Abstract

BackgroundMembers of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. Structural and biochemical characteristics of insect pacifastin-like peptides have been studied intensively, but only one inhibitor has been functionally characterised. Recent sequencing projects of metazoan genomes have created an unprecedented opportunity to explore the distribution, evolution and functional diversification of pacifastin genes in the animal kingdom.ResultsA large scale in silico data mining search led to the identification of 83 pacifastin members with 284 inhibitor domains, distributed over 55 species from three metazoan phyla. In contrast to previous assumptions, members of this family were also found in other phyla than Arthropoda, including the sister phylum Onychophora and the 'primitive', non-bilaterian Placozoa. In Arthropoda, pacifastin members were found to be distributed among insect families of nearly all insect orders and for the first time also among crustacean species other than crayfish and the Chinese mitten crab. Contrary to precursors from Crustacea, the majority of insect pacifastin members contain dibasic cleavage sites, indicative for posttranslational processing into numerous inhibitor peptides. Whereas some insect species have lost the pacifastin gene, others were found to have several (often clustered) paralogous genes. Amino acids corresponding to the reactive site or involved in the folding of the inhibitor domain were analysed as a basis for the biochemical properties.ConclusionThe absence of the pacifastin gene in some insect genomes and the extensive gene expansion in other insects are indicative for the rapid (adaptive) evolution of this gene family. In addition, differential processing mechanisms and a high variability in the reactive site residues and the inner core interactions contribute to a broad functional diversification of inhibitor peptides, indicating wide ranging roles in different physiological processes. Based on the observation of a pacifastin gene in Placozoa, it can be hypothesized that the ancestral pacifastin gene has occurred before the divergence of bilaterian animals. However, considering differences in gene structure between the placozoan and other pacifastin genes and the existence of a 'pacifastin gene gap' between Placozoa and Onychophora/Arthropoda, it cannot be excluded that the pacifastin signature originated twice by convergent evolution.

Highlights

  • Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins

  • Sequences were manually corrected, splice variants were determined and gene structures were verified, whenever possible, using EST sequences and/or conventional cloning techniques. By this combined approach pacifastin members were identified in 55 species, divided over three distinct animal phyla

  • Within Arthropoda, pacifastin-related precursors (PP) were identified in Insecta, Ellipura and Crustacea, suggesting a wide distribution of members of the pacifastin family in these branches of the phylum

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Summary

Introduction

Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. According to the NCBI platform (National Center for Biotechnology Information) more than 150 metazoan genome projects are in progress or completed and up to 36 million EST sequences are available http:// www.ncbi.nlm.nih.gov/ Comparative analysis of this huge dataset, covering numerous species of both vertebrates and invertebrates, has already proven to be a powerful tool to predict new gene products, facilitating their molecular characterization, illustrating cross species sequence homology, unraveling gene structures and revealing expansion or reduction of gene families during evolution. While in the MEROPS database 150 peptidase inhibitors are listed from humans, only 32, 11 and 42 have been annotated in the genomes of the Diptera A. aegypti, A. gambiae and D. melanogaster, respectively In spite of this apparent discrepancy, previous studies have reported several members of the pacifastin peptide inhibitor family (I19 in the peptidase/inhibitor MEROPS database), all of which have been identified in arthropod species

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