Cloning of a muscle-specific calpain from the American lobster Homarus americanus: expression associated with muscle atrophy and restoration during moulting
Department of Biology, Cell and Molecular Biology Program and Program in Molecular, Cellular and Integrative Neurobiology, Colorado State University, Fort Collins, CO 80523, USA
* Author for correspondence (e-mail: don{at}lamar.colostate.edu)
Accepted 24 October 2002
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Summary |
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Key words: calpain, calcium-dependent proteinase, muscle, atrophy, moulting, cDNA, gene expression, mRNA, lobster, Homarus americanus, Crustacea, Arthropoda
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Introduction |
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Lobster muscle contains four Ca2+-dependent cysteine proteinase
(CDPs I, IIa, IIb and III), or calpain, activities
(Mykles and Skinner, 1986).
All require Ca2+ at millimolar levels for full activity in
vitro and degrade myofibrillar protein
(Mattson and Mykles, 1993
;
Mykles and Skinner, 1982
,
1983
,
1986
). CDP IIb (195 kDa) is a
homodimer, consisting of a 95 kDa subunit, which appears to be the homolog of
Drosophila CalpA or Dm-calpain (Beyette et al.,
1993
,
1997
;
Beyette and Mykles, 1997
;
Emori and Saigo, 1994
;
Jékely and Friedrich,
1999
; Theopold et al.,
1995
). CDP IIb undergoes an autolytic cleavage at the N terminus,
but it has little effect on the Ca2+ sensitivity of the enzyme
(Beyette et al., 1993
;
Beyette and Mykles, 1997
). CDP
IIa (125 kDa) is a homodimer of a 60 kDa subunit
(Beyette et al., 1997
). The
subunit compositions of CDP I (310 kDa) and CDP III (59 kDa) are not known, as
neither has been purified to homogeneity.
Both Ca2+-dependent and ubiquitin/proteasome-dependent
proteolytic systems are stimulated during moult-induced claw muscle atrophy,
when at least 40% of the muscle protein is degraded
(Beyette and Mykles, 1999;
Mykles, 1998
,
1999
;
Skinner, 1966
). There is a
preferential degradation of thin myofilament proteins, resulting in a
reduction in the thin:thick myofilament ratio and increased thick myofilament
packing density (Ismail and Mykles,
1992
; Mykles and Skinner,
1981
,
1982
). It is estimated that 11
thin myofilaments are degraded for every thick myofilament degraded
(Mykles and Skinner, 1981
).
During postmoult, protein is resynthesized as the muscle is restored to its
original mass and myofilament composition
(Skinner, 1966
). Thus, there
is significant remodeling of the contractile apparatus as fibers undergo
atrophy and restoration during the moulting cycle. Calpains probably play an
important role, as they degrade the Z-line and myofibrillar proteins in
vitro and in situ and have higher activities in atrophic muscle
(Mattson and Mykles, 1993
;
Mykles, 1990
; Mykles and
Skinner, 1982
,
1983
). Moreover, the four
calpain activities may have specific functions, as each calpain has unique
substrate specificities when incubated with a mixture of myofibrillar proteins
(Mattson and Mykles, 1993
).
For example, CDP IIb preferentially degrades thin myofilament proteins and
thus may contribute to the loss of thin myofilaments in atrophic muscle
(Mattson and Mykles, 1993
).
Proteasome level and ubiquitin expression and conjugation are increased in
atrophic muscle, although the role of this proteolytic pathway in claw muscle
remains to be established (Koenders et
al., 2002
; Shean and Mykles,
1995
).
Here we report the cloning of a crustacean calpain gene with features that distinguish it from calpains isolated from other species. The cDNA encodes a non-EF-hand calpain with a unique N-terminal sequence and an extended acidic loop in the C2-like region of domain III. The transcript levels in various tissues and in claw muscle during the moulting cycle were quantified by northern blot analysis, relative reverse transcriptionpolymerase chain reaction (RT-PCR) and real-time PCR. The distribution of Ha-CalpM protein was determined by western blotting and immunocytochemistry. Since this calpain was highly expressed in skeletal muscles, we have named the gene Ha-CalpM (Homarus americanus muscle-specific calpain; GenBank accession no. AY124009).
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Materials and methods |
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For the first step, the deduced amino acid sequences of calpain catalytic subunits from diverse species were aligned to identify conserved sequences using the Clustal W method from the Lasergene biocomputing software (DNASTAR). Degenerate primer sequences were selected from regions of high amino acid sequence identity. Two pairs of primers were synthesized for nested PCR, covering a sequence segment of 553 base pairs (bp) (equivalent to bp 423-975 in Drosophila CalpA; accession number Z46891). The external primers were: 1F (sense): 5'-C(T)TN GGN GAC(T) TGC(T) TGG C(T)TN C(T)T-3' and 1B (antisense): 5'-C(G)T(A) CAT CCA G(A)AA T(C)TC NCC G(A)TC-3'. The internal primers were: 1F' (sense): 5'-TAC(T) GCN GGN ATI TTC(T) CAC(T) TT-3' and 1B' (antisense): 5'-NCC CCA NGG G(A)TT NCT(G) NAT(A)(G) NC-3'. The primer locations are indicated in Fig. 1.
