Functional specific binding of testosterone to Schistosoma haematobium 28-kilodalton glutathione S-transferase

Abstract

During parasitic disease such as schistosomiasis, sex hormones have an important influence on the age- and gender-dependent level of infection. Since mammal glutathione S-transferase (GST) has the ability to bind hormones and particularly sexual steroids to influence their transport, metabolism, and physiological action, we have evaluated the capacity of testosterone to bind the 28-kDa GST of the Schistosoma haematobium parasite (Sh28GST). For the first time, we have demonstrated a specific binding of testosterone to parasite GST protein with high affinity (K(d) = 2.57 x 10(-7) M). In addition, we have assessed the effect of this binding on Sh28GST enzymatic activity, a mechanism closely associated with the reduction of Schistosoma fecundity. We showed that testosterone has the functional ability to inhibit the Sh28GST enzymatic activity in a dose-dependent manner, suggesting that this hormone could be directly involved in an antifecundity mechanism. This effect seemed to be related to the binding of testosterone to one peptide involved in the enzymatic site (i.e., amino acids 24 to 43). During human infection, binding of sexual hormones to Schistosoma Sh28GST could play a key role in parasite metabolism, especially the decrease of fecundity, and could be involved in the sex-dependent immune response to Sh28GST that we have previously observed in infected adults.

FIG. 1.

Specific binding of testosterone to Sh28GST. Radioactivity counts were determined after incubation of ¹²⁵I-testosterone and Sh28GST protein (unlabeled testosterone at 0 M; see Materials and Methods). To determine the specificity of the binding, competition assay were performed using unlabeled testosterone incubated with increasing concentrations (10⁻⁷ to 10⁻³ M) and ¹²⁵I-testosterone at one fixed concentration. Triplicate determinations were performed at each concentration of unlabeled testosterone, and results are expressed as the mean cpm ± SD.

FIG. 2.

Inhibition of Sh28GST enzymatic activity by testosterone. Sh28GST protein was incubated in the presence of testosterone at increasing concentrations (0 to 5 × 10⁻¹ M). Enzymatic activity of Sh28GST was evaluated at each concentration of testosterone as described in Materials and Methods. Triplicate determinations were performed, and the results are expressed as the mean ± SD of ΔOD/minute reading values. Student’s t test was used to compare the mean between each testosterone concentration and the value without hormones. *, P < 0.05; **, P < 0.01.

FIG. 3.

Specific binding of testosterone to Sh28GST peptides. Synthetic peptides 24–43, 115–131, and 190–211 were incubated with testosterone (peroxidase labeled). Sh28GST protein was incubated as a positive control. Colorimetric development was carried out, and absorbance (OD) was measured at 405 nm. Results were expressed as ΔOD value after subtraction of background (as described in Materials and Methods). Triplicate determinations were performed, and the results are expressed as the mean ± SD of ΔOD reading values.

FIG. 4.

Sensorgrams of the binding of testosterone to Sh28GST peptides using Biacore technology. Biotinylated testosterone was immobilized onto an SA sensor chip coated with streptavidin. Synthetic peptides of Sh28GST (peptides 24–43, 115–131, and 190–211) were injected (100 μg/ml) on immobilized testosterone, and the sensorgrams corresponding to association time (180 s) and dissociation time (1,000 s) were evaluated for each peptide. Results are expressed as resonance units (RU) corresponding to the plateau value for the complex as described in Materials and Methods.