Abstract
Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that binds Ca2+ 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.
Highlights
We investigated whether Troponin C (TnC) plays a role in the catch contraction of molluscan smooth muscle in the bivalve Japanese pearl oyster Pinctada fucata
The binding of Ca2+ to TnC induces a conformational change in the troponin complex structure and enables myosin to interact with actin [1]-[6]
Multiple sequence alignments of Pifuc-TnC with TnCs from other organisms revealed that Pifuc-TnC has four potential EF-hand motifs, termed sites I, II, III and IV
Summary
It is distributed on thin filaments and inhibits the interaction between actin and myosin. All four vertebrate fast skeletal TnC EF-hand motifs are capable of binding Ca2+, only TnC sites II and IV are able to bind Ca2+ in arthropod and nematode striated muscles [7] [8]. In mollusks such as scallop and squid, only site IV is able to bind to Ca2+ [9] [10]. The ability of an EF-hand motif to bind Ca2+ is dependent upon its primary structure [11]
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