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The external primers were used for the first PCR reaction. A Perkin-Elmer DNA thermal cycler model 9600 was used for all reactions. The composition of the reaction mixture (25 µl) was 2.5 mmol l-1 MgCl2, 10 mmol l-1 Tris-HCl, pH 9.0, 50 mmol l-1 KCl, 0.1% Triton X-100, 200 µmol l-1 each of dNTPs, 1 µmol l-1 of each primer (1F and 1B), 0.75 µg plasmid, and 2.5 units of Taq polymerase (Promega). Initial denaturation was at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 54.8°C for 1 min, and extension at 72°C for 1 min. The final extension was at 72°C for 6 min. A 0.1 µl sample of the PCR reaction was used as template for subsequent amplification with the internal primers (1F' and 1B'). The reaction conditions were the same as those for the first-round PCR.
The Expand High Fidelity PCR System (Boehringer Mannheim) was used for IPCR. Inverse primers were synthesized based on the sequence of the nested PCR product: antisense, 5'-CAA GCC TTC CAT TGT ATG TG-3' (bp 600-619 in Fig. 1); sense, 5'-TCA TGC TTA CTC CAT CAC CC-3' (bp 909-928 in Fig. 1). The composition of the reaction mixture (50 µl) was 2 mmol l-1 Tris-HCl, pH 7.5, 10 mmol l-1 KCl, 0.1 mmol l-1 dithiothreitol (DTT), 0.01 mmol l-1 EDTA, 0.05% Tween-20, 200 µmol l-1 each of dNTPs, 300 nmol l-1 of each primer, 0.75 µg plasmid, and 2.6 units of Expand High Fidelity PCR enzyme. Initial denaturation was at 94°C for 2 min, followed by 10 cycles of denaturation at 94°C for 15 s, annealing at 59°C for 30 s, and extension at 68°C for 4 min. The reaction then underwent 20 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s and extension at 68°C for 4 min. The final extension was at 72°C for 6 min.
Based on the sequence from IPCR, specific primers to the 5' and 3' ends of Ha-CalpM were synthesized: sense, 5'-AGG AGC AAC AAC AGG ATG-3' (bp 1-18 in Fig. 1); antisense, 5'-TGC ACA GCC CAT CAT TAG-3' (bp 1959-1976 in Fig. 1). The PCR conditions were the same as those used for IPCR.
For cloning PCR products, reactions were separated electrophoretically on
1% agarose gels (70 V for 1.5 h); products were excized and purified using the
Qiagen Gel Purification kit. The DNA was concentrated in a Savant Speedvac and
ligated into the pGEM-T Easy plasmid vector (Promega). Ligation was achieved
by incubating 250 ng DNA, 50 ng vector and 3 Weiss units T4 DNA ligase in 30
mmol l-1 Tris-HCl, pH 7.8, 10 mmol l-1 MgCl2,
10 mmol l-1 DTT, 0.5 mmol l-1 ATP and 5% polyethylene
glycol, at room temperature for 1 h or overnight at 4°C (10 µl final
volume). DH5 electro-competent E. coli cells were transformed
with the ligation reaction by electro-transformation and plated on LB agar
plates containing 100 µg ml-1 ampicillin (Sigma). Positive
colonies were selected and grown overnight; plasmid DNA was isolated using a
Qiagen Plasmid Mini-Prep kit. Plasmid DNA containing inserts were sequenced in
both directions using the promoter primers Sp6 and T7 and gene-specific
primers (Davis Sequencing, Davis, CA, USA).
For sequence analysis, calpain sequences were retrieved from GenBank at the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). Sequence homologies were determined using the NCBI BLAST program. Multiple alignment and phylogenetic analyses were done using DNASTAR sequence analysis software.
Analysis of Ha-CalpM mRNA by northern blot and RT-PCR
Total RNA from lobster tissues was extracted using Trizol reagent (Gibco
BRL). The procedure was essentially the same as that recommended by the
manufacturer. Briefly, tissues were homogenized in 10 vol. Trizol (500 mg
tissue in 5 ml) using a Polytron homogenizer (Brinkmann Instruments, Westbury,
NY, USA) for about 1 min or until the tissues were completely homogenized.
Insoluble material was removed by centrifugation at 12 000 g
for 10 min at 4°C. 1 ml chloroform was added to each supernatant fraction
after incubation at room temperature for 5 min. Samples were vortexed and
centrifuged at 12 000 g for 15 min at 4°C. RNA in the
aqueous phase was precipitated with 2.5 ml isopropyl alcohol and pelleted by
centrifugation at 12 000 g for 10 min at 4°C. The
precipitated RNA was washed with 75% ethanol, dissolved in 50 µl deionized
formamide, and stored at -80°C.
