1, ATG
MGN MGN AAR TGY CAR; EcR F2, TGY MGN YTN VSN AAR TG); EcR R1, CNG YNA RNA RNG
CRT AYT C; EcR R2, TTN ARN ARN GYD ATY TGY TC. To obtain more of the 3'
sequence in the EcR ORF, a second round of nested PCR was done using two
sequence-specific forward primers (cEcR F1 and cEcR F2;
Table 1) and two degenerate
primers to highly conserved sequences (AEIWDV and PFLAEI) in domains E and F
(EcR R3, CNG YNA RNA RNG CRT AYT C; EcR R4, DAT YTC NGC NAR RAA NGG). All
primers were synthesized and purified by Integrated DNA Technologies, Inc.
(Des Moines, IA, USA).
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Total RNA was isolated from intermolt claw muscle or primary limb
regenerates (regeneration index 1316;
Skinner, 1985) using a Qiagen
RNeasy mini kit (Qiagen, Inc., Valencia, CA, USA). cDNA was synthesized
according to the manufacturer's protocol using the SuperScript II RNase
H-reverse transcriptase first strand synthesis system (Invitrogen, Inc.,
Carlsbad, CA, USA). Briefly, 12 µl of a mixture containing 1 µl oligo
(dT)1218 (500 µg ml1), 1 µg total RNA and 1
µl 10 mmol l1 dNTPs was heated to 65°C for 5 min and
chilled on ice for 1 min. 5x First-Strand Buffer (4 µl), 2 µl 0.1
mol l1 DTT and 1 µl RNaseOUT recombinant ribonuclease
inhibitor (40 units µl1; Invitrogen, Carlsbad, CA, USA)
were added, and the mixture was incubated at 42°C for 2 min. The reaction
was initiated by the addition of 1 µl (200 units) of SuperScript II and
incubated at 42°C for 50 min. The reaction was inactivated by heating at
70°C for 15 min.
PCR was performed using an ABI 9600 thermal cycler (Perkin-Elmer, Inc., Wellesley, MA, USA). Claw muscle cDNA was used for the EcR PCR, and limb regenerate cDNA was used for the calpain PCR. The reactions contained 3 µl cDNA, 3 µl 10x Takara EX Taq buffer (Takara, Inc., Madison, WI, USA), 2 µl 250 µmol l1 dNTPs, 1 µl forward primer (either CPN F1, EcR F1 or cEcR F1), 1 µl reverse primer (CPN R1, EcR R1 or EcR R3), 0.2 µl Takara EX Taq DNA polymerase (5 units µl1) and 18.8 µl PCR-grade deionized water. Initial denaturation (95°C for 5 min) was followed by 35 amplifying cycles (either 95°C for 30 s, 55°C for 30 s and 72°C for 30 s for calpain or 95°C for 30 s, 53°C for 30 s and 72°C for 1 min for EcR) and final extension at 72°C for 7 min. For the second PCR reaction, 0.2 µl of the first PCR reaction was used, in conjunction with one of four primer pairs (CPN F1/CPN R2, CPN F2/CPN R1, EcR F2/EcR R2 or cEcR F2/EcR R4). Other reaction components and PCR conditions were the same as those in the first reaction.
The PCR products were separated by 1.2% agarose gel electrophoresis and stained with ethidium bromide. The PCR products were purified from the gel slices using the Qiaquick Gel Extraction kit (Qiagen, Inc.), ligated into TA cloning vector with the TOPO TA Cloning kit (Invitrogen, Inc.) and transformed into One Shot TOP 10 E. coli strain (Invitrogen, Inc.). Transformants were first selected by bluewhite colony selection on LB agar plates containing 50 µg ml1 ampicillin (Sigma-Aldrich, Inc., St Louis, MO, USA) and subjected to PCR with T7 and M13-reverse vector primers to verify sizes of inserts. Plasmids were purified using the Qiagen Spin Mini prep kit and sequenced using T7 and M13-reverse vector primers (Davis Sequencing, Inc., Davis, CA, USA). If needed, gene-specific primers were used to obtain the complete sequences of both strands.
