FA2H-dependent fatty acid 2-hydroxylation in postnatal mouse brain

2-Hydroxy fatty acids are relatively minor species of membrane lipids found almost exclusively as N-acyl chains of sphingolipids. In mammals, 2-hydroxy sphingolipids are uniquely abundant in myelin galactosylceramide and sulfatide. Despite the well-documented abundance of 2-hydroxy galactolipids in the nervous system, the enzymatic process of the 2-hydroxylation is not fully understood. To fill this gap, we have identified a human fatty acid 2-hydroxylase gene (FA2H) that is highly expressed in brain. In this report, we test the hypothesis that FA2H is the major fatty acid 2-hydroxylase in mouse brain and that free 2-hydroxy fatty acids are formed as precursors of myelin 2-hydroxy galactolipids. The fatty acid compositions of galactolipids in neonatal mouse brain gradually changed during the course of myelination. The relative ratio of 2-hydroxy versus nonhydroxy galactolipids was very low at 2 days of age ( approximately 8% of total galactolipids) and increased 6- to 8-fold by 30 days of age. During this period, free 2-hydroxy fatty acid levels in mouse brain increased 5- to 9-fold, and their composition was reflected in the fatty acids in galactolipids, consistent with a precursor-product relationship. The changes in free 2-hydroxy fatty acid levels coincided with fatty acid 2-hydroxylase activity and with the upregulation of FA2H expression. Furthermore, mouse brain fatty acid 2-hydroxylase activity was inhibited by anti-FA2H antibodies. Together, these data provide evidence that FA2H is the major fatty acid 2-hydroxylase in brain and that 2-hydroxylation of free fatty acids is the first step in the synthesis of 2-hydroxy galactolipids.     

A novel method for the measurement of in vitro fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry

Fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is an enzyme responsible for the de novo synthesis of sphingolipids containing 2-hydroxy fatty acids. 2-Hydroxy sphingolipids are highly abundant in the brain, as major myelin galactolipids (galactosylceramide and sulfatide) contain a uniquely high proportion ( approximately 50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of the digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. The physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study the roles of FA2H and 2-hydroxy sphingolipids in various tissues, we developed a highly sensitive in vitro FA2H assay. FA2H-dependent fatty acid 2-hydroxylation requires an electron transfer system, which was reconstituted in vitro with an NADPH regeneration system and purified NADPH:cytochrome P-450 reductase. A substrate [3,3,5,5-D(4)]tetracosanoic acid was solubilized in alpha-cyclodextrin solution, and the 2-hydroxylated product was quantified by gas chromatography-mass spectrometry after conversion to a trimethylsilyl ether derivative. When the microsomes of FA2H-transfected COS7 cells were incubated with the electron transfer system and deuterated tetracosanoic acid, deuterated 2-hydroxy tetracosanoic acid was formed in a time- and protein-dependent manner. With this method, FA2H activities were reproducibly measured in murine brains and tissue culture cell lines.     

The human FA2H gene encodes a fatty acid 2-hydroxylase

2-Hydroxysphingolipids are a subset of sphingolipids containing 2-hydroxy fatty acids. The 2-hydroxylation occurs during de novo ceramide synthesis and is catalyzed by fatty acid 2-hydroxylase (also known as fatty acid alpha-hydroxylase). In mammals, 2-hydroxysphingolipids are present abundantly in brain because the major myelin lipids galactosylceramides and sulfatides contain 2-hydroxy fatty acids. Here we report identification and characterization of a human gene that encodes a fatty acid 2-hydroxylase. Data base searches revealed a human homologue of the yeast ceramide 2-hydroxylase gene (FAH1), which we named FA2H. The FA2H gene encodes a 372-amino acid protein with 36% identity and 46% similarity to yeast Fah1p. The amino acid sequence indicates that FA2H protein contains an N-terminal cytochrome b5 domain and four potential transmembrane domains. FA2H also contains the iron-binding histidine motif conserved among membrane-bound desaturases/hydroxylases. COS7 cells expressing human FA2H contained 3-20-fold higher levels of 2-hydroxyceramides (C16, C18, C24, and C24:1) and 2-hydroxy fatty acids compared with control cells. Microsomal fractions prepared from transfected COS7 cells showed tetracosanoic acid 2-hydroxylase activities in an NADPH- and NADPH: cytochrome P-450 reductase-dependent manner. FA2H lacking the N-terminal cytochrome b5 domain had little activity, indicating that this domain is a functional component of this enzyme. Northern blot analysis showed that the FA2H gene is highly expressed in brain and colon tissues. These results demonstrate that the human FA2H gene encodes a fatty acid 2-hydroxylase. FA2H is likely involved in the formation of myelin 2-hydroxy galactosylceramides and -sulfatides.     

