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

Human hookworms are among the most pathogenic soil-transmitted helminths. These parasitic nematodes have co-evolved with the host and are able to maintain a high worm burden for decades without killing the human host. However, it is possible to develop vaccines against laboratory-challenge hookworm infections using either irradiated third-state infective larvae (L3) or enzymes from the adult parasites. In an effort to control hookworm infection globally, the Human Hookworm Vaccine Initiative, a product-development partnership with the Sabin Vaccine Institute to develop new control tools including vaccines, has identified a battery of protein antigens, including surface-associated antigens (SAAs) from L3. SAA proteins are characterized by a 13 kDa conserved domain of unknown function. SAA proteins are found on the surface of infective L3 stages (and some adult stages) of different nematode parasites, suggesting that they may play important roles in these organisms. The atomic structures and function of SAA proteins remain undetermined and in an effort to remedy this situation recombinant Na-SAA-2 from the most prevalent human hookworm parasite Necator americanus has been expressed, purified and crystallized. Useful X-ray data have been collected to 2.3 A resolution from a crystal that belonged to the monoclinic space group C2 with unit-cell parameters a = 73.88, b = 35.58, c = 42.75 A, beta = 116.1 degrees .

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

BACKGROUND: 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. RESULTS: The crystal structures of Na-GST-1 and Na-GST-2, two major GSTs from Necator americanus the main human hookworm parasite, have been solved at the resolution limits of 2.4 A and 1.9 A respectively. The structure of Na-GST-1 was refined to R-factor 18.9% (R-free 28.3%) while that of Na-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 Na-GST-2. Na-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. CONCLUSION: The 3-dimensional structures of Na-GST-1 and Na-GST-2 show two views of human hookworm GSTs. While the GST-complex structure of Na-GST-2 reveals a typical GST G-site that of Na-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.

Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma.

The goal of this work was to identify the esterases in human plasma and to clarify common misconceptions. The method for identifying esterases was nondenaturing gradient gel electrophoresis stained for esterase activity. We report that human plasma contains four esterases: butyrylcholinesterase (EC 3.1.1.8), paraoxonase (EC 3.1.8.1), acetylcholinesterase (EC 3.1.1.7), and albumin. Butyrylcholinesterase (BChE), paraoxonase (PON1), and albumin are in high enough concentrations to contribute significantly to ester hydrolysis. However, only trace amounts of acetylcholinesterase (AChE) are present. Monomeric AChE is seen in wild-type as well as in silent BChE plasma. Albumin has esterase activity with alpha- and beta-naphthylacetate as well as with p-nitrophenyl acetate. Misconception #1 is that human plasma contains carboxylesterase. We demonstrate that human plasma contains no carboxylesterase (EC 3.1.1.1), in contrast to mouse, rat, rabbit, horse, cat, and tiger that have high amounts of plasma carboxylesterase. Misconception #2 is that lab animals have BChE but no AChE in their plasma. We demonstrate that mice, unlike humans, have substantial amounts of soluble AChE as well as BChE in their plasma. Plasma from AChE and BChE knockout mice allowed identification of AChE and BChE bands without the use of inhibitors. Human BChE is irreversibly inhibited by diisopropylfluorophosphate, echothiophate, and paraoxon, but mouse BChE spontaneously reactivates. Since human plasma contains no carboxylesterase, only BChE, PON1, and albumin esterases need to be considered when evaluating hydrolysis of an ester drug in human plasma.