Total RNA (20 µg) was separated electrophoretically on 1% agarose gels containing 0.5 mol l-1 formaldehyde in 1x Mops buffer (40 mmol l-1 Mops, pH 7.0, 10 mmol l-1 sodium acetate and 1 mmol l-1 EDTA) at 70 V for 3 h. RNA was transferred onto Hybond-N nylon membrane (Amersham) overnight in 20x SSC (3 mol l-1 NaCl and 0.3 mol l-1 sodium citrate, pH 7.0). The blot was UV cross-linked at 120 J for 30 s and hybridized in 20 ml hybridization buffer (Boehringer Mannheim) containing 50% deionized formamide, 5x SSC, 0.1% N-lauroylsarcosine, 0.02% SDS, 1% Blocking Reagent, and 1 µg digoxigenin (DIG)-labeled RNA probe overnight at 68°C. A DIG-RNA Labeling kit (Boehringer Mannheim) was used to synthesize the calpain probe by in vitro transcription from linearized plasmid containing a partial Ha-CalpM insert (bp 646-1129 in Fig. 1). Plasmid (1.8 µg) was linearized by digestion with NcoI (37°C for 1 h), separated electrophoretically on a 1% agarose gel, and purified with a Qiagen gel purification kit. The labeling reaction mixture (20 µl) contained 0.78 µg linearized plasmid, 1x DIG-RNA labeling mix (1 mmol l-1 ATP, 1 mmol l-1 CTP, 1 mmol l-1 GTP, 0.65 mmol l-1 UTP and 0.35 mmol l-1 DIG-11-UTP, pH 7.5), 1x transcription buffer, pH 7.5, and 30 units Sp6 RNA polymerase; the mixture was incubated at 37°C for 2 h. The membrane was washed twice (1 h each) with 0.5x SSC and 0.1% SDS at 68°C to remove unhybridized probe. Chemiluminescent detection of probe used anti-DIG antibody conjugated to alkaline phosphatase (Boehringer Mannheim) and disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD; Roche Molecular Biochemicals) according to Boehringer Mannheim's protocol.
For first-strand cDNA synthesis, total RNA in formamide was precipitated with 0.1 vol. 4 mol l-1 LiCl and 3 volumes of 100% ethanol. The RNA was washed in 75% ethanol and dissolved in diethyl pyrocarbonate (DEPC)-treated H2O. RNA was first treated with DNase (Gibco BRL) to remove contaminating DNA, then 2 µg of RNA was reverse-transcribed using SuperScriptTMII RNase H- reverse transcriptase (Gibco BRL). The reaction consisted of 2.5 µg of Oligo (dT) 12-18 (or 0.5 µg of random primers), 2.5 mmol l-1 dNTP, 1x First-Strand Buffer, 5 mmol l-1 DTT, 2.5 units RNase inhibitor and 200 units SuperscriptTM II RNase H- Reverse Transcriptase. The reaction mixture was incubated at 42°C for 50 min and stored at -20°C.
For the relative RT-PCR, a pair of 18S rRNA Primers and Competimers of
ratio 3:7 (Ambion) were included in the PCR reaction mixture together with the
calpain primer pairs directed to the 5' end of the Ha-CalpM cDNA: sense,
5'-AGG AGC AAC AAC AGG ATG-3' (bp 1-18 in
Fig. 1); antisense,
5'-CAA GCC TTC CAT TGT ATG TG-3' (bp 600-619 in
Fig. 1). The reaction mixture
contained 1x ExTaq buffer (2.5 mmol l-1 Mg2+, 200
µmol l-1 dNTP, 0.5 µmol l-1 of each primer, 1.5
units of Hot Start ExTaq polymerase (Takara). The final volume was 25 µl.
After synthesis of first-strand cDNA (see above), PCR amplification followed
at 92°C for 2 min, and 30 cycles of 94°C for 30 s, 60°C for 30 s
and 68°C for 45 s. The final extension time was 6 min at 72°C. The
conditions for amplifying lobster -actin (Ha-actin 1; accession no.
AF399872) were the same as that for Ha-CalpM and used the same primers
described previously (Koenders et al.,
2002
).
Real-time PCR quantification of Ha-CalpM transcript in lobster tissues used a SmartCycler instrument (Cepheid). A PCR master mix was assembled on ice containing 1x LightCycler FastStart Reaction Mix (SYBR Green I, dNTP mix with dUTP instead of dTTP, and FastStart Taq DNA polymerase), 3 mmol l-1 MgCl2 and 0.5 µmol l-1 of each primer (20 µl total volume). The sense primer (RTCalp-F1) was 5'-CAA CAA CAG GAT GTC GCT TAG TGA G-3' (bp 6-30 in Fig. 1) and the antisense primer (RT-Calp-B1) was 5'-TTC TGA AGG GAG ACG GAC TCT ATG-3' (bp 126-149 in Fig. 1). The mixtures were placed in reaction tubes, followed by the addition of 1 µl (100 ng) of first-strand cDNA. Plasmid containing Ha-CalpM cDNA insert was serially diluted (every tenfold) at a range of 0.28 fg to 28 pg to generate a standard curve. Both the Ha-CalpM plasmid and first-strand cDNA reactions were run simultaneously in a SmartCycler instrument using the same protocol. The protocol consisted of three stages: stage 1, holding at 95°C for 10 min; stage 2, 40 cycles at 95°C for 10 s, 57°C for 15 s with Optics on, and 72°C for 10 s; and stage 3, melting curve (60°C to 95°C at 0.2°C s-1). Cycle threshold analysis was used to quantify the absolute copy number of the Ha-CalpM transcript, as the cycle at which a specific PCR product begins to accumulate is directly proportional to the concentration of cDNA template in the reaction. A standard curve plotting the cycle threshold versus Ha-CalpM cDNA concentration was used to calculate the concentration of Ha-CalpM transcript in first-strand cDNA synthesized from tissue RNA.
