Phylogenetic Analysis Of Vertebrate Kininogen Genes

Kininogens, the precursors of bradykinins, vary extremely in both structure and function among different taxa of animals, in particular between mammals and amphibians. This includes even the most conserved bradykinin domain in terms of biosynthesis mode and structure. Besides the bradykinin domain, mammalian kininogen also contains three cystatin domains (the one in N-terminal part without canonical cysteine protease binding sites; the other two with those). To further elucidate the evolutionary dynamics of kininogen genes, in this work, we have identified 19 novel amino acid sequences from EST and genomic databases (for mammals, birds, and fishes), and explored their phylogenetic relationships using combined amino acid sequence and gene structure as markers.These mainly results were as follow:1) The topology of the neighbor-joining tree presented that all 57 kininogen amino acid sequences were classified into two clusters, one including mammals, birds, and fishes and the other including only amphibians. The high bootstrap values supported both the precision of topology and the reliability of predicted sequences.2) A total of 24 kininogen sequences from 16 species with clear intron–exon boundaries were used for gene structure analyses. Results suggested strong conservation in terms of both position and phase of most introns, though some introns were found only in certain mammals and birds or fishes.3) The conservative analyses of 57 kininogen amino acid sequences without signal peptides showed that all of the sequences shared the bradykinin domain, whereas other domains were conserved more or less in some taxa of animals.4) We also propose that the defense mechanism against specific predators in amphibian skin secretions has been bradykinin receptor dependent according to the detailed analysis of amphibian bradykinin domains.5) In addition, we also cloned a kininogen gene, which contained a 546bp open reading frame, and encoded 181 amino acids residues, from the liver of lamprey, Lampetra japonica, which is one of the most primitive jawless vertebrates still living today. Protein sequence analyses showed that there were a cystatin domain without canonical cysteine protease binding sites and a bradykinin domain with substitutions of amino acid residues.6) Using reverse transcription PCR, the kininogen mRNA was also detected in lamprey gut, kidney, and leukocyte, but absent in lamprey buccal gland. These above results seem to demonstrate that in lampreys, a kininogen gene encoded a cystatin domain and a bradykinin domain. During the process of vertebrate evolution, the gene was fused by some sequences and encoded proteins with more functions, gradually. Particularly, in amphibians, there was a special kininogen gene, only encoding the bradykinin domain, however, with multiple copies in different species. The canonical kininogen gene became a pseudogene and largely degenerated, but with gene structure conserved.Our findings may provide a foundation for identification and structural, functional, and evolutionary analyses of more kininogen genes and other gene families.