CDNA cloning and primary structure analysis of transglutaminase from bluefin tuna Thunnus orientalis

Abstract

A cDNA clone encoding a tissue-type transglutaminase (TGase) was isolated from the fast muscle of bluefin tuna Thunnus orientalis using a method that combines reverse transcription-PCR and rapid amplification of cDNA ends. The full-length cDNA was 2,872 bp with an open reading frame coding for a protein of 678 amino acid residues. The deduced amino acid sequence had a calculated molecular mass of 74,193 Da and a theoretical isoelectric point of 7.39. The bluefin tuna TGase showed a high amino acid identity of 75% to that of Atlantic salmon Salmo salar, and a low identity of 38% to that of red seabream Pagrus major. Three residues, namely, Cys269, His326, and Asp349, were conserved in a catalytic site, as has also been observed in TGases from other fish and mammals. In phylogenetic analysis, the bluefin tuna TGase cloned in this study was classified into tissue-type TGase2. Southern blot analysis of bluefin tuna genomic DNA digested with restriction enzymes PstI and KpnI using a digoxigenin-labeled probe revealed two bands of different sizes, suggesting the presence of multiple TGase genes in the bluefin tuna.