First published online August 4, 2005
Journal of Experimental Biology 208, 3177-3197 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01754
Three calpains and ecdysone receptor in the land crab Gecarcinus lateralis: sequences, expression and effects of elevated ecdysteroid induced by eyestalk ablation
H.-W. Kim1,
E. S. Chang2 and
D. L. Mykles1,*
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
View larger version (47K):
[in a new window]
|
Fig. 1. Domain organization of calpains from arthropods and nematode. Calpains from
land crab (Gl-CalpM, Gl-CalpB and Gl-CalpT), Drosophila melanogaster
(Dm-CalpA and Dm-CalpB) and Caenorhabditis elegans (TRA-3) are
depicted. All calpains share conserved proteolytic (II) and C2-like
(III) domains. The N-terminal domain I varies in sequence and length among
different calpains. Calpains differ in the C-terminal region. `Typical'
calpains (e.g. Gl-CalpB, Dm-CalpA and Dm-CalpB) have a calmodulin-like domain
(IV) containing five EF-hand motifs. `Atypical' calpains either lack domain IV
(Gl-CalpM and Ha-CalpM) or have domain IV replaced with a T domain (Gl-CalpT
and TRA-3). Gl-CalpM' and Dm-CalpA' are truncated proteins
resulting from alternative mRNA splicing of Gl-CalpM and Dm-CalpA,
respectively. Amino acid residues, numbered from the N-terminus, indicate the
boundaries between the domains.
|
|
View larger version (84K):
[in a new window]
|
Fig. 2. The complete sequence of calpain B cDNA from land crab (Gl-CalpB). The
sequence (2740 bp) contained a full open reading frame (167–2431 bp)
encoding a protein of 754 amino acids with a predicted mass of 88.89 kDa
(GenBank accession #AY639153). Locations of degenerate primer sites for
initial nested RT-PCR are underlined. Three amino acid residues comprising the
conserved catalytic triad (C, H, N) in domain II are in bold. The asterisk
indicates the stop codon. The poly(A) signal in the 3' UTR is in bold
and underlined.
|
|
View larger version (92K):
[in a new window]
|
Fig. 3. The complete sequence of calpain M cDNA from land crab (Gl-CalpM). The
sequence (2531 bp) contained a full open reading frame (220–1896 bp)
encoding a protein of 558 amino acids with a predicted mass of 65.23 kDa
(GenBank accession #AY639152). Locations of degenerate primer sites for
initial nested RT-PCR are underlined. Three amino acid residues comprising the
conserved catalytic triad (C, H, N) in domain II are in bold. The asterisk
indicates the stop codon. The poly(A) signal in the 3' UTR is in bold
and underlined. The arrow indicates the position of a putative alternative
splicing site that produces a truncated isoform (Gl-CalpM'; GenBank
accession #AY644674).
|
|
View larger version (97K):
[in a new window]
|
Fig. 4. The complete sequence of calpain T cDNA from land crab (Gl-CalpT). The
sequence (2639 bp) contained a full open reading frame (304–2223 bp)
encoding a protein of 639 amino acids with a predicted mass of 74.56 kDa
(GenBank accession #AY639154). Locations of degenerate primer sites for
initial nested RT-PCR are underlined. Three amino acid residues comprising the
conserved catalytic triad (C, H, N) in domain II are in bold. The asterisk
indicates the stop codon. The poly(A) signal in the 3' UTR is in bold
and underlined.
|
|
View larger version (111K):
[in a new window]
|
Fig. 5. Comparison of the deduced amino acid sequences in domains II and III of
calpains from arthropods, nematode and human. Amino acid sequences were
aligned using ClustalW (see Materials and methods). Broken lines indicate gaps
for optimizing alignment. Amino acid residues that are identical or similar in
all the sequences are highlighted in black; gray shading indicates identical
or similar amino acids in most of the sequences. Asterisks indicate residues
of the catalytic triad (C, H, N). Open inverted triangles indicate conserved
residues in two non-EF-hand Ca2+-binding regions in domain II.
