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Calponin Isoform Expression in the Japanese Pearl Oyster, Pinctada fucata

Calponin is a basic actin-binding protein found widely in invertebrate tissues including catch muscle and therefore may participate in catch contraction. There is limited information about molluscan calponin and molecular characterization to reveal its function in the regulatory system. We previously identified and partially sequenced three calponin isoforms of the Japanese pearl oyster, Pinctada fucata (Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3). In this study, the full-length nucleotide sequences of the three isoforms were determined. The primary structures revealed that Pifuc-CP-1 consists of 324 amino acids (aa) with a molecular mass (Mw) of 34.7 kDa and an isoelectric point (pI) of 9.40. Pifuc-CP-2 is 303 aa in length with a Mw of 33.3 kDa and a pI of 9.30, and Pifuc-CP-3 is 398 aa in length with a Mw of 43.8 kDa and a pI of 8.55. Domain architecture prediction showed that the three isoforms have a single calponin homology (CH) domain and multiple calponin (CN) domains. Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3 possess four, three and five CN domains, respectively. Tissue distribution analysis indicated the presence of additional calponin isoforms and these isoforms are distributed widely in muscle and non-muscle tissues. Results of cDNA cloning revealed further four calponin isoforms: Pifuc-CP-4 (402 aa, 42.8 kDa, pI = 9.10), Pifuc-CP-5 (285 aa, 30.7 kDa, pI = 9.45), Pifuc-CP-6 (286 aa, 31.1 kDa, pI = 9.60) and Pifuc-CP-7 (302 aa, 33.3 kDa, pI = 9.10). The domain architecture of these four isoforms also consists of a single CH domain and multiple CN domains. Pifuc-CP-4 possesses six CN domains, whereas Pifuc-CP-5, Pifuc-CP-6 and Pifuc-CP-7 contain three CN domains. Sequence alignment of P. fucata calponin isoforms showed that Pifuc-CP-1, Pifuc-CP-2, Pifuc-CP-3 and Pifuc-CP-4 have identical CH domain sequences, whereas Pifuc-CP-5, Pifuc-CP-6 and Pifuc-CP-7 have identical CH domain sequences. The CN repeats were not well conserved. These findings suggest that P. fucata calponin isoforms function differently in each tissue. How to cite this paper: Funabara, D., Osakabe, Y. and Kanoh, S (2019) Calponin Isoform Expression in the Japanese Pearl Oyster, Pinctada fucata. American Journal of Molecular Biology, 9, 154-172. https://doi.org/10.4236/ajmb.2019.94012 Received: July 18, 2019 Accepted: August 26, 2019 Published: August 29, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Mollusk bivalve adductor muscles are composed of two muscle types: phasic and catch. Phasic muscle is used for the quick closure of shells, whereas catch muscle functions in the sustain closure of shells. The contraction of both muscles is regulated by intracellular Ca 2+ concentrations [1]. Mollusks employ a thick filament-linked regulatory system where myosin directly binds Ca 2+ , leading to its activation and subsequent interaction with actin. Following a decrease in the intracellular Ca 2+ concentration, myosin is inactivated, and its interaction with actin in phasic muscle is abolished. In contrast, once Ca 2+ concentrations decrease to resting levels, catch muscles enter the high-tension catch state, which is maintained for long periods. Twitchin, a giant myosin-associated protein, tethers together the thin and thick filaments through its phosphorylation sites [2] [3] [4]. The involvement of the thin filament-linked regulatory system in catch contraction remains unresolved.
In contrast to molluscan muscles, vertebrate striated muscles employ a thin filament-linked regulatory system. Troponin (Tn) is the regulator of skeletal muscle contraction. Tn is distributed on thin filaments and inhibits the interaction between actin and myosin. Tn consists of three subunits: troponin C (TnC), troponin I (TnI) and troponin T (TnT). Since Tn is present in mollusk muscles, we have been investigating if there is a thin filament-linked regulatory system of catch contraction.
The Japanese pearl oyster, Pinctada fucata, is one of the most important molluscan species in the pearl culture industry. A genome database of P. fucata has recently been released and all the major muscle protein genes have been registered [5] [6] [7]. Therefore, we have used P. fucata as a model system to elucidate the molluscan muscle regulatory system. We recently performed molecular characterization of TnC and TnI from P. fucata, and suggested that Tn may participate in the regulation of the phasic adductor muscle not in catch muscle, because they are predominantly expressed in the phasic muscle [8] [9] [10].
We previously revealed that three calponin isoforms are expressed in P. fucata  Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3) by partial sequencing [7]. In this study, the molecular characterization of P. fucata calponin isoforms was performed by conducting 5' rapid amplification of cDNA ends (RACE) to determine the full-length sequences of the three isoforms. In addition, the structural and tissue distribution analysis was performed. Furthermore, we found four more isoforms (Pifuc-CP-4, Pifuc-CP-5, Pifuc-CP-6 and Pifuc-CP-7) using cDNA cloning.