RACE (rapid amplification of cDNA ends) was used to obtain full-length mRNA sequences. Poly (A+) RNA was isolated from claw muscle or limb regenerate total RNA using the Oligotex mRNA kit (Qiagen, Inc.). For the 3' sequence, the Invitrogen 3' RACE System was used. Briefly, first-strand cDNA synthesis reactions contained 200 ng claw muscle or limb regenerate poly (A+) RNA and adaptor primer (5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'). First-round PCR on 20 ng cDNA included a universal amplification primer (5'-CUACUACUACUAGGCCACGCGTCGACTAGTAC-3') and gene-specific forward primers (Table 1) at the following conditions: denaturation at 96°C for 5 min, 35 amplification cycles (96°C for 30 s, 60°C for 30 s and 72°C for 2 min) and extension at 72°C for 10 min. Nested PCR on 30 µl of each reaction was conducted with genespecific primers (Table 1) and abridged universal amplification primer (5'-GGCCACGCGTCGACTAGTAC-3') using the same conditions as the first-round PCR. Gel-purified PCR products were cloned into the TA vector and sequenced as described above.
SMARTTM RACE cDNA Amplification kit (BD Biosciences, Inc., Palo Alto, CA, USA) was used to obtain the 5' sequences of each gene. The first-strand cDNA synthesis reaction contained 3 µl poly(A+) RNA (50 ng for calpain primers and 100 ng for EcR primers), 1 µl 10 mmol l1 5' CDS primer [5'-(T)25N-1N-3'] and 1 µl 10 mmol l1 SMART II A oligo (5'-AAGCAGTGGTATCAACGCAGAGTACGCGGG-3') and was incubated at 68°C for 2 min. After chilling the reaction for 2 min on ice, 2 µl 5x First-Strand buffer (250 mmol l1 Tris-HCl, pH 8.3; 375 mmol l1 KCl; 30 mmol l1 MgCl2), 1 µl 20 mmol l1 DTT, 1 µl 10 mmol l1 dNTPs and 1 µl PowerScript reverse transcriptase (BD Biosciences Clontech, Mountain View, CA, USA) were added. The reaction was covered with 20 µl paraffin oil and incubated at 42°C for 1.5 h in an ABI 9600 thermal cycler. The reaction mixture was diluted 10-fold with autoclaved distilled water and used for first-round PCR with 10x Universal Primer A Mix (0.4 mmol l1 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3' and 2 mmol l1 5'-CTAATACGACTCACTATAGGGC-3') and gene-specific reverse primers (Table 1) under the following conditions: denaturation at 96°C for 5 min, 35 amplification cycles (either 96°C for 30 s, 65°C for 15 s and 72°C for 3 min for calpains or 96°C for 30 s, 66°C for 15 s and 72°C for 3 min for EcR) and extension at 72°C for 10 min. Second-round PCR was conducted using nested gene-specific primers (Table 1) and nested universal primer A (10 mmol l1, 5'-AAGCAGTGGTATCAACGCAGAGT-3'). The PCR conditions were the same as those used for first-round PCR. Gel-purified PCR products were cloned into the TA vector and sequenced as described above. Continuous sequences of three calpains were obtained by PCR using primer pairs to the start and stop codons to verify the sequence of each ORF.
Phylogenetic relationships between calpain sequences were determined with Treeview, which is a program that displays ClustalW result files (http://taxonomy.zoology.gla.ac.uk/rod/treeview/treeview_manual.html).
Calpain and EcR expression by RT-PCR
Total RNA was isolated from tissues using the RNeasy mini kit according to
the manufacturer's instructions (Qiagen, Inc.). Total RNA (1 µg) was
DNase-treated (Invitrogen, Inc.) and reverse-transcribed using the SuperScript
II RNase H-reverse transcriptase first strand synthesis system (Invitrogen,
Inc.). End-point PCR reactions were performed in an ABI 9600 thermal cycler
using TaKaRa Ex Taq HotStart polymerase (Takara, Inc.), gene-specific primers
(Table 2) and either 2 µl
(calpains) or 5 µl (EcR) of the first strand cDNA mixture as template. The
PCR conditions for calpains were an initial denaturation at 95°C for 4
min, 35 polymerization cycles (denaturation at 94°C for 30 s, annealing at
60°C for 30 s and extension at 72°C for 30 s), and final extension at
72°C for 2 min. PCR reactions were analyzed by separating all of the 20
µl reaction volume on 2% agarose ethidium bromide-stained gels. Primers
were designed using IDT BioTools program
(http://biotools.idtdna.com/gateway).
