Molecular Cloning And Evolution Analysis Of TnTLR1 And TnDIGIRR Genes In Teleost Fish

Members of the TLR-IL-1R superfamily, defined by the presence of an intracellular Toll-IL-1R (TIR) domain, are important in mediating immune responses. Based on extracellular domains, this superfamily can be deiveded into three subgroups. The first contains extracellular immunoglobulin (Ig) domains named IL-1R subfamily. The second is TLRs, which lack Ig domains, but have extracellular leucine-rich repeats. The third subgroup consists of upstream adapter molecules, including MyD88, MyD88 adapter-like (Mal, also known as TIRAP) and TIR domain-containing adaptor. TLR1 and SIGIRR are both members of TIR superfamily, belonging to the TLR subfamily and IL-1R subfamily respectively. As an important member of TLRs, TLR1 is known as PAMP recognition receptor, involving in the recognition of triacylated lipoproteins and mycobacterial products by binding to TLR2 to form a heterodimer. An orphan receptor, SIGIRR, has been identified as a novel member of the TIR superfamily, involving in the negative regulation of the TIR signaling pathways. DIGIRR is a homology of mammalian SIGIRR, and exists only in fish. Here we reported two novel genes TLR1 and DIGIRR in Tetraodon nigroviridi.The cloned TnTLR1 cDNA exhibited 2587 bp in length and contains a 122 bp 5' untranslated region (UTR), a 2391bp open reading frame (ORF) and a 74 bp 3'UTR. TnTLR1 is constitutively detectable in various tissues and organs selected, and the levels of the transcripts increased in spleen and heart after LPS stimulations. Gene organization of TnTLRl is similar to mammalian and avian, and a high degree of conservation of chromosome synteny exists between them. The protein sequence shares 33.7%～77.3% identity with known TLR1 members. Phylogenetic analysis grouped TnTLR1 with other fish TLRs on a separated branch, excluded from mammalian and avian TLR1s. TnTLR1 with 796 amino acids has a calculated molecular mass of 90.69 kDa and a theoretical pI of 8.43. Structurally, TnTLR1 has a Toll/interleukin-1 receptor (TIR) domain, a transmembrane domain and leucine-rich repeat (LRR) domain which are the hallmarks of TLR family. Phylogenetic analysis provided an evidence that the TnTLR1 was very close to mammalian TLR1, 6 and 10 and seemed to be their common ancestor.TnDIGIRR cDNA exhibited 1916 bp in length and contains a 112 bp 5' untranslated region (UTR), a 1575bp open reading frame (ORF) and a 229 bp 3'UTR. TnDIGIRR with 796 amino acids has a calculated molecular mass of 90.69 kDa and a theoretical pI of 8.43. Structurally, TnDIGIRR has a TIR domain, a transmembrane domain and two Ig domains which are the hallmarks of the second TIR subgroup. The protein sequence shares 31.9%～77.0% identity with known SIGIRR members. Phylogenetic analysis grouped TnDIGIRR with other fish DIGIRR on a separated branch, excluded from mammalian and avian SIGIRR. TnDIGIRR is highly expressed in the heart, brain, skin and liver; moderately expressed in the gill and intestine; and not expressed in musle. Gene organization, multiple alignment and signal peptide prediction shows that TnDIGIRR protein has a putative signal secretion peptide 1-23 aa in its N-terminal region while there is no predictable signal peptide in mammalian and avian; TnDIGIRR protein has a dramatically long extracellular region of 260 aa which corresponds two Ig domains instead of single one in high vertebrate just as its name goes; The specific amino acid Ser, which were characterized to be essential for the signalling of the IL-1RI, is conserved in all three fish DIGIRR but all changed to Cys in high vertebrate (including Danio). The dramatic features of structure indicated TnDIGIRR may possessed the ability of recognizing cytokine and be able to signal as IL-1R-like components.In conclusion, the overall characterizations of TnTLR1 and TnDIGIRR, including sequence, structure, synteny, location, functional domains and expression pattern were analyzed. The results indicated that the cloned TnTLR1 and TnSIGIRR are both orthologue of mammalian and avian TLR1 and DIGIRR respectively. This work will contribute to our further understanding of the function and evolution of this TIR superfamily.