Calponin-like Protein Research Articles

Two-dimensional gel and shotgun proteomics approaches to distinguish fresh and frozen-thawed curled octopus (Eledone cirrhosa)

The substitution and sale of frozen-thawed fish labeled as fresh is a widespread, difficult to unmask commercial fraud and a potential risk for consumer health. Proteomics could help to identify markers for the rapid screening of food samples and the identification of frozen-thawed seafood. Using two-dimensional electrophoresis (2-DE) and high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified biomarkers that are able to discriminate between fresh and frozen-thawed tissue samples of curled octopus (Eledone cirrhosa). The 2-DE analysis showed a significant reduction in two protein spots (molecular weight of 45–50 kDa, isoelectric point of 6.5–7) identified as transgelin. At shotgun analysis, nine proteins resulted modulated and transgelin was confirmed as down-regulated, making it a potentially useful marker for differentiating between fresh and frozen-thawed fish product samples. Biological significanceThis work, based on two different proteomics approaches, investigated differentially expressed proteins in the tentacles of the curled octopus (E. cirrhosa) after freezing-thawing processes. We were able to characterize the proteome of the tentacles, increasing our knowledge on this species, and a common down-regulated protein was identified by 2-DE and shotgun analysis, a calponin-like protein called transgelin, suggesting a potential use as a marker to distinguish different states of conservation in this species.

Recombinant Calponin of human filariid Brugia malayi : Secondary structure and immunoprophylactic potential

In the search for potential vaccine candidates for the control of human lymphatic filariasis, we recently identified calponin-like protein, that regulates actin/myosin interactions, in a proinflammatory fraction F8 (45.24-48.64kDa) of Brugia malayi adult worms. In the present study, the gene was cloned, expressed, and the recombinant Calponin of B. malayi (r-ClpBm) was prepared and characterized. r-ClpBm bears homology with OV9M of Onchocerca volvulus, a non-lymphatic filariid that causes loss of vision and cutaneous pathology. r-ClpBm was found to be a ∼45kDa protein that folds into a predominantly α-helix conformation. The protective efficacy of r-ClpBm against B. malayi infection in Mastomys coucha was investigated by assessing the course of microfilaraemia and adult worm burden in the host immunized with r-ClpBm and subsequently infected with infective third stage larvae (L3). Expression of the Calponin was detected in all life stages (microfilariae, L3, L4, L5 and adults) of the parasite and immunization with r-ClpBm partially protected M. coucha against establishment of infection as inferred by ∼42% inhibition in parasite burden. Upregulated cellular proliferation, TNF-α, IFN-γ, IL-1β, IL-4, nitric oxide (NO) release, expression of iNOS, and specific IgG, IgG1 and IgG2b in immunized animals correlated with parasitological findings. r-ClpBm immunization caused degranulation in majority of mast cells indicating possible involvement of mast cell products in reducing the parasite survival. It appears that complex mechanisms including Th1, Th2, NO and mast cells are involved in the clearance of infection. To the best of our knowledge this is the first report on cloning, expression of the gene and purification of r-ClpBm, determination of its secondary structure and its ability to partially prevent establishment of B. malayi infection. Thus, r-ClpBm may further be studied and developed in combination with other protective molecules of B. malayi as a component of potential filarial cocktail vaccine candidate.

Crenomytilus grayanus 40 kDa calponin-like protein: cDNA cloning, sequence analysis, tissue expression, and post-translational modifications