Fatty acid 2-hydroxylase regulates cAMP-induced cell cycle exit in D6P2T Schwannoma cells

Sphingolipids are ubiquitous components of eukaryotic cells that regulate various cellular functions. In many cell types, a fraction of sphingolipids contain 2-hydroxy fatty acids, produced by fatty acid 2-hydroxylase (FA2H), as the N-acyl chain of ceramide [hydroxyl fatty acid (hFA)-sphingolipids]. FA2H is highly expressed in myelin-forming cells of the nervous system and in epidermal keratinocytes. While hFA-sphingolipids are thought to enhance the physical stability of specialized membranes produced by these cells, physiological significance of hFA-sphingolipids in many other cell types is unknown. In this study, we report novel roles for FA2H and hFA-sphingolipids in the regulation of the cell cycle. Treatment of D6P2T Schwannoma cells with dibutyryl-cAMP (db-cAMP) induced exit from the cell cycle with concomitant upregulation of FA2H. Partial silencing of FA2H in D6P2T cells resulted in 60–70% reduction of hFA-dihydroceramide and hFA-ceramide, with no effect on nonhydroxy dihydroceramide and ceramide. Under these conditions, db-cAMP no longer induced cell cycle exit, and cells continued to grow and divide. Immunoblot analyses revealed that FA2H silencing prevented db-cAMP-induced upregulation of cyclin-dependent kinase inhibitors p21 and p27. These results provide evidence that FA2H is a negative regulator of the cell cycle and facilitates db-cAMP-induced cell cycle exit in D6P2T cells.     

Fatty acid 2-Hydroxylation in mammalian sphingolipid biology

2-Hydroxy fatty acids (hFA) are important components of a subset of mammalian sphingolipids. The presence of hFA in sphingolipids is best described in the nervous system, epidermis, and kidney. However, the literature also indicates that various hFA-sphingolipids are present in additional tissues and cell types, as well as in tumors. Biosynthesis of hFA-sphingolipids requires fatty acid 2-hydroyxlase, and degradation of hFA-sphingolipids depends, at least in part, on lysosomal acid ceramidase and the peroxisomal fatty acid α-oxidation pathway. Mutations in the fatty acid 2-hydroxylase gene, FA2H, have been associated with leukodystrophy and spastic paraparesis in humans, underscoring the importance of hFA-sphingolipids in the nervous system. In the epidermis, hFA-ceramides are essential for the permeability barrier function. Physiological function of hFA-sphingolipids in other organs remains largely unknown. Recent evidence indicates that hFA-sphingolipids have specific roles in cell signaling.     

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

FA 2-hydroxylase (FA2H) is an NAD(P)H-dependent enzyme that initiates FA α 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 stereospecific 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 FA2H knockdown were reversed by treatment with exogenous (R)-2-hydroxy palmitic acid, but not with the (S)-enantiomer. Further analysis of sphingolipids demonstrated that the (R)-enantiomer is enriched in hexosylceramide whereas the (S)-enantiomer is preferentially incorporated into ceramide, suggesting that the observed differential effects are in part due to synthesis of sphingolipids containing different 2-OH FA enantiomers. These results may help clarify the mechanisms underlying the recently identified diseases associated with FA2H mutations in humans and may lead to potential pharmaceutical and dietary treatments. This study also provides critical information to help study functions of 2-OH FA enantiomers in FA α oxidation and possibly other sphingolipid-independent pathways.     

