A fibroblast cell line defective in alkyl-dihydroxyacetone phosphate synthase: a novel defect in plasmalogen biosynthesis.
Using fluorescence-activated cytotoxicity selection, followed by colony autoradiographic screening of the surviving population, we have isolated a unique plasmalogen-deficient 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 32Pi. 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, alkyl-dihydroxyacetone 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.
Nagan
N
N
Department of Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.
Hajra
A K
AK
Das
A K
AK
Moser
H W
HW
Moser
A
A
Lazarow
P
P
Purdue
P E
PE
Zoeller
R A
RA
D019883:Alkyl and Aryl Transferases; D000818:Animals; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D005347:Fibroblasts / Q000166:cytology; D005808:Genes, Recessive; D005816:Genetic Complementation Test; D008830:Microbodies / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D016296:Mutagenesis; D009154:Mutation; D010955:Plasmalogens / Q000096:biosynthesis* / Q000378:metabolism; D012641:Selection, Genetic; D014166:Transferases / Q000235:genetics*; D014466:Ultraviolet Rays
D010955:Plasmalogens; C020791:phosphatidal ethanolamines; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C019654:alkylglycerone-phosphate synthase