Retinal very long-chain PUFAs: new insights from studies on ELOVL4 protein

Abstract

Compared with other mammalian tissues, retina is highly enriched in PUFA. Long-chain PUFA (LC-PUFA; C18-C24) are essential FAs that are enriched in the retina and are necessary for maintenance of normal retinal development and function. The retina, brain, and sperm also contain very LC-PUFA (VLC-PUFA; >C24). Although VLC-PUFA were discovered more than two decades ago, very little is known about their biosynthesis and functional roles in the retina. This is due mainly to intrinsic difficulties associated with working on these unusually long polyunsaturated hydrocarbon chains and their existence in small amounts. Recent studies on the FA elongase elongation of very long chain fatty acids-4 (ELOVL4) protein, however, suggest that VLC-PUFA probably play some uniquely important roles in the retina as well as the other tissues. Mutations in the ELOVL4 gene are found in patients with autosomal dominant Stargardt disease. Here, we review the recent literature on VLC-PUFA with special emphasis on the elongases responsible for their synthesis. We focus on a novel elongase, ELOVL4, involved in the synthesis of VLC-PUFA, and the importance of these FAs in maintaining the structural and functional integrity of retinal photoreceptors.

Fig. 1.

Structure of VLC-PUFA. Free FA form of VLC-PUFA 32:5n6 (A) and 34:5n3 (B). Note the polyunsaturated methyl end and the saturated carboxyl terminal ends. C: A typical phospholipid containing VLC-PUFA esterified to the sn-1 position of the glycerol backbone. LC-PUFA, either 22:6n3 or 20:4n6, or others can occupy the sn-2 position. The sn-3 position in this scenario is occupied by phosphocholine.

Fig. 2.

The FA elongation system. The pathway of malonyl-CoA mediated elongation of fatty acyl-CoAs involves four enzymes and fatty acyl-CoA intermediates. Each round of elongation consists of four successive steps. Step 1 involves condensation of malonyl-CoA with the acyl-CoA substrate and is catalyzed by the elongase enzyme (ELOVL). Enzymes in this initial condensation step determine chain length and degree of unsaturation of the substrate; hence, this is the rate-limiting step of the elongation system. The product of the condensation reaction, the 3-keto-acyl-CoA, then undergoes a reduction catalyzed by a β-ketoacyl-CoA reductase (step 2) to produce 3-hydroxyacyl-CoA. Dehydration of the 3-hydroxyacyl-CoA by β-hydroxyacyl-CoA dehydratase (step 3) results in formation of trans-2,3-acyl-CoA, which is then reduced by enoyl-CoA reductase (step 4). Depending on the need of the specific cell or the organism at any particular time, the product is either utilized or undergoes additional round(s) of elongation.

Fig. 3.

Fundus photographs of STGD3 family members who inherited the disease gene that illustrate how the typical phenotype changes over time. A: Right eye of a 5 year-old boy (B VI-9) with the disease haplotype and normal fundus. B: Left eye of a 9 year-old boy (B VI-6) with visual acuity of 20/20, 1-year course of hemeralopia, and early foveal atrophy. C: Left eye of a 29 year-old man (B V-23) with a typical early lesion without flecks. D: Right eye of a 58 year-old man (A IV-25) with a typical late lesion with flecks. Longitudinal follow-up of the left eye of a woman (B III-15) at 45 (E) and 53 (F); note the increasing macular atrophy and fundus flecks (157). [Reprinted from Edwards et al. (157) with permission of Elsevier Science].

Fig. 4.

Mutations in exon 6 of ELOVL4 sequence from STGD3 patients. A: DNA sequence from exon 6 of ELOVL4 from one unaffected (left) and one affected (right) family member. The affected family member is heterozygous. Clustering of the two DNA sequences demonstrate the deletion of the 5 bp segment AACTT in the affected patient (143). Reprinted from Edwards et al. (143) with permission from publishers. B: Complex deletions in the ELOVL4 gene in patients from the K4175 pedigree. The panel shows a partial sequence of exon 6 from patient III-1 with the double deletion mutations (ELOVL4 het). The nucleotide sequences and protein translations are shown above and below the direct-sequencing traces. The double deletion causes a frameshift that results in a premature stop codon after 10 amino acids (bottom trace). ELOVL4 wt, wild-type allele; ELOVL4 del (790ΔT+794ΔT), allele with the double deletion (142). Reprinted from Bernstein et al. (142) with permission from publishers.

Fig. 5.

Biosynthesis of VLC-PUFA in cardiomyocytes that express Elovl4 transgene. GC-MS was used to identify the VLC-PUFA derived from cardiomyocytes treated with either 20:5n3 or 22:5n3 for 72 h after transduction with recombinant Elovl4 or GFP viruses for 24 h. 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 mouse Elovl4 (red) minigene 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 the elongation of an 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 when 20:5n3 was used as the substrate. Note that each chromatogram was normalized to endogenous 20:1, which did not change among the sample groups. Reprinted from Agbaga et al. (39) with permission from publishers.

Fig. 6.

Pathways of VLC-PUFA biosynthesis. Schematic diagram of in vivo n3 VLC-PUFA biosynthetic pathway mediated by ELOVL4 and other ELO families [modified after Suh and Clandinin (100)]. 18:3n3, 20:5n3, or 22:5n3 can be converted to VLC-PUFA through the 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) (126). 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 FAs are formed only in ELOVL4-expressing cells. However, other ELOVL proteins may be responsible for elongations of some of these VLC-PUFA (C30–C38) by using products generated by the 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. Reprinted from Agbaga et al. (39) with permission from publishers.