出版社：American Society for Biochemistry and Molecular Biology
摘要：The synthesis of an -pyrene-labeled 1-O-alkyl-sn-glycerol wasperformed 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 covalentlyattached at the alkyl chain terminus. pAG was taken into CHO-K1cells 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 ethanolamineand choline phospholipids as well as a neutral lipid fractiontentatively identified as alkyldiacylglycerol. NRel-4 accumulatedmore fluorescence in the phospholipid fraction than CHO-K1,specifically in the ethanolamine phospholipids. Analysis ofthe fluorescent lipids showed that 93% of the pAG was incorporatedinto glycerolipids with the ether bond intact. Although theaddition of 20 µM 1-O-hexadecyl-sn-glycerol to the mediumfully restored PlsEtn biosynthesis in NRel-4 cells, pAG onlypartially restored PlsEtn synthesis. Incubation of cells withpAG followed by irradiation with long-wavelength (>300 nm)ultraviolet light resulted in cytotoxicity. NRel-4 cells displayedan increased sensitivity to this treatment compared with CHO-K1cells. This photodynamic cytotoxicity approach could be usedto select for mutants that are defective in downstream stepsin ether lipid biosynthesis.Supplementary key words pyrene • fluorescence • chemical synthesis • ether lipid • plasmalogen • alkylglycerol
Abbreviations: ADG, alkyldiacylglycerol; bT, bath temperature; DHAPAT, dihydroxyacetonephosphate acyltransferase; HG, 1-O-hexadecyl-sn-glycerol; pAG, 1-O-[9'-(1''-pyrenyl)]nonyl-sn-glycerol; Pi, inorganic phosphate; PlsCho, plasmenylcholine; PlsEtn, plasmenylethanolamine; P9OH, 9-(1'-pyrenyl)-1-nonanol; PtdEtn, phosphatidylethanolamine; TEA, triethylamine; UV, ultraviolet
-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.
Supplementary key words pyrene • fluorescence • chemical synthesis • ether lipid • plasmalogen • alkylglycerol
Abbreviations: ADG, alkyldiacylglycerol; bT, bath temperature; DHAPAT, dihydroxyacetonephosphate acyltransferase; HG, 1-O-hexadecyl-sn-glycerol; pAG, 1-O-[9'-(1''-pyrenyl)]nonyl-sn-glycerol; Pi, inorganic phosphate; PlsCho, plasmenylcholine; PlsEtn, plasmenylethanolamine; P9OH, 9-(1'-pyrenyl)-1-nonanol; PtdEtn, phosphatidylethanolamine; TEA, triethylamine; UV, ultraviolet