Purification and N-terminal sequencing of Ha-CalpM isoforms
Chromatographic isolation of Ha-CalpM followed the protocol described
previously (Mykles, 2000).
Briefly, claw and deep abdominal muscles were homogenized in Buffer A (20 mmol
l-1 Tris-acetate, pH 7.5, 20 mmol l-1 KCl, 1 mmol
l-1 EDTA and 1 mmol l-1 DTT) and centrifuged at 12 000
g for 15 min at 4°C. The supernatant was precipitated with
65% saturated ammonium sulfate, dialyzed overnight against 21 Buffer A without
DTT and KCl at 4°C, and chromatographed on an organomercurial-agarose
(OMA) column (5 cm x 5 cm). Active fractions from the OMA column were
concentrated by a stirred-cell ultrafiltration device (Model 8200, Amicon) and
dialyzed against two 21 changes of Buffer A. Protein was loaded onto a Q
Sepharose column (2.5 x 18 cm) and eluted with a linear NaCl gradient
from 0 to 1 mol l-1 in Buffer A (300 ml total volume). Western
blotting (see below) was used to identify Ha-CalpM-containing fractions, which
were pooled, concentrated to about 5 ml with Filtron Macrosep 50K devices, and
dialyzed against 21 Buffer B (20 mmol l-1 Pipes-NaOH, pH 6.8, 0.1
mol l-1 NaCl, 1 mmol l-1 EDTA, 20% glycerol and 1 mmol
l-1 DTT). The dialyzed sample was chromatographed on a Superdex 200
(Pharmacia) gel filtration column (2.6 cm x 86 cm) equilibrated with
Buffer B at 3 ml min-1. Fractions (1.5 ml) containing Ha-CalpM
(determined by western blotting) were pooled and loaded onto a Mono Q ion
exchange column (Pharmacia HR 5/5) equilibrated with Buffer B. Protein was
eluted from the column with a linear NaCl gradient from 0.1 to 0.5 mol
l-1 (20 ml total volume) at 1 ml min-1. Fractions (0.6
ml) were analyzed by western blotting using Ha-CalpM antiserum.
For N-terminal sequencing, the Ha-CalpM proteins (Mono Q fractions 23-25) were separated on a 6% SDS-polyacrylamide gel and transferred to PVDF (polyvinylidene difluoride) membrane using 1x CAPS, pH 11/10% methanol transfer buffer. The protein on the blot was stained with India ink and the 62 kDa and 68 kDa proteins were excized and submitted to the Colorado State University Macromolecular Resources Facility for sequencing by Edman degradation.
SDS-polyacrylamide gel electrophoresis and western blot analysis
For analysis of Ha-CalpM in lobster tissues, tissues were homogenized in 6
vol. ice-cold Buffer A containing 1x protease inhibitor cocktail {Sigma;
2 mmol l-1 AEBSF [4-(2-aminoethyl)benzene sulfonyl fluoride], 1
mmol l-1 EDTA, 130 µmol l-1 bestatin, 1.4 µmol
l-1 E-64, 1 µmol l-1 leupeptin and 0.3 µmol
l-1 aprotinin} and centrifuged at 12,000 g for 15 min at
4°C. The supernatant fractions were removed and stored on ice. Protein
concentration was determined by fluorescence emission with a Perkin-Elmer LS-2
Filter Fluorimeter using bovine serum albumin as standard
(Avruch and Wallach, 1971).
Protein samples (32 µg) were mixed with equal volumes of sample buffer
(150 mmol l-1 Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 20 mmol
l-1 DTT and 0.01% Bromophenol Blue) and incubated for 5 min at
95°C. Proteins were separated electrophoretically by discontinuous
SDS-polyacrylamide gel electrophoresis (SDS-PAGE;
Laemmli, 1970). For the column
chromatography samples, 10 µl of each fraction was analyzed. Proteins in
gels were either stained with ammoniacal silver
(Mykles, 1988
) or
electrophoretically transferred to PVDF membrane (Hybond-P, Amersham)
(Towbin et al., 1979
).
For immunochemical studies, a polyclonal antibody was raised against a 28-amino acid (aa) sequence (53-80) in the N-terminal region of the Ha-CalpM deduced sequence. Peptide (NH2-SNDYTQKRIAKGGLKIPKKGFRTLRDEC-COOH) conjugated to keyhole limpet hemocyanin was used to raise antibody in rabbits by the Macromolecular Resource Facility at Colorado State University.
In some cases anti-Ha-CalpM IgG was affinity-purified using the Ha-CalpM peptide covalently-bonded to Immobilon-AV membrane (Millipore). Briefly, a 2 cm x 2 cm square of Immobilon-AV membrane was incubated with 1 mg peptide in 2 ml 0.5 mol l-1 potassium phosphate, pH 7.4, at room temperature for 2 h. The membrane was washed with 1 mol l-1 NaHCO3 for 2 h, followed by two 30 min washes in PBS (phosphate-buffered saline; 80 mmol l-1 disodium hydrogen orthophosphate, 20 mmol l-1 sodium dihydrogen orthophosphate and 100 mmol l-1 NaCl, pH 7.5) containing 0.1% Tween-20. The membrane was incubated with 2 ml of the Ha-CalpM antiserum for 3.5 h, rinsed three times with PBS, and bound antibody was eluted in 0.9 ml 100 mmol l-1 glycine, pH 2.5, for 10 min. The solution was neutralized with 0.1 ml 1 mol l-1 Tris-HCl, pH 8.0, and stored at 0°C with 0.02% sodium azide. The concentration of immunoglobulin G (IgG) was determined by dot-blot analysis comparing serial dilutions of affinity-purified IgG with serial dilutions of known concentrations of rabbit IgG; detection used the Biotin/Avidin System (see below).
