Cloning, Expression And Functional Analysis Of Amphialt In Amphioxus Branchiostoma Japonicus

Alanine aminotransferase (ALT) is a kind of vitamin-B6-dependent aminotransferases and it is the member of the subgroup I which are all have the conserved domains named AAT like superfamily. The structures, functions, sequences and expressions of many kinds of ALT have been studied in deep. Yet little is known to date about it in the amphioxus Branchiostoma japonicus. Amphioxus or lancelet, a cephalochordate, located at the base of vertebrates, and is becoming an emerging model organism for insights into the origin and evolution of vertebrates. In this paper, we report the cloning, characterization, expression, phylogenetic analysis and functional characterization of amphioxus alt gene (Amphialt).The full-length cDNA of Amphialt was 2371 bp long, containing an open reading frame (ORF) of 1503 bp which encoded a protein of 500 amino acids with a predicted molecular mass of approximately 54.9 kDa. The deduced protein had the complete conserved domain of transaminase class I (AAT like, cd00609), including the catalytic residue (Lys317) and pyridoxal 5'-phosphate (PLP) binding sites (Tyr193-Asn245-Asp276-Tyr279-Ser316-Lys317-Arg326) which are both characteristic of ALT. Sequence analysis demonstrated that AmphiALT shared 62.1-64.7% and 62.7～64.5% sequence identity to vertebrate ALT1 and ALT2, respectively, and 52.7～57.7% identity to invertebrate ALT. The phylogenetic tree found that AmphiALT formed an independent cluster with Florida amphioxus ALT and ascidian ALT, which was intermediary from invertebrate ALT and vertebrate ALT and located at the base of vertebrate ALT1 and ALT2 isoenzymes, suggesting that AmphiALT may represent the archetype of vertebrate ALT1 and ALT2 isoenzymes.Northern blot revealed the presence of an approximate 2370 bp transcript corresponding in size to the full-length Amphialt cDNA we cloned in B. japonicus. In situ hybridization demonstrated that Amphialt transcripts were highest in the hepatic caecum, and at a lower level in the hind-gut, gill, ovary and testis, but no signal was observed in the epidermis, muscle, neural tube and notochord. These indicated that Amphialt was expressed in a tissue-specific manner. Challenge with LPS soon (6,12 and 24 h) resulted in an up-regulation of Amphialt expression in the hepatic caecum, suggesting that Amphialt may be an immune defense-relevant molecule and involve in the host immune defense.The purified AmphiALT was used to test its activities. Strikingly, recombinant AmphiALT, with a specific activity of 0.114±0.02 U/mg, was capable of specifically binding to the Gram-negative bacterium Escherichia coli and to the conserved molecule LPS, as well as inhibiting its growth and causing its lysis. The recombinant AmphiALT has catalytic activity and bacteriostatic activity. Taken together, it is proposed that AmphiALT is a novel sensor capable of identifying LPS and an effector molecule capable of causing damage to Gram-negative bacteria like E.coli. It also bolsters the notion that the hepatic caecum of amphioxus is the precursor of vertebrate liver, acting as a major tissue in acute phase response.