J Vet Sci.  2012 Mar;13(1):49-58. 10.4142/jvs.2012.13.1.49.

Cloning and characterization of a selenium-independent glutathione peroxidase (HC29) from adult Haemonchus contortus

Affiliations
  • 1College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China. lixiangrui@njau.edu.cn

Abstract

The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus.

Keyword

glutathione peroxidase; Haemonchus contortus; HC29 cDNA

MeSH Terms

Amino Acid Sequence
Animals
Base Sequence
Cloning, Molecular
DNA, Complementary/genetics/isolation & purification
Glutathione Peroxidase/*genetics/*metabolism
Goat Diseases/parasitology
Goats
Haemonchiasis/parasitology/prevention & control/*veterinary
Haemonchus/*enzymology/*genetics
Hydrogen Peroxide/metabolism
Molecular Sequence Data
Phylogeny
RNA, Helminth/chemistry/genetics
Random Amplified Polymorphic DNA Technique
Rats
Rats, Sprague-Dawley
Sequence Alignment
Sequence Analysis, DNA

Figure

  • Fig. 1 Alignment of nucleotide and predicted amino acid sequences of HC29 cDNA. Nucleotides consisting of the non-coding regions are in lowercase letters while nucleotides of the presumed coding region are in uppercase letters. The initiation ATG and stop TAG codons are shaded. Distal polyadenylation signal sequence AATGAA is shaded. The nucleotides and amino acids are numbered along the left margins. Conserved residues of the sequence are shown in boxes. The catalytic triad active sites are shaded. The spliced leader sequence is indicated in shadow.

  • Fig. 2 Phylogenetic tree based on the predicted amino acid sequences of selected glutathione peroxidases (GPXs) against HC29 protein. Sequences were derived from GenBank or EMBL databases.

  • Fig. 3 Alignment of the critical GPX residues in the amino acid sequences of GPX homologues. The GPX motif and catalytic triad sites are boxed.

  • Fig. 4 Alignment of nucleic acid sequences of HC29 and GPX. The sliced leader sequences and codons of the catalytic triad sites are boxed. hc29: HC29, bag: reported GPX by Bagnall and Kotze [2].

  • Fig. 5 Alignment of amino acid sequences between HC29 and GPX. The GPX motifs and catalytic triad sites are boxed. hc29: HC29, pre: reported GPX by Bagnall and Kotze [2].

  • Fig. 6 Expression of HC29 protein and Western blotting. Gels were stained with Coomassie blue, and 15 µL samples were loaded per lane. Lane PM: Standard protein molecular weight marker, Lane 1: Extracts of Escherichia (E.) coli (BL21 strain) transformed with pET-28a empty expression vector before induction with isopropyl-β-D-thiogalactopyranoside (IPTG, negative control), Lane 2: Extracts of E. coli (BL21 strain) transformed with pET-28a empty expression vector after 5 h of induction with IPTG (negative control), Lane 3: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 before induction with IPTG, Lanes 4~8: Extracts of E. coli (BL21 strain) transformed with pET-28a/HC29 after 1~5 h of induction with IPTG, Lane 9: Purified recombinant HC29 protein supernatants, Lane 10: HC29 natural protein in soluble proteins of Haemonchus contortus was identified using rat sera as a primary antibody.


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