The detection of Ha-CalpM used the Biotin/Avidin System (Vector Laboratories). The membrane was blocked with 5% non-fat milk in TBS (Tris-buffered saline; 20 mmol l-1 Tris-HCl, pH 7.5, and 137 mmol l-1 NaCl) at room temperature for 1 h. The primary antibody incubation was 1 h with Ha-CalpM antiserum or affinity-purified IgG (1:10,000 dilution) in 3% non-fat milk in TBS-T (0.01% Tween-20 in TBS). The secondary antibody incubation was 1 h with biotinylated goat anti-rabbit IgG (1:10,000 dilution in TBS-T). The membrane was incubated with ABC solution (10 µl avidin and 10 µl biotinylated horseradish peroxidase in 10 ml TBS-T) for 30 min. ECL Plus (Amersham Pharmacia) was used for chemiluminescent detection of peroxidase activity.
Immunocytochemistry
Tissues were fixed in 4% paraformaldehyde in lobster saline (0.5 mol
l-1 NaCl, 15 mmol l-1 KCl, 10 mmol l-1 EDTA,
25 mmol l-1 Hepes-NaOH, pH 7.5), dehydrated through an ethanol
series, and embedded in paraffin. Immunodetection used the Vectastain ABC
Elite kit (Vector laboratories) with minor modifications
(Mykles et al., 2000). After
hydration, sections (10 µm) were washed twice in PBS buffer containing 15
mmol l-1 glycine (PBS/Gly) for 5 min and blocked with 1.5% normal
goat serum in PBS/Gly for 1 h. Sections were incubated with Ha-CalpM antiserum
or preimmune serum (1:5000 dilutions in 1.5% normal goal serum in PBS/Gly)
overnight. After washing with PBS/Gly, sections were incubated with
biotinylated goat anti-rabbit IgG (1:200 dilution in 1.5% normal goat serum in
PBS/Gly) for 2 h, followed by incubation with ABC reagent (avidin/biotinylated
horseradish peroxidase complex) for 30 min. Antibody was detected
colormetrically by incubating the sections in a solution containing 0.8 mg
ml-1 diaminobenzidine tetrahydrochloride, 0.4 mg ml-1
NiCl, 0.01% H2O2, and 0.1 mol l-1 Tris-HCl,
pH 7.4. The sections were rinsed with deionized H2O, dehydrated
through an ethanol series, cleared in xylene, and mounted in Permount (Fisher
Scientific). Sections were photographed under bright-field illumination.
Modeling of lobster calpain
The Ha-CalpM sequence (aa residues 61-575) was modeled using the crystal
structure of mammalian m-calpain as template
(Hosfield et al., 1999;
Strobl et al., 2000
).
Swiss-Model and Sybyl programs were used for initial tracing of the backbone.
Further refinement of the putative secondary structure used the algorithm from
Kabsch and Sander (1983
) using
UCSF MidasPlus software with these parameters: energy cutoff for hydrogen
bonding, -0.5 kcal mol-1; minimum helix length, 3; minimum sheet
length, 3.
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Results |
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The full-length Ha-CalpM cDNA consists of 1977 nucleotides
(Fig. 1). The position of the
initiation codon is similar to that of Drosophila calpains
(Jékely and Friedrich,
1999; Theopold et al.,
1995
). The Ha-CalpM cDNA has a 5' UTR of 15 bp and a
3' UTR of 234 bp. The open reading frame (ORF; bp 16-1743) encodes a
protein of 575 amino acids with a predicted molecular mass of 66.3 kDa.
Alignment of the deduced aa sequence of Ha-CalpM with Drosophila
and human calpains is shown in Fig.
2. Ha-CalpM has the highest sequence identity with
Drosophila calpains A and B (CalpA and CalpB; 50% identity, 67%
similarity). In addition to Drosophila calpains and human m-calpain,
Ha-CalpM also has significant sequence similarities to other mammalian
calpains, such as digestive tract-specific calpain (CAPN9 or nCL-4; AF022799;
64% similarity) and skeletal muscle-specific calpain (CAPN3 or p94; AF127765;
62% similarity). The similarity to human m-calpain is 60%. The domain
organization of Ha-CalpM was assigned according to that of the
Drosophila calpain sequences
(Sorimachi and Suzuki, 2001).
Domain I (aa 1-110) has a unique N-terminal sequence (aa 1-75) that has no
sequence identity with any other sequence in the NCBI database. Domain II (aa
111-396) consists of a highly-conserved cysteine proteinase sequence
containing the catalytic triad (Cys141, His299 and Asn329) and two aspartates
(Asp132 and Asp366) involved in Ca2+ binding. Domain III (aa
397-575) contains a short insertion sequence (aa 408-417) and an acidic loop
with an expanded stretch of an additional 11 aspartates (aa 454-470) that may
bind Ca2+ and help activate the proteinase. Ha-CalpM lacks domain
IV (a calmodulin-like domain) found in EF-hand calpains.
