April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Elongation of Very Long Chain Fatty Acids by Elovl4
Author Affiliations & Notes
  • S. Logan
    Cell Biology, University of Oklahoma HSC, Oklahoma City, Oklahoma
  • M.-P. G. Agbaga
    Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, Oklahoma
    Opthalmology, Dean A. McGee Eye Institute, Oklahoma City, Oklahoma
  • R. S. Brush
    Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, Oklahoma
    Opthalmology, Dean A. McGee Eye Institute, Oklahoma City, Oklahoma
  • A. O. Edwards
    Institute of Molecular Biology, University of Oregon, Eugene, Oregon
  • R. E. Anderson
    Ophthalmology/Cell Biology,
    Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, Oklahoma
    Opthalmology, Dean A. McGee Eye Institute, Oklahoma City, Oklahoma
  • Footnotes
    Commercial Relationships  S. Logan, None; M.-P.G. Agbaga, None; R.S. Brush, None; A.O. Edwards, None; R.E. Anderson, None.
  • Footnotes
    Support  NIH Grants EY014467, EY00871, EY04149, EY12190, and RR17703; Foundation Fighting Blindness and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 2495. doi:
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    • Get Citation

      S. Logan, M.-P. G. Agbaga, R. S. Brush, A. O. Edwards, R. E. Anderson; Elongation of Very Long Chain Fatty Acids by Elovl4. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2495.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Mutations in the ELOVL4 gene lead to autosomal dominant Stargardt-like macular dystrophy (STGD3). These frame-shift mutations result in premature termination of ELOVL4 protein, loss of its carboxy-terminal endoplasmic reticulum (ER) retention motif, and subsequent mislocalization. Whether the nonsense mutation (5 bp deletion) contributes to disease progression by altering the enzymatic activity of ELOVL4 is unknown and thereby limits the development of therapeutic interventions. The purpose of this study is to establish the condensation activity of wild type ELOVL4, which is the step specifically catalyzed by ELOVL4 during fatty acid elongation, and determine if mutant ELOVL4 retains this activity.

Methods: : Wild type (WT), STGD3 mutant, and a fusion construct containing the 5 bp deletion and the WT ER localization signal were N-terminally tagged with hemagglutinin (HA) and expressed in HEK293 cells. Cellular expression and localization were confirmed by immunohistochemistry and Western blotting. In vitro elongation assay was performed by supplementing transfected cells with 20:5n3 or 34:5n3. Following treatment, lipids were converted to fatty acid methyl esters (FAMES) and analyzed by gas chromatography-mass spectrometry (GC-MS). Microsomal fractions from cells over-expressing ELOVL4 were isolated and elongase activity was assayed in the presence of NADPH/NADH, 26:0-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.

Results: : Wild-type ELOVL4 localized to the ER while mutant protein was aggregated and mislocalized to other cellular compartments. Addition of the WT ER localization signal in the mutant protein resulted in localization of the mutant to the ER. Wild type ELOVL4 showed elongation of 20:5n3 and 34:5n3 in culture, whereas no elongation was seen in cells expressing either STGD3 or ER fusion of the mutant. In microsomal reactions, wild type ELOVL4 mediated condensation and elongation of 26:0 and 34:5n3.

Conclusions: : ELOVL4 is involved in the condensation reaction in very long chain fatty acid biosynthesis. Further experiments determining mutant activity is crucial to gain a better understanding of its role in Stargardt3 pathogenesis.

Keywords: proteins encoded by disease genes • retinal degenerations: cell biology • retina 

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