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

Abstract

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.

Modified Phosphatidylethanolamine as the Active Component of Oxidized Low Density Lipoprotein Promoting Platelet Prothrombinase Activity

“…The extracted lipids were separated with the solvent CHCl 3 /CH 3 OH (9:1). The spots representing underivatized PE and ethanolamine phospholipids with reduced Schiff base (18) were eluted from the silica. Phosphate analysis indicated that 9.1% of the total PE recovered had been reduced by NaCNBH 3 .…”

An Animal Cell Mutant with a Deficiency in Acyl/Alkyl-dihydroxyacetone-phosphate Reductase Activity

“…FAA.K1B is a variant of CHO-K1 that was isolated from a population of cells that were defective in the ability to take up long chain fatty alcohol (20). All cell lines were routinely cultured in Ham's F12 nutrient mix (BioWhittaker) containing 10% fetal bovine serum (BioWhittaker), glutamine (1 mM), penicillin G (100 units/ml), and streptomycin (75 units/ ml).…”

“…After drying, 0.5 ml of 0.5 N NaOH was added to solubilize the cell material at 37°C for 2 h. Aliquots were used for liquid scintillation counting. [9,10-3 H]Hexadecanol into Complex Lipids-FAA.K1B was isolated from a mutagenized population of CHO-K1 that had survived a tritium suicide selection protocol designed to select cells that were less able to take up [9,10-3 H]hexadecanol into stable pools of lipid (20). All, except FAA.K1B, were characterized as having a defect in fatty-alcohol oxidase activity.…”

Genetics and prospective therapeutic targets for Sjögren-Larsson Syndrome

“…[62,63] To date, however, very few studies have focused on the toxic effects of the longer chain aldehydes (C16–C18) that are seen in SLS. [64,65] Because of their lipophilic properties, long-chain aldehydes are expected to partition into membranes of various intracellular compartments, most notably the ER and peroxisomes where FALDH is located.…”