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A phylogenetic tree was generated comparing the domain II amino acid sequence of Ha-CalpM (Ha-CalpM-II) with the domain II sequences of selected calpains representing diverse phyla (Fig. 3). The Shistosoma calpain domain II sequence served as the outgroup in the analysis. As expected, Ha-CalpM clustered with Drosophila CalpA and CalpB. The human p94 (Capn3), human m-calpain (Capn2), and mouse nCL-4 (Capn9) formed a distinct, but related group; mouse Capn5 and C. elegans tra-3 calpains were more divergent.
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Expression of Ha-CalpM
Northern blotting was used initially to determine the tissue distribution
and sizes of Ha-CalpM mRNAs. The membrane was hybridized with a DIG-labeled
RNA probe synthesized from the domain II region (equivalent to aa 171-332) of
Ha-CalpM under high stringency. The results show that Ha-CalpM was highly
expressed in skeletal muscle (Fig.
4, lanes ac). Two major transcripts (6.1 kb and 3.3 kb)
were detected. A minor 4 kb mRNA was also present in fast muscles (cutter claw
and deep abdominal). The probe hybridized to mRNAs of varying sizes from
intestine (Fig. 4, lane f).
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RT-PCR was used to detect low levels of expression and to determine whether
the hybridization of the probe to intestine RNA
(Fig. 4) was specific to
Ha-CalpM. Primers were synthesized to amplify the sequence encoding the unique
N-terminal sequence of Ha-CalpM. As expected, a large amount of PCR product
(619 bp) was synthesized from muscle RNAs
(Fig. 5, lanes ac).
Little or no Ha-CalpM PCR product was obtained from digestive gland, gill,
intestine, nerve cord/thoracic ganglion, integument and antennal gland
(Fig. 5, lanes di),
indicating that the hybridization in the northern blot to intestine RNA was
not due to high levels of Ha-CalpM mRNA. PCR amplification of 18S rRNA (324
bp) and -actin (Ha-Actin1; 400 bp) products indicated that each lane
contained the same amount of total RNA and that the RNA was not degraded
(Fig. 5). A negative PCR
control using total RNA as template produced no products, indicating no DNA
contamination (data not shown). The
-actin was expressed at a higher
level in the crusher claw muscle than in the cutter claw and deep abdominal
muscles, which corresponds to the higher ratio of actin to myosin in slow
fibers than in fast fibers (Mykles,
1985
,
1988
). The same RNA was used
to quantify the copy number of Ha-CalpM mRNA in the tissues by real-time PCR
(Table 1). The highest copy
numbers were in skeletal muscles, although the amount in slow muscle (crusher
claw) was about 25% that in fast muscle (cutter claw and deep abdominal).
Other tissues expressed Ha-CalpM at much lower copy numbers (0.4% to 3% that
of deep abdominal muscle; Table
1).
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|
An antibody raised against a 28-aa sequence in the N-terminal domain of Ha-CalpM (aa 53-80) was used to determine Ha-CalpM protein levels and distribution in lobster tissues. In western blots, the antibody detected two proteins in skeletal muscles: a 62 kDa isoform in cutter and crusher claw muscles and a 68 kDa isoform in deep abdominal muscle (Fig. 6, lanes ac). The isoforms were not detected in gill, intestine, heart or testis (Fig. 6, lanes dg). Immunocytochemistry of transverse sections of the cutter and crusher claw muscles showed that Ha-CalpM was distributed throughout the cytoplasm (Fig. 7). Ha-CalpM was also localized in some, but not all, nuclei (Fig. 7, compare the Hematoxylin staining of nuclei in B with the staining of fewer nuclei in C and D).
|
|
As calpains are involved in moult-induced claw muscle atrophy, the
expression of Ha-CalpM was determined in claw muscle from lobsters at
different moult stages. For real-time PCR, total RNA was isolated from crusher
claw muscle at intermoult, early premoult (D0), and postmoult (2-4
days postecdysis) stages, reverse-transcribed and PCR-amplified in the
presence of SYBR Green I fluorescent dye
(Fig. 8). There was no
significant effect of moult stage on Ha-CalpM mRNA levels (P<0.28;
ANOVA single factor analysis). Parallel PCR reactions using Ha-actin1 primers
showed no changes in Haactin1 mRNA (data not shown), which was consistent with
the constitutive expression of actin during the moutling cycle reported
recently (Koenders et al.,
2002). The 62 kDa Ha-CalpM isoform was expressed in claw muscles
at all stages of the moulting cycle (Fig.
9). As levels were quite variable in intermoult muscle
(Fig. 9, lanes a,b), the higher
levels of Ha-CalpM in postmoult claw muscles (lanes e,f) were not
significantly different.
|
|
Purification of Ha-CalpM
Four calpain activities (CDPs I, IIa, IIb and III) have been characterized
biochemically in lobster skeletal muscles
(Mykles and Skinner, 1986).