Calponin-like protein (CaP-40), a third major protein after actin and tropomyosin, has recently been identified by us in the Ca2+-regulated thin filaments of mussel Crenomytilus grayanus. It contains calponin homology domain, five calponin family repeats and possesses similar biochemical properties as vertebrate smooth muscle calponin. In this paper, we report a full-length cDNA sequence of CaP-40, study its expression pattern on mRNA and protein levels, evaluate CaP-40 post-translational modifications and perform protein-protein interaction analysis.The full-length sequence of CaP-40 consists of 398 amino acids and has high similarity to calponins among molluscan species. CaP-40 gene is widely expressed in mussel tissues, with the highest expression in adductor and mantle. Comparison of these data with protein content established by mass-spectrometry analysis revealed that the high mRNA content is mirrored by high protein levels for adductor smooth muscles. To provide unbiased insight into the function of CaP-40 and effect of its over-expression in adductor smooth muscle, we built protein-protein interaction network of identified Crenomytilus grayanus proteome. In addition, we showed that CaP-40 is subjected to post-translational N- and C-terminal acetylation at N127, G229 and G349 sites which potentially regulates its function in vivo.

Calponin-Like Protein from Mussel Smooth Muscle Is a Competitive Inhibitor of Actomyosin ATPase.

The goal of this work was to elucidate the mechanism of inhibition of the actin-activated ATPase of myosin subfragment-1 (S1) by the calponin-like protein from mussel bivalve muscle. The calponin-like protein (Cap) is a 40-kDa actin-binding protein from the bivalve muscle of the mussel Crenomytilus grayanus. Kinetic parameters Vmax and KATPase of actomyosin ATPase in the absence and the presence of Cap were determined to investigate the mechanism of inhibition. It was found that Cap mainly causes increase in KATPase value and to a lesser extent the decrease in Vmax, which indicates that it is most likely a competitive inhibitor of actomyosin ATPase. Analysis of Vmax and KATPase parameters in the presence of tropomyosin revealed that the latter is a noncompetitive inhibitor of the actomyosin ATPase.

Identification of putative ecdysteroid and juvenile hormone pathway genes in the shrimp Neocaridina denticulata

Although the sesquiterpenoid juvenile hormone (JH) and the steroidal ecdysteroids are of vital importance to the development and reproduction of insects, our understanding of the evolution of these crucial hormonal regulators in other arthropods is limited. To better understand arthropod hormone evolution and regulation, here we describe the hormonal pathway genes (e.g. those involved in hormone biosynthesis, degradation, regulation and signal transduction) of a new decapod model, the shrimp Neocaridina denticulata. The majority of known insect sesquiterpenoid and ecdysteroid pathway genes and their regulators are contained in the N. denticulata genome. In the sesquiterpenoid pathway, these include biosynthetic pathway components: juvenile hormone acid methyltransferase (JHAMT); hormone binding protein: juvenile hormone binding protein (JHBP); and degradation pathway components: juvenile hormone esterase (JHE), juvenile hormone esterase binding protein (JHEBP) and juvenile hormone epoxide hydrolase (JHEH), with the JHBP, JHEBP and JHEH genes being discovered in a crustacean for the first time here. Ecdysteroid biosynthetic pathway genes identified include spook, phantom, disembodied, shadow and CYP18. Potential hormonal regulators and signal transducers such as allatostatins (ASTs), Methoprene-tolerant (Met), Retinoid X receptor (RXR), Ecdysone receptor (EcR), calponin-like protein Chd64, FK509-binding protein (FKBP39), Broad-complex (Br-c), and crustacean hyperglycemic hormone/molt-inhibiting hormone/gonad-inhibiting hormone (CHH/MIH/GIH) genes are all present in the shrimp N. denticulata. To our knowledge, this is the first report of these hormonal pathways and their regulatory genes together in a single decapod, providing a vital resource for further research into development, reproduction, endocrinology and evolution of crustaceans, and arthropods in general.

