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A trypsin homolog in amphioxus: expression, enzymatic activity and evolution

Molecular Biology Reports volume 39, pages 1745–1753 (2012)Cite this article

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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Acknowledgments

This work was supported by a grant (2008AA09260) of Ministry of Science and Technology (MOST) of China.

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  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

    Wenrong Feng & Shicui Zhang

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  1. Wenrong Feng
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  2. Shicui Zhang
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Correspondence to Shicui Zhang.

Electronic supplementary material

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11033_2011_915_MOESM1_ESM.tif

Supplementary Fig. 1 Nucleotide and deduced amino acid sequences of amphioxus (Branchiostoma japonicum) trypsinogen cDNA (GenBank accession number: ADO08222). The start codon is boxed. The asterisk (*) represents the stop codon. The predicted signal sequence at the N-terminus is underlined. The polyadenylation signal (AATAAA) and polyadenylation tail is marked with double underlines. The Tryp_SPc domain is between two arrowheads and His, Asp and Ser residues of the catalytic triad are marked by closed squares (■). The number of the nucleotide and amino acid sequences is shown on the left. (TIFF 23595 kb)

11033_2011_915_MOESM2_ESM.tif

Supplementary Fig. 2 Diagram of the genomic structures of trypsin genes from human, cattle, chicken, zebrafish, amphioxus and ascidians. Boxes represent the exons. Thin joining lines indicate the sequences of introns. Solid boxes represent the region coding for the structural sequences of the protein. Blank boxes represent the untranslated region. The values below the lines and boxes indicate the sizes of the introns and exons, respectively. (TIFF 42826 kb)

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Feng, W., Zhang, S. A trypsin homolog in amphioxus: expression, enzymatic activity and evolution. Mol Biol Rep 39, 1745–1753 (2012). https://doi.org/10.1007/s11033-011-0915-y

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  • DOI: https://doi.org/10.1007/s11033-011-0915-y

Keywords

  • Amphioxus
  • Lancelet
  • Branchiostoma
  • Trypsin
  • Evolution