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 Adjuvanticity of an O. volvulus-Derived rOv-ASP-1 Protein in Mice Using Sequential Vaccinations and in Non-Human Primates

Adjuvants potentiate antigen-specific protective immune responses and can be key elements promoting vaccine effectiveness. We previously reported that the Onchocerca volvulus recombinant protein rOv-ASP-1 can induce activation and maturation of naïve human DCs and therefore could be used as an innate adjuvant to promote balanced Th1 and Th2 responses to bystander vaccine antigens in mice. With a few vaccine antigens, it also promoted a Th1-biased response based on pronounced induction of Th1-associated IgG2a and IgG2b antibody responses and the upregulated production of Th1 cytokines, including IL-2, IFN-γ, TNF-α and IL-6. However, because it is a protein, the rOv-ASP-1 adjuvant may also induce anti-self-antibodies. Therefore, it was important to verify that the host responses to self will not affect the adjuvanticity of rOv-ASP-1 when it is used in subsequent vaccinations with the same or different vaccine antigens. In this study, we have established rOv-ASP-1's adjuvanticity in mice during the course of two sequential vaccinations using two vaccine model systems: the receptor-binding domain (RBD) of SARS-CoV spike protein and a commercial influenza virus hemagglutinin (HA) vaccine comprised of three virus strains. Moreover, the adjuvanticity of rOv-ASP-1 was retained with an efficacy similar to that obtained when it was used for a first vaccination, even though a high level of anti-rOv-ASP-1 antibodies was present in the sera of mice before the administration of the second vaccine. To further demonstrate its utility as an adjuvant for human use, we also immunized non-human primates (NHPs) with RBD plus rOv-ASP-1 and showed that rOv-ASP-1 could induce high titres of functional and protective anti-RBD antibody responses in NHPs. Notably, the rOv-ASP-1 adjuvant did not induce high titer antibodies against self in NHPs. Thus, the present study provided a sound scientific foundation for future strategies in the development of this novel protein adjuvant

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)

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