Cloning and nucleotide sequence of a novel 28-kDa protein from the mantle muscle of the squid Todarodes pacificus with homology to tropomyosin

In recent studies, we found autodegradation of collagen from the mantle muscle of the squid Todarodes pacificus and also that the 28- and 25-kDa proteins are closely related to this phenomenon [Connect. Tissue Res. 45 (2004) 109-121]. We obtained partial sequences of three internal portions of this protein, which suggested that 25-kDa protein is a partially degraded form of the 28-kDa protein. We determined the full cDNA sequence of this protein by the degenerate polymerase chain reaction (PCR) using the information of amino acid sequences. The deduced amino acid sequence corresponding to the 212-bp cDNA contained all of the amino acid identified from the 28-kDa protein. Rapid amplification of cDNA ends (RACE) and squid mantle muscle RNA allowed cloning of the full 522-bp sequence, corresponding to a protein of 174 amino acids. A database search indicated that this is a new protein that shares 27-34% identity with tropomyosins from various animals. Structural prediction suggested that it possesses heptad repeats that form coiled-coil structures. We expressed a recombinant protein encoded by the 212-bp cDNA in Escherichia coli and used it to generate a polyclonal antibody. Western blotting with this antibody showed that the 28-kDa protein is expressed in fin, tentacle, and mantle muscle, but not in liver.