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Cloning of a muscle-specific calpain from the American lobster Homarus americanus: expression associated with muscle atrophy and restoration during moulting

Xiaoli Yu and Donald L. Mykles^*

Department of Biology, Cell and Molecular Biology Program and Program in Molecular, Cellular and Integrative Neurobiology, Colorado State University, Fort Collins, CO 80523, USA

View larger version (69K): [in a new window]   Fig. 1. The complete sequence of Ha-CalpM cDNA cloned from a lobster fast muscle library. The sequence (1977 bp) contains an open reading frame (bp 16-1743) encoding a protein of 575 amino acids (aa) with a predicted mass of 66.3 kDa. The three conserved aa (Cys 141, His 299, Asn 329) essential for hydrolase activity are indicated in bold. A stretch of 17 aspartates (aa 454-470) is underlined. The numbers on the left indicate aa positions; those on the right indicate nucleotides. The highly conserved aa sequences used for initial cloning with nested PCR are underlined and italicized. GenBank accession no. AY124009.

View larger version (75K): [in a new window]   Fig. 2. Comparison of Ha-CalpM amino acid (aa) sequence with calpains from Drosophila and human. The deduced aa sequence of Ha-CalpM was aligned with Drosophila (CalpA and CalpB) and human (m-Calp) calpain sequences (see Materials and methods). Gaps (broken lines) were introduced to optimize the alignment. Aa residues that are identical to the Ha-CalpM sequence are highlighted. Ha-CalpM is highly homologous to Drosophila calpains (50% identity, 67% similarity). Ha-CalpM has three domains: domain I (aa 1-110) has a unique N-terminal sequence comprising the first 75 aa residues, domain II (aa 111-386) consists of a highly conserved cysteine proteinase sequence and domain III (aa 387-575) has a C2-like region that includes an acidic loop containing a stretch of 17 aspartate residues (aa 454-470). Ha-CalpM lacks a calmodulin-like domain (Domain IV) found in the human m-calpain and Drosophila sequences. Ha-CalpM has a short insertion sequence (aa 398-407) in domain III near the boundary with domain II that is not found in the other sequences. The boundaries between domains I and II, II and III, and III and IV are indicated by I/II, II/III and II/IV, respectively. The open triangles indicate the three amino acid residues of the catalytic triad (Cys141, His299, Asn329) in domain II. The asterisks indicate two conserved aspartate residues (Asp132 and Asp366) in domain II that bind Ca2+. Domain IV in the Drosophila and human sequences contains five EF-hand motifs (EF-1, EF-2, etc.). GenBank accession numbers are Z46891 for Drosophila calpain A (CalpA), AF062404 for Drosophila calpain B (CalpB), and M23254 for human m-calpain (m-Calp).

View larger version (7K): [in a new window]   Fig. 3. Phylogenetic relationship of Ha-CalpM to other calpains. Amino acid sequences of the proteinase domain (domain II) of calpains from selected species were compared using the Clustal V method (see Materials and methods). Branch lengths are proportional to the inferred phylogenetic distances, with the Shistosoma mansoni domain II sequence serving as the outgroup. Ha-CalpM was grouped with Drosophila calpains (Dm-CalpA and Dm-CalpB), reflecting the high sequence identity among the three sequences. GenBank accession numbers for Drosophila calpains and human m-calpain are given in the legend of Fig. 2; the accession numbers for the other calpains are X92523 for human p94 (CAPN3), AF022799 for mouse nCL-4 (CAPN9), M74233 for S. mansoni, Y10656 for mouse calpain 5 (CAPN5), and U14480 for C. elegans tra-3.

