Purification and characterization of recombinant human acid sphingomyelinase expressed in insect Sf21 cells.

Publisher Summary Human acid sphingomyelinase (haSMase, EC 3.1.4.12) is a lysosomal enzyme catalyzing the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. An inherited deficiency of the enzyme activity results in Niemann-Pick disease type A and B, an autosomal recessive lipid storage disorder accompanied by a massive accumulation of sphingomyelin in lysosomes. In 1987, acid sphingomyelinase was isolated from human urine. The enzyme was a monomeric 72-kDa glycoprotein containing a protein core of about 61 kDa. The purified urinary haSMase had a specific enzyme activity of about 2.5 mmol sphingomyelin cleaved per hour and milligram protein using a micellar assay system. Several alternatively spliced haSMase cDNAs were isolated and the genomic sequence encoding haSMase was characterized. Ceramide, the product of sphingomyelin degradation, is found to be an important cellular signaling molecule involved in apoptosis, cell differentiation, and proliferation. Recent studies indicate a direct involvement of aSMase in certain receptor-mediated cell signaling events. Studies on the biosynthesis and processing of haSMase in fibroblasts and transfected COS-1 cells reveal extensive posttranslational processing during transport to lysosomes. Many functional and structural studies on haSMase require milligram amounts of enzymatically active protein. However, human tissue is not a suitable enzyme source due to the low aSMase expression level. To obtain essential amounts of the enzyme, this chapter discusses a baculovirus-mediated expression of recombinant haSMase (r-haSMase) in Spodoptera frugiperda 21 (Sf21) cells and develops an efficient purification procedure.