CLONING OF A HUMAN ACID SPHINGOMYELINASE CDNA WITH A NEW MUTATION THAT RENDERS THE ENZYME INACTIVE

A cDNA encoding human acid sphingomyelinase was initially obtained by screening a placental cDNA library in lambda gt11 with a synthetic oli gonucleotide probe and subsequently with partial cDNA. The full-length cDNA, hPSM55, comprised 2,376 nucleotides, with a 5' untranslated seq uence of 122 nucleotides, an open reading frame of 1,884 nucleotides e ncoding a protein of 627 amino acids, and a 3' untranslated region of 370 bases. hPSM55 was almost identical to pASM-1FL reported by Schuchm an et al. (J. Biol. Chem. 266,8531-8539, 1991) except for a 6 base pai r deletion in the signal peptide, which indicated the possible removal of valine and leucine residues between positions 36 and 37, and a 463 T- to C-transition, which indicated a possible substitution of 155argi nine for cystine. This cDNA was expressed in both COS-7 cells and Chin ese hamster ovary cells. There was no increase in acid sphingomyelinas e activity in either cell line following transfection. However, the co rrection of a single base change, 463C to T, in hPSM55 caused increase d acid sphingomyelinase activity in transfectants. These results sugge st that the mutation of nucleotide 463C to T plays an important role i n the catalytic activity of acid sphingomyelinase.