Stereospecificity of fatty acid 2-hydroxylase and differential functions of 2-hydroxy fatty acid enantiomers

Abstract

(FA2H) is a recently identifi ed NAD(P)H-dependent enzyme that catalyzes the hydroxylation of FA at the C-2 position to produce 2-hydroxy FAs (2-OH FAs) ( Scheme 1 ) Scheme 1( 2, 3 ). In addition to undergoing ␣ oxidation to form oddchain-length FAs ( 4 ), the FA2H-generated 2-OH FAs are incorporated into a long-chain sphingosine base via an amide bond to generate 2-hydroxy sphingolipids (2-OH sphingolipids) ( Scheme 1 ) ( 5 ). The 2-OH sphingolipids are important components of plasma membrane lipid rafts, which host a variety of signaling and traffi cking events. Specifi c functions of 2-OH sphingolipids in membrane endocytosis were fi rst demonstrated in Saccharomyces cerevisiae yeast strains with defective SCS7/FA2H ( 6, 7 ). Recent genetic studies suggest a critical role of FA2H and possibly 2-OH sphingolipids in multiple neurodegenerative diseases. Loss of FA2H in mice causes signifi cant demyelination and profound axonal loss ( 8,9 ). Mutations in the FA2H gene in humans are associated with leukodystrophy ( 10 ), spastic paraplegia ( 11 ), and neurodegeneration with brain iron accumulation ( 12 ). FA2H may also play a signifi cant role in metabolism. We recently showed decreased levels of glucose transporter 4 (GLUT4) proteins and thus cellular glucose uptake in FA2H knockdown adipocytes ( 13 ). A recent study also suggested a role of FA2H in synthesis of specifi c sebaceous gland or sebum lipids and in regulating the hair follicle homeostasis ( 14 ).2-OH FAs are chiral due to the asymmetric carbon bearing the hydroxyl group, and a specifi c 2-hydroxylase usually generates enantiopure products. Biological samples and diets therefore have different enantio-enrichments of Abstract FA 2-hydroxylase (FA2H) is an NAD(P)H-dependent enzyme that initiates FA ␣ oxidation and is also responsible for the biosynthesis of 2-hydroxy FA (2-OH FA)-containing sphingolipids in mammalian cells. The 2-OH FA is chiral due to the asymmetric carbon bearing the hydroxyl group. Our current study performed stereochemistry investigation and showed that FA2H is stereospecifi c for the production of ( R )-enantiomers. FA2H knockdown in adipocytes increases diffusional mobility of raft-associated lipids, leading to reduced GLUT4 protein level, glucose uptake, and lipogenesis. The effects caused by Manuscript received 23 February 2012 and in revised form 29 March 2012. Published, JLR Papers in Press, April 19, 2012 DOI 10.1194 Stereospecifi city of fatty acid 2-hydroxylase and differential functions of 2-hydroxy fatty acid enantiomers Abbreviations: Cer, ceramide; CHO, Chinese hamster ovary; CTxB, cholera toxin subunit B; D, diffusion coeffi cient; FA2H, FA 2-hydroxylase; FAME, FA methyl ester; FRAP, fl uorescence recovery after photobleaching; GLUT4, glucose transporter 4; HexCer, hexosylceramide; Mf, mobile fraction; MTPA, ␣ -methoxy-␣ -trifl uoromethylphenylacetyl; NCI-MS, negative chemical ionization mass spectrometry; NL, neutral loss; 2-OH Cer, 2-hydroxy ceramide; 2-OH FA, 2-hydroxy FA; 2-OH PA, 2-OH palmitic acid; 2-OH sphingo...

2′-Hydroxy ceramide in membrane homeostasis and cell signaling

“…Interestingly, the N -acyl chain of mammalian 2′-hydroxy sphingolipids were reported to be exclusively in the 2′R configuration (Karlsson et al, 1969; Mislow and Bleicher, 1954). This stereo-specificity is consistent with the sterospecific 2-hydroxylation by FA2H (Guo et al, 2012). There is some evidence of 2′S -hydroxy fatty acid in mammalian sphingolipids in foodstuff (Jenske and Vetter, 2008).…”

Uncovering biologically significant lipid isomers with liquid chromatography, ion mobility spectrometry and mass spectrometry

“…The final standards studied were the R and S orientations of the 2-hydroxyl group in ceramides (i.e. Cer(d18:1/18:0(2R-OH)) and Cer(d18:1/18:0(2S-OH))) which are thought to be formed from different hydroxylases and be important in membrane fluidity [28]. In the IMS-MS analyses, the R and S isomers did not separate in the negative mode, but in positive mode upon sodium ion binding, structural differences were observed.…”

2-Hydroxy Fatty Acid Enantiomers of Gb 3 Impact Shiga Toxin Binding and Membrane Organization

“…Previous studies have demonstrated that the unsaturation and the a-hydroxylation of the fatty acid play pivotal roles in STxB-induced membrane reorganization and membrane scission (5,7,8). However, the stereochemistry of the 2OH group at the fatty acid has not yet been addressed, even though Guo et al (9) showed that it influences the diffusional mobility of raft-associated lipids in adipocytes. A recently developed route to enantiomerically pure a-hydroxylated nervonic acid (10) enabled us to synthesize the naturally occurring Gb 3 -C24:1-2(R)OH (Gb 3 -R) and the nonnatural Gb 3 -C24:1-2(S)OH (Gb 3 -S) (Fig.…”