CONICET | Buscador de Institutos y Recursos Humanos

The giant kidney worm, Dioctophyme renale, is a debilitating and potentially lethal parasite that typically inhabits and destroys, the hosts right kidney, and may also be found in ectopic sites. It is circumglobally distributed, mainly in wild carnivores that live close to rivers and is increasingly regarded as a threat to other domestic animals and humans. Previous results from our laboratory identified two major proteins present in the pseudocelomic fluid (PF), one of 17 kDa (P17), that has a prosthetic heme group and is possibly related to oxygen transport, and another of 44 kDa (P44), that binds lipids and is secreted. Both proteins would play a crucial role in the biochemistry of the parasite because lacking the metabolic pathways for the syntheses of the heme group, fatty acids, and cholesterol, this organism must obtain these compounds from its host. The aim of this work is to carry out a structural and biophysical characterization of P44, emphasizing its possible biological functions. Therefore, the protein was purified from PF by SEC and then delipidated by HPLC. Purity was checked by SDS-PAGE followed by Western blotting. Glycosylated residues were evidenced using a Schiff based dyeing and by an enzymatic deglycosylation assay. P44 presents both far and near UV CD spectra consistent with a well folded protein. Additionally, denaturation curves were studied by fluorescence, showing high stability towards denaturing agents such urea and guanidinium chloride. To assess function, fluorescence displacement experiments were carried out. Affinity for oleic acid (Kdapp 6.9 µM) and cholesterol (Kdapp 27.6 µM) was measured by their ability to displace DAUDA or ANS probes, respectively. In summary, P44 emerges as a novel nematode lipid binding protein likely involved in the modulation of the host´s immune response.