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

Abstract

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.