Cloning of a Human Acid Sphingomyelinase cDNA with a New Mutation That Renders the Enzyme Inactive¹

A cDNA encoding human acid sphingomyelinase was initially obtained by screening a placental cDNA library in λ gt11 with a synthetic oligonucleotide probe and subsequently with partial cDNA. The full-length cDNA, hPSM55, comprised 2, 376 nucleotides, with a 5' untranslated sequence of 122 nucleotides, an open reading frame of 1, 884 nucleotides encoding a protein of 627 amino acids, and a 3' untranslated region of 370 bases. hPSM55 was almost identical to pASM-lFL reported by Schuchman et al. (J. Biol. Chem 266, 8531–8539, 1991) except for a 6 base pair deletion in the signal peptide, which indicated the possible removal of valine and leucine residues between positions 36 and 37, and a ⁴⁶³T- to C-transition, which indicated a possible substitution of ¹⁵⁵arginine for cystine. This cDNA was expressed in both COS-7 cells and Chinese hamster ovary cells. There was no increase in acid sphingomyelinase activity in either cell line following transfection. However, the correction of a single base change, ⁴⁶³C to T, in hPSM55 caused increased acid sphingomyelinase activity in transfectants. These results suggest that the mutation of nucleotide ⁴⁶³C to T plays an important role in the catalytic activity of acid sphingomyelinase.