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

Troponin (Tn) is the sarcomeric Ca²⁺-dependent regulator for striated muscle contraction in vertebrates. It is distributed on thin filaments and inhibits the interaction between actin and myosin. Troponin consists of three subunits: troponin C (TnC), troponin I (TnI), and troponin T (TnT). 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, the authors investigated whether TnC played a role in the catch contraction of molluscan smooth muscle in the bivalve Japanese pearl oyster Pinctada fucata. They obtained live specimens of the Japanese pearl oyster, Pinctada 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. Total RNA was extracted from the phasic part of the adductor muscle using a conventional method. The primary structure of Pifuc-TnC was deduced from the nucleotide sequence determined by cDNA cloning and compared with those from mollusks using ClustalW. The 3D structure of Pifuc-TnC was predicted with SWISS-MODEL. The genome sequence including the Pifuc-TnC gene was obtained by BLAST searching the Pifuc-TnC nucleotide sequence against the P. fucata genome database. Gene expression patterns of Pifuc-TnC in the catch and phasic muscles, gill, mantle and foot were analyzed by quantitative real-time PCR.

The full-length primary structure of the TnC protein from the Pifuc-TnC was determined and found it was 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. And the Pifuc-TnC gene was predominantly expressed in adductor phasic muscle and rarely in adductor catch muscle, gill, mantle and foot.

In conclusion, these findings suggest that TnC may not have a role in catch muscle contraction. Further molecular characterization studies on Pifuc-TnI and Pifuc-TnT are required to clarify the function of troponin in Pinctada fucata.

Article by Daisuke Funabara, et al, from Mie University, Tsu, Japan.

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