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Sreemathi Logan, Martin-Paul G. Agbaga, Michael D. Chan, Richard S. Brush, Robert E. Anderson; STGD3 Mutant Exerts A Dominant Negative Effect On Elovl4 Enzymatic Activity During VLC-PUFA Biosynthesis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6890.
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Autosomal dominant Stargardt3 macular dystrophy results from mutations in the ELOVL4 gene. ELOVL4 mediates the rate-limiting condensation reaction during very long chain (VLC, ≥C28) fatty acid biosynthesis. The STGD3 mutant protein (MUT) is truncated, which results in mislocalized protein aggregation. The mechanism by which the MUT contributes to disease progression is unknown. The purpose of this study is to determine if MUT is enzymatically active and if it has any effect on wild type (WT) activity.
Untagged (ELO) or HA-tagged (WT) Elovl4, MUT, and ER retention fusion of MUT (mERET) Elovl4 were expressed individually or co-expressed, and analyzed by immunohistochemistry and Western blotting. Cells (either HEK293 or Arpe19) co-expressing WT/ELO and MUT, or WT/ELO and GFP, or WT/ELO and mERET were supplemented in culture with 20:5n3 (EPA), a VLC fatty acid precursor. Following treatment, total lipids were extracted, converted to fatty acid methyl esters (FAMES), and analyzed by gas chromatography-mass spectrometry. Microsomes from cells over-expressing ELOVL4 were isolated and elongase activity was assayed in the presence of NADPH/NADH, 20:5n3-CoA, 34:5n3-CoA, and 2[14C]-malonyl-CoA. Omitting NADPH/NADH from the reaction revealed condensation activity alone. FAMES were generated and separated by C-18 reverse phase HP-TLC, and incorporated radioactivity was visualized by autoradiography and quantified by counting in a liquid scintillation counter.
WT forms aggregates when co-expressed with MUT, but not when co-expressed with mERET. In culture, WT activity is significantly decreased in the presence of MUT or mERET. ELO activity is also decreased in the presence of MUT, mERET, or H161Q (active site mutant of WT Elovl4), confirming tag-independent oligomerization of Elovl4. Neither MUT nor mERET have any activity when expressed individually compared to controls. In microsome assay, MUT did not show any condensation activity when compared to WT, similar to GFP and H161Q.
1) WT is an enzyme with innate elongase (ELO) activity, 2) MUT does not have ELO activity, even though the active site is preserved, and 3) MUT has a dominant negative effect on WT activity. Further experiments to explore mutant effect on WT activity and the downstream effects on cellular function are crucial to generate targeted therapy.
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