April 2011
Volume 52, Issue 14
ARVO Annual Meeting Abstract  |   April 2011
Enzymatic Activity Of Elovl4 Is Dependent On HVYHH Motif In The Active Site
Author Affiliations & Notes
  • Sreemathi Logan
    Cell Biology,
    University of Oklahoma HSC, Oklahoma City, Oklahoma
  • Martin-Paul Agbaga
    University of Oklahoma HSC, Oklahoma City, Oklahoma
  • Michael D. Chan
    University of Oklahoma HSC, Oklahoma City, Oklahoma
  • Richard S. Brush
    University of Oklahoma HSC, Oklahoma City, Oklahoma
  • Albert O. Edwards
    Institute of Molecular Biology, University of Oregon, Eugene, Oregon
  • Robert E. Anderson
    Cell Biology and Ophthalmology,
    University of Oklahoma HSC, Oklahoma City, Oklahoma
    Dean McGee Eye Institute, Oklahoma City, Oklahoma
  • Footnotes
    Commercial Relationships  Sreemathi Logan, None; Martin-Paul Agbaga, None; Michael D. Chan, None; Richard S. Brush, None; Albert O. Edwards, None; Robert E. Anderson, None
  • Footnotes
    Support  NIH Grants EY00871, EY04149, EY12190, EY014467 and RR17703; Foundation Fighting Blindness and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1770. doi:
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      Sreemathi Logan, Martin-Paul Agbaga, Michael D. Chan, Richard S. Brush, Albert O. Edwards, Robert E. Anderson; Enzymatic Activity Of Elovl4 Is Dependent On HVYHH Motif In The Active Site. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1770.

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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). ELOVL4 is involved in the elongation of very long chain fatty acids >C26 in the endoplasmic reticulum (ER). The purpose of this study is to establish the condensation activity of wild type Elovl4, the step specifically catalyzed by Elovl4 during fatty acid elongation, and determine if the histidine rich motif in the proposed active site is involved in the condensation reaction.

Methods: : Four putative active site mutants were generated by PCR-based mutagenesis; 3 mutants had single histidine residues mutated to glutamine, while the fourth mutant incorporated all three histidine mutations. Adenovirus constructs of HA-tagged wild type (WT) Elovl4 and active site mutants of the WT were generated and expressed in HEK293 cells. Cellular expression and localization were confirmed by immunohistochemistry and Western blotting. In vitro elongation assay was performed by supplementing transduced cells with 20:5n3 or 34:5n3. Following treatment, lipids were extracted, converted to fatty acid methyl esters (FAMES) and analyzed by gas chromatography-mass spectrometry (GC-MS).

Results: : Active site mutants and WT Elovl4 showed subcellular co-localization with the ER marker Calnexin. Western blotting showed expression of the active site mutants at the same molecular size as the WT. When supplemented with fatty acid PUFA precursors 20:5n3 (EPA) and 34:5n3, all cells internalized the precursors to similar extents; however, only WT ELOVL4 showed elongation of the precursors to the very long chain fatty acids >C34 in culture. No significant elongation was seen in cells over-expressing active site mutants of ELOVL4 with respect to controls (GFP over-expressing or untransduced cells).

Conclusions: : Herein we have, for the first time, established the active site of Elovl4 enzyme and its participation in very long chain fatty acid biosynthesis. Since the Stargardt3 mutant retains this active site despite the C-terminal truncation, it may still perform the condensation reaction without yielding the final elongated product. Thus, reactive keto-intermediates of fatty acid biosynthesis may be generated by the STGD3 mutant, which could contribute to the disease process. Further experiments determining mutant activity is crucial to gain a better understanding of its role in Stargardt3 pathogenesis.

Keywords: macula/fovea • adenovirus • enzymes/enzyme inhibitors 

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