Animal Samples
We obtained live specimens of two-year-old P. fucata that were cultured in Ago Bay, Mie Prefecture, Japan. The adductor muscle, gill, mantle and foot were dissected from each oyster body, immediately frozen in liquid nitrogen and stored at −80˚C until use.

cDNA Cloning of Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3
Total RNA was extracted from the phasic part of the adductor muscle using a conventional method [22]. Partial nucleotide sequences of Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3, as determined by 3' RACE, were reported previously [7]. To determine the full-length sequence of each, 5' RACE was carried out using the 5' RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Invitrogen, Carlsbad, CA, USA) using total RNA as a template. Primers were designed using the known sequences of Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3.

Protein Expression Analysis of Calponin in Tissues of P. fucata
Protein expression patterns of calponin in tissues of P. fucata were analyzed by immunoblotting using the anti-Yesso scallop calponin antiserum prepared in our previous study [23]. Catch and phasic muscles, gill, mantle and foot were homogenized in phosphate-buffered saline and subjected to 10% SDS-PAGE, followed by electro-blotting onto a polyvinylidene difluoride membrane. After blocking, the membrane was hybridized with an anti-Yesso scallop calponin antiserum. Horseradish peroxidase-conjugated goat anti-rabbit IgG was used as the secondary antibody. Detection was carried out with 0.2 mg/ml 3,3'-diaminobenzidine and 0.005% hydrogen peroxide in Tris-buffered saline.

cDNA Cloning of P. fucata Calponin Isoforms
Protein expression analysis revealed the possibility that other isoforms were expressed in P. fucata tissues. Thus, we carried out reverse transcriptase (RT)-PCR to obtain cDNA clones encoding calponin isoforms. cDNA was synthesized from total RNA of catch and phasic muscles with the 3'-Full RACE Core Set

Gene and Protein Expression Analyses of Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3
Gene expression analysis showed that the Pifuc-CP-1, Pifuc-CP-2 and Pifuc-CP-3 genes were expressed predominantly in adductor phasic muscle, whereas relatively weaker expression was detected in catch muscle (Figure 4). Gene expression of the three genes was barely detectable in gill, mantle and foot. Immunoblotting analysis of the protein expression profiles in P. fucata tissues detected multiple proteins
We tried tissue distribution analysis for the Pifuc-CP-4, Pifuc-CP-5, Pifuc-CP-6 and Pifuc-CP-7 genes, but there was no region specific to respective genes by nucleotide sequences. As the position of the primers and TaqMan probe for Pifuc-CP-1 was shared by Pifuc-CP-4 and Pifuc-CP-5, the gene expression of Pifuc-CP-1 shown in Figure 4 includes that of Pifuc-CP-4 and Pifuc-CP-5. In the same way, the gene expression of Pifuc-CP-2 includes that of Pifuc-CP-7, and the gene expression of Pifuc-CP-3 includes that of Pifuc-CP-6.

Phylogenetic Analysis of Calponin
Phylogenetic tree analysis showed that Pifuc-CP isoforms are grouped into the same clade ( Figure 11). Calponin from the Mediterranean mussel Mytilus    galloprovincialis, which is found in catch muscle, were separated into the same clade, implying that bivalve calponin works in the same fashion in muscle contraction [17].

Discussion
In this study, we found that seven calponin isoforms (Pifuc-CP  calponin inhibits actomyosin Mg-ATPase activity in vitro and interacts with F-actin [17] [18] [29]. Therefore, Pinctada calponin may interact with F-actin in the same fashion and its affinity for F-actin may depend on the number of CN domains. Protein expression analysis revealed that P. fucata calponin isoforms are expressed in muscle tissues and in non-muscle tissues, gill, mantle and foot ( Figure 5).  [3]. Mammalian smooth muscles exhibit latch contraction similar to catch contraction [11]. The molecular mechanism of the tension maintenance of the latch contraction remains unresolved but it has been suggested that calponin participates in the tethering of thick-and thin-filaments, like molluscan twitchin [13]. In the resting stage of mammalian smooth muscle, calponin interacts longitudinally with two actin monomers that involve its low and high affinity binding sites. Upon increasing Ca 2+ concentration within the sti- whereas the central fragment of calponin (residues 145-163) remains bound to F-actin. In this scenario, calponin acts to tether thick-and thin-filaments and slows down the detachment rate of activated cross-bridges. This reaction introduces an internal load that triggers maximal contraction [13]. This model reminds us that thick-and thin-filaments are tethered by calponin besides twitchin in molluscan catch muscle. A question for the twitchin model described above is that the amount of twitchin (molar ratio to myosin = 1:15) [34] seems to be too small to tether thick-and thin-filaments to maintain the tension in the catch state. To answer this question, the calponin model might be used to catch contraction together with the twitchin model. Further studies on proteins that interact with molluscan calponin are required to elucidate the calponin function in catch contraction.