Autoproteolytic Cleavage and Activation of Human Acid Ceramidase

Abstract

Herein we report the mechanism of human acid ceramidase (AC; N-acylsphingosine deacylase) cleavage and activation. A highly purified, recombinant human AC precursor underwent self-cleavage into ␣ and ␤ subunits, similar to other members of the N-terminal nucleophile hydrolase superfamily. This reaction proceeded with first order kinetics, characteristic of selfcleavage. AC self-cleavage occurred most rapidly at acidic pH, but also at neutral pH. Site-directed mutagenesis and expression studies demonstrated that Cys-143 was an essential nucleophile that was required at the cleavage site. Other amino acids participating in AC cleavage included Arg-159 and Asp-162. Mutations at these three amino acids prevented AC cleavage and activity, the latter assessed using BODIPY-conjugated ceramide. We propose the following mechanism for AC self-cleavage and activation. Asp-162 likely forms a hydrogen bond with Cys-143, initiating a conformational change that allows Arg-159 to act as a proton acceptor. This, in turn, facilitates an intermediate thioether bond between Cys-143 and Ile-142, the site of AC cleavage. Hydrolysis of this bond is catalyzed by water. Treatment of recombinant AC with the cysteine protease inhibitor, methyl methanethiosulfonate, inhibited both cleavage and enzymatic activity, further indicating that cysteine-mediated self-cleavage is required for ceramide hydrolysis. Human acid ceramidase (AC;3 N-acylsphingosine deacylase; EC 3.5.1.23) hydrolyzes the sphingolipid, ceramide, into sphingosine and free fatty acid. AC is considered a lysosomal enzyme since it has optimal in vitro activity at acidic pH, and most of the lipid storage in Farber disease patients (the genetic disorder resulting from the deficiency of this enzyme) occurs within late endosomes and/or lysosomes (1). It is therefore thought that the main function of AC is to participate in lysosomal membrane turnover. A low level, secreted form of AC also has been described (2-4), although its biological function remains unknown.In addition to its important housekeeping function in sphingolipid metabolism, AC participates in signal transduction pathways that regulate various physiological and pathological processes. Recently, it was shown that the AC gene (Asah1) is among the first genes expressed in newly formed mouse embryos, and its deficiency causes embryo death at the two-cell stage (5). In addition, many studies have reported the involvement of AC in complex diseases. For example, AC is overexpressed in several types of human cancer (prostate, head and neck squamous cell, etc.) (6, 7), and cancer therapy based on the inhibition of AC activity has recently been proposed (8, 9). Moreover, impaired ceramide metabolism has been implicated in the pathogenesis of diabetes, Alzheimer disease, atherosclerosis, thrombosis, and cardiomyocyte apoptosis (10 -14). Although the precise role of AC in these common diseases is unknown, the enzyme is likely to act as a rheostat controlling the levels of ceramide, sphingosine, and sphingosine-1-phosphate in cel...

Acid ceramidase as a therapeutic target in metastatic prostate cancer

“…AC belongs to the N -terminal nucleophile (Ntn) hydrolase family ( 48 ). This family of enzymes shares the common feature of having an N -terminal nucleophile, which is generated by autoproteolytic processing ( 49 ).…”

Acid Ceramidase (ASAH1) Represses Steroidogenic Factor 1-Dependent Gene Transcription in H295R Human Adrenocortical Cells by Binding to the Receptor

“…Five human ceramidases have been cloned and are categorized by their optimal pH for in vitro activity as acid (ASAH1) and neutral (ASAH2) as well as three isoforms of alkaline (ACER1 to ACER3) (26,53,78). ASAH1 is a glycoprotein processed from a 55-kDa precursor into a heterodimeric protein formed by ␣ (14-kDa) and ␤ (40-kDa) subunits via autoproteolytic cleavage (3,66). In vivo studies demonstrated that ASAH1 requires sphingolipid activator proteins (SAP; saposin), mainly SAP-D, as cofactors for maximal activity (39).…”

Identification of Cystatin SA as a Novel Inhibitor of Acid Ceramidase

“…We have previously reported that the inactive AC precursor undergoes self-cleavage and activation, similar to other N-terminal nucleophile hydrolase family members (7). To the best of our knowledge this is the only mammalian ceramidase that is known to undergo this activation mechanism.…”

“…We have recently shown that the inactive AC precursor undergoes self-cleavage to form a mature, active enzyme, and that the mechanism of this transformation is similar to other members of the N-terminal nucleophile hydrolase superfamily (7). Typically, the activity of one N-terminal nucleophile hydrolase subfamily member, the cysteine protease, is inhibited by small proteins known as cystatins (8).…”