Biosynthesis of the Unsaturated 14-Carbon Fatty Acids Found on the N Termini of Photoreceptor-specific Proteins

Abstract

In the vertebrate retina, a number of proteins involved in signal transduction are known to be N-terminal acylated with the unusual 14 carbon fatty acids 14:1n-9 and 14:2n-6. We have explored possible pathways for producing these fatty acids in the frog retina by incubation in vitro with candidate precursor fatty acids bearing radiolabels, including [3H]14:0, [3H]18:1n-9, [3H]18:2n-6, and [3H]18:3n-3. Rod outer segments were prepared from the radiolabeled retinas for analysis of protein-linked fatty acids, and total lipids were extracted from the remaining retinal pellet. Following saponification of extracted lipids, fatty acid phenacyl esters were prepared and analyzed by high pressure liquid chromatography (HPLC) with detection by continuous scintillation counting. Transducin, whose alpha-subunit (Gt alpha) is known to bear N-terminal acyl chains, was extracted from the rod outer segments and subjected to SDS-polyacrylamide gel electrophoresis and fluorography to detect radiolabeled proteins. Gt alpha was also subjected to methanolysis, and the resulting fatty acyl methyl esters were analyzed by HPLC. The identities of HPLC peaks coinciding with unsaturated species of both phenacyl esters and methyl esters were confirmed by reanalyzing them after catalytic hydrogenation. The results showed that 14:1n-9 can be derived in the retina from 18:1n-9 and 14:2n-6 from 18:2n-6, most likely by two rounds of beta-oxidation, but that neither is produced in detectable amounts from 14:0. Retroconversion of unsaturated 18 carbon fatty acids to the corresponding 14 carbon species showed specificity, in that 18:3n-3 was not converted to 14 carbon fatty acids in detectable amounts. Myristic acid (14:0), 14:1n-9, and 14:2n-6 were all incorporated into Gt alpha. A much less efficient incorporation of 18:1n-9 into Gt alpha was also observed, but no radiolabeling of Gt alpha was observed in retinas incubated with 18:3n-3. Thus, retroconversion by limited beta-oxidation of longer chain unsaturated fatty acids appears to be the most likely metabolic source of the unusual fatty acids found on the N termini of signal transducing proteins in the retina.

Conversion of eicosapentaenoic acid to chain-shortened omega–3 fatty acid metabolites by peroxisomal oxidation

“…These metabolites are formed through peroxisomal ␤oxidation, and the main product is the 16-carbon metabolite, 16:3n-6 (1). Additional studies have shown that the retina can convert linoleic acid to the 14:2n-6 that is utilized for acylation of photoreceptor proteins (25). The present work indicates that EPA is converted to similar products, and as in the case of arachidonic acid, the most abundant metabolite that accumulates under all of the conditions tested is 16:4n-3, the omega-3 analogue of 16:3n-6.…”

Role of Elovl4 Protein in the Biosynthesis of Docosahexaenoic Acid

“…FAMEs were analyzed by gas-liquid chromatography as described (Tanito et al, 2008). Fatty acid phenacyl esters (FAPES) were prepared from total lipid extracts for high performance liquid chromatography (HPLC) analysis as previously described (DeMar and Anderson 1997; DeMar et al, 1996). …”

Biosynthesis of fatty acids and related metabolites

“…Anderson and co-workers have found that frog retina outer rod segments converted radiolabelled oleic acid 18:1 (n 9) 76 to 152 and 18:2 (n 6) 114 to 153, presumably by two rounds of -oxidation, and that 18:3 (n 3) or 14:0 151 were not incorporated into either 14:1 (n 9) 152 or 14:2 (n 6) 153 (Scheme 40). 209 There does not appear to be a 5Z desaturase operative in this system.…”