A trypsin homolog in amphioxus: expression, enzymatic activity and evolution |
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Authors: | Wenrong Feng Shicui Zhang |
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Institution: | (1) 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; |
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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 His72-Asp118-Ser215 and the S1 substrate binding residue Asp209, 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. |
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