Purification, characterization, and biosynthesis of human acid ceramidase

Abstract

Acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) is the lysosomal enzyme catalyzing the hydrolysis of ceramide to sphingosine and free fatty acid. Its inherited deficiency causes ceramide accumulation in Farber's disease. The enzyme was purified to apparent homogeneity from human urine by sequential chromatography on octyl-Sepharose, concanavalin A-Sepharose, blue-Sepharose, and DEAE-cellulose. The final preparation, which was enriched approximately 4450-fold over the starting material, resulted in a polypeptide of approximately 50 kDa and could be reduced into two subunits of approximately 13 (alpha) and approximately 40 (beta) kDa. Treatment of the purified enzyme with endoglycosidase H or peptido-N-glycanase F reduced the molecular mass of the beta subunit to approximately 30-35 and approximately 27 kDa, respectively. In contrast, the molecular mass of the alpha subunit was unchanged. The purified enzyme had an apparent Km of 149 microM and a Vmax of 136 nmol/mg/h using N-lauroylsphingosine as substrate. Polyclonal antibodies were raised against the purified urinary enzyme and used to investigate the biosynthesis of acid ceramidase. Immunoprecipitation studies on metabolically labeled skin fibroblasts indicated that both subunits arose from a single precursor of approximately 55 kDa. A minor portion of newly synthesized acid ceramidase was secreted into the medium as a monomeric 47-kDa protein, indicating that generation of the mature heterodimeric enzyme occurred in endosomal and/or lysosomal compartments.