TY - JOUR
T1 - A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase
T2 - A novel defect in plasmalogen biosynthesis
AU - Nagan, Narasimhan
AU - Hajra, Amiya K.
AU - Das, Arun K.
AU - Moser, Hugo W.
AU - Moser, Ann
AU - Lazarow, Paul
AU - Purdue, P. Edward
AU - Zoeller, Raphael A.
PY - 1997/4/29
Y1 - 1997/4/29
N2 - Using fluorescence-activated cytotoxicity selection, followed by colony autoradiographic screening of the surviving population, we have isolated a unique plasmalogendeficient Chinese hamster ovary (CHO) cell line. The mutant, NZel-1, showed a dramatic (90%) reduction in the rate of biosynthesis and levels of plasmalogens, as determined using short- and long-term labeling with 32P(i). Enzymatic assays and lipid supplementation studies showed that NZel-1 was defective in a single step in the biosynthetic pathway for plasmalogens. This step, catalyzed by the peroxisomal enzyme, alkyldihydroxyacetone phosphate (DHAP) synthase, is responsible for the introduction of the ether bond found in plasmalogens. The activity of alkyl- DHAP synthase was reduced in whole-cell homogenates from NZel-1 to 18% of wild-type values. Unlike previously described plasmalogen-deficient mutants, NZel-1 contained peroxisomes, as confirmed by immunofluorescence microscopy and catalase release by digitonin. Peroxisomal functions, including the breakdown of very long-chain (>20 carbons) fatty acids, phytanic acid oxidation, and the acylation of DHAP, were normal. Cell fusion studies revealed that the mutation is recessive and belongs to a new complementation group. To our knowledge this is the first report describing the isolation and characterization of a mutant CHO cell line defective in plasmalogen biosynthesis which contains intact, functional peroxisomes. These cells will allow us to examine the role of ether lipids in cellular functions without complications associated with peroxisome deficiency.
AB - Using fluorescence-activated cytotoxicity selection, followed by colony autoradiographic screening of the surviving population, we have isolated a unique plasmalogendeficient Chinese hamster ovary (CHO) cell line. The mutant, NZel-1, showed a dramatic (90%) reduction in the rate of biosynthesis and levels of plasmalogens, as determined using short- and long-term labeling with 32P(i). Enzymatic assays and lipid supplementation studies showed that NZel-1 was defective in a single step in the biosynthetic pathway for plasmalogens. This step, catalyzed by the peroxisomal enzyme, alkyldihydroxyacetone phosphate (DHAP) synthase, is responsible for the introduction of the ether bond found in plasmalogens. The activity of alkyl- DHAP synthase was reduced in whole-cell homogenates from NZel-1 to 18% of wild-type values. Unlike previously described plasmalogen-deficient mutants, NZel-1 contained peroxisomes, as confirmed by immunofluorescence microscopy and catalase release by digitonin. Peroxisomal functions, including the breakdown of very long-chain (>20 carbons) fatty acids, phytanic acid oxidation, and the acylation of DHAP, were normal. Cell fusion studies revealed that the mutation is recessive and belongs to a new complementation group. To our knowledge this is the first report describing the isolation and characterization of a mutant CHO cell line defective in plasmalogen biosynthesis which contains intact, functional peroxisomes. These cells will allow us to examine the role of ether lipids in cellular functions without complications associated with peroxisome deficiency.
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U2 - 10.1073/pnas.94.9.4475
DO - 10.1073/pnas.94.9.4475
M3 - Article
C2 - 9114014
AN - SCOPUS:0030903458
SN - 0027-8424
VL - 94
SP - 4475
EP - 4480
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 9
ER -