Troponin C (TnC) is one of the subunits of troponin. Troponin, which is activated by Ca 2+ 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 Ca 2+ -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.

Troponin is a thin filament-associated regulator of vertebrate striated muscle contraction. Troponin changes its structure upon Ca 2+ 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 Ca 2+. To confirm the number of Ca 2+-binding sites in Pifuc-TnC and whether Ca 2+ binding induces a conformational change, we purified the full-length protein and a variant, Pifuc-TnC-E142Q (that has a mutation in the predicted Ca 2+-binding site of site IV), following their expression in laboratory E. coli. Isothermal titration calo

Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca 2+ 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.

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Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca 2+ 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.