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Molecular Cloning and Tissue Distribution of Troponin I from the Japanese Pearl Oyster, Pinctada fucata

American Journal of Molecular Biology 2019年第2期9卷 29-40页

作者： daisuke funabara Yoshinori Urakawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ and is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of troponin I (TnI) in vertebrates has been extensively characterized, but its role in molluscan muscles has not yet been elucidated. Our previous work suggested that the troponin C subunit has a role in adductor phasic muscle but not in catch muscle. Here, we investigated the molecular characteristics of TnI from the bivalve Japanese pearl oyster, Pinctada fucata to aid the elucidation of the function of molluscan muscle troponin. We determined the primary structure of the full-length TnI protein from the P. fucata adductor muscle (Pifuc-TnI) and found that it is composed of 286 amino acid residues with a predicted molecular weight of 33,737. Motif structure predictions and multiple sequence alignments revealed that Pifuc-TnI has a 138 residue extension at its N-terminus compared with rabbit TnI. This is analogous to characterized TnIs from other mollusks. However, unlike scallop TnI, Pifuc-TnI is predicted to contain two cAMP-dependent protein kinase phosphorylation sites, at residues 39 - 45 (RRGTEDD) and 145 - 151 (KKKSKRK). Phylogenetic analysis indicated that Pifuc-TnI and molluscan TnIs were grouped into the same clade. Pifuc-TnI gene structure predictions using Splign alignment of our obtained cDNA and genome sequences indicated that Pifuc-TnI consists of fifteen exons, with the start and stop codons located in exon 2 and exon 11, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnI gene is predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, and is not expressed in the gill, mantle or foot. These findings suggest that TnI, as a component of the troponin complex, plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction.

关键词： Adductor Muscle Catch Contraction Pinctada fucata Troponin Troponin I

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Phosphorylation Properties of the N-Terminal Region of Twitchin from Molluscan Catch Muscle

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American Journal of Molecular Biology 2019年第3期9卷 110-120页

作者： daisuke funabara Yuuki Nishimura Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin phosphorylation and dephosphorylation. Twitchin from the ABRM of the Mediterranean mussel, Mytilus galloprovincialis, is phosphorylated by cAMP-dependent protein kinase (PKA), and PKA phosphorylation sites are located in both the N- and C-terminal regions of the twitchin molecule. The D2 site, which is adjacently located to the C-terminus, participates in forming a myosin, actin, and twitchin complex that is thought to contribute towards the maintenance of tension in the catch state. In contrast, although it has been reported to interact with thin-filaments, the molecular function of the region including the D1 site has remained largely unstudied. Three additional PKA consensus sequences were identified near the D1 site;however, it was not known if these sites could be directly phosphorylated by PKA. Here, we performed phosphorylation assays to identify phosphorylation sites near the D1 site using recombinant protein variants (TWD1-SSSS, TWD1-AAAS, TWD1-AASA, TWD1-ASAA, TWD1-SAAA, and TWD1-AAAA). All variants, except TWD1-AAAA (where all phosphorylatable serine residues were replaced by alanines), were phosphorylated by PKA. The four phosphorylation sites were named D1-1, D1-2, D1-3, and D1-4 (the originally identified D1) in order from the N-terminus. Phosphorylation assays using a 1/12.5 weight ratio of PKA to each TWD1 variant revealed that D1-4 was the most rapidly phosphorylated, closely followed by D1-1. However, D1-2 and D1-3 were phosphorylated at a lower level under equivalent conditions and were not phosphorylated when PKA was incubated with each TWD1 variant at a 1/100 weight ratio. Furthermore, we observed that TWD1-SSSS was phosphorylated in a stepwise fashion. These findings contribute towards the elucidation of the function of the twitchin D1

关键词： Catch Contraction Phosphorylation Phos-Tag Gel Electrophoresis PKA Twitchin

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Troponin T from the Japanese Pearl Oyster Pinctada fucata: Molecular Cloning, Tissue Distribution, Gene Structure, and Interaction Analysis with Tropomyosin

