Ascaris lumbricoides ß carbonic anhydrase: a potential target enzyme for treatment of ascariasis.

BACKGROUND: A parasitic roundworm, Ascaris lumbricoides, is the causative agent of ascariasis, with approximately 760 million cases around the world. Helminthic infections occur with a high prevalence mostly in tropical and developing xcountries. Therefore, design of affordable broad-spectrum anti-helminthic agents against a variety of pathogens, including not only A. lumbricoides but also hookworms and whipworms, is desirable. Beta carbonic anhydrases (ß-CAs) are considered promising targets of novel anthelminthics because these enzymes are present in various parasites, while completely absent in vertebrates. METHODS: In this study, we identified an A. lumbricoides ß-CA (AIBCA) protein from protein sequence data using bioinformatics tools. We used computational biology resources and methods (including InterPro, CATH/Gene3D, KEGG, and METACYC) to analyze AlBCA and define potential roles of this enzyme in biological pathways. The AlBCA gene was cloned into pFastBac1, and recombinant AIBCA was produced in sf-9 insect cells. Kinetics of AlBCA were analyzed by a stopped-flow method. RESULTS: Multiple sequence alignment revealed that AIBCA contains the two sequence motifs, CXDXR and HXXC, typical for ß-CAs. Recombinant AIBCA showed significant CA catalytic activity with kcat of 6.0 × 10(5) s(-1) and kcat/KM of 4.3 × 10(7) M(-1) s(-1). The classical CA inhibitor, acetazolamide, showed an inhibition constant of 84.1 nM. Computational modeling suggests that the molecular architecture of AIBCA is highly similar to several other known ß-CA structures. Functional predictions suggest that AIBCA might play a role in bicarbonate-mediated metabolic pathways, such as gluconeogenesis and removal of metabolically produced cyanate. CONCLUSIONS: These results open new avenues to further investigate the precise functions of ß-CAs in parasites and suggest that novel ß-CA specific inhibitors should be developed and tested against helminthic diseases.

Proteomic and immunochemical characterization of glutathione transferase as a new allergen of the nematode Ascaris lumbricoides.

Helminth infections and allergy have evolutionary and clinical links. Infection with the nematode Ascaris lumbricoides induces IgE against several molecules including invertebrate pan-allergens. These antibodies influence the pathogenesis and diagnosis of allergy; therefore, studying parasitic and non-parasitic allergens is essential to understand both helminth immunity and allergy. Glutathione transferases (GSTs) from cockroach and house dust mites are clinically relevant allergens and comparative studies between them and the GST from A. lumbricoides (GSTA) are necessary to evaluate their allergenicity. We sought to analyze the allergenic potential of GSTA in connection with the IgE response to non-parasitic GSTs. IgE to purified GSTs from Ascaris (nGSTA and rGSTA), house dust mites (rDer p 8, nBlo t 8 and rBlo t 8), and cockroach (rBla g 5) was measured by ELISA in subjects from Cartagena, Colombia. Also, multidimensional proteomic approaches were used to study the extract of A. lumbricoides and investigate the existence of GST isoforms. We found that among asthmatics, the strength of IgE levels to GSTA was significantly higher than to mite and cockroach GSTs, and there was a strong positive correlation between IgE levels to these molecules. Specific IgE to GSTA was found in 13.2% of controls and 19.5% of asthmatics. In addition nGSTA induced wheal and flare in skin of sensitized asthmatics indicating that it might be of clinical relevance for some patients. Frequency and IgE levels to GSTA were higher in childhood and declined with age. At least six GST isoforms in A. lumbricoides bind human IgE. Four isoforms were the most abundant and several amino acid substitutions were found, mainly on the N-terminal domain. In conclusion, a new allergenic component of Ascaris has been discovered; it could have clinical impact in allergic patients and influence the diagnosis of mite and cockroach allergy in tropical environments.

Immunolocalization of arginine kinase (AK) in Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides.