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For quantitative RT-PCR, transcript levels of the three calpains (Gl-CalpB,
Gl-CalpM and Gl-CalpT) and ecdysone receptor (Gl-EcR) were determined by
real-time PCR using a Cepheid Smart Cycler instrument (Cepheid, Sunnyvale, CA,
USA) and sequence-specific primers (Medler
and Mykles, 2003; Yu and
Mykles, 2003
). Transcript levels of the four genes were normalized
to the mRNA level of elongation factor 2 (EF2), which was constitutively
expressed. The PCR products were ligated into TOPO 2.1 vector using TOPO TA
Cloning kit (Invitrogen, Inc.). Standard curves were generated using serial
dilutions (10 fg to 10 ng) of plasmids containing either Gl-CalM, Gl-CalpB,
Gl-CalpT or Gl-EF2 inserts (Medler and
Mykles, 2003
; Yu and Mykles,
2003
). Reaction mixtures contained 2 µl LightCycler FastStart
Reaction Mix (Roche, Indianapolis, IN, USA; 10x buffer, Fast Taq DNA
polymerase and dNTPs), 2 µl 25 mmol l1 MgCl2,
2 µl cDNA template, 1 µl forward primer
(Table 3; 10 pmol
µl1), 1 µl reverse primer
(Table 3; 10 pmol
µl1) and 12 µl PCR-grade water. The PCR conditions
were denaturation at 96°C for 5 min, 40 amplification cycles (96°C for
20 s, 65°C for 15 s and 72°C for 30 s), and melting curve detection
(60°C+0.2 deg. s1). PCR products were evaluated by
melting temperature analysis and separation on 2% agarose ethidium
bromide-stained gels (Medler and Mykles,
2003
; Yu and Mykles,
2003
).
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Statistical analyses
The Statview 5.0.1 Program (SAS Institute, Inc., Cary, NC, USA) was used
for statistical analyses. One-way analysis of variance (ANOVA) was used to
compare the copy numbers determined by real-time PCR. Values were
log-transformed due to high levels of variability between samples and to
correct for the correlation between mean and variance
(Medler and Mykles, 2003).
Pair-wise post-ANOVA comparisons used a Bonferroni test with an
experiment-wise error rate of 0.05.
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Results |
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The characteristics of the cDNAs encoding the three land crab calpains are summarized in Table 4. The domain organization of the land crab calpains is compared with that of calpains from lobster (Ha-CalpM), Drosophila (Dm-CalpA and Dm-CalpB) and nematode (TRA-3) in Fig. 1. The DNA and deduced amino acid sequences of Gl-CalpB, Gl-CalpM and Gl-CalpT are presented in Figs 2, 3 and 4, respectively. The deduced amino acid sequence of Gl-CalpB was 61% identical to Dm-CalpB, 51% identical to Dm-CalpA and 50% identical to human Capn3 (accession #NP-775110.1). The deduced amino acid sequence of Gl-CalpM was most similar to that of Ha-CalpM (66% identity) and Dm-CalpA (48% identity). The amino acid sequence of Gl-CalpT was 47% identical to human Capn5 (accession #O15484) and 41% identical to C. elegans TRA-3 (accession #AAB60256).
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Gl-CalpB had the four-domain organization of typical calpains: N-terminal (dI), catalytic (dII), C2-like (dIII) and calmodulin-like (dIV) (Figs 1, 2). Gl-CalpM had the conserved catalytic and C2-like domains but lacked the C-terminal calmodulin-like domain (Figs 1, 3). A minor, alternatively spliced variant of Gl-CalpM (Gl-CalpM') was obtained with 3' RACE; the deduced polypeptide is truncated 958 bp from the start codon, which results in the absence of the catalytic asparagine in domain II (Fig. 3). Gl-CalpT resembled other calpains from domains I to III, but dIV was replaced with a T domain found in Capn5 and TRA-3 (Figs 1, 4).
