Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses

To examine the intracellular localization of neutral sphingomyelinase 1 (nS Mase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E. coli. It has been reported that, in rat live r or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amou nt in nucleus and cytoplasm. The biochemical properties, dithiothreitol req uirement and high salt concentration dependency, of cloned and expressed nS Mase I resemble those of previously described nuclear neutral SMase of AH79 74. The present study was therefore focused on the nuclear localization of this enzyme. Western blotting of subcellular fractions using anti-rat nSMas e 1 antibody revealed most nSMase 1 to be associated with the nuclei and so me with microsomes, but not with plasma membranes. Consistently, neutral SM ase activity in nuclear extract was immunoprecipitated by the antibody, whi le that of plasma membranes was not. The results indicate that nSMase 1 mai nly resides in the nucleus and may thus differ from neutral SMase in plasma membrane. On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive pro tein bands on western blotting, suggesting that a large part of nuclear neu tral SMase may be nSMase 1. Removal of the nuclear envelope by treatment wi th Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e. nuclear envelope, ch romatin, and nuclear matrix) revealed nSMase I signal exclusively in the nu clear matrix. Immunocytochemistry with AH7974, as well as rat fibroblast ce ll line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, w ith some in the cytoplasm. Moreover, immuno-electron microscopy clearly sho wed the signal of nSMase 1 to be more dense in the nucleus than in the cyto plasm of AH7974.