Kazuharu Takeuchi

A hyperosmotic stress-induced mRNA of carp cell encodes Na+- and Cl−-dependent high affinity taurine transporter

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.

Expression and functional analysis of mussel taurine transporter, as a key molecule in cellular osmoconforming

SUMMARY Most aquatic invertebrates adapt to environmental osmotic changes primarily by the cellular osmoconforming process, in which osmolytes accumulated in their cells play an essential role. Taurine is one of the most widely utilized osmolytes and the most abundant in many molluscs. Here, we report the structure, function and expression of the taurine transporter in the Mediterranean blue mussel (muTAUT), as a key molecule in the cellular osmoconforming process. Deduced amino acid sequence identity among muTAUT and vertebrate taurine transporters is lower (47-51%) than that among vertebrate taurine transporters (>78%). muTAUT has a lower affinity and specificity for taurine and a requirement for higher NaCl concentration than vertebrate taurine transporters. This seems to reflect the internal environment of the mussel; higher NaCl and taurine concentrations. In addition to the hyperosmotic induction that has been reported for cloned taurine transporters, the increase in muTAUT mRNA was unexpectedly observed under hypoosmolality, which was depressed by the addition of taurine to ambient seawater. In view of the decrease in taurine content in mussel tissue under conditions of hypoosmolality reported previously, our results lead to the conclusion that muTAUT does not respond directly to hypoosmolality, but to the consequent decrease in taurine content. By immunohistochemistry, intensive expression of muTAUT was observed in the gill and epithelium of the mantle, which were directly exposed to intensive osmotic changes of ambient seawater.

The existence of aspolin and its trimethylamine‐N‐oxide demethylating activity in the muscle of freshwater fish

Aspolin is a polyaspartic acid-like protein, which is originally isolated from walleye pollack Theragra chalcogramma muscle as trimethylamine-N-oxide (TMAO) demethylase. Although carp Cyprinus carpio muscle contains a trace amount of the enzyme substrate, TMAO, aspolin can be extracted and purified by acid treatment, successive chromatographies and polyacrylamide gel electrophoresis, and has twice the amount of that in walleye pollack muscle. Carp aspolin showed a low enzymatic activity in the presence of Fe2+ and reductants, and its Km value (100 mM) to TMAO was extremely high. It was a thermostable protein and had an unfolded conformation. The amino acid sequence of carp aspolin 1 deduced from cDNA revealed that it contained a long Asp polymer, an uninterrupted stretch of 138 Asp residues, followed by four amino acid residues, His-Glu-Glu-Leu, in C-terminus. The chain length was shorter by 42 Asp residues than that of its walleye pollack counterpart.

Taurine transporter: hyperosmotic stress-responsive gene

To study osmotic stress response of fish cells, we screened osmotic stress-inducible mRNA in a common carp cell line (EPC) by RNA arbitrarily primed polymerase chain reaction and isolated a cDNA encoding a Na+- and Cl+- dependent highhigh affinity taurine transporter as a hyperosmotic stress-inducible gene. Northern blot analysis revealed the marked increase in taurine transporter mRNA by hyperosmotic stress. To study changes in taurine transporter gene expression in an individual fish, we measured the mRNA level in Mozambique tilapia (Oreochromis mossambicus) during high-salinity adaptation. By the transfer of tilapia cultured in freshwater to 70% artificial seawater, plasma osmolality increased by as much as 100–135 mOsm/kg H2O along with the marked increase in taurine transporter mRNA level in all the tissues examined. Since an upregulation of taurine transporter expression leads to the activation of accumulation of taurine in the cells, these results suggest the significant role of taurine as an osmolyte in fish.