Multiple amino acid sequence alignment showed high similarity in domains II and III in calpains from nematode (TRA-3), arthropods (Gl-CalpM, Gl-CalpB, Gl-CalpT, Dm-CalpA and Dm-CalpB) and mammals (calpains 1 and 3) (Fig. 5). All have the conserved catalytic triad (C, H, N) and two non-EF hand Ca2+-binding sites in dII. A C2-like sequence containing an acidic loop in dIII was common to all calpains. The major difference between the two CalpM sequences was that Ha-CalpM had two acidic amino acid insertions, one (DDSDD) near the end of dII and the other (DDDDDDDDDDRG) in the C2 acidic loop region, that were absent in Gl-CalpM. Unlike a muscle-specific mammalian Capn3, calpains from arthropods and nematode lacked an insertion sequence in dII. Domain IV, when present, was well conserved from arthropods to mammals, although Dm-CalpA had an insertion sequence between EF-1 and EF-2 not found in any other calpain (Fig. 6). The sequence of the T domain of Gl-CalpT was similar to that of other `T domain' calpains (Fig. 7).
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To study the sequence relationships of the three land crab calpains, a phylogenetic analysis was done based on the deduced amino acid sequences of dII and dIII of calpains from arthropods, nematode and mammals (Fig. 8). The sequences clustered in four groups. TRA-3, Capn5 and Gl-CalpT were clustered as a distinct group, even though the T domain sequences were not included in the analysis. Mammalian Capn1 and Capn3 were grouped together and were more closely related to calpains with an EF-hand domain. Crustacean-specific Ha-CalpM and Gl-CalpM were grouped separately from arthropod A/B-type calpains (Gl-CalpB and Dm-CalpA and B).
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cDNA encoding ecdysone receptor
The initial 700-bp product from nested PCR using degenerate EcR primers was
95% identical to the deduced amino acid sequence of the EcR from fiddler crab
(accession #AAC33432). 3' RACE using sequence-specific primers failed.
If the 3' UTR in the Gl-EcR is as long as it is in fiddler crab EcR
(4 kb), it may be difficult to amplify with 3' RACE. Additional
3' sequence was obtained by PCR using gene-specific forward primers
(cEcR F1 and cEcR F2; Table 1)
and degenerate primer sets (EcR F3 and EcR F4; see Materials and methods).
5' RACE yielded a 300-bp product, which extended the 5' sequence
another 100 bp. The consensus partial sequence of Gl-EcR was 1005 bp and
encoded a deduced 335-amino acid sequence encompassing part of the C domain
and all of the D and E domains (Fig.
9). The partial amino acid sequence of Gl-EcR was aligned with
full-length sequences of EcR-encoding genes from fiddler crab (Up-EcR) and
locust (Lm-EcR) (Fig. 10). The
Gl-EcR sequence was 93% identical with homologous regions of Up-EcR and 66%
identical with Lm-EcR.
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Effects of eyestalk ablation on calpain and EcR expression in skeletal muscles
As claw muscle atrophy coincides with elevated ecdysteroid levels during
premolt, the effects of ecdysteroids, induced by eyestalk ablation, on the
expression of the EcR and the three calpains were determined in claw and
thoracic muscles. The thoracic muscle served as a non-induced control, as it
does not undergo premolt atrophy (Moffett,
1987). EcR mRNA was used as an indicator of tissue sensitivity to
molt induction, as it is upregulated in response to elevated ecdysteroids in
insects (Sun et al.,
2003
).
The X-organ/sinus gland complex in the eyestalk is the primary source of
molt-inhibiting hormone (MIH), a neuropeptide that inhibits ecdysteroid
synthesis and secretion by the Y-organs. Eyestalk ablation, therefore, is used
as an effective strategy to rapidly stimulate the Y-organs and chronically
increase the level of ecdysteroids in the hemolymph
(Skinner, 1985). Ecdysteroid
levels were significantly higher one day after eyestalk ablation
(80.8±22.3 ng ml1, mean ± 1 S.D.,
N=4) compared with intact controls (31.9±5.9 ng
ml1, N=5; P=0.002). Ecdysteroid levels in
3-day eyestalk-ablated animals were not determined, although previous work
showed that ablation results in chronically elevated ecdysteroid levels
(McCarthy and Skinner,
1977
).
