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

PLoS Negl Trop Dis. 2014 Aug 28;8(8):e3097. doi: 10.1371/journal.pntd.0003097. eCollection 2014 Aug.

Abstract

Background: Deoxyribonuclease 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 findings: In 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.

Conclusions: 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.

MeSH terms

  • Animals
  • Endodeoxyribonucleases* / chemistry
  • Endodeoxyribonucleases* / genetics
  • Endodeoxyribonucleases* / metabolism
  • Helminth Proteins* / chemistry
  • Helminth Proteins* / genetics
  • Helminth Proteins* / metabolism
  • Molecular Sequence Data
  • Recombinant Proteins* / chemistry
  • Recombinant Proteins* / genetics
  • Recombinant Proteins* / metabolism
  • Trichinella spiralis* / enzymology
  • Trichinella spiralis* / genetics

Substances

  • Helminth Proteins
  • Recombinant Proteins
  • Endodeoxyribonucleases
  • deoxyribonuclease II

Associated data

  • GENBANK/KF984291

Grants and funding

This work was supported by grants from the Ministry of Science and Technology of China (MOST 2011AA10A215) and the National Natural Science Foundation of China (NSFC 31030064, 30972177, 81070311, 31072124). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.