Isolation and characterization of a Chinese hamster ovary cell line deficient in fatty alcohol:NAD+ oxidoreductase activity.

James, Paul F.; Rizzo, William B.; Lee, Jongsoon; Zoeller, Raphael A.

doi:10.1073/pnas.87.16.6102

Cited by 14 publications

(17 citation statements)

References 32 publications

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“…Accumulation of fatty alcohols has been reported in several cell lines defective in plasmalogen biosynthesis (7,16). Given these findings, it seems likely that the enzymes involved in metabolism of fatty alcohols are coordinately regulated to make these substrates available depending on the biosynthetic needs of the cell.…”

Section: Discussionmentioning

confidence: 96%

“…In contrast, the converse is true in liver, where plasmalogens are less abundant (6) and fatty alcohol oxidase activity is high. The latter enzyme activity is not altered in plasmalogen-deficient cells including a peroxisome assemblydefective CHO cell mutant, PEX2-deficient ZR-82 (7,16), implying that the accumulation of fatty alcohols in these cells is conferred by a combination of impaired incorporation of fatty alcohols in plasmalogen biosynthesis and elevated stability of Far1.…”

Section: Discussionmentioning

confidence: 99%

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Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Honsho

Asaoku

Fujiki

2010

Journal of Biological Chemistry

114

132

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Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.Plasmalogens are a major subclass of choline and ethanolamine glycerophospholipids that link a long chain fatty alcohol at the sn-1 position by a vinyl ether bond. Plasmalogen synthesis is accomplished via a seven-step reaction pathway (1), which is initiated by the peroxisomal matrix protein, dihydroxyacetone phosphate acyltransferase (DHAPAT) 2 (2). Formation of the ether bond is catalyzed by another peroxisomal matrix enzyme, alkyl dihydroxyacetone phosphate synthase (ADAPS), which replaces the acyl chain of 1-acyl-DHAP with a long chain fatty alcohol (1, 3). In animal cells, fatty alcohols are primarily produced by reduction of the corresponding fatty acyl-CoAs (4) and are utilized as substrates for the synthesis of ether lipids and wax esters. Fatty acyl-CoA reductase (Far) converts a fatty acyl-CoA into a fatty alcohol and CoA-SH and is enriched in subcellular fractions containing catalase, a peroxisomal marker enzyme (4, 5). Tissues such as the brain and intestine mucosa synthesize large amounts of plasmalogens, and correspondingly, contain abundant Far activity. In contrast, Far activity is low in liver, which contains little ether lipid (6). Far activity is increased 4 -5-fold in plasmalogen-deficient fibroblasts from patients with rhizomelic chondrodysplasia punctata (RCDP), apparently resulting in accumulation of fatty alcohol (7).Recently, FAR1 and FAR2 cDNAs were isolated from human and mouse (8). Far1 prefers saturated and unsaturated fatty acylCoAs of 16 -18 carbon atoms as substrates, consistent with the composition of fatty alcohols in the sn-1 position of plasmalogens, whereas Far2 prefers saturated fatty acyl-CoA substrates of 16 -18 carbons. These studies and those mentioned above suggest that Far1 is responsible for supplying fatty alcohols for ether bond formation and th...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Honsho

Asaoku

Fujiki

2010

Journal of Biological Chemistry

114

132

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“…A block in fatty alcohol oxidation in SLS (28) or impaired incorporation of alcohol into ether lipid biosynthesis, as seen in patients with rhizomelic chondrodysplasia punctata (29), results in fatty alcohol accumulation. In genetically mutant cultured Chinese hamster ovary cells, the combination of FAO deficiency and defective ether glycerolipid synthesis leads to a much greater accumulation of fatty alcohol than either metabolic defect alone (30), suggesting that fatty alcohol can be diverted through alternative pathways when one pathway is blocked. This conclusion is supported by our studies demonstrating preferential channeling of radioactive 18:0-OH into wax esters and 1-O-alkyl-2,3-diacylglycerol in SLS keratinocytes.…”