The EcR, calpain and EF2 mRNA levels in thoracic and claw muscles were quantified by real-time PCR. EF2, which is constitutively expressed, was used as an internal standard to normalize the PCR reactions. In thoracic muscle, eyestalk ablation had no significant effect on expression of the Gl-EcR and the three calpains (Fig. 13B). By contrast, Gl-EcR expression in claw muscle increased about 15-fold one day after eyestalk ablation but then decreased to about 2.8-fold above the level in intact animals three days after eyestalk ablation (Fig. 13A). Gl-CalpT mRNA paralleled the expression of Gl-EcR in claw muscle; it increased about 19.3-fold above the level in intact animals one day after eyestalk ablation and about 4.3-fold higher three days after eyestalk ablation. Gl-CalpM and Gl-CalpB expression was not affected by eyestalk ablation. The relationship between the three calpain mRNAs and the EcR mRNA is presented in Fig. 14. The expression of Gl-EcR and CalpT was highly correlated in both claw muscle and thoracic muscle from intact and eyestalk-ablated animals (Fig. 14, bottom panels). There was no correlation between EcR expression and either Gl-CalpM or Gl-CalpB in either muscle (Fig. 14, top and middle panels, respectively).
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Discussion |
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Gl-CalpB appears to be the only typical calpain in crustaceans, as it is
the only land crab calpain with the C-terminal EF-hand domain IV
(Fig. 2). Sequence alignment
analysis of domains II and III and its estimated mass (89 kDa) indicate
that Gl-CalpB is most closely related to Drosophila calpains A and B
(Farkas et al., 2004
;
Jékely and Friedrich,
1999
; Pinter et al.,
1992
). Gl-CalpB combines features found in Dm-CalpA or Dm-CalpB.
It has no insertion sequence in dIV, like Dm-CalpB, but has a dI length
similar to that in Dm-CalpA (Fig.
1; Emori and Saigo,
1994
; Jékely and
Friedrich, 1999
; Theopold et
al., 1995
). These data suggest that Dm-CalpA and Dm-CalpB are
derived from duplication of a common ancestral gene and that no such
duplication occurred in crustaceans.
The M-type calpains are unique to crustaceans. Gl-CalpM from land crab has
the highest structural similarity with Ha-CalpM, the first calpain cloned from
a crustacean (Yu and Mykles,
2003). Both encode polypeptides of about 66 kDa that lack dIV.
Unlike other truncated forms, crustacean CalpM does not appear to be produced
by alternate splicing of a typical calpain gene. Dm-CalpA', for example,
is an alternatively spliced transcript of Dm-CalpA
(Theopold et al., 1995
). In
mammals, nCL-2', which lacks domains III and IV, is produced by
alternative splicing of the nCL-2 (Capn8) gene
(Sorimachi et al., 1993
). We
were not successful in obtaining longer cDNAs containing the dIV region using
3' RACE PCR with various sequence-specific primer sets. The absence of a
longer mRNA is supported by immunoblot results using an antibody raised
against a 28-amino acid N-terminal sequence of Ha-CalpM; only two proteins
with masses of 62 and 68 kDa were detected in lobster muscles
(Yu and Mykles, 2003
). A
truncated, alternatively spliced isoform of Gl-CalpM, Gl-CalpM', was
isolated by 3' RACE. Gl-CalpM' is likely to be catalytically
inactive, as the alternative transcript has a stop codon inserted before the
Asn residue of the catalytic triad in dII. As it is expressed at very low
levels, it was not characterized further.
The Gl-CalpT is a member of the T domain calpain family. Its amino acid
sequence has the highest sequence identity with human Capn5 and nematode TRA-3
(Figs 5,
7). TRA-3 was first isolated as
a sex determinant of the soma and germ line in hermaphrodites of C.
elegans (Barnes and Hodgkin,
1996). TRA-2A is a substrate for TRA-3, and cleavage of TRA-2A by
TRA-3 generates a peptide that has feminizing activity
(Sokol and Kuwabara, 2000
).
Two mammalian calpains, Capn5 and Capn6, have the same structure as Gl-CalpT
and TRA-3. However, Capn6 is catalytically inactive, as one or two residues of
the catalytic triad (C, H, N) are mutated in human (K, H, N) and mouse (K, Y,
N) (Dear et al., 1997
;
Matena et al., 1998
).
Interestingly, the Drosophila genome apparently lacks a CalpT-like
gene (Friedrich et al., 2004
;
Goll et al., 2003
).
