title: A trypsin homolog in amphioxus: expression, enzymatic activity and evolution.
abstract: Trypsin has been documented in a variety of species including both vertebrates and invertebrates, but little is known about it in amphioxus, a model organism for insights into the origin and evolution of vertebrates. Here we identified a trypsin gene in Branchiostoma japonicum. The cDNA was 978 bp long with an ORF encoding a deduced protein of 272 amino acids. The deduced protein had an N-terminal signal peptide of 15 amino acids, a 16 activation peptide with the typical cleavage site Arg/Ile, a Tryp_SPc domain with the catalytic triad His(72)-Asp(118)-Ser(215) and the S1 substrate binding residue Asp(209), which are all characteristic of trypsinogens. The recombinant trypsin protein was able to hydrolyse the trypsin prototypic substrate BAEE, which was inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. Both northern blotting and tissue-section in situ hybridization demonstrated that trypsin gene was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum, mid-gut and ovary. And the whole mount in situ hybridization showed that it began to express in the middle third of the full-length primitive gut in 2-day larvae, where the hepatic caecum will form later during development. Phylogenetic analysis indicated that both amphioxus and ascidian trypsins are more closer to each other than to vertebrate trypsins, suggesting a continuous evolutionary divergence of vertebrate trypsins after split from protochordate/vertebrate common ancestor.
authors.affiliation: Institute of Evolution & Marine Biodiversity and Department of Marine Biology, Ocean University of China, Room 205, Ke Xue Guan, 5 Yushan Road, Qingdao 266003, China.
authors.forename: Wenrong
authors.initials: W
authors.lastname: Feng
authors.forename: Shicui
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002816:Chordata, Nonvertebrate / Q000235:genetics*; D003001:Cloning, Molecular; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D019143:Evolution, Molecular; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D014357:Trypsin / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D001120:Arginine; C009727:benzoylarginine ethyl ester; D014357:Trypsin