Differential expression of sex-related fat body proteins during the larval–pupal developmental stages of the silkworm (Bombyx mori)

The silkworm fat body is the site of many intermediary metabolic processes, and a source of sustenance for growth throughout the life cycle. Fat body proteins are responsible for storing nutrients, providing energy, and regulating hormones, and they have been identified using proteomic approaches. However, detailed differential expression of sex-related fat body proteins has not previously been evaluated. In the present study, we characterized the differential expression of sex-related fat body proteins, by using 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry identification and bioinformatics methods. We extracted the fat body proteins from 5-day-old fifth instar larvae (L5), 10-day-old fifth instar larvae (corresponding to the end of spinning [LE]), and 0-day-old pupae (P0) of the multivoltine silkworm variety “Da Zao”. We confirmed the presence of 11 important sex-specific expression proteins and 14 stage-specific expression proteins. We accurately identified 13 of these specific expression proteins, including actin, calponin-like protein, 75kDa subunit NADH, receptor for activated protein kinase C from Bombyx mori (BmRACK), IMP (inosine monophosphate) cyclohydrolase, tropomyosin 1, β-tubulin, hypothetical protein, antichymotrypsin precursor, and 30K protein precursor. We showed that BmRACK was differentially expressed between male and female silkworms. We discuss the biological roles of the specific expression proteins during the larval–pupal developmental stages.

Differential Expression of Stage-Specific Fat Body Proteins during Larval-Pupal Period in Silkworm (<i>Bombyx mori</i>)

The silkworm fat body is the organ of many intermediary metabolic processes and sustenance of growth throughout the life, and its proteins are responsible for storing nutrients, providing energy and regulating hormones. To date, detailed differential expressions of fat body proteins have not been clearly analyzed. In this research, fat body proteins were extracted from the larvae of the 5th day of the 5th instar, un-pupated larvae of spinning end and pupae of pupating day of the multivoltine silkworm variety Da Zao, and the differential expressed proteins were characterized by two-dimensional gel electrophoresis, followed by mass spectrometry identification and informatics method. 14 stage-specific expression proteins were detected and confirmed. Among them, 7 proteins were accurately identified, such as actin, calponin-like protein, beta-tubulin, hypothetical protein, antichymotrypsin precursor and 30K protein precursor etc. The biological roles of these important proteins during the larval-pupal developmental stages were discussed.

Expressed sequence tags 454 sequencing and biomineralization gene expression for pearl sac of the pearl oyster,Pinctada fucata martensii

Pearl sac is the tissue that secretes proteins resulting into artificial pearl formation, a highly controlled biomineralization process similar to development of inner shell regulated by the mantle. We present here the pearl sac transcriptome of Pinctada fucata martensii using massively parallel pyrosequencing. A total of 21 729 putative unique transcripts were assembled and annotated. Twenty-three candidate biomineralization genes were identified, such as calmodulin, calponin-like protein (CLP), mantle gene, pearlin, nacrein and Pif. The sequence data contained 4303 potential SNP loci and 4291 probable indels, providing a resource for molecular biomarker, population genetics and gene function. Expression levels of six potential biomineralization genes were also characterized with real-time PCR. The expression level of mantle gene 4 was the highest, followed by lysine-rich matrix protein 4. Nearly all detected genes maintained the highest expression levels in mantle edge and the lowest in pearl sac. In both tissues of mantle centre and pearl sac, the six genes had an obvious higher expression activity in host oyster producing nacre pearls than those producing plain nuclei, but the expression level of CLP was similar in the two host groups. These results will notably advance biomineralization and transcript-wide study in P. fucata martensii and other Pinctada species.

Molluscan smooth catch muscle contains calponin but not caldesmon

We isolated Ca(2+)-regulated thin filaments from the smooth muscle of the mussel Crenomytilus grayanus and studied the protein composition of different preparations from this muscle: whole muscle, heat-stable extract, fractions from heat-stable extract, thin filaments and intermediate stages of thin filaments purification. Among the protein components of the above-listed preparations, we did not find caldesmon (CaD), although two isoforms of a calponin-like (CaP-like) protein, which along with CaD is characteristic of vertebrate smooth muscle, were present in thin filaments. Thus, CaD is not Ca(2+)-regulator of thin filaments of this muscle. On the other hand, the mussel CaP-like protein is also not such Ca(2+)-regulator since we have shown that this protein can be selectively removed from isolated mussel thin filaments without loss of their Ca(2+)-sensitivity. We suggest that thin filaments in the smooth catch muscle possess other type of Ca(2+)-regulation, different from that in vertebrate smooth muscles.

