Role of Stargardt-3 macular dystrophy protein (ELOVL4) in the biosynthesis of very long chain fatty acids

Abstract

Stargardt-like macular dystrophy (STGD3) is a dominantly inherited juvenile macular degeneration that eventually leads to loss of vision. Three independent mutations causing STGD3 have been identified in exon six of a gene named Elongation of very long chain fatty acids 4 (ELOVL4). The ELOVL4 protein was predicted to be involved in fatty acid elongation, although evidence for this and the specific step(s) it may catalyze have remained elusive. Here, using a gain-of-function approach, we provide direct and compelling evidence that ELOVL4 is required for the synthesis of C28 and C30 saturated fatty acids (VLC-FA) and of C28-C38 very long chain polyunsaturated fatty acids (VLC-PUFA), the latter being uniquely expressed in retina, sperm, and brain. Rat neonatal cardiomyocytes and a human retinal epithelium cell line (ARPE-19) were transduced with recombinant adenovirus type 5 carrying mouse Elovl4 and supplemented with 24:0, 20:5n3, or 22:5n3. The 24:0 was elongated to 28:0 and 30:0; 20:5n3 and 22:5n3 were elongated to a series of C28-C38 PUFA. Because retinal degeneration is the only known phenotype in STGD3 disease, we propose that reduced VLC-PUFA in the retinas of these patients may be the cause of photoreceptor cell death.

Fig. 1.

Transgenic expression of mouse ELOVL4 in rat cardiomyocytes and ARPE-19 cells. (A) Comparison of quantitative expression of Elovl4 gene in different rat tissues and in ARPE-19 cells by qRT-PCR, and presented relative to the expression of the housekeeping gene Rpl 19. The values represent the means (± SEM) of n = 3 after normalizing with Rpl 19 calculated by the comparative threshold cycle (C_t) method. Significant expression of Elovl4 was observed in transduced cells. B, brain; R, retina; S, skin; T, testis; H, heart; L, liver; C, cardiomyocytes; C-E, Elovl4-expressing cardiomyocytes; R-C, RPE-Choroid; A, ARPE-19; A-E, Elovl4-expressing ARPE-19 cells. (B) Western blots showing expression of ELOVL4, GFP, and β-actin in cardiomyocytes transduced with or without recombinant adenoviruses. The nontransduced and GFP-expressing controls did not show ELOVL4 expression, whereas Elovl4-transduced cells showed abundant ELOVL4 protein expression. (C) ELOVL4 immunolabeling is detected in mouse and rat retinas, using affinity-purified ELOVL4 antibodies (green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Images were captured by using an Olympus FluoView Confocal Microscope with 20× and 60× objective lenses. IS, inner segments of rod and cone photoreceptors; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cells layer.

Fig. 2.

Biosynthesis of 28:0 and 30:0 from 24:0 in cardiomyocytes and ARPE-19 cells expressing Elovl4 transgene. Cardiomyocytes or ARPE-19 cells were transduced with or without recombinant Elovl4 or GFP viruses for 24 h and then cultured in 24:0-supplemented complete media for 72 h. Total lipids extracted from an aliquot of homogenate equivalent to 500 μg of protein were converted to FAME and analyzed by GC-MS in the SIM mode. (A and B) Cardiomyocytes without (A) and with (B) 24:0. (C and D) ARPE-19 cells without (C) and with (D) 24:0. The 26:0, 28:0 and 30:0 response values were obtained by using the m/z ratios 410.4, 438.4, and 466.5, respectively. Sample concentrations were determined by comparison to external standards, using 25:0 and 27:0 as internal standards. Multivariate ANOVA with posthoc Scheffé test was used to determine statistical significance. ●, no significant difference; **, P < 0.01; ***, P < 0.001 (n = 3).

Fig. 3.

Biosynthesis of VLC-PUFA in cardiomyocytes expressing Elovl4 transgene. GC-MS allowed identification of the VLC-PUFA derived from sample equivalent to 2.0 mg of protein from cardiomyocytes treated with 20:5n3 or 22:5n3 for 72 h after transduction with recombinant Elovl4 or GFP viruses for 24 h (n = 3). The PUFA response values were obtained by using the m/z ratios 79.1, 108.1, and 150.1 in SIM mode and abundances were compared by normalizing the chromatograms to the response of 20:1. (A) Rat cardiomyocytes expressing ELOVL4 (red) or GFP (green) and nontransduced cells (blue) were cultured without precursors for 72 h. All cells, irrespective of ELOVL4 expression, synthesized C22-C26 PUFA. ELOVL4 expression in the absence of precursors resulted in elongation of endogenous precursor to C28-C38 VLC-PUFA. (B) Cardiomyocytes in A above cultured with 20:5n3 synthesized C24-C26 in all treatment groups. Significant biosynthesis of C28-C38 n3 VLC-PUFA occurred in Elovl4-transduced cells (red), but not in GFP (green) and nontransduced cells (blue), with accumulation of 34:5n3 and 36:5n3. (C) Cardiomyocytes in A above cultured with 22:5n3 synthesized C24-C26 in all treatment groups. They also synthesized the same n3 VLC-PUFA as found for 20:5n3. Note that each chromatogram was normalized to endogenous 20:1, which did not change among the sample groups.

Fig. 4.

Schematic diagram of in vivo n3 VLC-PUFA biosynthetic pathway mediated by ELOVL4 and other ELO families [modified after Suh and Clandinin (38)]. 18:3n3, 20:5n3, or 22:5n3 can be converted to VLC-PUFA through consecutive enzymatic activities of desaturases and elongases. Although some elongases are specific for a single step, others are nonspecific or multifunctional and act at several steps (e.g., human ELOVL5 and murine ELOVL2) (11). We propose that ELOVL4 is essential for elongation of saturated 26:0 to 28:0 and of 26:5n3 to 28:5n3. It is also possible that ELOVL4 is necessary for generating (C30–C38):5n3 because these fatty acids are formed only in ELOVL4 expressing cells. However, other ELOVLs may be responsible for elongations of some of these VLC-PUFA (C30–C38) by using products generated by ELOVL4 elongase activity. The synthesis of 34:6n3 and 36:6n3 in our study suggests desaturase activity on their shorter chain precursors. However, the specific desaturase(s) involved is not known.