FIGURE 7.
Sphingomyelinase activity assays of recombinant human SMPDL3A. A, sphingomyelinase activity assay of lysates of CHO-SMPDL3A cells treated ± tetracycline (1 μm, 16 h), using [3H]choline methyl-labeled sphingomyelin/Triton X-100 micelles as substrate under acidic (pH 5.5; 100 mm sodium acetate, 5 mm Zn2+) or neutral (pH 7.5; 100 mm Tris, 5 mm Mg2+) conditions. B, sphingomyelinase activity assay of conditioned serum-free media from CHO-SMPDL3A cells treated ± tetracycline at pH 7.5 under conditions described in A. C, sphingomyelinase activity assay of HI pooled human serum containing 1 μCi if [3H]sphingomyelin (SM). HI serum/sphingomyelin mixtures were supplemented (50% v/v) either with water, 1 unit of B. cereus neutral sphingomyelinase, or conditioned serum-free media from tetracycline-induced (1 μm, 16 h) CHO-SMPDL3A cells. D, lipidomic MS/MS analysis of SMPDL3A-overexpressing human cells. Inset, HEK-SMPDL3A cells were grown to near-confluency in 75-cm2 flasks and treated with or without 1 μm tetracycline for 24 h (n = 6 flasks for each treatment), and whole cell lysates were prepared. Expression of SMPDL3A was examined by Western blotting with 6E3G4A1 anti-SMPDL3A antibodies. Total cellular lipids were extracted from lysates, and lipidomic profiles were generated by tandem LC-MS/MS. Lipid concentrations for each class are normalized to that of the noninduced control cells. No significant differences in the concentrations of any examined lipid class exist between SMPDL3A-induced and -uninduced cells (unpaired two-tailed t test). Lipid class abbreviations are as follows: BMP, bis(monoacylglycerol)phosphate; CE, cholesterol esters; Cer, ceramide; COH, free cholesterol; DHC, dihexosylceramide; dhCer, dihydroceramide; GM, gangliosides; MHC, monohexosylceramide; PG, phosphatidylglycerol; THC, trihexosylceramide; DG, diacylglycerol; TG, triacylglycerol; LPC, lysophosphatidylcholine; LPAF, lyso-platelet-activating factor; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; oddPC, odd chain phosphatidylcholine; PC, phosphatidylcholine; PC(O), alkyphosphatidylcholine; PC(P), phosphatidylcholine plasmalogen; PI, phosphatidylinositol; SM, sphingomyelin; PE, phosphatidylethanolamine; PE(O), alkylphosphatidylethanolamine; PE(P), phosphatidylethanolamine plasmalogen; PS, phosphatidylserine.