Identification and expression of a novel carbonic anhydrase isozyme in the pufferfish Takifugu vermicularis

• Carbonic anhydrase (CA) is required for maintaining the acid-base balance in fish.
• Homologous sequence PCR identified a pufferfish Takifugu vermicularis pCAn isozyme.
• Phylogenetic analysis revealed that pCAn was a membrane bound CA protein.
• Similar motif distribution exist between pCAn and the predicted CA IV-like isozyme.
• pCAn isozyme mRNA is highly in the gills as are other CA IV-like isozymes.

Carbonic anhydrase (CA) is a key element for maintaining acid base balance in fish. In our present experiment, novel CA isozymes were identified from the pear puffer (Takifugu vermicularis). Based on the high homology of two predicted CA sequences of the tiger puffer (Takifugu rubripes), a 1715 bp novel cDNA was obtained from T. vermicularis. The open reading frame showed a complete coding sequence of 552 bp with a deduced peptide sequence of 183 amino acids that exhibited highest (97%) identity with pufferfish putative CA III and CA IV-like sequences. In addition, this translated protein sequence showed 36–37% identity with zebrafish CA IV-like, CA XVa, CA XVb, and CA XVc proteins. Phylogenetic analysis revealed that the pufferfish novel protein (pCAn) was a membrane-bound CA protein. Alignment of multiple CA sequences illustrated that most of the putative active site residues of the pCAn isozyme were situated at highly conserved regions of the CA sequences. Examination of motif distribution suggested that the pCAn isozyme was very similar to the puffer predicted CA IV-like isozyme. Reverse transcription-polymerase chain reaction (PCR) analysis showed highly differential expression in the brain, gills, kidney, and muscle, whereas CA mRNA expression was almost absent in heart, liver, and intestine. Quantitative PCR expression of CA mRNA abundance suggested several-fold higher expression of pCAn isozymes in the gills compared to other tissues tested. Our results suggest that the pCAn isozyme might be related to CA IV-like isozymes. Further functional studies are needed to investigate the function of the pCAn isozyme in T. vermicularis.