It is likely that all three crustacean calpains have
Ca2+-dependent proteinase activity. The deduced amino acid
sequences of the protease domain are highly conserved, including the three
amino acid residues essential for catalytic activity
(Fig. 5). In addition, the
three calpains contain two well-conserved non-EF hand Ca2+-binding
sites in dII and a C2-like Ca2+/phospholipid-binding
site in dIII (Fig. 5), both of
which are important in Ca2+-dependent activation
(Alexa et al., 2004; Moldoveanu
et al., 2002
,
2004
). In the absence of
Ca2+, the catalytic residues are misaligned and the substrate
binding cleft is disrupted; binding of Ca2+ to domains II and III
activates the enzyme by driving the realignment of the active site residues
(Moldoveanu et al., 2002
,
2004
;
Strobl et al., 2000
). The
interaction of residues R104 and E333 in dII of mammalian calpain provides
cooperativity between the two Ca2+-binding sites
(Moldoveanu et al., 2004
);
these two residues are conserved in the crustacean genes. Moreover, calpains
lacking the calmodulin-like domain retain Ca2+-dependent activity
(Hata et al., 2001
;
Sokol and Kuwabara, 2000
).
Four Ca2+-dependent proteinase (CDP) activities have been
characterized biochemically in crustacean muscle
(Mykles and Skinner, 1986).
These activities differ in native mass and subunit composition
(Table 6). Ha-CalpM appears to
encode CDP III, based on similar masses and chromatography properties
(Yu and Mykles, 2003
); the
native enzyme consists of a single polypeptide
(Table 6). Gl-CalpB may encode
CDP IIb, as the subunit mass (95 kDa) of the purified lobster CDP IIb
(Beyette et al., 1993
;
Beyette and Mykles, 1997
) is
similar to the estimated mass (89 kDa) of the deduced Gl-CalpB amino acid
sequence. In addition, CDP IIb and the Dm-CalpA 95-kDa gene product share
immunological properties. Polyclonal antibodies raised against lobster CDP IIb
and Dm-CalpA protein cross-react, while a polyclonal antibody raised against a
conserved 20-amino acid sequence around the cysteine residue in the active
site of mammalian µ- and m-calpains (GATRTDICQGALGDCWLLAA) does not react
with either Gl-CalpB or Dm-CalpA (Beyette
et al., 1997
). Analysis of this same sequence in Gl-CalpB
(GATRFDVKQGELGDCWLLAA) indicates that four residues (Thr, Ile, Cys and Ala) in
mammalian calpains are replaced by Phe, Val, Lys and Glu in Gl-CalpB
(Fig. 5). The replacement of
two uncharged residues with two charged residues may explain why the antibody
does not react with lobster CDP IIb. The Gl-CalpB polypeptide probably does
not associate with a regulatory subunit, as the sequence in the putative EF-5
is the least conserved with vertebrate heterodimeric calpains (e.g. Capn1; see
Fig. 6). This is consistent
with the homodimeric structure of the native CDP IIb
(Beyette and Mykles, 1997
).
|
The identity of Gl-CalpT with CDP I or IIa is less certain. A polyclonal
antibody raised against the 20-residue mammalian active site sequence reacts
with a 60-kDa protein in immunoblots of a partially purified preparation of
lobster CDP IIa (Beyette et al.,
1997). However, eight of the 20 residues around the catalytic
cysteine differ between Capn1 and Gl-CalpT
(Fig. 5), making it less likely
that the antibody would recognize the CalpT sequence. Furthermore, the deduced
amino acid sequence of Gl-CalpT has an estimated mass of
74 kDa, which is
significantly greater than the 60 kDa estimated from immunoblots. CDP I is not
well characterized. Its large native mass (310 kDa) suggests it is a multimer
of the proteins encoded by CalpB, CalpT and/or an additional unidentified
calpain gene. CalpM can be excluded as a component of CDP I, as the Ha-CalpM
antibody does not react with any proteins in CDP I fractions eluting from a
gel filtration column (Yu and Mykles,
2003
). Further work is required to reconcile the CalpB and CalpT
cDNAs with the calpain activities.
Three general patterns of calpain expression are observed in the nine
tissues from adult intermolt land crabs analyzed by real-time PCR. The first
pattern, in which both Gl-CalpB and Gl-CalpM are expressed at higher levels
than Gl-CalpT, is found in skeletal muscles
(Fig. 12A,B). The second
pattern, in which Gl-CalpB is dominant, is found in heart, gill, thoracic
ganglion, digestive gland and testis (Fig.
