Necator americanus Ancylostoma Secreted Protein-2 (Na-ASP-2) Binds an Ascaroside (ascr#3) in Its Fatty Acid Binding Site

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.

Keywords: CAP [cysteine-rich secretory protein (CRISP)/antigen 5/pathogenesis related-1 (PR-1)]; TAPs [testis specific proteins (Tpx)/antigen 5 (Ag5)/pathogenesis related-1 (PR-1)/Sc7]; lipid binding; sperm coating protein (SCP); venom allergen-like (VAL).

Figure 1

Targeted ascarosides and their fatty acid moieties. The corresponding ascarosides are ascr#3 (1); oscr#10 (2) and their side chain moieties are 3-5. Compound names are 3 = (R)-8-hydroxynonanoic acid, 4 = (R, E)-8-hydroxynon-2-enoic acid, and 5 = 9-hydroxynonanoic acid.

Figure 2

Synthesis of ascarosides. The synthetic pathway designed for protected ascarylose 8, ascr#3 (1), oscr#10 (2) are illustrated. Detailed synthesis methods are described in the supplementary methods.

Figure 3

Na-ASP-2 binds both cholesterol and free palmitic acid. (A) Ligand binding of [³H]-cholesterol to Na-ASP-2. Purified Na-ASP-2 (100 pmol) was incubated with increasing concentrations of [³H]-cholesterol (100–400 pmol), in absence and presence of 400 pmol of unlabeled cholesterol (chol). The protein was separated from the unbound ligand by adsorption to an anion-exchange matrix and the protein-bound radioligand was quantified by scintillation counting. The background curve shows values obtained in the absence of added protein. Data represent mean ± SD of 3 independent experiments. (B) Competitive binding of unlabeled cholesterol (50 or 5,000 pmol) to Na-ASP-2. Binding of [³H]-cholesterol (50 pmol) to Na-ASP-2 (100 pmol) was assessed in the presence of the indicated concentrations of unlabeled cholesterol (chol). Each data point is the average of duplicate assays and represents the amount of [³H]-cholesterol bound relative to a control containing no unlabeled cholesterol. (C) Ligand binding of [³H]-palmitic acid to Na-ASP-2. Purified Na-ASP-2 (100 pmol) was incubated with increasing concentrations of [³H]-palmitic acid (100–400 pmol), in absence and presence of 400 pmol of unlabeled palmitic acid (pal). (D) Competitive binding of unlabeled palmitic acid (50 or 5,000 pmol) to Na-ASP-2. Binding of [³H]-palmitic acid (50 pmol) to Na-ASP-2 (100 pmol) was assessed in the presence of the indicated concentration of unlabeled palmitic acid (pal). Each data point is the average of duplicate assays and represents the amount of [³H]-palmitic acid bound relative to a control containing only labeled palmitic acid. Data represent mean ± SD of three independent experiments. Asterisks denote statistical significance relative to the control containing only the radiolabeled ligand and either purified Na-ASP-2 or Pry1. (**p < 0.001; *p < 0.01).

Figure 4

Binding of ligands to Na-ASP-2 and Pry1. (A) Free fatty acids and ascarosides fail to compete with [³H]-cholesterol for binding to Na-ASP-2. Binding of [³H]-cholesterol (50 pmol) to Na-ASP-2 (100 pmol) was assessed in the presence of (50 pmol and 500 pmol) of unlabeled ascarosides or fatty acid moieties (1-5) (B) Fatty acids moieties and ascr#3 compete with [³H]-palmitic acid for binding to Na-ASP-2. (C) Only fatty acids moieties compete [³H]-palmitic acid for binding to Pry1. Competitive binding was tested with either 50 or 500 pmol of the unlabeled ligands and 50 pmol of [³H]-palmitic acid for binding to 100 pmol purified Na-ASP-2 or Pry1. The ascarosides tested are (1) (ascr#3) and (2) (oscr#10) while the fatty acids are 3 [(R)-8-hydroxynonanoic acid], 4 [(R, E)-8-hydroxynon-2-enoic acid], and 5 (9-hydroxynonanoic acid). Data represent mean ± SD of 3 independent experiments. Asterisks denote statistical significance relative to the control containing only the radiolabeled ligand and either purified Na-ASP-2 or Pry1. (**p < 0.001; *p < 0.01). n.s., not significant.

Figure 5

Na-ASP-2 selectively binds ascr#3 but not oscr#10. Binding of ascarosides and their fatty acid moieties by Pry1 and Na-ASP-2 as measured by microscale thermophoresis. (A,G) Palmitic acid; (B,H) ascr#3; (C,I) oscr#10; (D,J) (R)-8-hydroxynonanoic acid; (E,K) (R, E)-8-hydroxynon-2-enoic acid; (F,L) 9-hydroxynonanoic acid. Pry1 binds palmitic acid and free hydroxylated nanonoic acids with similar affinities but binds neither the ascarosides ascr#3 and oscr#10. Na-ASP-2 binds palmitic acid, ascr#3 and free hydroxylated nanonoic acids with similar affinities but not oscr#10. The K_d values are indicated in each figure with N/A (not applicable) where there is no binding. Data represent mean ± SD of three independent experiments.

Figure 6

Comparison of fatty acid binding cavities of Na-ASP-2 and Pry1. (A) Structure based alignment of Na-ASP-2, Pry1, and three N. brasilensis SCP/TAPs proteins (genbank codes VDL79275.1; VDL83979.1; and VDL79274.1). The sequences are aligned with ClustalW Omega and the secondary structural features are illustrated with the coordinates of Hp-VAL-4 and Pry1 using ESPript (Gouet et al., 2003). The alpha helices (alpha 1 and alpha 3) that form the palmitate-binding cavity have similar lengths for Na-ASP-2 and the N. brasilensis proteins whereas Pry1 has shorter helices. The secondary structure elements shown are alpha helices (α), 3₁₀-helices (h), beta strands (β), and beta turns (TT). Identical residues are shown in solid red, and conserved residues are in red. The locations of the cysteine residues involved in disulfide bonds are numbered in green. (B) Both of the helices (α1 and α3) forming the palmitic acid binding cavity of Pry1 (cyan) are shorter than those from Na-ASP-2 (gray). Also shown in magenta is the stick structure of palmitate superposed from the X-ray structure of the complex of tablysin-15 with palmitate (Ma et al., 2011).