Differential regulation of taurine biosynthesis in rainbow trout and Japanese flounder

Abstract

Animals have varied taurine biosynthesis capability, which was determined by activities of key enzymes including cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSD). However, whether CDO and CSD are differentially regulated across species remains unexplored. In the present study, we examined the regulations of CDO and CSD in rainbow trout and Japanese flounder, the two fish species with high and low taurine biosynthesis ability respectively. Our results showed that the expression of CDO was lower in rainbow trout but more responsive to cysteine stimulation compared to that in Japanese flounder. On the other hand, both the expression and catalytic efficiency (k(cat)) of CSD were higher in rainbow trout than those of Japanese flounder. A three-residue substrate recognition motif in rainbow trout CSD with sequence of F126/S146/Y148 was identified to be responsible for high k(cat), while that with sequence of F88/N108/F110 in Japanese flounder led to low k(cat), as suggested by site-directed mutagenesis studies. In summary, our results determined new aspects of taurine biosynthesis regulation across species.

Figure 1. Multiple sequence alignment of CDOs.

Highly conserved residues are indicated with black backgrounds and similar residues are shaded. The cupin superfamily motifs are indicated in the top line. Dashes indicate gaps. Asterisks show highly conserved cysteine and tyrosine residues formation of cross-linked cofactor of CDO. From top to bottom, the sequences are from Paralichthys olivaceus (KP739882), Oncorhynchus mykiss (KP739883), Danio rerio (Q6NWZ9), Mus musculus (NP_149026), Rattus norvegicus (AAH70509), Homo sapiens (AAH24241), and Xenopus laevis (NP_001083506)

Figure 2. Multiple sequence alignment of CSDs.

Highly conserved residues are indicated with black backgrounds and similar residues are shaded. Dashes indicate gaps. The conserved DOPA decarboxylase domain are indicated in the top line. The substrate recognition motif are indicated in the bottom line. Asterisks show the conserved residues in PLP binding site. From top to bottom, the sequences are from Paralichthys olivaceus (KP739884), Takifugu rubripes (ABF22453), Danio rerio (NP_001007349), Cyprinus carpio (BAE73113), Oncorhynchus mykiss (KP739885), Mus musculus (NP_659191), Rattus norvegicus (NP_068518), and Homo sapiens (NP_057073)

Figure 3. Sequence alignment of the substrate recognition motif in CSD across species.

The three key residues in motif are bold. From top to bottom, the sequences are from insect, echinoderm, fish, amphibian, sauropsida, chicken and mammals

Figure 4. Hepatic expression of CDO and CSD in rainbow trout and Japanese flounder.

Copy numbers of CDO and CSD in livers of rainbow trout (RT) and Japanese flounder (JF) were measured by a method of absolute quantification of cDNAs. The results are expressed as copies per μg oligo-dT primed cDNA and the data are presents as means ± S.E.M.(n = 6) (a–b). Protein abundance of CDO and CSD in fish livers was analysed using western blot. Aliquots of 20 μg protein was loaded in each line and β-tubulin was used as a loading control (c). The relative protein abundance of CDO and CSD in rainbow trout (RT) livers were normalized to the total protein loading and the expression levels in Japanese flounder (JF) were expressed as relative expression values to those in RT group. The data are expressed as means ± S.E.M.(n = 4) (d,e). The differences between experimental groups are tested using independent t tests. **p < 0.01, ***p < 0.001

Figure 5. Enzyme activities of CDO and CSD in fish livers.

The enzyme activities of CDO (a) are expressed as nmol CSA per min per mg protein and the enzyme activities of CSD (b) are expressed as nmol hypotaurine per min per mg protein. The data are expressed as means ± S.E.M.(n = 6). The differences between experimental groups of rainbow trout (RT) and Japanese flounder (JF) are tested using independent t tests. **p < 0.01; ***p < 0.001

Figure 6. CDO expression in response to cysteine stimulation.

Rainbow trout (RT) and Japanese flounder (JF) CDO were transfected into HepG2 cells and the expression in response to cysteine levels (0, 0.05, 0.1, 0.3, 0.6, 1 mM) was analyzed with western bolt. Aliquots of 20 μg protein was loaded in each line and β-tubulin was used as the loading control. The overexpressed CDO was detected with antibody against FLAG (a). CDO with Cys-Tyr cofactor (mature form, the lower band) and the cofactor-free form (immature form, the upper band) were quantified using NIH Image 1.63 software respectively. Relative protein abundance was expressed as relative expression value to the cysteine-free group (b–c). The data are expressed as means ± S.E.M. (n = 3). At least triplicates were conducted for each data point. The different expression of total CDO in response to cysteine were tested using one-way ANOVA and Tukey’s multiple-range test. Different letters above the bars denote significant differences between groups at the p < 0.05 level

Figure 7. Kinetic characterization of recombinant CDO and CSD proteins.

Recombinant rainbow trout (RT) and japanese flounder (JF) CDO were separated by 4-20% TruPAGE^TM Precast Gels (a). The wild-type (wt) and site-mutants (sm) of CSD proteins were separated by 10% SDS-PAGE gel (b). Activity of recombinant rainbow trout (RT) CDO and Japanses flounder (JF) CDO (0.2uM) was investigated using a wide substrate range (0-20 mM), which revealed a two phase kinetics (c). The kinetic parameters of fish CDOs were analyzed according to Michaelis-Menten results of CDO activities at cysteine concentrations below 4 mM (d). Activity of recombinant fish CSDs (0.5uM) at a CSA concentration of 0–15 mM were measured and it was with well-behaved Michaelis-Menten kinetics. Kinetic parameters of the wild type (WT) and the S146N/Y148F mutant of CSD from rainbow trout were analyzed according to Michaelis-Menten results (e). The kinetic parameters of the wild type (WT) and the N108S/F110Y mutant of CSD from Japanese flounder were analyzed according to Michaelis-Menten results (f). The kinetic characterization of recombinant proteins were estimated using nonlinear fitting by Prism 5 software (Graphpad software). The data are expressed as means ± S.E.M. (n = 3)