12CG). The third pattern, in which Gl-CalpM mRNA level is
highest, is found in ovary and integument
(Fig. 12H,I). Differences in
calpain expression are also observed in insect. Although Drosophila
calpains are expressed throughout development, Dm-CalpA, Dm-CalpB and Dm-CalpC
differ in their mRNA levels and tissue distribution. In early embryos,
Dm-CalpA is localized in the anterior pole and posterior surface regions
(Emori and Saigo, 1994), while
Dm-CalpB has a uniform distribution
(Farkas et al., 2004
). During
nuclear division, Dm-CalpA is more evenly distributed and becomes associated
with the precleavage furrows before cellularization
(Emori and Saigo, 1994
). In
late embryos, Dm-CalpB is preferentially expressed in the trachea and foregut
(Farkas et al., 2004
). In
larvae, Dm-CalpB mRNA and protein levels decline, whereas Dm-CalpC mRNA is
moderately elevated (Farkas et al.,
2004
; Spadoni et al.,
2003
). Both Dm-CalpB and Dm-CalpC are expressed highly in the
salivary glands of third instar larvae
(Farkas et al., 2004
;
Spadoni et al., 2003
). In
adults, Dm-CalpA mRNA is present in many tissues, such as ovary, brain,
ventral ganglion, midgut and heart, but not in the indirect flight muscles
(Amano et al., 1997
;
Theopold et al., 1995
).
Dm-CalpB is also expressed in ovary
(Farkas et al., 2004
). The
tissue distribution of Dm-CalpC in adults has not been reported. Such dynamic
changes in localization and distribution suggest that the different calpains
carry out specialized functions in arthropod tissues.
Ha-CalpM was first identified as a muscle-specific calpain, as it is
expressed at the highest levels in lobster skeletal muscles
(Yu and Mykles, 2003). The
analysis of Gl-CalpM, however, shows that it is expressed in ovary and
integument at levels higher than that in skeletal muscle. It might first
appear that the tissue expression pattern of Gl-CalpM deviates more from that
of Ha-CalpM. However, the apparent differences are much less, when one
considers the fiber-type compositions of the muscles. The levels of the
Ha-CalpM mRNA are about 4-fold higher in fast muscle (cutter claw closer and
deep abdominal flexor muscles) than in slow muscle (crusher claw closer)
(Yu and Mykles, 2003
). The
claw closer and thoracic muscles in the land crab are composed of only slow
fibers (Mykles, 1988
;
Mykles and Skinner, 1981
). If
one excludes the fast muscles, the ratios of Gl-CalpM expression in slow
muscle in relationship with that in most of the other tissues are similar to
those of Ha-CalpM. An exception is the difference in CalpM expression in the
integument. In lobster, Ha-CalpM mRNA level in the integument is about 13%
that in crusher claw closer muscle (Yu and
Mykles, 2003
), whereas Gl-CalpM mRNA level is about 18-fold and
43-fold greater in integument than that in the thoracic muscle and claw
muscle, respectively (Fig. 12,
compare A, B and I). Gl-CalpM was also highly expressed in ovary.
Unfortunately, the lobster ovary was not analyzed for Ha-CalpM expression
(Yu and Mykles, 2003
).
A cDNA encoding a partial sequence of EcR was obtained from land crab claw
muscle mRNA. The domain organization of Gl-EcR is similar to that of fiddler
crab EcR (Chung et al., 1998a;
Durica et al., 2002
), as well
as other nuclear steroid receptors (Renaud
and Moras, 2000
). The N-terminal A/B domain is the least conserved
in amino acid sequence and length (Cherbas
et al., 2003
; Hu et al.,
2003
; Onate et al.,
1998
). In insects, receptors differing in A/B domains are
generated by alternate transcriptional start sites or alternative splicing
(Schubiger et al., 2003
;
Segraves and Woldin, 1993
;
Talbot et al., 1993
). By
contrast, the fiddler crab EcR has one A/B domain isoform but has several
alternative spliced isoforms around domain E
(Durica et al., 2002
). The C
domain is highly conserved among different nuclear steroid receptors and
primarily serves as a DNA-binding domain (DBD) containing two zinc-finger
motifs. The E domain is a moderately conserved region of about 250 amino acids
that serves as the ligand-binding domain (LBD)
(Billas et al., 2003
;
Grebe et al., 2003
;
Wang et al., 2000
). Domain E
of many nuclear streroid receptors can also interact with numerous proteins,
such as homo/heterodimeric partners, co-repressors and co-activators
(Hu et al., 2003
;
Shibata et al., 1997
). Both
the DBD and LBD of Gl-EcR and Up-EcR are closely related to those domains in
insect EcR (Fig. 10) and
therefore probably have the same function. The D domain is a hinge region
connecting the DBD with the LBD and is important in ligand binding
(Grebe et al., 2003
).
