Molecular Cloning and Characterization of a Full-length Complementary DNA Encoding Human Acid Ceramidase

Abstract

Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1.23) hydrolyzes the sphingolipid ceramide into sphingosine and free fatty acid. Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule. Moreover, an inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease. Human AC was purified from urine, and 117 amino acid residues were determined by microsequencing. Degenerative oligonucleotide probes were then constructed and used to screen for human fibroblast and pituitary cDNA libraries. Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA containing a 17-base pair (bp) 5-untranslated sequence, a 1185-bp open reading frame encoding 395 amino acids, a 1110-bp 3-untranslated sequence, and an 18-bp poly(A) tail. Transient expression of the full-length cDNA in COS-1 cells led to a 10-fold increase in AC activity. In addition, biosynthetic studies carried out in the transfected cells demonstrated that 13-kDa (␣) and 40-kDa (␤) AC subunits were derived from a common 55-kDa precursor encoded by the full-length cDNA. This protein pattern was identical to that seen in normal human skin fibroblasts. A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming the authenticity of the full-length cDNA.Human acid ceramidase ((AC) 1 N-acylsphingosine amidohydrolase, EC 3.5.1.23) catalyzes the hydrolysis of ceramide to free fatty acid and sphingosine (1). An inherited deficiency of AC activity leads to the lysosomal storage disorder known as Farber disease (FD), also called Farber lipogranulomatosis (2). Patients with FD accumulate ceramide in most tissues, leading to painful swelling of the joints and tendons, pulmonary insufficiency, and a shortened life-span. The clinical diagnosis of FD is usually confirmed by biochemical methods, including the determination of lysosomal ceramide accumulation and/or the deficiency of AC activity. To date, seven FD subtypes have been described with varying degrees of clinical involvement; notably, a direct correlation between the amount of ceramide accumulation and the clinical severity of FD patients has recently been demonstrated (3).In addition to its central role in disease pathogenesis, sphingolipid biosynthesis, and membrane formation, ceramide is an important cell-signaling molecule involved in a variety of diverse processes such as neurite growth, monocyte differentiation, and Fas (Apo1/CD95)-induced apoptosis (for reviews, see Refs. 4 and 5). Moreover, sphingosine (the catabolic product of ceramide degradation) has been shown to inhibit protein kinase C activity and can exert a variety of effects on cell growth and differentiation (6 -8). Since ceramide degradation is the only catabolic source of intracellular sphingosine (9, 10), AC activity may be the rate-limiting step in determining the intracellular levels of this compound.About 2 years ago, AC was purified...

Bone marrow transplantation for infantile ceramidase deficiency (Farber disease)

“…5 Molecular analyses have identified specific mutations of the acid ceramidase gene among the different disease phenotypes. 4,5 The infantile phenotype of acid ceramidase deficiency bears the eponym Farber disease and is associated with painful swelling around joints, multiple subcutaneous nodules (lipogranulomata), hoarseness due to lipogranulomata in the upper respiratory tract, psychomotor retardation, and failure to thrive. 6,7 Recurrent pulmonary infections, aspiration pneumonitis, swallowing difficulties and hepatomegaly are common.…”

Molecular Characterization of N-Acylethanolamine-hydrolyzing Acid Amidase, a Novel Member of the Choloylglycine Hydrolase Family with Structural and Functional Similarity to Acid Ceramidase

“…These primers were designed according to the reported nucleotide sequence of human AC (GenBank TM accession number U70063) (47). The amplified 1.2-kb fragment was digested with KpnI and BamHI and inserted into pcDNA 3.1(ϩ).…”

The role of ceramide in receptor- and stress-induced apoptosis studied in acidic ceramidase-deficient Farber disease cells

“…Farber disease, also called Farber lipogranulomatosis, is a rare lysosomal storage disorder characterized by an inherited de®ciency of acidic ceramidase, which catalyzes the hydrolysis of ceramide to free fatty acid and sphingosine (Koch et al, 1996). Patients with Farber disease accumulate ceramide in most tissues, leading to painful swelling of the joints and tendons, pulmonary insu ciency, and a shortened life-span (Fujiwaki et al, 1992;Moser et al, 1969).…”