RESULTS Characterization of SM-deficient and functionally restored cells by transfection with the SM synthase gene

Lysenin is an SM-directed cytolysin purified from the earthworm (14), which binds to membrane SM and induces pore formation in the plasma membrane and subsequent cell death (14). During investigation of the sphingolipid metabolism in SM synthase–defective WR19L mouse lymphoid cells transfected with the human Fas gene (WR19L/Fas; reference 15), we established membrane sphigomyelin-deficient cells, WR/Fas-SM (), which were resistant to lysenin-mediated cell lysis. Recently, we established a functional revertant cell line, designated WR/Fas-SMS1 cells, by transfection of SMS1 (12). WR/Fas-SMS1 cells exhibited restored SM synthesis assayed by radiolabeling of cellular lipids with [14C] serine (Fig. 1 A) and recovered sensitivity against lysenin-mediated cytolysis (not depicted; reference 12). Recently, Yamaji-Hasegawa et al. produced a mutant lysenin, which specifically binds to SM without induction of cell death (16). Using the mutant lysenin conjugated with maltose-binding protein (MBP), we examined SM expression on the plasma membrane of WR/Fas-SM () and WR/Fas-SMS1 cells by confocal microscopy. Expression of SM detected by lysenin–MBP plus FITC-labeled anti-MBP antibody was positive in WR/Fas-SMS1 but not in WR/Fas-SM () cells (Fig. 1 B). Membrane expression of ganglioside GM1 detected by FITC-labeled CTx was observed on both cells (not depicted; reference 12).