Arginine kinase (AK) is a member of the phosphagen kinase family. AK plays a major role in cellular energy metabolism in invertebrates including nematodes. In the present study, we performed the direct immunofluorescence test to determine the immunolocalization of AK in different stages of the life cycle (eggs, larvae, and adult worms) of Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides. Our results indicated variable levels of expression of AK in different stages. Moreover, strong fluorescence was observed in cleaving eggs than in dormant eggs. The highest activity of the enzyme was observed in the fully developed eggs. This may be due to high expression of AK in embryonic development, which is associated with increased energy demand due to cleavage and cellular differentiation. Surprisingly, expression of AK is significantly higher in the middle part and posterior end compared to anterior end of the larvae. In addition, AK is highly concentrated in cellular and metabolically active parts of the body such as hypodermis, muscle, intestine, ovaries, oviducts, and uterus, while it is absent in noncellular areas like cuticle. The present study revealed the presence of AK in T. canis, A. lumbricoides, and T. vitulorum and that it plays a major role in energy metabolism of these nematodes. Interestingly, antiserum was prepared against the recombinant T. canis AK and reacts with the native AKs of T. canis, A. lumbricoides, and T. vitulorum. AK levels could vary in relation to maximum potential rates of ATP turnover, oxidative capacity, and energy output. Further studies on subcellular localization of AK in these important helminths provide new information for researchers to develop effective anthelmintics against the parasites of veterinary and of public health importance.

Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides.

The cysteine protease inhibitor from Ascaris lumbricoides, a roundworm that lives in the human intestine, may be involved in the suppression of human immune responses. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of the cysteine protease inhibitor from A. lumbricoides are reported. The rod-shaped crystal belonged to space group C2, with unit-cell parameters a = 99.40, b = 37.52, c = 62.92 Å, ß = 118.26°. The crystal diffracted to 2.1 Šresolution and contained two molecules in the asymmetric unit.

Ascaris haemoglobin is a nitric oxide-activated 'deoxygenase'.

The parasitic nematode Ascaris lumbricoides infects one billion people worldwide. Its perienteric fluid contains an octameric haemoglobin that binds oxygen nearly 25,000 times more tightly than does human haemoglobin. Despite numerous investigations, the biological function of this molecule has remained elusive. The distal haem pocket contains a metal, oxygen and thiol, all of which are known to be reactive with nitric oxide. Here we show that Ascaris haemoglobin enzymatically consumes oxygen in a reaction driven by nitric oxide, thus keeping the perienteric fluid hypoxic. The mechanism of this reaction involves unprecedented chemistry of a haem group, a thiol and nitric oxide. We propose that Ascaris haemoglobin functions as a 'deoxygenase', using nitric oxide to detoxify oxygen. The structural and functional adaptations of Ascaris haemoglobin suggest that the molecular evolution of haemoglobin can be rationalized by its nitric oxide related functions.

Conservation, evolution, and specificity in cellular control by protein phosphorylation.

The glycolytic control enzyme phosphofructokinase from the parasitic nematode Ascaris lumbricoides is regulated by reversible phosphorylation. The enzyme is phosphorylated by an atypical cyclic adenosine monophosphate (cAMP)-dependent protein kinase whose substrate specificity deviates from that of the mammalian protein kinase. This variation is explained by structural peculiarities on the surface part of the catalytic groove of the protein kinase. Also, the protein phosphatases responsible for the reversal of phosphorylation appear to act specifically in glycolysis and are different from those participating in regulation of glycogenolysis.

Enzyme polymorphisms in Ascaris lumbricoides in Bangladesh.

Fourteen enzymes of Ascaris lumbricoides were analysed electrophoretically on cellulose acetate plates; 8 stained well and 3 were found to be polymorphic. Allelic and genotypic frequencies at the 3 polymorphic enzyme loci, Mpi, Pgi and 6-Pgd, were determined in a sample of 117 worms from 8 children living in different houses in an urban slum in Bangladesh. Allele frequencies in samples of parasites from the different children were compared to test for the possibility of non-random distribution of parasite genotypes between people. No strong evidence of differences was found. Diploid genotype frequencies did not deviate significantly from those expected from the Hardy-Weinberg equilibrium. Significant linkage disequilibrium was observed in one of 3 pairs of enzyme loci tested, which might suggest that some genetic subdivision exists in the local A. lumbricoides population, although no strong interference should be made from this single result. Overall, the results suggest that the worms sampled formed part of a single population which appears to be randomly mating.

[The presence of antioxidative defense enzymes in the ascarid Ascaris lumbricoides]. / Nalichie fermentov antiokisilitel'noi zashchity u askardiy Ascaris lumbricoides.

Superoxide dismutase, glutathione peroxidase and glutathione have been discovered in muscles of the ringworm A. lumbricoides. Superoxide dismutase activity was also revealed in eggs and larvae. Using polyacrylamide gel electrophoresis and electrofocusing, it was shown that this enzyme exists in ringworm muscles in multiple molecular forms. Oxygen poisoning of the ringworm and aerobic incubation of muscle homogenates in the presence of Fe+2 do not result in accumulation of malonyldialdehyde. Biochemical defence mechanisms are discussed against oxygen poisoning of the organisms which live under the conditions of reduced and changing pO2.