2-Hydroxy-ceramide synthesis by ceramide synthase family: enzymatic basis for the preference of FA chain length

Ceramide is unusually abundant in epidermal stratum corneum and is important for permeability barrier function. Ceramides in epidermis also comprise an unusual variety, including 2-hydroxy (alpha-hydroxy)-ceramide. Six mammalian ceramide synthase/longevity assurance homologue (CerS/LASS) family members have been identified as synthases responsible for ceramide (CER) production. We reveal here that of the six, CerS3/LASS3 mRNA is the most predominantly expressed in keratinocytes. Moreover, its expression is increased upon differentiation. CerS family members have known substrate specificities for fatty acyl-CoA chain length and saturation, yet their abilities to produce 2-hydroxy-CER have not been examined. In the present study, we demonstrate that all CerS members can produce 2-hydroxy-CER when overproduced in HEK 293T cells. Each produced a 2-hydroxy-CER with a chain length similar to that of the respective nonhydroxy-CER produced. In HeLa cells overproducing the FA 2-hydroxylase FA2H, knock-down of CerS2 resulted in a reduction in total long-chain 2-hydroxy-CERs, confirming enzyme substrate specificity for chain length. In vitro CerS assays confirmed the ability of CerS1 to utilize 2-hydroxy-stearoyl-CoA as a substrate. These results suggest that all CerS members can synthesize 2-hydroxy-CER with specificity for 2-hydroxy-fatty acyl-CoA chain length and that CerS3 may be important in CER and 2-hydroxy-CER synthesis in epidermis.     

Myelin-Associated Galactolipids in Primary Cultures from Dissociated Fetal Rat Brain: Biosynthesis, Accumulation, and Cell Surface Expression

Galactolipid metabolism was investigated as a function of development in primary cultures initiated from 19-21-day-old dissociated fetal rat brain. Significant amounts of galactocerebrosides, sulfatides, and monogalactosylglycerides were synthesized and accumulated by 8 days in culture. Thereafter the synthetic rates and levels of these galactolipids increased rapidly, reaching maximal values approximately 22-29 days in culture. Galactolipids containing nonhydroxy or 2-hydroxy fatty acid were both synthesized at approximately equal rates. The initial rates of synthesis, investigated at 15, 29, and 50 days in culture, were three- to fivefold higher for galactocerebrosides than for sulfatides and two- to threefold higher than for monogalactosylglycerides. The total number of cells staining with antisera against galactocerebroside of sulfatide also increased very rapidly between 8 and 22 days in culture, reaching levels of 4-5 million cells per seeded fetal brain. The amount of galactocerebroside or sulfatide per cell stained with the corresponding antiserum increased severalfold from 10 to 27 days in culture and remained high until at least 36 days in culture (the latest time point examined). Thus, the temporal expression of galactolipid accumulation in the cell cultures was comparable to that occurring in rat brain, but some important quantitative reductions in the levels of accumulation per cell in culture were noted. In addition, in contrast to normal brain in which galactolipid synthetic rates are reduced after the period of most active myelination, in culture both synthesis and turnover of these galactolipids remained high, suggestive of a partial arrest in myelin maturation.     

