Binding of hematin by a new class of glutathione transferase from the blood-feeding parasitic nematode Haemonchus contortus

Abstract

The phase II detoxification system glutathione transferase (GST) is associated with the establishment of parasitic nematode infections within the gastrointestinal environment of the mammalian host. We report the functional analysis of a GST from an important worldwide parasitic nematode of small ruminants, Haemonchus contortus. This GST shows limited activity with a range of classical GST substrates but effectively binds hematin. The high-affinity binding site for hematin was not present in the GST showing the most identity, CE07055 from the free-living nematode Caenorhabditis elegans. This finding suggests that the high-affinity binding of hematin may represent a parasite adaptation to blood or tissue feeding from the host.

FIG. 1.

Structure homology model of H. contortus GST (HcGST-1) with GSH constructed with SWISS-MODEL and viewed with Rasmol. In the model, the GSH thiol is within hydrogen-bonding distance of Tyr 8, the GSH Gly carboyl is within hydrogen-bonding distance of Trp 39, the γ-Glu carboxyl is within hydrogen bonding distance of Gln 63, and the GSH backbone hydrogen bonds to Leu 51.

FIG.2.

Phylogenetic tree showing the relationship of HcGST-1 to all C. elegans proteins containing an N-terminal and/or C-terminal GST domain, as indicated by InterPro (domains IPR004045 and IPR004046, respectively). The initial alignment of protein sequences was achieved with BioEdit (16) and CLUSTAL W (28). The final tree was constructed by cluster analysis with TreeCon (30). The numbers indicate the bootstrap values for 100 replicates. All C. elegans protein sequences were obtained from the Sanger Institute (http://www.sanger.ac.uk/Projects/C_elegans/wormpep/), and accession numbers are indicated by the prefix “CE.” The nucleotide sequence of the H. contortus-excreted GST (HcGST-E) was obtained from GenBank (National Center for Biology Information; http://www.ncbi.nlm.nih.gov/) (accession no. BM138779).

FIG. 3.

Production and purification of rHcGST-1: Coomassie-stained SDS-12% PAGE. Lane 1, somatic extract of E. coli BL21(DE3) plus pET23d (control); lane 2, somatic extract of E. coli BL21(DE3) plus pAR10 (containing HcGST-1); lane 3, GSH-agarose affinity-purified rHcGST-1; lane 4, molecular mass protein standard.

FIG. 4.

Electrospray mass spectrometer analysis of purified recombinant H. contortus GST, establishing the mass at 23,603 Da.

FIG. 5.

Western blot probed with polyclonal antibodies to H. contortus GST-1, showing the expression of GST in native H. contortus tissue. Lane 1, somatic extract of H. contortus; lanes 2 and 4, molecular mass protein standard; lane 3, somatic extract of E. coli BL21(DE3) plus pET23d (control); lane 5, somatic extract of E. coli BL21(DE3) plus pAR10 (containing HcGST-1); lane 6, GSH-agarose affinity-purified rHcGST-1.

FIG. 6.

Southern blots on H. contortus (A) and C. elegans (B and C) genomic DNA digests, confirming a close relationship between H. contortus and C. elegans at the genomic level. Lanes 1, BamHI; lanes 2, EcoRI; lanes 3, HindIII; lanes 4, BamHI and EcoRI; lanes 5, BamHI and HindIII; lanes 6, EcoRI and HindIII; lanes 7, positive control (HcGST-1 gene). (A) H. contortus genomic DNA digests probed with radiolabeled HcGST-1 gene (60°C; high-stringency washes: twice with wash A, twice with wash B, and twice with wash C). (B) C. elegans genomic DNA digests probed with radiolabeled HcGST-1 gene (60°C; low-stringency washes: twice with wash A). (C) C. elegans genomic DNA digests probed with radiolabeled HcGST-1 gene (60°C; high-stringency washes: twice with wash A, twice with wash B, and twice with wash C).

FIG. 7.

Two-dimensional gel and Western blot on somatic H. contortus protein, showing the expression of up to eight similar GSTs, or modified forms of GST(s), in adult worms. (A) Two-dimensional gel of somatic H. contortus protein. The first dimension was run at 11 cm with an IPG of pH 3 to 10, and the second dimension was run on SDS-8 to 18% PAGE. (B) Western blot on two-dimensional gel of somatic H. contortus protein probed with polyclonal antibodies to rHcGST-1.

FIG. 8.

Two-dimensional gel and Western blot on somatic C. elegans protein, supporting the presence of nematode generic classes of GSTs. (A) Two-dimensional gel of glutathione-agarose affinity-purified C. elegans protein extract. The first dimension was run at 11 cm with an IPG of pH 3 to 10, and the second dimension was run on SDS-8 to 18% PAGE. (B) Western blot on two-dimensional gel of affinity-purified C. elegans protein extract probed with polyclonal antibodies to HcGST-1.

FIG. 9.

Binding of hematin to HcGST-1 as demonstrated by intrinsic fluorescence and competitive fluorescence spectrometry. (A) Double-reciprocal plot of the quenching of intrinsic fluorescence in rHcGST-1 (Q) against the concentration of free hematin (H_f) (calculated as previously described [31]), with an intrinsic fluorescence K_d value (rHcGST-1) of 1.72 ± 0.10 μM for hematin. Shown are averages of at least three determinations, with error bars representing standard deviations. (B) Competitive fluorescence of rHcGST-1 with the fluorescent ligand ANS and hematin. Lines 1 and 2 indicate the fluorescence of the rHcGST-1 protein with and without ANS, respectively. Lines 3 to 9 show the quenching of fluorescence due to the addition of hematin (with increments of 0.2 μM). (C) Competitive fluorescence of rHcGST-1 with ANS presented as a double-reciprocal plot, with a competitive fluorescence K_d value (rHcGST-1) of 1.13 ± 0.07 μM for hematin. Shown are averages of at least three determinations, with error bars representing standard deviations.