A novel FADS1 isoform potentiates FADS2-mediated production of eicosanoid precursor fatty acids

Abstract

The fatty acid desaturase (FADS) genes code for the rate-limiting enzymes required for the biosynthesis of long-chain polyunsaturated fatty acids (LCPUFA). Here we report discovery and function of a novel FADS1 splice variant. FADS1 alternative transcript 1 (FADS1AT1) enhances desaturation of FADS2, leading to increased production of eicosanoid precursors, the first case of an isoform modulating the enzymatic activity encoded by another gene. Multiple protein isoforms were detected in primate liver, thymus, and brain. In human neuronal cells, their expression patterns are modulated by differentiation and result in alteration of cellular fatty acids. FADS1, but not FADS1AT1, localizes to endoplasmic reticulum and mitochondria. Ribosomal footprinting demonstrates that all three FADS genes are translated at similar levels. The noncatalytic regulation of FADS2 desaturation by FADS1AT1 is a novel, plausible mechanism by which several phylogenetically conserved FADS isoforms may regulate LCPUFA biosynthesis in a manner specific to tissue, organelle, and developmental stage.

Fig. 1.

Currently accepted pathway for LCPUFA synthesis from precursors. Alternating desaturation and elongation occurs on the ER. Δ8-desaturation of 20:2n-6 and 20:3n-3 yield 20:3n-6 and 20:4n-3, intermediates in the conventional pathway to 20:4n-6 and 20:5n-3 (18), which can be further elongated and desaturated.

Fig. 2.

Novel alternative mRNA isoforms of baboon FADS1 identified by RACE. Exons are numbered 1–12. Transcripts b, c, d, e, f, and g code for a 444 amino acid protein (collectively FADS1CS). Transcripts h, i, and j code for a 360 amino acid protein (collectively FADS1AT1). Transcript k (FADS1AT2) codes for 270 amino acid protein. (A) Transcripts a is a recent NCBI annotation. (B) Transcript b is the classical transcript (CS). (C–G) In transcripts c through g, the difference is in the length of 3′ UTR and poly(A) tail. (H) In transcript h, exon 1 is truncated and the 5′ UTR is altered. The three histidine motifs are retained but the cytochrome b5 domain is lost. (I, J) In transcripts i and j, exon 1 is deleted and exon 2 is truncated. The 5′ UTR region is embedded within exon 2; the three histidine motifs are retained but the cytochrome b5 domain is lost. (K) In transcript k, multiple exons are missing via alternative splicing. The ATG start site is within exon 1.

Fig. 3.

GC results of transfection assay of MCF7 cells with 20:3n-6 substrate. Cells stably expressing (A) vector only or (B) FADS1AT1 show no desaturase activity. Cotransfection of both the (C) vector-only cells and (D) FADS1AT1 cells with FADS1 shows enhanced 20:4n-6 (via 20:3→20:4 catalyzed by FADS1) but no difference in 20:4n-6, indicating FADS1AT1 is not a modifier of FADS1 activity.

Fig. 4.

GC results of transfection assay of MCF7 cells with 18:2n-6. Transfection of (A) vector-only cells or (B) FADS1AT1 cells shows no desaturation activity (C) Cotransfection of the vector-only cells with FADS2 shows 18:3n-6 and 20:3n-6 product synthesis. (D) Cotransfection of FADS1AT1 with FADS2 produces 18:3n-6 and 20:3n-6 enhanced by more than 2-fold.

Fig. 5.

(A) RT-PCR analysis of FADS1CS and FADS1AT1 expression shown in 9 baboon neonate tissues (lane 1, DNA 100 bp ladder; lane 2, liver; lane 3, kidney; lane 4, retina; lane 5, occipital lobe; lane 6, lung; lane 7, spleen; lane 8, pancreas; lane 9, ovary; lane 10, skeletal muscle; lane 11, no RT). The amplified products were resolved on 2% agarose gel and visualized with ethidium bromide. (B) FADS1CS and FADS1AT1 expression in HepG2, MCF7, and human SK-N-SH neuroblastoma (NB) cells (lane 1, DNA 100 bp ladder; lane 2, FADS1CSHepG2; lane 3, FADS1AT1HepG2; lane 4, β-actinHepG2; lane 5, FADS1CSMCF7; lane 6, FADS1AT1MCF7; lane 7, β-ActinMCF7; lane 8, FADS1CSNB; lane 9, FADS1AT1NB; lane 10, β-actinNB). The amplified products were resolved on 2% agarose gel and visualized with ethidium bromide. FADS1CS and FADS1AT1 generate 143 base pair (bp) products; β-actin, 200 bp products.

Fig. 6.

FADS1 protein detection in neonate baboon tissues and mammalian cells. (A) Western blot analysis of FADS1 and β-actin (loading control) using total protein from thymus, brain, and liver tissues. (B) Western blot analysis of FADS1 and β-actin using total protein from HepG2 and MCF7 cells.

Fig. 7.

FADS1 protein isoform expression and fatty acid changes upon NB cell differentiation. (A) Western blot analysis of FADS1 and β-actin using total protein from undifferentiated and differentiated NB cells. (B) Fatty acid analysis of undifferentiated (open bars) and differentiated (black bars) NB cells.

Fig. 8.

Subcellular localization of FADS1 isoforms. SK-N-SH NB cells were transfected with (A) pEFADS1 and (B) pEFADS1AT1; stained with MitoTracker Red (Mt) or ER-tracker Blue-White DPX (ER); and visualized with an inverted confocal microscope. (C) Western blot analysis using GFP, β-actin, and COXIV antibodies.

Fig. 9.

Translation of all FADS trapped in ribosomes of mouse MEF cells. FADS1 mRNA is translated into protein at similar to levels FADS2-3.