Abstract
During their infective stages, hookworms release excretory-secretory (E-S) products, small molecules, and proteins to help evade and suppress the host's 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. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). In this study, two ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using both a ligand competition assay and microscale thermophoresis. The fatty acid moieties of both ascarosides tested and ascr#3, an ascaroside found in rat hookworm E-S products, bind to Na-ASP-2's palmitate binding cavity. These molecules were confirmed to bind to the palmitate but not the sterol binding sites. An ascaroside, oscr#10, which is not found in hookworm E-S products, does not bind to Na-ASP-2. More studies are required to determine the structural basis of ascarosides binding by Na-ASP-2 and to understand the physiological significance of these observations.
Highlights
Necator americanus and Ancylostoma duodenale are hookworms causing a disease burden of over 22 million disability-adjusted life years (de Silva et al, 2003; Hotez, 2007; Murray et al, 2013; Diemert et al, 2018)
Our studies showed that the binding of [3H]-palmitic acid by Na-Ancylostoma secreted protein (ASP)-2 was competed by the ascaroside, ascr#3 (1) and by all the fatty acid moieties 3-5 tested with the same order of magnitude, but not by the ascaroside, oscr#10 (2), Figure 4B
Our results reveal that the fatty acid moieties of ascarosides, ascr#3 and oscr#10 bind Na-ASP-2 and Pry1, with the latter SCP/TAPS protein from Saccharomyces cerevisiae serving as a control
Summary
Necator americanus and Ancylostoma duodenale are hookworms causing a disease burden of over 22 million disability-adjusted life years (de Silva et al, 2003; Hotez, 2007; Murray et al, 2013; Diemert et al, 2018). The most abundant proteins secreted by third-stage infective larvae (L3) of N. americanus upon host entry are N. americanus Ancylostoma secreted protein 1 (Na-ASP-1) and N. americanus Ancylostoma secreted protein 2 (Na-ASP-2) (Hotez et al, 2003) These Ancylostoma secreted proteins are the major protein components of the L3 excretory-secretory (E-S) products that facilitate the evasion and suppression of the host’s immune system and have been found in parasitic nematodes (Hawdon et al, 1995, 1996, 1999; Hawdon and Hotez, 1996; Gao et al, 2001; Zhan et al, 2003; Asojo et al, 2018; Darwiche et al, 2018). Sterols and fatty acid bind at these two different and independent binding sites on the CAP domain of SCP/TAPS proteins have been confirmed for multiple CAP proteins and with mutagenesis studies (Darwiche and Schneiter, 2017; Darwiche et al, 2017a; Asojo et al, 2018)
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