The Major Cat Allergen Fel d 1 Binds Steroid and Fatty Acid Semiochemicals: A Combined In Silico and In Vitro Study

Abstract

The major cat allergen Fel d 1 is a tetrameric glycoprotein of the secretoglobin superfamily. Structural aspects and allergenic properties of this protein have been investigated, but its physiological function remains unclear. Fel d 1 is assumed to bind lipids and steroids like the mouse androgen-binding protein, which is involved in chemical communication, either as a semiochemical carrier or a semiochemical itself. This study focused on the binding activity of a recombinant model of Fel d 1 (rFel d 1) towards semiochemical analogs, i.e., fatty acids and steroids, using both in silico calculations and fluorescence measurements. In silico analyses were first adopted to model the interactions of potential ligands, which were then tested in binding assays using the fluorescent reporter N-phenyl-1-naphthylamine. Good ligands were fatty acids, such as the lauric, oleic, linoleic, and myristic fatty acids, as well as steroids like androstenone, pregnenolone, and progesterone, that were predicted by in silico molecular models to bind into the central and surface cavities of rFel d 1, respectively. The lowest dissociation constants were shown by lauric acid (2.6 µM) and androstenone (2.4 µM). The specific affinity of rFel d 1 to semiochemicals supports a function of the protein in cat's chemical communication, and highlights a putative role of secretoglobins in protein semiochemistry.

Keywords: 2D interaction maps; N-phenyl-1-naphthylamine; chemical communication; in silico docking; ligand-binding assays; molecular modeling; odorant-binding protein; pheromone; protein–ligand interactions; secretoglobin.

Figure 1

1-NPN binding to rFel d 1. To a 1-µM solution of the protein in 50 mM Tris-HCl, pH 7.4, aliquots of 1 mM solution of 1-NPN in methanol were added to final concentrations of 1–20 µM. (a) The representative emission curves experimentally obtained. No significant fluorescence emission was recorded in the same conditions with the protein alone (not shown). (b) The saturation binding curve obtained from the average of three experiments. Data were analyzed with GraphPad software and gave a value of 5.8 µM for the binding constant (SD 0.62). The relative Scatchard plot (inset) shows a linear behavior, apparently indicating the presence of a single binding site without cooperativity effects.

Figure 2

Competitive binding of selected fatty acids (a) and steroids (b) to rFel d 1. Fluorescence emission spectra were recorded at 25 °C in the presence of 1 µM of rFel d 1 and 2 μM of 1-NPN; excitation and emission wavelengths were 337 and 407 nm, respectively. Fluorescence of probe-protein complexes in the absence of a competitor was normalized to 100%.

Figure 3

Molecular residue interactions of Fel d 1 with the best ligands, lauric acid (a) and androstenone (b). The interactions are shown in molecular ligand binding view (surface mesh) with a 2D-interaction map of the selective best-fitting ligands to the central and surface binding cavities of Fel d 1. The 2D map reports H-bond interactions in green color and hydrophobic interactions (van der Waals and alkyl/pi-alkyl) in pink color. All the amino acid residue interactions within 4 Å from the ligand are shown.