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 Sjögren-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 microM 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 Sjögren-Larsson syndrome.
Highlights
§ To whom correspondence should be addressed: Dept. of Biophysics, Center for Advanced Biomedical Research, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118
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, this was not observed in wild-type cells
We propose a possible role for fatty aldehydes, or other aldehydic species, in mediating some of the symptoms associated with Sjogren-Larsson syndrome
Summary
Materials—[9,10-3H]Hexadecanoic acid (28 Ci/mmol; 1 Ci ϭ 37 Gbq), [methyl-3H]thymidine (248 Gbq/mmol), and EN3HANCE spray were purchased from NEN Life Science Products. [9,10-3H]Hexadecanol was synthesized from [9,10-3H]hexadecanoic acid by the method of Davis and Hajra [16]. [1-14C]Hexadecanol (12.5 mCi/mmol) was purchased from Sigma. [1-14C]Hexadecanal was synthesized from [1-14C]hexadecanol, and other aldehydes were synthesized from the corresponding fatty alcohols (purchased from Sigma) by the method of Corey and Suggs [17]. Twice during the selection period, the cells were harvested with trypsin, pelleted by centrifugation, and replated in 100-mm tissue culture dishes This was followed, the day, with a medium change to remove dead cells. The medium was removed, the cells were washed once with 2 ml of PBS, and 0.5 ml of 0.5% (w/v) Coomassie Blue in methanol:H2O:acetic acid (45: 45:10) was added for 30 min. 0.1 ml of the cell suspension was used for protein determination, and 0.8 ml was added to a glass tube containing 2 ml of methanol, 1 ml of chloroform, and 300 g of carrier lipid (total mouse liver lipid) to form a single phase solution [24]. Bands of interest were scraped into scintillation vials and quantitated by liquid scintillation spectrometry
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