MOLECULAR-CLONING AND CHARACTERIZATION OF A FULL-LENGTH COMPLEMENTARY-DNA ENCODING HUMAN ACID CERAMIDASE - IDENTIFICATION OF THE FIRST MOLECULAR LESION CAUSING FARBER-DISEASE

Human acid ceramidase ((AC) N-acylsphingosine amidohydrolase, EC 3.5.1 .23) hydrolyzes the sphingolipid ceramide into sphingosine and free fa tty acid, Ceramide is an essential component of all sphingolipids and an important cell-signaling molecule, Moreover, an inherited deficienc y of AC activity leads to the lysosomal storage disorder known as Farb er disease, Human AC was purified from urine, and 117 amino acid resid ues were determined by microsequencing. Degenerative oligonucleotide p robes were then constructed and used to screen for human fibroblast an d pituitary cDNA libraries, Several partial cDNA clones were obtained, and two of these were combined to construct a full-length cDNA contai ning a 17-base pair (bp) 5'-untranslated sequence, a 1185-bp open read ing frame encoding 395 amino acids, a 1110-bp 3'-untranslated sequence , and an 18-bp poly(A) tail. Transient expression of the full-length c DNA in COS-1 cells led to a 10-fold increase in AC activity, In additi on, biosynthetic studies carried out in the transfected cells demonstr ated that 13-kDa (alpha) and 40-kDa (beta) AC subunits were derived fr om a common 55-kDa precursor encoded by the full length cDNA. This pro tein pattern was identical to that seen in normal human skin fibroblas ts, A homoallelic point mutation (T222K) was also identified in the AC gene of a patient suffering from Farber disease, further confirming t he authenticity of the full-length cDNA.