Abstract
Troponin (Tn) is composed of three subunits (TnI, TnC and TnT) that bind Ca2+ 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.
Highlights
Bivalve adductor muscles are composed of phasic and catch muscles
These findings suggest that troponin T (TnT) plays a regulatory role in adductor phasic muscle contraction, but not in catch contraction
We have added the molecular characterization of Pifuc-TnT to our previous studies of Pifuc-troponin C (TnC) and Pifuc-troponin I (TnI) [14] [15], completing the subunit characterization of Pifuc-Tn
Summary
Bivalve adductor muscles are composed of phasic and catch muscles. The larger phasic muscle is used to close shells quickly and the smaller catch muscle is used to keep shells closed. Catch muscles can maintain their tension for long periods with little energy consumption after developing the contraction [1]. They start to contract following an increase in intracellular Ca2+ concentrations, which activates myosin and creates tension, and they subsequently enter the catch state once Ca2+ concentrations decrease to resting levels. A thin filament-linked regulatory system is used by vertebrate striated muscles. Troponin (Tn) is a regulator of skeletal muscle contraction It is arranged over thin filaments and prevents actin-myosin interaction. Ca2+ binding to TnC leads to a conformational change in the complex structure of Tn that allows myosin to interact with actin [5] [6] [7] [8]. While Tn is present in molluscan muscles, whether it is involved in a thin filament-linked regulatory system similar to that in vertebrates is currently unclear
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