Gl-EcR and Gl-CalpT expression are upregulated by eyestalk ablation in claw
muscle but not in thoracic muscle. Premolt atrophy occurs specifically in the
claw muscle (Moffett, 1987;
Mykles and Skinner, 1982a
),
suggesting that sensitivity to ecdysteroid may be important in initiating
muscle protein degradation. The expression of Gl-EcR and Gl-CalpT was highly
correlated in claw and thoracic muscles in intact and eyestalk-ablated animals
(Figs 13,
14). These results suggest
that expression of Gl-EcR and Gl-CalpT is linked. One possibility is that the
EcR/RXR complex binds directly to the promoter of the Gl-CalpT gene,
which induces its expression. Another possibility is that ecdysone early
response genes, such as E75 or E74 are induced by the
EcR/RXR complex, and they, in turn, induce the expression of
Gl-CalpT. In Manduca sexta, the EcR/RXR complex induces
E75 within 30 min (Zhou et al.,
1998
). The lower expression of Gl-EcR and Gl-CalpT in claw muscle
three days after eyestalk ablation (Fig.
13A) suggests a feedback inhibition in response to sustained
elevated ecdysteroids that is not mediated by the neurosecretory center in the
eyestalk. In insects, 20-hydroxyecdysone inhibits ecdysteroid production in
the molting gland (Beydon and Lafont,
1983
; Sakurai and Williams,
1989
), which is associated with changes in the expression and
phosphorylation state of certain USP isoforms
(Song and Gilbert, 1998
). In
crustaceans, ecdysteroid inhibits ecdysteroidogenesis in the Y-organ, but the
mechanism is not known (Dell et al.,
1999
).
The role of each calpain in premolt claw muscle atrophy remains to be
established. Unlike mammalian skeletal muscle, in which calpains are
restricted to initial disassembly of sarcomeric elements
(Goll et al., 2003;
Jackman and Kandarian, 2004
),
crustacean calpains can carry out both disassembly and subsequent degradation
of myofibrillar proteins (Mykles,
1998
). Only Gl-CalpT is upregulated in response to elevated
ecdysteroids, suggesting it is involved in initiating the atrophy program
through the proteolytic modification of signaling proteins. This is analogous
to the modification of TRA-2A by TRA-3 in mediating feminization of C.
elegans embryos (Sokol and Kuwabara,
2000
). In mammals, limited proteolysis of protein kinases and
phosphatases by calpain often alters their biochemical properties
(Goll et al., 2003
;
Mykles, 1998
). The ubiquitous
tissue expression and lack of ecdysteroid regulation of Gl-CalpB suggests that
it has a housekeeping function. There is a preferential degradation of thin
filaments during atrophy (Ismail and
Mykles, 1992
; Mykles and
Skinner, 1981
). CDP I and CDP IIb more efficiently degrade thin
filaments than CDP IIa and CDP III
(Mattson and Mykles, 1993
).
CDP III is encoded by CalpM (Yu and
Mykles, 2003
). Gl-CalpB probably encodes CDP IIb
(Table 6), but Gl-CalpT cannot
be identified with either CDP I or CDP IIa without further analysis. Neither
Ha-CalpM mRNA nor protein levels in lobster claw muscle change during the
molting cycle (Yu and Mykles,
2003
). If CalpB and CalpM are involved, their activities are not
controlled at the transcriptional level. Calpains are regulated
post-translationally by phosphorylation and/or endogenous activators or
inhibitors (Friedrich, 2004
;
Goll et al., 2003
;
Mykles, 1998
). We propose that
an increase in the relative activity of Gl-CalpB (CDP IIb) compared with the
other calpains mediates the preferential degradation of thin filaments during
premolt.
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Acknowledgments |
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