Abstract
Purpose :
ELOVL2 encodes an enzyme that elongates polyunsaturated fatty acids (LC-PUFAs). It’s crucial for synthesis of omega-3 docosahexaenoic acid (DHA) and VLC-PUFAs, all playing critical role in retina biology as integral parts of photoreceptor disk membranes. The RPE cells are important in regulation and delivery of LC-PUFAs to the adjacent photoreceptors. However, the practical role of ELOVL2 in RPE structure and function remain unclear.
Methods :
To understand the biological role of ELOVL2 in RPE, we used siRNAs directed against ELOVL2 in ARPE19 cells and investigated the effect of ELOVL2 in ARPE19 cells morphology and biology functions. Fluorescently labeled WGA was used to visualize cell membranes. Mass spectrometry-based lipidomic and proteomic analyses were used to understand the molecular role of ELOVL2 in ARPE19 cells.
Results :
WGA staining of normal ARPE19 cells reveals a highly ordered pattern of cells, with cell boundaries strongly stained and sharply demarcated. In ELOVL2 KD ARPE19 cells, the staining becomes more diffuse, stronger in cytoplasm and staining perinuclear regions in the cell. Moreover, the borders of ELOVL2 KD ARPE19 cells are disordered and there are more discontinuities between cells. The quantitative results of the fatty acid composition of membrane lipidsshowed that there was a significant decrease of FA 22:4, 22:5 and 26:4 in ELOVL2 KD ARPE19 cells. Among them, FA22:4 and FA22:5 are substrates of ELOVL2, and FA26:4 are elongation products of FA24:4, which is a product of ELOVL2. Therefore, the lack of ELOVL2 leads to a decline in VLC-PUFA synthesis. Then the molecular mechanisms of the role of ELOVL2 in cell membrane were assessed by proteomics,revealing that among all the 153 significantly changed proteins (out of 2381 proteins in total, (|Fold change|≥1.5, P-Value<0.05), 48 proteins were identified as membrane proteins (DAVID database), including membrane magnesium transporter 1 (MMGT1, up-regulated, p=0.01), caveolin 2 (CAV2, down-regulated, p=0.02), platelet derived growth factor receptor beta (PDGFRB, down-regulated, p=0.004), et al.
Conclusions :
Our data revealed that ELOVL2 depletion disrupted membrane compositions on lipid and protein levels and may further alter the membrane structural and functional properties. These changes of the cell membrane will most probably affect transmembrane transport, transmembrane receptors’ activity and other membrane related biological functions.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.