X-ray structures of Na-GST-1 and Na-GST-2 two glutathione s-transferase from the human hookworm Necator americanus

<p>Abstract</p> <p>Background</p> <p>Human hookworm infection is a major cause of anemia and malnutrition of adults and children in the developing world. As part of on-going efforts to control hookworm infection, The Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective L3 larval stages and adult stages of the parasite. Adult stage antigens include the cytosolic glutathione-S-transferases (GSTs). Nematode GSTs facilitate the inactivation and degradation of a variety of electrophilic substrates (drugs) via the nucleophilic addition of reduced glutathione. Parasite GSTs also play significant roles in multi-drug resistance and the modulation of host-immune defense mechanisms.</p> <p>Results</p> <p>The crystal structures of <it>Na</it>-GST-1 and <it>Na</it>-GST-2, two major GSTs from <it>Necator americanus </it>the main human hookworm parasite, have been solved at the resolution limits of 2.4 Å and 1.9 Å respectively. The structure of <it>Na</it>-GST-1 was refined to R-factor 18.9% (R-free 28.3%) while that of <it>Na</it>-GST-2 was refined to R-factor 17.1% (R-free 21.7%). Glutathione usurped during the fermentation process in bound in the glutathione binding site (G-site) of each monomer of <it>Na</it>-GST-2. <it>Na</it>-GST-1 is uncomplexed and its G-site is abrogated by Gln 50. These first structures of human hookworm parasite GSTs could aid the design of novel hookworm drugs.</p> <p>Conclusion</p> <p>The 3-dimensional structures of <it>Na</it>-GST-1 and <it>Na</it>-GST-2 show two views of human hookworm GSTs. While the GST-complex structure of <it>Na</it>-GST-2 reveals a typical GST G-site that of <it>Na</it>-GST-1 suggests that there is some conformational flexibility required in order to bind the substrate GST. In addition, the overall binding cavities for both are larger, more open, as well as more accessible to diverse ligands than those of GSTs from organisms that have other major detoxifying mechanisms. The results from this study could aid in the design of novel drugs and vaccine antigens.</p

The evaluation of recombinant hookworm antigens as vaccines in hamsters (Mesocricetus auratus) challenged with human hookworm, Necator americanus

We have previously reported the successful adaptation of human hookworm Necator americanus in the golden hamster, Mesocricetus auratus. This animal model was used to test a battery of hookworm (N. americanus and Ancylostoma caninum) recombinant antigens as potential vaccine antigens. Hamsters immunized a leading vaccine candidate N. americanus–Ancylostoma secreted protein 2 (Na-ASP-2) and challenged with N. americanus infective larvae (L3), resulted in 30–46.2% worm reduction over the course of three vaccine trials, relative to adjuvant controls. In addition, significant reduction of worm burdens was also observed in the hamsters immunized with adult hookworm antigens A. caninum aspartic protease 1 (Ac-APR-1); A. caninum-glutathione-S transferase 1 (Ac-GST-1) and Necator cysteine proteases 2 (Na-CP-2) (44.4%, 50.6%, and 29.3%, respectively). Our data on the worm burden reductions afforded by these hookworm antigens approximate the level of protection reported previously from dogs challenged with A. caninum L3, and provide additional evidence to support these hookworm antigens as vaccine candidates for human hookworm infection. The hamster model of N. americanus provides useful information for the selection of antigens to be tested in downstream vaccine development

Ancylostoma caninum MTP-1, an Astacin-Like Metalloprotease Secreted by Infective Hookworm Larvae, Is Involved in Tissue Migration

