Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol.
We have developed a two-step selection protocol to generate a population of Chinese hamster ovary (CHO) cell variants that are plasmalogen-deficient, but contain intact, functional peroxisomes (plasmalogen-/peroxisome+). This involved sequential exposures of a mutagenized cell population to photodynamic damage by using two different pyrene-labelled sensors, 9-(1'-pyrene)nonanol and 12-(1'-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of which displayed a severe decrease in plasmalogen biosynthesis. Further characterization of one of the plasmalogen-deficient isolates, NRel-4, showed that it contained intact, functional peroxisomes. Whole-cell homogenates from NRel-4 displayed severely decreased dihydroxyacetone phosphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRel-4 and another, recently described, plasmalogen-deficient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Purdue and Zoeller (1997) Proc. Natl. Acad. Sci. U.S. A. 94, 4475-4480] were hypersensitive to singlet oxygen, supporting the notion of plasmalogens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRel-4 upon restoration of plasmalogen content by supplementation with a bypass compound, sn-1-hexadecylglycerol. NRel-4 and other plasmalogen-/peroxisome+ strains will allow us to examine further the role of ether lipids in cellular functions without complications associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.
Nagan
N
N
Department of Biophysics, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118, USA.
Hajra
A K
AK
Larkins
L K
LK
Lazarow
P
P
Purdue
P E
PE
Rizzo
W B
WB
Zoeller
R A
RA
D000217:Acyltransferases / Q000172:deficiency*; D000818:Animals; D016466:CHO Cells; D002374:Catalase / Q000276:immunology; D002470:Cell Survival / Q000187:drug effects; D018902:Chondrodysplasia Punctata, Rhizomelic / Q000235:genetics; D006224:Cricetinae; D005227:Fatty Acids / Q000494:pharmacology; D007150:Immunohistochemistry; D008830:Microbodies / Q000201:enzymology* / Q000473:pathology; D016296:Mutagenesis / Q000235:genetics; D010743:Phospholipids / Q000032:analysis; D010955:Plasmalogens / Q000096:biosynthesis*; D011721:Pyrenes / Q000494:pharmacology; D014466:Ultraviolet Rays / Q000009:adverse effects
D005227:Fatty Acids; D010743:Phospholipids; D010955:Plasmalogens; D011721:Pyrenes; D002374:Catalase; D000217:Acyltransferases; C020051:glycerone-phosphate O-acyltransferase