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

Abstract

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.

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

“…Pifuc-TnC and Pifuc-TnI are predominantly expressed in phasic muscle, suggesting that Pifuc-Tn proteins in phasic muscle operate within a regulatory system [14] [15]. Therefore, we speculated that Pifuc-TnT interacts with Pifuc-TM1 and not Pifuc-TM2.…”

“…To examine the molecular mechanism of thin filament-linked regulation of molluscan muscle contraction, our studies have centered on the adductor muscle Tn subunits. To this end, P. fucata TnC (Pifuc-TnC) and TnI (Pifuc-TnI) have recently been described [13] [14] [15]. Pifuc-TnC has four EF-hand motifs, sites I-IV, and, like other molluscan TnCs [16] [17] [18], only site IV is able to bind Ca 2+ [12].…”

“…Molluscan TnIs have an extension sequence at their N-terminus that differentiates them from mammalian TnIs [15] [19] [20]. Like other molluscan TnI proteins, Pifuc-TnI has a 138-residue extension at its N-terminus that is not present in rabbit TnI [19] [20].…”

“…Software for predicting motif structures suggests that Pifuc-TnI comprises two coils (4 -25 and 60 -152 residues) and a troponin motif (161 -286 residues). Pifuc-TnI also includes consensus sequences for cAMP-dependent protein kinase in residues 39 -45 (RRGTEDD) and 145 -151 (KKKSKRK), which are D. Funabara et al unique from scallop TnIs [15]. A number of different protein kinases in vertebrate cardiac muscle have been shown to phosphorylate TnI [21], increasing the probability that Pifuc-Tn controls the phasic adductor muscle contraction in a similar fashion.…”

Calponin Isoform Expression in the Japanese Pearl Oyster, <i>Pinctada fucata</i>

“…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].…”