Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2) selectively binds an ascaroside (ascr#3)

During their infective stages, hookworms release excretory-secretory (E-S) products, including small molecules and proteins, to help evade and suppress the hosts immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. Ascarosides play vital roles in signaling, development, reproduction, and survival. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). ASP belongs to the SCP/TAPS (sperm-coating protein / Tpx / antigen 5 / pathogenesis related-1 / Sc7) superfamily of proteins, members of which have previously been shown to bind to eicosanoids and fatty acids. These molecules are structurally similar to the fatty acid moieties of ascarosides. The objective of this study was to determine if the hookworm ASP; N. americanus Ancylostoma secreted protein 2 (Na-ASP-2) binds to the ascarosides or their fatty acid moieties. We describe investigations of our hypothesis that there is a functional relationship between the major secreted proteins and signaling small molecules found in hookworm E-S products. To accomplish this, several ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using a ligand competition assay and microscale thermophoresis. Our results reveal that the fatty acid moieties of the ascarosides, bind specifically to the palmitic acid binding cavity of Na-ASP-2. Additionally, ascr#3, an ascaroside that is present in mammalian hookworm E-S products binds to the palmitic acid binding cavity of Na-ASP-2, whereas oscr#10 which is not found in hookworm E-S products does not bind. Future studies are required to determine the structural basis of ascaroside binding by Na-ASP-2 and to understand the physiological significance of these observations.