Changes in the fatty acid composition of cerebrosides and sulfatides of human nervous tissue with age

Sphingogalactolipids (galactocerebrosides and sulfatides) have been isolated in almost quantitative yields from normal human nervous tissue (mostly brain) at different ages and their fatty acid compositions have been determined by gas-liquid chromatography. The ratio of hydroxy acids to normal acids increased slightly during myelination and then remained rather constant; in adults the ratio for cerebrosides was about 2, and for sulfatides, 0.6-0.8. In adult nervous tissue the two predominant fatty acids of cerebrosides and sulfatides were the C(24) monounsaturated and 2-hydroxy saturated acids. The infant brain galactolipids had (compared with child and adult) a lower percentage of C(22)-C(26) fatty acids and a much lower percentage of monoenoic acids, both of normal and hydroxy acids. Low activities of fatty acid elongation and desaturation systems during myelination are inferred. Fatty acid changes with age were the same for cerebrosides and sulfatides but occurred later in the sulfatides, which supports the hypothesis that the cerebrosides are precursors of the sulfatides. The adult pattern of fatty acid composition with regard to degree of unsaturation and total percentage of C(22)-C(26) acids was reached as early as at 2 yr of age, but the percentage of odd-numbered (C(23) and C(25)) fatty acids continued to increase up to the age of 10-15 yr. The fatty acid composition of the galactolipids of peripheral nerves differed mainly in its lower percentages of C(25) and C(26) acids and higher percentages of C(22) and C(16) acids. This composition is thus intermediate between those of brain and of extraneural organs.     

The Crystal Structure of an Integral Membrane Fatty Acid α-Hydroxylase

Neuronal electrical impulse propagation is facilitated by the myelin sheath, a compact membrane surrounding the axon. The myelin sheath is highly enriched in galactosylceramide (GalCer) and its sulfated derivative sulfatide. Over 50% of GalCer and sulfatide in myelin is hydroxylated by the integral membrane enzyme fatty acid 2-hydroxylase (FA2H). GalCer hydroxylation contributes to the compact nature of the myelin membrane, and mutations in FA2H result in debilitating leukodystrophies and spastic paraparesis. We report here the 2.6 Å crystal structure of sphingolipid α-hydroxylase (Scs7p), a yeast homolog of FA2H. The Scs7p core is composed of a helical catalytic cap domain that sits atop four transmembrane helices that anchor the enzyme in the endoplasmic reticulum. The structure contains two zinc atoms coordinated by the side chains of 10 highly conserved histidines within a dimetal center located near the plane of the cytosolic membrane. We used a yeast genetic approach to confirm the important role of the dimetal-binding histidines in catalysis and identified Tyr-322 and Asp-323 as critical determinants involved in the hydroxylase reaction. Examination of the Scs7p structure, coupled with molecular dynamics simulations, allowed for the generation of a model of ceramide binding to Scs7p. Comparison of the Scs7p structure and substrate-binding model to the structure of steroyl-CoA desaturase revealed significant differences in the architecture of the catalytic cap domain and location of the dimetal centers with respect to the membrane. These observations provide insight into the different mechanisms of substrate binding and recognition of substrates by the hydroxylase and desaturase enzymes.     

Functional association of cell death suppressor, Arabidopsis Bax inhibitor-1, with fatty acid 2-hydroxylation through cytochrome b5

Bax inhibitor-1 (BI-1) is a widely conserved cytoprotective protein localized in the endoplasmic reticulum (ER) membrane. We identified Arabidopsis cytochrome  b ₅ (AtCb5) as an interactor of Arabidopsis BI-1 (AtBI-1) by screening the Arabidopsis cDNA library with the split-ubiquitin yeast two-hybrid (suY2H) system. Cb5 is an electron transfer protein localized mainly in the ER membrane. In addition, a bimolecular fluorescence complementation (BiFC) assay and fluorescence resonance energy transfer (FRET) analysis confirmed that AtBI-1 interacted with AtCb5 in plants. On the other hand, we found that the AtBI-1-mediated suppression of cell death in yeast requires Saccharomyces cerevisiae fatty acid hydroxylase 1 (ScFAH1), which had a Cb5-like domain at the N terminus and interacted with AtBI-1. ScFAH1 is a sphingolipid fatty acid 2-hydroxylase localized in the ER membrane. In contrast, AtFAH1 and AtFAH2, which are functional ScFAH1 homologues in Arabidopsis, had no Cb5-like domain, and instead interacted with AtCb5 in plants. These results suggest that AtBI-1 interacts with AtFAHs via AtCb5 in plant cells. Furthermore, the overexpression of AtBI-1 increased the level of 2-hydroxy fatty acids in Arabidopsis, indicating that AtBI-1 is involved in fatty acid 2-hydroxylation.     

Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids,GLUT4 Level,and Lipogenesis in 3T3-L1 Adipocytes

Straight chain fatty acid α-oxidation increases during differentiation of 3T3-L1 adipocytes, leading to a marked accumulation of odd chain length fatty acyl moieties. Potential roles of this pathway in adipocyte differentiation and lipogenesis are unknown. Mammalian fatty acid 2-hydroxylase (FA2H) was recently identified and suggested to catalyze the initial step of straight chain fatty acid α-oxidation. Accordingly, we examined whether FA2H modulates adipocyte differentiation and lipogenesis in mature adipocytes. FA2H level markedly increases during differentiation of 3T3-L1 adipocytes, and small interfering RNAs against FA2H inhibit the differentiation process. In mature adipocytes, depletion of FA2H inhibits basal and insulin-stimulated glucose uptake and lipogenesis, which are partially rescued by the enzymatic product of FA2H, 2-hydroxy palmitic acid. Expression of fatty-acid synthase and SCD1 was decreased in FA2H-depleted cells, and levels of GLUT4 and insulin receptor proteins were reduced. 2-Hydroxy fatty acids are enriched in cellular sphingolipids, which are components of membrane rafts. Accelerated diffusional mobility of raft-associated lipids was shown to enhance degradation of GLUT4 and insulin receptor in adipocytes. Consistent with this, depletion of FA2H appeared to increase raft lipid mobility as it significantly accelerated the rates of fluorescence recovery after photobleaching measurements of lipid rafts labeled with Alexa 488-conjugated cholera toxin subunit B. Moreover, the enhanced recovery rates were partially reversed by treatment with 2-hydroxy palmitic acid. In conclusion, our findings document the novel role of FA2H in adipocyte lipogenesis possibly by modulation of raft fluidity and level of GLUT4.     

Lipid composition of Quaking mouse myelin: comparison with normal mouse myelin in the adult and during development

Abstract— In the ‘Quaking’ mouse, a deficiency in the long chain fatty acid content of galactolipids has been shown to occur. Myelin in the mutant has been compared to myelin in adult and in 12-day-old controls. We have shown that myelin is not only quantitatively reduced but also qualitatively modified, with a higher protein and a lower galactolipid content. Cerebrosides contain only a small amount of kerasin, lacking long chain nonhydroxylated fatty acids in comparison to both controls; the relative percentage of phrenosin is increased. Although many similarities exist between adult Quaking myelin and myelin at 12 days, differences have been shown to occur which may be in relation to a genetic block at an earlier stage of development.     

Effects of Brefeldin A on Galactosphingolipid Synthesis in an Immortalized Schwann Cell Line: Evidence for Different Intracellular Locations of Galactosylceramide Sulfotransferase and Ceramide Galactosyltransferase Activities

Abstract: Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6-h labeling period, a dose-dependent inhibition of [³⁵S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 µg/ml BFA. Labeling of newly synthesized galactosphingolipids with [³H]-palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA-GalCer) to 162% of control values, whereas labeling of GalCer containing 2-hydroxy fatty acids (HFA-GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA-GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA-GalCer synthesis. Examination of [³H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2-hydroxylation or the formation of HFA ceramide. Incorporation of [³H]palmitic acid into glucosylceramide and GM3 was increased over control levels whereas labeling of GM2 was inhibited, consistent with what has been reported previously for the effect of BFA on these glycolipids in other cell types. These results suggest that there are at least two separate intracellular sites for the galactosylation of HFA and NFA ceramide, respectively, which can be distinguished by their sensitivity to BFA. Our results also indicate that the site of GalCer sulfation is not redistributed to the endoplasmic reticulum in the presence of BFA and therefore may be localized to the distal Golgi or trans-Golgi network.     