Characterization of the Pearl Oyster (Pinctada martensii) Mantle Transcriptome Unravels Biomineralization Genes

Pearl oyster, Pinctada martensii, is a marine bivalve species widely distributed in tropic and subtropic marine coasts. Mantle is the special tissue of P. martensii that secretes biomineralization proteins inducing shell deposition as well as iridescent nacre both in the inner shell and artificial nucleus. The pearl oyster is very efficient for artificial pearl production and is therefore an ideal organism for studies into the processes of biomineralization. However, deficiency of transcriptome information limits the insight into biomineralization mechanisms and pearl formation. In this study, we sequenced and characterized the P. martensii mantle transcriptome using 454 pyrosequencing. A total of 25,723 unique transcripts were assembled from 220,824 quality reads, followed by annotation and Gene Ontology classification analysis. A total of 146 unique transcript segments homologous to 49 reference biomineralization genes were identified, including calcineurin-binding protein, amorphous calcium carbonate binding protein 1, calmodulin, calponin-like protein, carbonic anhydrase 1, glycine-rich shell matrix protein, lysine-rich matrix protein, mantle gene or protein, nacrein, pearlin, PIF, regucalcin, and shematrin. The sequence data enabled the identification of 10,285 potential single nucleotide polymorphism loci and 7,836 putative indels, providing a resource for molecular biomarker, population genetics, and functional genomic studies. A large number of candidate genes for biomineralization were identified, considerably enriching resources for the study of shell formation. These sequence data will notably advance biomineralization and transcriptome study in pearl oyster and other Pinctada species.

Filamin isoforms in molluscan smooth muscle

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS as homologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to their apparent molecular weight determined by SDS-PAGE: 270kDa, 230kDa and 105kDa, respectively. Both FLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domain typical of filamins. These findings, together with the data from peptide mass fingerprints, indicate that FLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoform in the mussel catch muscle. De novo sequencing data revealed structural differences between both filamin isoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its binding partners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As for the FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or a C-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with mussel catch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coelutes with the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction between both proteins.

Differential Expression Analysis of Fat Body Proteome during Pupation in Silkworm (<i>Bombyx mori</i>)

In order to investigate the differential expression of proteins related to pupation in silkworm, the fat body proteins were extracted from larvae of 5th day of 5th instar, un-pupated larvae and pupae of pupating day of multivoltine variety “da zao”. The optimized IPG-IEF/SDS-PAGE (2-DE) was used to analyze the differential expression of proteome in different developmental stage during pupation. The results showed that 66 proteins exhibited a more than two-fold differential expression, in which 43 proteins were down-regulated while 23 proteins were up-regulated. Additionally, 14 proteins with stage-specific expression characteristics were also observed. From 25 proteins with three-fold differential expression, 12 proteins were identified by MALDI-TOF/TOF MS, such as actin, calponin-like protein, NADH, beta-tubulin, receptor for activated protein kinase C, IMP cyclohydrolase, tropomyosin, antichymotrypsin precursor and 30K protein precursor etc. These results suggest that the differentially expressed proteins (enzymes) are related to the regulation of silkworm metamorphosis or sex differentiation, which provides a new experimental basis for better understanding the physiological functions and the mechanism of gene expression in silkworm fat body.

