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

Abstract

This article is available online at http://www.jlr.org hydrolase encoded by the ASAH1 gene. The main clinical features include painful and progressively deformed joints, subcutaneous nodules, a hoarse cry due to laryngeal involvement, and premature death. Hepatosplenomegaly and nervous system dysfunction may also occur ( 1, 2 ). Although the pathogenesis of FD is still unclear in terms of the molecular lesions caused by ceramide storage, the involvement of aCDase defi ciency is unquestionable. Recent interest in aCDase also stems from the fact that this enzyme appears to modulate cell functions by controlling the levels of ceramide and sphingoid bases, which are both considered as putative bioactive molecules ( 3 ).Diagnosis of FD must be biochemically confi rmed by the demonstration of defi cient activity of aCDase, which can then be further documented by characterization of the ASAH1 molecular defects. Although aCDase is a very well known enzyme, existing methods for determining its activity and for FD diagnosis still exhibit many disadvantages. The methods that have been used for FD diagnostic purposes can be classifi ed into three groups: i ) aCDase enzymatic assays ( 4-16 ), classically performed with radiolabeled substrates ( 4-13 ), which are rather water-insoluble and require at least one detergent (see Table 1 ) ; ii ) loading tests, consisting of the addition of exogenous radiolabeled sphingolipids, e.g., ceramide ( 17, 18 ), sulfatide ( 19,20 ), or sphingomyelin ( 21 ), on cultured living cells and the study of their metabolism; and iii ) determination of accumulated ceramide, either by sophisticated chromatographic methods ( 22,23 ) or through the use of the diacylglycerol kinase assay in the presence of ␥ [ Farber disease (FD) is a rare inherited lipid storage disorder, also known as lipogranulomatosis, which is characterized by accumulation of ceramide in the cells and tissues of patients ( 1, 2 ). This accumulation is the consequence of a defi cient intracellular activity of aCDase, a lysosomal

Activity of neutral and alkaline ceramidases on fluorogenic N-acylated coumarin-containing aminodiols

“…In agreement with the AC activity of NAAA, in this article, we show that NAAA hydrolyzes acceptor FRET pairs are located as part of the acyl chain and/or the sphingoid base of the substrate, and fl uorescence is displayed by the ceramidase hydrolysis-promoted fl uorescence quencher release. On the other hand, a series of fl uorogenic coumarin-containing ceramidase substrates has also been described ( 10,11 ). These compounds (RBM14 compounds, Fig.…”

“…1 ), to determine AC activity. Other analogs of RBM14C16 with different N -acyl chain lengths were later reported as AC substrates and for use in diagnosis of Farber disease ( 11 ). Among the several analogs, RBM14C12 was the preferred substrate for AC.…”

“…2E ). Interestingly, the N -C8 analog was also hydrolyzed by mNC, in contrast to AC, which does not accept this compound as substrate ( 11 ), and the hNC and bacterial NC, for which the C8 and the C10 derivatives are poor substrates.…”

“…Most ceramidase inhibitors have been discovered after either rational design or screening of a small series of compounds. Although a number of procedures for the determination of ceramidase activities have been reported ( 9 ), massive screening relies on the availability of high-throughput methods, only a few of which have been described (10)(11)(12). These include a fl uorescent sphingolipid fl uorescence resonance energy transfer (FRET) probe that allows homogeneous ratiometric determination of enzyme activity in real-time ( 12 ).…”

Acid ceramidase as a therapeutic target in metastatic prostate cancer

“…For the assay, 75 µl of reaction buffer (100 mM sodium acetate buffer, pH 4.5, for acid ceramidase activity), containing 40 µM RBM14C12 fl uorogenic substrate ( 22 ) (with or without test compounds), was mixed with 25 µl of the cell lysates (20 µg of total protein content) and incubated at 37°C for 3 h. For time dependence of inhibition, incubations were carried out for 0.5, 1, 2, and 3 h with different amounts of protein. The reaction was stopped by addition of methanol followed by NaIO 4 (2.5 mg/ml in 200 mM glycine/NaOH buffer, pH 10.6).…”