出版社：American Society for Biochemistry and Molecular Biology
摘要：The synthesis of an ω-pyrene-labeled 1- O -alkyl- sn -glycerol was performed using a chirospecific method starting from R -(−)-2,3- O -isopropylidene- sn -glycerol. The product, 1- O -[9′-(1″-pyrenyl)]nonyl- sn -glycerol (pAG), is a fluorescent ether lipid that has a pyrene moiety covalently attached at the alkyl chain terminus. pAG was taken into CHO-K1 cells and a plasmalogen-deficient variant of CHO-K1, NRel-4. This variant is defective in dihydroxyacetonephosphate acyltransferase, which catalyzes the first step in plasmenylethanolamine (PlsEtn) biosynthesis. pAG was incorporated primarily into ethanolamine and choline phospholipids as well as a neutral lipid fraction tentatively identified as alkyldiacylglycerol. NRel-4 accumulated more fluorescence in the phospholipid fraction than CHO-K1, specifically in the ethanolamine phospholipids. Analysis of the fluorescent lipids showed that 93% of the pAG was incorporated into glycerolipids with the ether bond intact. Although the addition of 20 μM 1- O -hexadecyl- sn -glycerol to the medium fully restored PlsEtn biosynthesis in NRel-4 cells, pAG only partially restored PlsEtn synthesis. Incubation of cells with pAG followed by irradiation with long-wavelength (>300 nm) ultraviolet light resulted in cytotoxicity. NRel-4 cells displayed an increased sensitivity to this treatment compared with CHO-K1 cells. This photodynamic cytotoxicity approach could be used to select for mutants that are defective in downstream steps in ether lipid biosynthesis.
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