Due to its involvement in the human genetic disorder Farber Lipogranulomatosis (“FD”), Acid ceramidase (“AC;” N-acylsphingosine deacylase, I.U.B.M.B. Enzyme No. EC 3.5.1.23) is the most extensively studied member of the ceramidase enzyme family. The protein has been purified from several sources, and the human and mouse cDNAs and genes have been obtained (Bernardo et al., “Purification, Characterization, and Biosynthesis of Human Acid Ceramidase,” J. Biol. Chem. 270:11098-102 (1995); Koch et al., “Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase. Identification of the First Molecular Lesion Causing Farber Disease,” J. Biol. Chem. 2711:33110-5 (1996); Li et al., “Cloning and Characterization of the Full-length cDNA and Genomic Sequences Encoding Murine Acid Ceramidase,” Genomics 50:267-74 (1998); Li et al., “The Human Acid Ceramidase Gene (ASAH): Chromosomal Location, Mutation Analysis, and Expression,” Genomics 62:223-31 (1999)). Growing interest in the biology of this and other ceramidases stems from the fact that these enzymes play a central role in ceramide metabolism.
Ceramide is a signaling lipid that is produced in response to various stimuli and extrinsic factors, including serum deprivation and treatment with many chemotherapy drugs, as well as in many human diseases (Hannun, “Function of Ceramide in Coordinating Cellular Responses to Stress,” Science 274:1855-9 (1996); Spiegel et al., “Signal Transduction Through Lipid Second Messengers,” Curr. Opin. Cell. Biol. 8:159-67 (1996)). Inside cells, ceramide can influence growth and differentiation, regulate protein secretion, induce DNA fragmentation and apoptosis, and increase the synthesis and secretion of cytokines Normally present in low amounts, in response to these factors, ceramide is rapidly produced at the cell surface, leading to membrane re-organization and downstream signaling that results in apoptosis. After stimulation, AC and/or other ceramidases may then hydrolyze ceramide into the individual fatty acid and sphingosine components (Gatt, “Enzymic Hydrolysis and Synthesis of Ceramide,” J. Biol. Chem. 238:3131-3 (1963); Gatt, “Enzymatic Hydrolysis of Sphingolipids. 1. Hydrolysis and Synthesis of Ceramides by an Enzyme from Rat Brain,” J. Biol. Chem. 241:3724-31 (1966); Hassler & Bell, “Ceramidase: Enzymology and Metabolic Roles,” Adv. Lip. Res. 26:49-57 (1993)). Because ceramide degradation is the only source of intracellular sphingosine (Rother et al., “Biosynthesis of Sphingolipids: Dihydroceramide and Not Sphinganine Is Desaturated by Cultured Cells,” Biochem. Biophys. Res. Commun. 189:14-20 (1992)), these enzymes may also be rate-limiting steps in determining the intracellular levels of this compound. Importantly, a derivative of sphingosine, sphingosine-1-phosphate (“S1P”), can counteract the apoptotic effects of ceramide (Cuvillier et al., “Suppression of Ceramide-mediated Programmed Cell Death by Sphingosine-1-phosphate,” Nature 381:800-3 (1996)), leading to the suggestion that ceramidases can be “rheostats” that maintain a proper balance between cell growth and death (Spiegel & Merrill, “Sphingolipids Metabolism and Cell Growth Regulation,” FASEB J. 10:1388-97 (1996)).
AC hydrolyzes the amide bond linking the sphingosine and fatty acid moieties of the lipid ceramide (Park and Schuchman, “Acid Ceramidase and Human Disease,” Biochim. Biophys. Acta. 1758(12): 2133-2138 (2006)). Ceramide, sphingosine (and its phosphorylated derivative S1P) are bioactive lipids, and thus the activity of AC must be carefully regulated in cells (Young et al., “Sphingolipids: Regulators of Crosstalk Between Apoptosis and Autophagy,” J. Lipid Res. 54:5-19 (2013). One important mechanism by which AC activity is regulated is the cleavage of the inactive precursor polypeptide into the active enzyme consisting of an alpha and beta subunit linked via disulfide bonds (Shtraizent et al., “Autoproteolytic Cleavage and Activation of Human Acid Ceramidase,” J. Biol. Chem. 283:11253-11259 (2008)). It has previously been shown that recombinant AC produced in Chinese Hamster ovary (“CHO”) cells and secreted into the media is a mixture of inactive precursor and active (cleaved) enzyme (He et al., “Purification and Characterization of Recombinant, Human Acid Ceramidase,” J. Biol. Chem. 278:32978-32986 (2003)).
The present invention is directed to overcoming these and other deficiencies in the art.