Characterisation of a plancitoxin-1-like DNase II gene in Trichinella spiralis.

Chengshui Liao,Tingting Li,Xiaolei Liu,Pascal Boireau,He Gao,Bin Tang,Xiuping Wu,Mingyuan Liu,Xidong Liu,Qingsong Sun,Feng Wang,Xuelin Wang,Pan Liu,Ying Zhao,Xue Bai,Aaron R Jex

doi:10.1371/journal.pntd.0003097

Chengshui Liao, Tingting Li + Show 14 more

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https://doi.org/10.1371/journal.pntd.0003097

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Abstract

BackgroundDeoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain.Methodology/Principal FindingsIn this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.ConclusionsThis study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.

Highlights

Deoxyribonucleases (DNases) are typically divided into two distinct categories, namely, Deoxyribonuclease I (DNase I) and Deoxyribonuclease II (DNase II), based their biochemical properties during DNA degradation [1]
Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML)
This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity

Summary

Introduction

Deoxyribonucleases (DNases) are typically divided into two distinct categories, namely, Deoxyribonuclease I (DNase I) and Deoxyribonuclease II (DNase II), based their biochemical properties during DNA degradation [1]. The nucleotide and amino acid sequences of these genes were unclear until the human DNase II gene was cloned in 1998 [6]. Soon after this initial report, DNase II or DNase II homologues were identified in vertebrates, invertebrates, and nonmetazoans [7]. Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. XM_003370715.1) contains the HKD motif in its Cterminus domain

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