Binding of Hematin by a New Class of Glutathione Transferase from the Blood-Feeding Parasitic NematodeHaemonchus 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.The gastrointestinal blood-feeding nematode Haemonchus contortus represents a major economic burden to agricultural communities worldwide, causing infections resulting in anemia, weight loss, and ultimately death in small ruminants. There are presently no commercial vaccines available for H. contortus infections, and the most effective method of control is a combination of pasture management and the use of chemical agents (anthelmintics). Increasing reports of drug-resistant H. contortus indicate that this current control strategy is not sustainable (26), with chemicals no longer effectively controlling H. contortus infections in several parts of the world (33). Understanding the host-parasite relationship at the mucosal feeding surface is important in identifying new therapeutic approaches. The levels of the phase II detoxification system glutathione transferase (GST) have been shown to increase in parasitic helminths during chronic infection (3). Previous research has attempted to correlate this overexpression with the ability of GST to detoxify immune-initiated cytotoxic products of lipid peroxidation (8, 10) or has associated the overexpression of GST, including H. contortus GST, with drug resistance (18,19). In this paper, we analyze a new GST from the sheep strongylid H. contortus and show that this GST does not appear to have a broad immune defense or drug metabolism role but possibly has a more focused detoxification function within the nematode. MATERIALS AND METHODSIsolation, recombinant expression, and purification of H. contortus GST. mRNA was isolated from adult H. contortus nematodes (CAVR, a drug-resistant strain) with a Quickprep Micro mRNA purification kit (Pharmacia). H. contortus cDNA was obtained with a First Strand cDNA synthesis kit (Pharmacia). The H. contortus GST-encoding DNA was isolated by an established strategy with an upstream primer derived from the N-terminal sequence of the native H. contortus GST protein (27) and a downstream oligo(dT)-based anchor primer (1, 5). The PCR product (approximately 650 bp) was cloned into pUC18 (SureClone ligation kit; Pharmacia), and the insert was sequenced. The H. contortus GST was directionally cloned into pET23d and sequenced in both directions (Long-read LI-COR, GenBank accession number AF281663). The recombinant H. contortu...

Molecular Cloning, Biochemical Characterization, and Partial Protective Immunity of the Heme-Binding GlutathioneS-Transferases from the Human HookwormNecator americanus

“…Sequence comparison with GSTs from other organisms demonstrated that GSTs from hookworms are from a novel family of nematode-specific GSTs designated the nu class (54). The nematode-specific nu-class GSTs are functionally and structurally different from other classes of GSTs and are characterized by a high-affinity binding site for heme and its related products and limited activity with other substrates (59,69). The crystal structures of nu-class GSTs contain long/deep and large clefts FIG.…”

“…The IC 50 of hematin for rNa-GST-2-conjugating activity was 3-to 5-fold lower than those of the other hookworm GSTs, implying that Na-GST-2 exhibited the highest affinity for heme. The hookworm GSTs' heme-binding K d values were about 10-fold larger than their corresponding IC 50 s, suggesting that quenching of intrinsic fluorescence detects a heme-binding site distinct from the active site, as seen for Hc-GST-1 from Haemonchus contortus (59).…”

“…Ligand binding to hookworm GSTs was determined by measuring changes in intrinsic protein fluorescence, as described previously (59). In the binding assays, 1 M recombinant hookworm GST was added to 20 mM potassium phosphate buffer (pH 6.5) containing 100 mM sodium chloride at 25°C.…”

“…Each absorbed rabbit anti-Na-GST serum was then used to determine whether the corresponding native protein was present in larval or adult hookworms, by Western blotting as previously described (5,58). The somatic extracts of N. americanus adult worms were also separated by two-dimensional gel electrophoresis (25,59) and silver-stained or electrotransferred for Western blotting as described above.…”

“…Our previous studies have identified a nematode-specific class of parasitic GSTs exhibiting a high affinity for heme (59,69). X-ray structural data suggest that heme binding is due to the association of tetrapyrrole compounds with a large hydrophobic binding pocket that forms during homodimerization of two hookworm GST subunits (3).…”