Complementary DNA sequences of the constant regions of T-cell antigen receptors [alpha], [beta] and [gamma] in mandarin fish, Siniperca chuatsi Basilewsky, and their transcriptional changes after stimulation with Flavobacterium columnare

In this study, the constant-region genes (C[alpha], C[beta] and C[gamma]) that encode the T-cell antigen receptor (TCR) [alpha], [beta] and [gamma] chains were cloned from mandarin fish, Siniperca chuatsi Basilewsky, an important freshwater fish species in China. The complementary DNA sequences of C[alpha], C[beta] and C[gamma] were 843, 716 and 906 base pairs (bp) in length and had a 465-, 289- and 360-bp 3' untranslated region, encoding 125, 142 and 182 amino acids, respectively. The amino-acid sequences of the constant regions of mandarin fish TCR [alpha], [beta] and [gamma] chains (encoded by C[alpha], C[beta] and C[gamma], respectively) were most similar to those of their teleost counterparts, showing 60% similarity with pufferfish, 48% similarity with Atlantic salmon and 57% similarity with flounder, respectively. The phylogenetic analysis revealed that the mandarin fish C[alpha], C[beta] and C[gamma] were clustered, respectively, with their vertebrate counterparts. The mandarin fish C[alpha], C[beta] and C[gamma] could also be separated into four domains: immunoglobulin; connecting peptide (CP); transmembrane (TM); and cytoplasmic tail. Several conserved features in mammalian TCRs were also found in those of mandarin fish, such as a conserved cysteine residue in the CP domain of C[alpha], necessary for creating an interchain disulphide bond with the TCR [beta] chain, and a conserved antigen receptor TM motif in C[alpha] and C[beta]. Meanwhile, transcripts of C[alpha], C[beta] and C[gamma] were detectable in all examined organs, with a stronger signal observed in lymphoid organs. In addition, the temporal transcriptional changes for C[alpha] and C[gamma] were investigated, 1, 2, 3, 4, 5, 6 and 8 weeks after stimulation with Flavobacterium columnare, in head kidney, spleen, blood, thymus, gill and intestine, using real-time polymerase chain reaction. The results demonstrated stimulation-dependent up-regulations in almost all tissues examined, which indicates that T cells may play important roles in preventing mandarin fish from bacterial invasion. In particular, apart from thymus, T cells were distributed mainly in gill and intestine, where striking up-regulation of C[gamma] was also observed. These results will facilitate functional studies of teleost TCRs and T cells. [PUBLICATION ABSTRACT]