View larger version (91K): [in a new window]   Fig. 4. Northern blot analysis of Ha-CalpM expression in lobster tissues. Total RNA (20 µg) was separated electrophoretically on a 1% agarose gel and blotted overnight. The membrane (A) was hybridized under high stringency with a DIG-labeled RNA probe synthesized from a plasmid with an insert encoding the region encompassing amino acid residues 171-332 in domain II of Ha-CalpM (Fig. 1; see Materials and methods). Three Ha-CalpM transcripts were detected (3.3, 4.0 and 6.1 kb) in cutter (Cu) and crusher (Cr) claw muscles and deep abdominal (DA) muscle (A, lanes a—c). The fast muscles expressed all three mRNAs (lanes a and c), while the slow muscle (lane b) expressed primarily the 3.3 kb mRNA. Intestine (In; lane f) showed a strong hybridization to mRNA in the 3.3-6.1 kb range, as well as to mRNAs greater than 6.1 kb. Little or no Ha-CalpM mRNA was detected in digestive gland (DG; lane d), gill (Gi; lane e), nerve cord (NC; lane g), integument (Ig; lane h), heart (Ht; lane i) or antennal gland (AG; lane j). RNA loading was determined by staining the gel with Ethidium Bromide (B). Lane M, molecular mass markers (kb) are indicated.

View larger version (65K): [in a new window]   Fig. 5. RT-PCR analysis of Ha-CalpM and -actin expression in lobster tissues. Total RNA from various tissues were DNase-treated, reverse-transcribed, and PCR-amplified using primers directed to the 5' ends of Ha-CalpM (bp 1-18 and 600-619 in Fig. 1) and Ha-Actin1 (bp 1-23 and 379-400; see Koenders et al., 2002). PCR amplification of 18S rRNA served as an internal standard. (A) The PCR products synthesized from Ha-CalpM (619 bp) and 18S rRNA (324 bp). (B) The PCR product (400 bp) synthesized from -actin. Shown are negative images of ethidium bromide-stained agarose gels. There were high amounts of Ha-CalpM mRNA in cutter claw, crusher claw and deep abdominal muscles (lanes a—c). Little or no Ha-CalpM product was amplified from other tissues (lane e—i). -Actin was expressed in all tissues except antennal gland (lane i). Abbreviations as in Fig. 4.

View larger version (41K): [in a new window]   Fig. 6. SDS-PAGE and western blot analysis of Ha-CalpM in lobster tissues. Supernatant fractions (32 µg protein) from various tissues were electrophoresed on 6% SDS-polyacrylamide gels and either stained with Coomassie Blue (C) or transferred onto PVDF membrane and probed with affinity-purified anti-Ha-CalpM IgG (A) or pre-immune IgG (B). The Ha-CalpM antibody detected a 62 kDa isoform in claw muscles (lanes a and b) and a 68 kDa isoform in deep abdominal muscle (lane c). Non-specific binding of the antibody (B, lanes e—g) is indicated by asterisks in A. Hemocyanin, an extracellular 78 kDa oxygen-binding protein in the hemolymph, is a major contaminant in all tissues. Positions of molecular mass standards (kDa) are indicated. Te, testis; all other abbreviations as in Fig. 4.

View larger version (99K): [in a new window]   Fig. 7. Immunocytochemical localization of Ha-CalpM in claw muscles from adult lobster. Transverse sections were stained with Hematoxylin/Eosin (B) or incubated with pre-immune serum (A) or Ha-CalpM antiserum (C,D). Nuclei (arrowheads) are associated with the sub-sarcolemmal cytoplasm of sarcolemal clefts (arrows), which are large infoldings of the cell membrane. The Ha-CalpM antiserum stained the cytoplasm uniformly and some nuclei. Each field of view contains portions of two muscle fibers. ES, extracellular space. Scale bar, 100 µm.

View larger version (15K): [in a new window]   Fig. 8. Real-time PCR analysis of Ha-CalpM expression in lobster crusher claw muscles during the moulting cycle. Total RNA from intermoult, early premoult (stage D0), and early postmoult (2-4 days post-ecdysis) lobsters was DNase-treated, reverse-transcribed, and PCR-amplified in reactions containing SYBR Green I fluorescent dye (see Materials and methods). Values are means ±1 S.D. (N=4 for each moult stage). Ha-CalpM transcript copy number did not change significantly (P<0.28; ANOV A single factor analysis).