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American Journal of Molecular Biology 2020年第1期10卷 61-73页

作者： daisuke funabara Yoshinori Urakawa daisuke Ishikawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ and regulate striated muscle contraction in vertebrates. TnT’s function has been extensively described in vertebrates, but its role has been obscure in molluscan muscles. Our previous work indicated that the TnC and TnI subunits work in adductor phasic muscle, but not in catch muscle. Here, we have characterized TnT from the Japanese bivalve pearl oyster Pinctada fucata to start to explain the function of Tn in molluscan muscle contraction. We determined the primary structure of the full-length TnT protein from the P. fucata adductor muscle (Pifuc-TnT), and found that it is composed of 316 amino acid residues with a predicted molecular mass of 37.4 kDa. Multiple sequence alignment showed that Pifuc-TnT has an extension of >60 residues at the C-terminus that are not present in vertebrate TnTs, including known TnTs from other mollusks. Pifuc-TnT gene structure predictions using Splign alignment of the cDNA generated in this study and genome sequences indicated that Pifuc-TnT consists of 13 exons. Start and stop codons are located in exons 2 and 12, respectively. Quantitative real-time PCR revealed that the Pifuc-TnT gene was predominantly expressed in adductor phasic muscle, weakly in adductor catch muscle, slightly in gill, and not at all in mantle and foot. These findings suggest that TnT plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction. Isothermal titration calorimetry revealed that unlike vertebrate TnTs, Pifuc-TnT does not interact with P. fucata tropomyosin-1 nor with tropomyosin-2. These findings in P. fucata imply that Tn functions differently in molluscan muscle than it does in vertebrates.

关键词： Adductor Muscle Catch Contraction Pinctada fucata Troponin T Tropomyosin

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Calponin Isoform Expression in the Japanese Pearl Oyster, Pinctada fucata

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American Journal of Molecular Biology 2019年第4期9卷 154-172页

作者： daisuke funabara Yuuki Osakabe Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

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

关键词： Adductor Muscle Calponin Calponin Isoform Catch Contraction Pearl Oyster

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Ca2+-Induced Conformational Change of Troponin C from the Japanese Pearl Oyster, Pinctada fucata

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American Journal of Molecular Biology 2018年第4期8卷 205-214页

作者： daisuke funabara daisuke Ishikawa Yoshinori Urakawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca2+ binding to troponin C, one of the subunits of troponin, allowing myosin to interact with actin. We recently elucidated the molecular characteristics of the Japanese pearl oyster Pinctada fucata troponin C (Pifuc-TnC), revealing the possibilities that Pifuc-TnC and vertebrate muscle TnC play dissimilar roles in muscle contraction. Pifuc-TnC has four EF-hand motifs, but, unlike vertebrate TnC, only one (site IV) was predicted to bind Ca2+. To confirm the number of Ca2+-binding sites in Pifuc-TnC and whether Ca2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calorimetry demonstrated Ca2+ binding to Pifuc-TnC, whereas Pifuc-TnC-E142Q was unable to bind Ca2+, confirming that site IV is the only Ca2+-binding site in Pifuc-TnC. Pifuc-TnC eluted in a later fraction from a gel filtration column in the presence of Ca2+ compared with the condition when Ca2+ was absent. In contrast, the elution profiles of Pifuc-TnC-E142Q were equivalent in both the presence and absence of Ca2+, suggesting that Ca2+ binding to Pifuc-TnC induces a conformational change that delays its elution from the column. UV-absorption spectral analysis revealed that binding of Ca2+ to Pifuc-TnC caused an increase in absorption at a wavelength of approximately 250 nm, possibly because phenylalanine residues had been exposed on the surface of the molecule as a result of a conformational change. Differential scanning calorimetric analyses of Pifuc-TnC showed aggregation in the presence of Ca2+ in accordance with an increase of temperature, but no aggregation was seen in the absence of Ca2+. In combination, these findings suggest that Ca2+ binding to site IV induces a conformational