The mass of the deduced Ha-CalpM sequence (66.3 kDa) and masses of the
Ha-CalpM isoforms in western blots (62 kDa and 68 kDa) suggested that Ha-CalpM
cDNA encoded either CDP IIa (60 kDa subunit mass; 125 kDa native mass) or CDP
III (59 kDa native mass). A soluble extract of intermoult lobster muscles
(claws and deep abdominal) containing all four calpain activities was
subjected to a series of chromatographic separations on organomercurial
agarose, Q Sepharose, Superdex-200 gel filtration and Mono Q columns
(Mykles, 2000
). The 62 kDa and
68 kDa Ha-CalpM isoforms co-eluted from each column and the results are shown
for the final two separations (Figs
10,
11). The Ha-CalpM isoforms
eluted from the Superdex-200 gel filtration column at a position corresponding
to the native mass of CDP III (Fig.
10, lanes hn). The gel filtration fractions containing
Ha-CalpM were concentrated and chromatographed in a Mono Q anion exchange
column; the Ha-CalpM proteins eluted as a single peak (fractions 23-25;
Fig. 11, lanes i,j). Edman
degradation of the purified 62 kDa and 68 kDa isoforms did not yield
N-terminal sequences for either polypeptide.
|
|
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Discussion |
---|
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---|
It is likely that Ha-CalpM has Ca2+-dependent proteinase
activity, even though it lacks domain IV. Ca2+-binding regions in
domains II and III in other calpains
(Andresen et al., 1991;
Hosfield et al., 2001
;
Moldoveanu et al., 2002
) are
highly conserved in Ha-CalpM (Fig.
2). Expressed recombinant mammalian calpains lacking domain III or
IV or only containing domain II retain Ca2+ dependency
(Hata et al., 2001
;
Vilei et al., 1997
). Tra-3, a
non-EF-hand calpain involved in sex determination in C. elegans, is
activated by Ca2+ (Sokol and
Kuwabara, 2000
). These data suggest that Ha-CalpM has
Ca2+-dependent proteolytic activity, although this must be
demonstrated on expressed recombinant protein using functiional assays.
The Ha-CalpM cDNA appears to encode CDP III, one of four calpain activities
identified in lobster skeletal muscles
(Mykles and Skinner, 1986).
The Ha-CalpM isoforms elute from a gel filtration column at the same position
as CDP III (59 kDa). The other three calpains (CDP I, 310 kDa; CDP IIa, 125
kDa; and CDP IIb, 195 kDa) elute earlier from the column
(Mykles, 2000
). The elution
position of the Ha-CalpM proteins from the Mono Q column differed from those
of CDPs IIa and IIb; under identical conditions, CDP IIa elutes at fractions
17-18 and CDP IIb at fractions 27-28
(Beyette et al., 1997
;
Mykles, 2000
). Moreover, the
68 kDa isoform is similar to the predicted mass (66.3 kDa) of the deduced
amino acid sequence of Ha-CalpM. These data indicate that the native CDP III
comprises a single polypeptide encoded by the Ha-CalpM gene. CDP IIb is a
homodimer of a 95 kDa subunit, while CDP IIa is a homodimer of a 60 kDa
subunit; both calpains appear to be products of distinct genes
(Beyette et al., 1997
). The
subunit composition of CDP I is not known. However, the identity of Ha-CalpM
and CDP III remains to be established. We were unable to obtain N-terminal
sequences of the 62 kDa and 68 kDa isoforms by Edman degradation, suggesting
that the N termini were chemically blocked.
The 62 kDa and 68 kDa isoforms are probably encoded by the same gene, since
both proteins react specifically to antibody raised against the unique
N-terminal sequence and coelute through several chromatographic separations.
The expression of the two isoforms is not correlated with the fiber type
composition of the muscles. The 62 kDa isoform occurs in both crusher claw
closer muscle, which contains only slow fibers, and cutter claw closer muscle,
which contains mostly fast fibers (Mykles,
1985). The 62 kDa isoform could either be the active form of
calpain produced by autolysis of the 68 kDa isoform or an isoform generated by
alternative splicing or post-translational processing.
A three-dimensional model of Ha-CalpM, using the crystal structure of
mammalian m-calpain (Capn2) as template, is presented in
Fig. 12. The colors denote aa
residues that are identical between Ha-CalpM and human m-calpain. The first
75-aa acid sequence of domain I is unique, showing no sequence identity with
any other gene in the NCBI database. Consequently, the first 60 residues could
not be accommodated in the model. The last 35-aa sequence of domain I has 40%
and 49% identity with the same region of Drosophila calpains A and B,
respectively. Domain II is divided into two subdomains (IIa and IIb), which
form the active site (Fig.
12A) (Hosfield et al.,
1999; Strobl et al.,
2000
). The Cys141 in domain IIa and the His299 and Asn329 in
domain IIb form a catalytic triad essential for cysteine proteinase activity
(Arthur et al., 1995
). Many of
the conserved residues are located in the vicinity of the catalytic site
(Fig. 12A). The most striking
feature of Ha-CalpM exists in domain III, which contains a highly acidic
sequence consisting of a stretch of 17 aspartate residues
(Fig. 12B). All calpains have
an acidic loop as part of the C2-like domain, but the expansion of this acidic
stretch is unique to Ha-CalpM. A repeat sequence of 44 aspartate residues has
been reported in calsequestrin, a high-capacity, moderate-affinity,
Ca2+-binding protein that acts as an internal Ca2+ store
in fast muscle fibers (Treves et al.,
1992
). Analysis of the crystal structure of m-calpain suggests
that the acidic region interacts with Ca2+ and functions as an
`electrostatic switch' for regulating proteinase activity
(Hosfield et al., 2001
;
Strobl et al., 2000
). The
X-ray structures of rat and human m-calpain also show that domain III is
engaged in extensive ionic interactions with all the other domains within the
large subunit and thus seems to function as an organizing center of calpain
(Hosfield et al., 1999
;
Strobl et al., 2000
). These
data suggest that domain III of Ha-CalpM plays a significant role for full
activation of the enzyme. Ca2+ binding would result in movement of
the Asn329 and His299 on one side of the catalytic cleft toward the Cys141 on
the other, thus forming of a functional catalytic site
(Fig. 12A).