Roman numerals indicate boundaries between domains. Numbers at the right
indicate amino acid positions, numbered from the N-terminus of each protein.
Boxes with broken lines indicate locations of two acidic amino acid expansions
found in Ha-CalpM, but not in Gl-CalpM; one (DDSDD) is positioned near the end
of domain II and the other (DDDDDDDDDDRG) is located in the acidic loop region
in domain III. Human calpain 3 contains a unique insertion sequence in domain
II. Accession numbers: C. elegans TRA-3, NP502751; Dm-CalpA,
NP477047; Dm-CalpB, NP524016; human calpain 1, AAH08751 human calpain 3,
NP058813; human calpain 5, JC5772; Ha-CalpM, AAM88579
|
|
View larger version (76K):
[in a new window]
|
Fig. 6. Comparison of amino acid sequences in domain IV of `typical' calpains from
arthropods and human. Deduced amino acid sequences were aligned using ClustalW
(see Materials and methods). Broken lines indicate gaps for optimizing
alignment. Amino acid residues that are identical or similar in all the
sequences are highlighted in black; gray background indicates identical or
similar amino acids in most of the sequences. Numbers at the right indicate
amino acid positions, numbered from the N-terminus of each protein. Domain IV
is a calmodulin-like domain that contains five EF-hand motifs (boxes with
broken line). Dm-CalpA contains a unique insertion sequence between EF-1 and
EF-2. The putative EF-5 region was the least conserved between the calpain
sequences. Accession numbers are the same as those given in the legend to
Fig. 5.
|
|
View larger version (42K):
[in a new window]
|
Fig. 7. Comparison of amino acid sequences of the domain T in T-type calpains from
land crab (Gl-CalpT), nematode (TRA-3) and human (calpain 5). Deduced amino
acid sequences were aligned using ClustalW (see Materials and methods). Broken
lines indicate gaps for optimizing alignment. Amino acid residues that are
identical or similar in all the sequences are highlighted in black; gray
background indicates identical or similar amino acids in most sequences.
Numbers at the right indicate amino acid positions, numbered from the
N-terminus of each protein. Accession numbers: calpain 5, JC5772; TRA-3,
NP502751.
|
|
View larger version (7K):
[in a new window]
|
Fig. 8. Phylogenetic relationships of selected calpains from arthropods, nematode
and human. The deduced amino acid sequences of the catalytic (II) and
C2-like (III) domains in land crab (Gl-CalpM, Gl-CalpB and
Gl-CalpT), lobster (Ha-CalpM), fruit fly (Dm-CalpA and Dm-CalpB), nematode
(TRA-3) and human (Capn1, Capn3 and Capn5) calpains were analyzed using
ClustalW and Treeview software (see Materials and methods). The calpains
cluster into four groups: T-type calpains (TRA-3, Gl-CalpT and Capn5),
mammalian `typical' calpains (Capn1 and Capn3), crustacean M-type calpains
(Gl-CalpM and Ha-CalpM) and arthropod A/B-type calpains (Gl-CalpB, Dm-CalpA
and Dm-CalpB).
|
|
View larger version (38K):
[in a new window]
|
Fig. 9. Nucleotide and deduced amino acid sequence of partial cDNA encoding
ecdysone receptor (Gl-EcR) from land crab. The cDNA sequence (1005 bp) encoded
a partial protein sequence containing 335 amino acids (GenBank accession
#AY642975). Locations of degenerate primers used for nested RT-PCR to obtain
the initial cDNA are indicated in bold and with a dashed line with solid
arrowhead. Locations of sequence-specific forward primers (cEcR F1 and cEcR
F2) and degenerate nested reverse primer (EcR R4) used to obtain more of the
3' sequence are indicated in bold and with a dashed line with open
arrowhead. Solid lines with solid arrowheads indicate locations of
sequence-specific primers used for 5' RACE to obtain additional 5'
sequence in the ORF (Table
1).
