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S. Logan, M.-P. Agbaga, R. S. Brush, K. R. Henry, J. Tran, R. E. Anderson; Substrate Specificity of ELOVL4 in Very Long Chain Fatty Acid Biosynthesis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6004.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the ELOVL4 gene result in the pathogenesis of Stargardt-like macular dystrophy or STGD3. Previous studies from our lab have shown that ELOVL4 is involved in the biosynthesis of very long chain saturated (VLC-FA) and polyunsaturated fatty acids (VLC-PUFA) in cultured cells. However, the specificity of substrates >C28 catalyzed by ELOVL4 is not conclusively established. The purpose of this study is to determine substrate specificity as well as the particular steps in the elongation process catalyzed by ELOVL4 using human embryonic kidney (HEK293) and hepatocyte (HEPG2) cell lines overexpressing the protein.
Cell culture-ELOVL4 protein was expressed in HEK293 and HEPG2 cells via adenoviral transduction. Non-transduced and GFP expressing cells were used as controls. Following treatment of HEPG2 cells with 20:5n3 and HEK293 with 26:0, 28:0 and 30:0, total lipids were extracted, converted to fatty acid methyl esters (FAMES) and analyzed by gas chromatography-mass spectrometry (GC-MS).Microsomal elongase assay-Cells overexpressing ELOVL4, retinal and brain tissue were homogenized and the microsomal fraction was recovered via differential centrifugation. Elongase activity was assayed in the presence of NADPH/NADH, 26:0 CoA and 2[14C]-malonyl-CoA. Omitting NADPH/NADH from the reaction revealed condensation activity. FAMES were generated and analyzed by C-18 reverse phase HPTLC. Incorporated radioactivity was visualized using a Packard Cyclone Storage Phosphor System
HEK293 cells transduced with ELOVL4 elongated the precursors 26:0, 28:0 and 30:0 to VLCFA (28:0 to 32:0). Control cells internalized the precursor with the same efficiency as ELOVL4-transduced cells, but did not elongate the precursors. HEPG2 cells transduced with ELOVL4 also elongated 20:5n3 up to 34:5n3. Microsomal reactions showed condensation and elongation of the 26:0 CoA substrate.
We have shown that ELOVL4 is involved in elongation steps in VLCFA biosynthesis, specifically from C26 to C28, C28 to C30 and C30 to C32.
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