Infective larvae (L3) of nematodes secrete macromolecules that are critical to infection and establishment of the parasite in the host. The dog hookworm Ancylostoma caninum secretes an astacin-like metalloprotease, Ac-MTP-1, upon activation in vitro with host serum. Recombinant Ac-MTP-1 was expressed in the baculovirus/insect cell system as a secreted protein and was purified from culture medium by two separate methods, cation-exchange fast-performance liquid chromatography and gelatin-affinity chromatography. Recombinant MTP-1 was catalytically active and digested a range of native and denatured connective tissue substrates, including gelatin, collagen, laminin, and fibronectin. A dog was immunized with recombinant Ac-MTP-1 formulated with AS03 adjuvant, and the antiserum was used to immunolocalize the anatomic sites of expression within A. caninum L3 to secretory granules in the glandular esophagus and the channels that connect the esophagus to the L3 surface and to the cuticle. Antiserum inhibited the ability of recombinant MTP-1 to digest collagen by 85% and inhibited larval migration through tissue in vitro by 70 to 75%, in contrast to just 5 to 10% inhibition obtained with preimmunization serum. The metalloprotease inhibitors EDTA and 1,10-phenanthroline also reduced the penetration of L3 through skin in vitro by 43 to 61%. The data strongly suggest that Ac-MTP-1 is critical for the invasion process of hookworm larvae, and moreover, that antibodies against the enzyme can neutralize its function and inhibit migration

Nu class GSTs a) -GST-2, c) HpolGST have larger binding cavity than sigma class GST b) HsGST

<p><b>Copyright information:</b></p><p>Taken from "X-ray structures of -GST-1 and -GST-2 two glutathione s-transferase from the human hookworm "</p><p>http://www.biomedcentral.com/1472-6807/7/42</p><p>BMC Structural Biology 2007;7():42-42.</p><p>Published online 26 Jun 2007</p><p>PMCID:PMC1924862.</p><p></p> d) Overlay of the cavities reveals the considerable reduction in the active site size between sigma class (blue) and nu class (cyan). The structure of HpolGST is missing a loop in close proximity to the binding cavity and we modeled it as cartoon from the -GST-2 structure. The glutathione in the G-site is shown as red stick model

Sequence and structural alignment of Nu class GSTs with a Sigma Class GST (HsGST, human GST or hematopoietic prostagladin D synthase 37) and other parasite GSTs (SjGST, 43, (-GST-1) 42

<p><b>Copyright information:</b></p><p>Taken from "X-ray structures of -GST-1 and -GST-2 two glutathione s-transferase from the human hookworm "</p><p>http://www.biomedcentral.com/1472-6807/7/42</p><p>BMC Structural Biology 2007;7():42-42.</p><p>Published online 26 Jun 2007</p><p>PMCID:PMC1924862.</p><p></p> (a) The alignment reveals that firstly N-terminal alpha beta domain is more conserved than the C-terminal alpha domain. Furthermore, -GST-1 has higher sequence identity with HpolGST than -GST-2 and the lowest similarity is with the HsGST. This figure was generated with ESPript [55, 56]. (b) Structural alignment of monomers of Nu class GSTs (-GST-1, magenta; -GST-2, gold; HpolGST, green) with a sigma class GST (HsGST, cyan)

Molecular Cloning, Biochemical Characterization, and Partial Protective Immunity of the Heme-Binding Glutathione S-Transferases from the Human Hookworm Necator americanus

Hookworm glutathione S-transferases (GSTs) are critical for parasite blood feeding and survival and represent potential targets for vaccination. Three cDNAs, each encoding a full-length GST protein from the human hookworm Necator americanus (and designated Na-GST-1, Na-GST-2, and Na-GST-3, respectively) were isolated from cDNA based on their sequence similarity to Ac-GST-1, a GST from the dog hookworm Ancylostoma caninum. The open reading frames of the three N. americanus GSTs each contain 206 amino acids with 51% to 69% sequence identity between each other and Ac-GST-1. Sequence alignment with GSTs from other organisms shows that the three Na-GSTs belong to a nematode-specific nu-class GST family. All three Na-GSTs, when expressed in Pichia pastoris, exhibited low lipid peroxidase and glutathione-conjugating enzymatic activities but high heme-binding capacities, and they may be involved in the detoxification and/or transport of heme. In two separate vaccine trials, recombinant Na-GST-1 formulated with Alhydrogel elicited 32 and 39% reductions in adult hookworm burdens (P < 0.05) following N. americanus larval challenge relative to the results for a group immunized with Alhydrogel alone. In contrast, no protection was observed in vaccine trials with Na-GST-2 or Na-GST-3. On the basis of these and other preclinical data, Na-GST-1 is under possible consideration for further vaccine development

Crystallization and preliminary X-ray analysis of Na-SAA-2 from the human hookworm parasite Necator americanus

The purification, crystallization and preliminary X-ray diffraction analysis of a surface-associated antigen from the major human hookworm N. americanus is presented