关键词： Ca2+-Binding Catch Muscle Conformational Change EF-Hand Troponin C

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Molecular Cloning and Tissue Distribution of Troponin C from the Japanese Pearl Oyster, Pinctada fucata

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American Journal of Molecular Biology 2018年第3期8卷 166-177页

作者： daisuke funabara Yoshinori Urakawa Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

Troponin C (TnC) is one of the subunits of troponin. Troponin, which is activated by Ca2+ binding, is a thin filament-associated regulator of vertebrate striated muscle contraction. The function of TnC in vertebrates has been characterized in detail, but the role of TnC in molluscan muscles is still unclear. In this work, we investigated whether TnC plays a role in the catch contraction of molluscan smooth muscle in the bivalve Japanese pearl oyster Pinctada fucata. We determined the full-length primary structure of the TnC protein from the P. fucata adductor muscle (Pifuc-TnC), and found it is composed of 150 amino acid residues with a predicted molecular weight of 17,400. Multiple sequence alignments indicated that it had four EF-hand motifs, but only one (site IV) was predicted to have Ca2+-binding ability. This is analogous to characterized TnCs from other mollusks. Three-dimensional modeling of Pifuc-TnC using SWISS-MODEL indicated the presence of a short loop within the α-helix connecting the site II and III EF-hand motifs. We predicted the gene structure of Pifuc-TnC using Splign alignment of our obtained cDNA and genome sequences and elucidated that Pifuc-TnC consists of five exons, with the start and stop codons located in exon 1 and exon 5, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TnC gene is predominantly expressed in adductor phasic muscle and rarely in adductor catch muscle, gill, mantle and foot. These findings suggest that TnC may not have a role in catch muscle contraction.

关键词： Adductor Muscle Catch Contraction EF-Hand Troponin Troponin C

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Tropomyosin Isoform Expression in the Adductor Muscle of the Japanese Pearl Oyster, Pinctada fucata

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American Journal of Molecular Biology 2019年第1期9卷 16-27页

作者： daisuke funabara Ayaka Ohta Jungo Sueyoshi Satoshi Kanoh Graduate School of Bioresources Mie University Tsu Japan

We determined the full-length primary structure of the tropomyosin (TM)-1 and -2 proteins from the adductor muscle of the Japanese pearl oyster Pinctada fucata (Pifuc-TM-1 and Pifuc-TM-2), and found that they are each composed of 284 amino acid residues. We predicted the gene structure of P. fucata TM (Pifuc-TM) using Splign alignment of our cDNA with genomic sequences and elucidated that Pifuc-TM consists of 10 exons. Exons 1 - 3 and 5 - 10 are used to transcribe Pifuc-TM-1 mRNA, and exons 1 - 4 and 6 - 10 are used to transcribe Pifuc-TM-2 mRNA. Both genes share the same start and stop codons located in exon 1 and exon 10, respectively. Using quantitative real-time PCR, we determined that the Pifuc-TM-1 gene was mainly expressed in adductor phasic muscle, and at a relatively weaker level in adductor catch muscle, whereas the Pifuc-TM-2 gene was expressed equally in both phasic and catch muscles. They were weakly expressed in gill and mantle. Immunoblot analysis using anti-Pifuc-TM-1 and anti-Pifuc-TM-2 antibodies revealed that adductor phasic muscle contained Pifuc-TM-1, while adductor catch muscle contained both Pifuc-TM-1 and Pifuc-TM-2. Differential scanning calorimetry (DSC) analysis was carried out for Pifuc-TM-1 and Pifuc-TM-2 expressed in bacteria, as well as TM purified from P. fucata phasic and catch muscle tissues (phasic-TM and catch-TM). The DSC data indicated that phasic-TM was mainly composed of Pifuc-TM-1, whereas catch-TM contained Pifuc-TM-1 and Pifuc-TM-2. These findings suggest that the distribution of Pifuc-TM-1 and Pifuc-TM-2 in adductor muscle is specific to the muscle fiber type, and reflects the properties of each.

关键词： Adductor Muscle Alternative RNA Processing Catch Contraction Pearl Oyster Tropomyosin

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