Thin filaments of bivalve smooth muscle may contain a calponin-like protein

A novel 40-kDa calponin-like protein (CaP) was detected in thin filaments from catch muscles of the mussel Crenomytilus grayanus. The content of CaP in thin filaments depends on isolation conditions and varies from complete absence to the presence in amounts comparable with that of tropomyosin. The most significant factor that determines the CaP content in thin filaments is the temperature of solution in which thin filaments are sedimented by ultracentrifugation during isolation. At 22°C and optimal values of pH and ionic strength of the extraction solution, all CaP co-sediments with thin filaments. At 2°C it does not interact with thin filaments and remains in the supernatant. Like vertebrate smooth muscle calponin (33 kDa), the mussel CaP is thermostable, inhibits the Mg2+-ATPase activity of actomyosin, and can be phosphorylated, which is performed by endogenous (co-isolated) kinases in a Ca2+-independent manner. Thus, the C. grayanus CaP is a new member of the family of calponins, the function of which in muscle and nonmuscle cells is still obscure. We suggest that CaP is involved in Ca2+-independent regulation of smooth muscle contraction.

Immunolocalization of the 38.3 kDa calponin-like protein in stratified muscles of the tail of Schistosoma japonicum cercariae

Calponins are proteins present in vertebrate smooth musculature where they occur in association with thin myofilaments. Calponins are not present in vertebrate or invertebrate striated muscles. The blood fluke Schistosoma japonicum expresses a 38.3-kDa protein that bears substantial homology with vertebrate calponin and occurs entirely within smooth musculature of adults. Calponin-like immunoreactivity has been demonstrated in smooth muscles of many invertebrate phyla. The Schistosoma japonicum calponin has been localised in smooth myofibrils of adults where it is associated with myofilaments and sarcoplasmic reticulum. In this study, the ultrastructural localisation of the protein in muscles of S. japonicum cercariae is described. The protein is present in smooth muscles of the forebody and the stratified muscle of the tail. Within the stratified layer, the protein occurs predominantly in transverse arrays of sarcoplasmic reticulum. The localisation data suggest that the calponin-like protein of S. japonicum is involved in contraction of the stratified tail muscle. Furthermore, the presence of a calponin system in the stratified muscle suggests that this muscle is simply a superior form of muscle, closely related to smooth muscles that use a caldesmin-calponin system in contraction.

Isolation and characterization of a novel 45 kDa calponin-like protein from anterior byssus retractor muscle of the mussel Mytilus galloprovincialis

SUMMARY: A calponin-like protein of 45 kDa was isolated from mussel anterior byssus retractor muscle (ABRM) and its inhibitory effects on actomyosin Mg2+-ATPase was demonstrated. The 2-D electrophoresis for ABRM myofibrils gave a spot of 45 kDa protein in addition to myofibrillar proteins such as myosin and actin. The 45 kDa protein, which was more basic and showed a slightly higher molecular weight than actin, was isolated by ion-exchange chromatography and subjected to chymotryptic digestion. N-terminal amino acid sequencing of polypeptide fragments produced gave two sequences, ASQKGMTSFGAVRHH and GMDRALISKMGSKYDSGL, both of which showed a high homology to those of vertebrate calponins and invertebrate calponin-related proteins. Furthermore, the 45 kDa protein strongly reacted with commercially available antibody raised against chicken smooth muscle calponin, demonstrating that the mussel ABRM 45 kDa protein is a new member of the calponin family. Then, actomyosin Mg2+-ATPase activity of ABRM was measured in the presence and absence of the 45 kDa protein. The 45 kDa protein clearly inhibited actomyosin Mg2+-ATPase activity in a dose-dependent manner as in the case of other vertebrate calponins. These results indicate that the 45 kDa calponin-like protein is involved in the thin filament-associated regulation of molluscan smooth muscle contraction, possibly of a unique contraction called catch.