Section: Discussionmentioning

confidence: 99%

Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome

Rizzo

Craft

Somer

et al. 2008

Journal of Lipid Research

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Sjögren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). FALDH is an enzyme component of fatty alcohol:NAD oxidoreductase (FAO), which is necessary for fatty alcohol metabolism. To better understand the biochemical basis for the cutaneous symptoms in this disease, we investigated lipid metabolism in cultured keratinocytes from SLS patients. Enzyme activities of FALDH and FAO in SLS cells were ,10% of normal. SLS keratinocytes accumulated 45-fold more fatty alcohol (hexadecanol, octadecanol, and octadecenol) than normal, whereas wax esters and 1-O-alkyl-2,3-diacylglycerols were increased by 5.6-fold and 7.5-fold, respectively. SLS keratinocytes showed a reduced incorporation of radioactive octadecanol into fatty acid (24% of normal) and triglyceride (13% of normal), but incorporation into wax esters and 1-O-alkyl-2,3-diacylglycerol was increased by 2.5-fold and 2.8-fold, respectively. Our results indicate that FALDH deficiency in SLS keratinocytes causes the accumulation and diversion of fatty alcohol into alternative biosynthetic pathways. The striking lipid abnormalities in cultured SLS keratinocytes are distinct from those seen in fibroblasts and may be related to the stratum corneum dysfunction and ichthyosis in SLS.

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“…We have previously reported the isolation of a CHO mutant cell line, FAA.1, that also displayed a deficiency in FAldDH activity (13). However, the FAA.1 variant was a double mutant, in that it was isolated from a CHO mutant cell line, ZR-82 (14), that was deficient in ether lipid biosynthesis (EL Ϫ ) and perox-isome function.…”

mentioning

confidence: 99%

“…While FAA.1 is phenotypically similar to SLS fibroblasts with respect to the FAldDH deficiency, the EL Ϫ phenotype and loss of functional peroxisomes complicates analyses concerning the relationship between the FAldDH deficiency and changes in cellular functions. For example, EL Ϫ cells can accumulate significant amounts of long chain fatty alcohols (13,15), a phenotype that has also been reported in FAldDH Ϫ cells (8). In this study, we used a selection process, similar to that used previously (13), to isolate an FAldDH Ϫ mutant cell line, FAA.K1A, from a wild-type (EL ϩ ) background.…”

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confidence: 99%

Isolation of Animal Cell Mutants Defective in Long-chain Fatty Aldehyde Dehydrogenase

James¹,

Zoeller

1997

Journal of Biological Chemistry

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Using tritium suicide, we have isolated a variant of the Chinese hamster ovary cell line, CHO-K1, that is deficient in long-chain fatty alcohol:NAD ؉ oxidoreductase (FAO; EC 1.1.1.192). Specifically, it was the fatty aldehyde dehydrogenase component that was affected. The enzymatic deficiency found in this mutant strain, designated FAA.K1A, was similar to that displayed by fibroblasts from patients with Sjö gren-Larsson syndrome (SLS), an inheritable neurocutaneous disorder. Complementation analyses suggested that the deficiency in fatty alcohol oxidation in the FAA.K1A cells and the SLS fibroblasts is a result of lesions in homologous genes. The FAA.K1A cells were unable to convert long chain fatty aldehydes to the corresponding fatty acids. This resulted in a hypersensitivity of the FAA.K1A cells to the cytotoxic effects of long chain fatty aldehydes. The difference between the mutant and wild-type cells was most obvious when using fatty aldehydes between 14 and 20 carbons, with the greatest difference between wild-type and mutant cells found when using octadecanal. Fibroblasts from a patient with SLS also displayed the hypersensitivity phenotype when compared with FAldDH ؉ human fibroblasts. In both CHO and human FAldDH ؊ cell lines, addition of long chain fatty aldehydes to the medium caused a dramatic increase in aldehyde-modified phosphatidylethanolamine, presumably through Schiff's base addition to the primary amine of the ethanolamine head group. When 25 M hexadecanal was added to the growth medium, approximately 10% of the phosphatidylethanolamine was found in the fatty aldehyde-modified form in FAA.K1A, although this was not observed in wild-type cells. Modified phosphatidylethanolamine could be detected in FAldDH ؊ cells even when exogenous fatty aldehydes were not added to the medium. We propose a possible role for fatty aldehydes, or other aldehydic species, in mediating some of the symptoms associated with Sjö gren-Larsson syndrome.

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Isolation and characterization of a Chinese hamster ovary cell line deficient in fatty alcohol:NAD+ oxidoreductase activity.

Cited by 14 publications

References 32 publications

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome

Isolation of Animal Cell Mutants Defective in Long-chain Fatty Aldehyde Dehydrogenase

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