|
Calpain genes show a range of tissue expression, from ubiquitous to highly
tissue-specific. The mammalian calpains 1 (µ-calpain), 2 (m-calpain), 5, 7,
10, 12 and 13, are ubiquitously expressed, although calpain 5 is highly
expressed in colon, small intestine and testis and calpain 12 is highly
expressed in hair follicle (Huang and
Wang, 2001; Sorimachi and
Suzuki, 2001
). The preferential expression of Ha-CalpM in skeletal
muscle is not unusual. Alternatively spliced isoforms of mammalian CAPN3 are
restricted to skeletal muscle (p94 calpain), lens (Lp82 and Lp85 calpains) and
retina (Rt88 calpain) (Huang and Wang,
2001
; Sorimachi and Suzuki,
2001
). Mammalian calpain 6 is expressed in placenta, calpain 8 in
stomach mucosa, calpain 9 in digestive tract, and calpain 11 in intestine
(Dear and Boehm, 1999
;
Lee et al., 1999
). In
Drosophila, Dm-CalpA is differentially expressed during development
(Emori and Saigo, 1994
;
Theopold et al., 1995
).
Although the Dm-CalpA mRNA is ubiquitously expressed in adult flies,
immunocytochemistry shows the protein is restricted to cells in the brain,
ventral ganglion and blood (hemocytes)
(Theopold et al., 1995
). These
data suggest that certain calpains mediate tissue-specific functions.
The size (about 2 kb) of the Ha-CalpM cDNA is smaller than the transcript sizes in northern blots. Two major transcripts of Ha-CalpM (6.1 kb and 3.3 kb) were detected at high stringency. An additional minor transcript (4 kb) was expressed in fast muscle. This suggests that the three transcripts contain the Ha-CalpM ORF but differ in the lengths of the UTRs. The 3' UTR of the Ha-CalpM cDNA lacks a polyadenylation signal and a poly(A) tail. Given the high percentage (40%) of adenosines in the 3' UTR, the oligo(dT) primer used for the initial cDNA synthesis may have annealed to this region, rather than to a poly(A) tail located further downstream. This suggests that the 3' UTR of the Ha-CalpM mRNA is longer than 234 bp. The three transcript sizes may arise from alternative polyadenylation signals in the 3' UTR.
Crustacean muscles contain four calpain activities that are involved in the
degradation of myofibrillar proteins during programmed claw muscle atrophy
(Mykles, 1999). Coincident
with this degradation is a substantial restructuring of the myofibrils.
Myofibrillar cross-sectional area decreases 78%, the thin:thick myofilament
ratio decreases from 9:1 to 6:1, and thick myofilament packing increases by
51% to 72% (Ismail and Mykles,
1992
; Mykles and Skinner,
1981
). Although Ha-CalpM mRNA and protein levels were not elevated
in premoult muscle, the muscle-specific expression of Ha-CalpM
(Table 1; Figs
4,5,6),
and the ability of Ha-CalpM/CDP III to degrade myofibrillar proteins
(Mattson and Mykles, 1993
),
suggests that it has a role in remodeling and/or maintaining the structure of
the contractile apparatus. This is analogous to the p94 isoform of CAPN3 in
mammalian skeletal muscle. The activity of p94 is necessary to maintain muscle
fiber viability and myofibrillar protein composition. Loss-of-function
mutations in the CAPN3 gene are the underlying cause of limb-girdle muscular
dystrophy type 2A in humans (Chae et al.,
2001
). Expression of CAPN3 responds rapidly to conditions that
induce muscle atrophy or degeneration. Denervation or muscle injury result in
a rapid decrease in CAPN3 mRNA in mouse skeletal muscle
(Stockholm et al., 2001
),
whereas sepsis causes an increase in CAPN3 mRNA in rat skeletal muscle
(Williams et al., 1999
). The
p94 appears to maintain the structural integrity of the contractile apparatus
through its interaction with connectin/titin
(Herasse et al., 1999
).
In summary, the full-length cDNA encoding a non-EF-hand calpain expressed in lobster skeletal muscle has been characterized. This is the first calpain gene isolated from a crustacean species. It appears to encode the CDP III activity characterized in biochemical studies. The Ha-CalpM is expressed as a 62 kDa isoform in claw muscles and a 68 kDa isoform in deep abdominal muscle; the functional significance of the two isoforms is not known. Although the four crustacean calpains appear to be products of different genes, the precise relationship between Ha-CalpM and CDPs I, IIa and IIb will not be established until the full-length sequences of the remaining calpains are determined. Our data suggest that Ha-CalpM plays a role in restructuring the skeletal muscle during the moulting cycle. However, its specific role will remain undefined until endogenous substrates are identified.
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Acknowledgments |
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