Molecular characterization of a calponin-like protein from Schistosomajaponicum

The gene for a Schistosoma japonicum (Philippine strain origin) (Sjp) calponin-like protein has been cloned and characterised. The clone, designated P14, was isolated from a Sjp adult worm lambda ZAP cDNA library by immunoscreening, and was shown to contain a full-length cDNA encoding a 38.3 kDa protein that shared significant sequence similarity to a number of previously reported calponins and 22 kDa smooth-muscle proteins. Northern analysis indicated the P14 transcript was ≈2.2 kb in both Sjp and Chinese strain S. japonicum (Sjc) adult worms. Southern blot analysis of genomic DNA suggested that several copies of the P14 gene are present in the Sjc and Sjp genomes but only one copy was evident in the S. mansoni (Sm) genome. Western blot analysis indicated that the product of P14 occurs as a 38 kDa protein in adult Sjp worms and homologues are present in adult worms of Sjc and Sm. At least six isoforms, all with a similar molecular size of ≈38 kDa and isoelectric points ranging from 8.1 to 9.5, were present in adult Sjc worms. The protein was immunolocalized to the muscle of male and female Sjc adult worms. Recombinant protein was expressed in E. coli and purified under denaturing conditions, and in yeast to produce a soluble protein in purified form. The availability of purified, correctly folded protein will allow investigations into its biological functions and potential involvement in host immunity.

Expression of an acidic isoform of calponin in rat brain: western blots on one- or two-dimensional gels and immunolocalization in cultured cells.

Calponin, an actin- and Ca(2+)-calmodulin-binding protein characterized as an inhibitory factor of the smooth-muscle actomyosin activity, has also been shown to be present in some non-muscle cells. However, there is a controversy as to whether calponin is present or not in brain. Several laboratories indicate that this protein is absent in chicken or bovine brains, while Applegate et al. [Applegate, Feng, Green and Taubman (1994) J. Biol. Chem. 269, 10683-10690] have recently reported the identification of an mRNA specific for a 36 kDa non-muscle calponin analogue in homogenates of rat brains. For the first time we demonstrate, by Western blots and in situ immunofluorescence localization using monoclonal as well as affinity-purified polyclonal antibody to gizzard calponin, that a 36-37 kDa and a 35-36 kDa calponin-like proteins are expressed respectively in pig and rat brains and in rat cerebellar cultured cells. The acidic pI (5.2-5.4) of the rat brain protein revealed by isoelectric focusing is in good agreement with that of the protein coded for by the calponin isoform mRNA described by Applegate et al. and is different from that of the protein from chicken gizzard (pI 9.9). Brain calponin-like protein is different from two other Ca(2+)-calmodulin-binding proteins previously identified in brain, namely caldesmon and adducin, and from tropomyosin.

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a highly immunogenic calponin-like protein

The identification and characterization of a recombinant cDNA clone, designated OV9M, expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Clone OV9M was identified by screening a λgt11 cDNA expression library derived from adult O. volvulus mRNA using pooled rabbit antisera raised against the third (L3) and fourth (L4) stage larvae of the parasite. The cDNA clone encodes an open reading frame of 238 amino acids corresponding to a 27-kDa polypeptide. This polypeptide contains a series of five highly conserved repeats of 25 amino acids that are similar to repeats found in calponin, a protein previously only identified in vertebrate smooth muscle. Extension of the 5′ end of the cDNA clone revealed two additional repeats extending the sequence to 378 amino acids, encoding a 41.8-kDa protein. Affinity purified antibodies, which bound specifically to the glutathione S-transferase-OV9M fusion polypeptide, recognize a series of antigens in extracts of O. volvulus microfilariae, L3, L4 and adult stages. The apparent molecular weight of the native OV9M protein in the adult is 45 kDa. Similar proteins are present in extracts of other nematodes including Caenorhabditis elegans, and antibodies from other filarial infections are cross reactive with glutathione S-transferase-OV9M fusion polypeptide. Immunoelectron microscopy revealed that the antigen encoded by this clone is present in the longitudinal muscles of the various larval stages and adult worms. Antibodies to the OV9M protein are present in 40–60% of both patently infected and non-patent individuals residing in onchocerciasis endemic areas.