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

摘要

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 fo und it is composed of 150 amino acid residues with a predicted molecular weight of 17,400. Multiple sequence alignments indicated that it ha d 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.