A simple fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease

Abstract

Acid ceramidase (aCDase) is one of several enzymes responsible for ceramide degradation within mammalian cells. As such, aCDase regulates the intracellular levels of the bioactive lipid ceramide. An inherited deficiency of aCDase activity results in Farber disease (FD), also called lipogranulomatosis, which is characterized by ceramide accumulation in the tissues of patients. Diagnosis of FD is confirmed by demonstration of a deficient aCDase activity and the subsequent storage of ceramide. Existing methods include extremely complex assays, many of them using radiolabeled compounds. Therefore, the aCDase assay and the in vitro enzymatic diagnosis of FD are still performed in only a very limited number of specialized laboratories. Here, the new fluorogenic substrate Rbm14-12 was synthesized and characterized as a new tool to determine aCDase activity. The resulting optimized assay was performed in 96-well plates, and different fibroblast and lymphoid cell lines derived from FD patients and controls were tested to measure aCDase activity. As a result, the activity in cells of FD patients was found to be very low or even null. This new fluorogenic method offers a very easy and rapid way for specific and accurate determination of aCDase activity and, consequently, for diagnosis of FD.

Fig. 1.

A: General chemical structure of Rbm14 compounds, (e.g., for Rbm14-12, n = 10). B: Comparison of the hydrolysis rates of the seven substrate analogs. The ceramide analogs (all at 40 µM) with the indicated side chain length were incubated for 3 h in the presence of FD1 AcCer10× (empty bars) or FD1 (solid bars) cell lysates (20 µg protein) in the absence of detergents. The activities measured in FD1 cells (solid bars) are too low to be seen. Results are the means of three independent experiments.

Fig. 2.

pH-dependence of Rbm14-12 hydrolysis by human cells. The ceramide analog (at 20 µM) was incubated for 3 h with lysates (20 µg protein) from three cell lines exhibiting different degrees of aCDase activity: FD1 AcCer10× fibroblasts (△), normal lymphoid cells (○), and FD1 fibroblasts (□). The buffers were: glycine-HCl (pH 2.5 to 3), sodium acetate (pH 3.5 to 5.5), sodium phosphate (pH 6 to 8.1), and glycine-NaOH (pH 8.9 to 9.8). Results are representative of at least four independent experiments.

Fig. 3.

Characteristics of Rbm14-12 hydrolysis. A: Effect of protein concentration on Rbm14-12 hydrolysis. The substrate (at 20 µM) was incubated for 3 h in the presence of different amounts of FD1 AcCer10× fibroblast cell lysates. Results are representative of two independent experiments. B: Time-dependence of Rbm14-12 hydrolysis. The substrate (at 20 µM) was incubated for the indicated time periods in the presence of FD1 AcCer10× cell lysate (20 µg of protein). Data are representative of two independent experiments. C: Effect of detergents on Rbm14-12 hydrolysis. The substrate (at 20 µM) was incubated for 3 h at different pH in the presence or absence (♦) of 0.5% sodium cholate (▪), 0.05% Tween-20 (▴) or 0.1% Triton X-100 (X). Results represent the mean of two independent experiments performed in duplicate. D: Kinetics of Rbm14-12 hydrolysis. The substrate was incubated for 3 h at different concentrations in the presence of FD1 AcCer10× cell lysate (10 to 25 µg of protein). Lineweaver-Burk representation and the resulting parameters are shown. Results are the means of five independent experiments.