|
|
View larger version (58K):
[in a new window]
|
Fig. 10. Comparison of deduced amino acid sequences of ecdysone receptor (EcR) cDNAs
from land crab, fiddler crab and locust. The partial Gl-EcR sequence is
aligned with EcR sequences from fiddler crab, Celuca (Uca)
pugilator (Up-EcR; #AAC33432), and locust, Locusta
migratoria (Lm-EcR; #AAD19828). Gl-EcR had the highest sequence identity
with the other two EcR sequences in the DNA-binding (domain C) and
ligand-binding (domain E) domains. Overall identities were 93% between Gl-EcR
and Up-EcR and 66% between Gl-EcR and Lm-EcR. Amino acid identities or
similarities in all three sequences are highlighted in black. Boundaries
between domains are indicated by capital letters.
|
|
View larger version (55K):
[in a new window]
|
Fig. 11. Tissue expression of land crab calpain B, M and T mRNAs using end-point
RT-PCR. Total RNA from each tissue was DNase-treated, reverse-transcribed and
PCR-amplified using primers specific for Calpains M, B and T
(Table 2). Shown is a reversed
image of an ethidium bromide-stained agarose gel of PCR products. The sizes of
the products were 310 bp for CalpM, 538 bp for CalpB and 536 bp for CalpT.
Gl-CalpB was expressed at varying levels in all tissues; Gl-CalpM was
expressed in all tissues except Y-organ; Gl-CalpT was expressed in all tissues
except eyestalk ganglia and Y-organ. Lane a, claw muscle (CM); b, leg muscle
(LM); c, limb regenerate (LR); d, thoracic muscle (TM); e, gill (Gi); f, heart
(Ht); g, hind gut (HG); h, thoracic ganglion (TG); i, eyestalk ganglia (EG);
j, Y-organ (YO).
|
|
View larger version (82K):
[in a new window]
|
Fig. 12. Quantification of calpain B, M and T mRNAs in tissues from intermolt land
crabs using real-time PCR. Transcript levels, expressed as log copy number,
were determined in nine tissues. Elongation factor 2 (EF2) mRNA (GenBank
accession #AY552550) served as an internal standard to normalize the PCR
reactions. P-values between means for each calpain within each tissue
are given at the top of each graph (see
Table 3 for primers and
Materials and methods for statistical analysis). There were three general
patterns of expression. In skeletal muscle (A,B), CalpM and CalpB were
expressed at similar levels, which were at least an order of magnitude higher
than CalpT expression. In heart, gill, thoracic ganglion, digestive gland and
testis (C–G), CalpB was expressed at higher levels than CalpM and CalpT.
In ovary and integument (H,I), CalpM was expressed at higher levels than
CalpB, which was expressed at higher levels than CalpT.
|
|
View larger version (30K):
[in a new window]
|
Fig. 13. Effect of eyestalk ablation on expression of land crab ecdysone receptor
(EcR) and calpains in skeletal muscles. Transcript levels, expressed as log
copy number, in claw (A) and thoracic (B) muscles were quantified using
real-time PCR. Elongation factor 2 (EF2) mRNA served as an internal standard
to normalize the PCR reactions. P-values between means for each gene
are given at the top of each graph (N=13 for claw muscle;
N=14 for thoracic muscle; see Materials and methods for statistical
analysis). In claw muscle, eyestalk ablation, which increases hemolymph
ecdysteroid levels, significantly increased the level of EcR and CalpT mRNAs;
CalpM and CalpB mRNA levels were unaffected. In thoracic muscle, there was no
significant effect of eyestalk ablation on EcR and calpain expression.
|
|
View larger version (34K):
[in a new window]
|
Fig. 14. The relationship between EcR and calpain expression in skeletal muscles
from intact and 1- or 3-day eyestalk-ablated land crabs. Correlations between
each calpain mRNA and EcR mRNA were determined with Statview (see Materials
and methods). The expression of CalpT was significantly correlated with EcR
expression in claw (P<0.001) and thoracic (P=0.0105)
muscles. CalpB and CalpM mRNA levels were not correlated with EcR mRNA.
|
|
© The Company of Biologists Ltd 2005