View larger version (83K): [in a new window]   Fig. 9. Western blot analysis of Ha-CalpM in claw muscles during the moulting cycle. Supernatant fractions (32 µg protein) of claw muscles at different moulting stages were electrophoresed on 6% SDS-polyacrylamide gels and either stained with Coomassie Blue (B) or transferred onto PVDF membrane and probed with Ha-CalpM antiserum (A) or pre-immune serum (data not shown). The 62 kDa isoform (arrow) was present at all moulting stages: intermoult (lanes a,b), premoult (lanes c,d), and postmoult (lanes e,f). Proteins binding non-specifically to the antibody are indicated by asterisks in A. Positions of molecular mass standards (kDa) are indicated. I-1 and I-2, intermoult stage at 20% and 30% of the intermoult interval, respectively; D1, premoult stage D1; D3, premoult stage D3; P-1 and P-2, postmoult stage at 7% and 13% of the intermoult interval, respectively.

View larger version (82K): [in a new window]   Fig. 10. Gel filtration chromatography of Ha-CalpM. A calpain fraction from Q Sepharose ion exchange chromatography was separated on a Superdex-200 gel filtration column (see Materials and methods). 10 µl samples of odd-numbered fractions were separated on 10% SDS-polyacrylamide gels and either stained with silver (A) or blotted onto PVDF membrane and probed with Ha-CalpM antiserum (B). The antibody recognized two Ha-CalpM isoforms of 62 kDa and 68 kDa (B, arrows) that co-eluted from the column. The smaller molecular mass immunoreactive polypeptides are probably degradation products. The arrow in A indicates the position of the 62 kDa isoform in the silverstained gel. The elution position of 62 kDa and 68 kDa isoforms (maximum at fraction 21) corresponded to that of CDP III (estimated mass 59 kDa) (Mykles, 2000; Mykles and Skinner, 1986). The peak CDP IIa activity (125 kDa) elutes at fraction 15 and the peak CDP IIb activity (195 kDa) elutes at fraction 12 (Mykles, 2000). Positions of molecular mass standards (kDa) are indicated.

View larger version (73K): [in a new window]   Fig. 11. Ion-exchange chromatography of Ha-CalpM. Fractions 17-23 from gel filtration chromatography (Fig. 10) were pooled and separated on a Mono Q column (see Materials and methods). 10 µl samples of odd-numbered fractions between fractions 7 and 35 were separated on 10% SDS-polyacrylamide gels and either stained with silver (A) or blotted onto PVDF membrane and probed with Ha-CalpM antiserum (B). The arrows indicate the positions of the 62 kDa and 68 kDa Ha-CalpM isoforms. The elution position of Ha-CalpM (fractions 23-25; 0.35-0.37 moll-1 NaCl) differed from that of CDP IIb (fractions 27-28; 0.40-0.41 moll-1 NaCl) and CDP IIa (fractions 17-18; 0.28 moll-1 NaCl) under identical conditions (Beyette et al., 1997; Mykles, 2000). Positions of molecular mass standards (kDa) are indicated.

View larger version (64K): [in a new window]   Fig. 12. Model of Ha-CalpM, based on the crystal structure of mammalian m-calpain (CAPN2) in the absence of Ca2+. Identical residues between the lobster and human deduced amino acid sequences are colored (red, acidic residues; blue, basic; yellow, hydrophobic; lavender, uncharged polar). (A) `Top' view of the catalytic domain (domain II) containing the three conserved residues (C141, H299 and N329, in green) forming the catalytic triad of the active site. The residues of the triad are surrounded by conserved nonpolar residues, which form a hydrophobic environment within the catalytic cleft. (B) `Side' view showing an acidic loop (magenta and red) containing 19 acidic residues in a stretch of 20 residues (D452, D455, D457 and D458 are not colored because they did not align with identical residues in the human m-calpain; see Fig. 2). The acidic loop, which includes a stretch of 11 aspartate residues (magenta; residues 460-470) unique to the lobster sequence, is part of a putative calcium-dependent phospholipid-binding C2-like region in domain III. Binding of Ca2+ to the acidic loop and to two aspartates (Fig. 2; D232 and D366) in domain II results in closure of the catalytic cleft to allow cooperation between C141, H299 and N329 necessary for catalytic activity (Hosfield et al., 2001; Moldoveanu et al., 2002). The first 60 residues of the N-terminal sequence were excluded, due to its high divergence from the mammalian sequence (Fig. 2). The lobster sequence lacks a calmodulin-like domain (domain IV).

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