Changes in fatty acid composition of human brain myelin lipids during maturation

Abstract— The variation with age of the fatty acid composition of the major lipids in human brain myelin was compared with that of cerebral white matter from the same region. The myelin was isolated from the semiovale centre of the cerebrum of 27 subjects neonatal to old aged. The phospholipid, cholesterol and galactolipid concentrations were determined in all the samples, as were the proportions of the major phospholipid classes. The proportions of cholesterol and especially of the galactolipids increased in myelin during the first 6 months, and in cerebral white matter up to 2 years. During this period the individual phospholipids also varied substantially. Serine phosphoglycerides and especially sphingomyelins increased, and choline phosphoglycerides decreased. The fatty acid patterns of ethanolamine phosphoglycerides (EPG) and sphingomyelins underwent the largest changes. The proportions of saturated fatty acids in EPG diminished rapidly, and there was an increase of monoenoic acids. Fatty acids of the linoleic acid series showed a peak between 4 and 12 months, after which time their proportion slowly diminished to old age. The major fatty acid of this series was docosatetraenoic acid, 22:4 (n-6), which constituted more than 25% of total fatty acids at the maximum level. The fatty acid changes were larger in cerebral white matter, but from 2 years of age the EPG fatty acid pattern in myelin was similar to that in white matter. The fatty acid changes in serine and choline phosphoglycerides of myelin with maturation were much less striking than in EPG but of a similar type. In myelin sphingomyelin the proportion of saturated long-chain fatty acids, C₁₆-C₂₂, diminished, while that of monoenoic acids increased and continued to do so up to old age. From 2 years of age the fatty acid patterns in myelin and cerebral white matter were quite similar. Also the fatty acid patterns of cerebrosides and sulphatides in cerebral white matter and myelin were the same except for the first 2 months of life. The same fatty acid changes occurred in cerebrosides and sulphatides as in the sphingomyelins, i.e. increased proportions of unsaturated (monoenoic) acids. The proportions of 24:1 and 24h:1 and of the odd-numbered fatty acids 25:1 and 23h:1 continued to increase to old age. The variations of the individual lipid fatty acid patterns were small except in the youngest age classes, in which the variations were presumably ascribable to the difficulty in determining the gestational age.     

The fatty acid composition of sphingolipids from bovine CNS axons and myelin

Abstract— Cerebrosides, sulphatides and sphingomyelin were isolated from bovine CNS myelin and from myelin-free axons derived from myelinated axons. The fatty acid composition of each sphingolipid was determined by gas-liquid chromatography of the fatty acid methyl esters. In each case the fatty acids of the axonal sphingolipids were of shorter average chain length than those from the corresponding myelin lipids. These differences, however, were small and the fatty acids of the axonal cerebrosides and sulphatides were similar in average chain length to those reported previously for bovine myelin. The principal unsubstituted acid of both cerebroside and sulphatide from axons was 24: 1, with the total long chain acids (> C₁₈) amounting to 80 and 85 per cent, respectively. The corresponding figures for myelin galactolipids were 94 and 95 per cent long chain acids. The principal α-hydroxy acid of both axonal galactolipids was 24 h:0, with cerebroside having 80 per cent and sulphatide 92 per cent long chain acids, compared to the figures of 87 and 97 per cent for the corresponding myelin lipids. In axonal sphingomyelin the major acid was 18:0 (compared to 24:1 in myelin) and the long chain acids were 61 per cent of the total vs 76 per cent of the total for myelin sphingomyelin. The non-identity of axonal and myelin sphingolipid fatty acids substantiates the belief that they are intrinsic axonal constituents. These findings do not rule out the possibility of a close metabolic relationship between the sphingolipids of the axon and its myelin sheath.     

Changes in CLIP3 expression after sciatic nerve injury in adult rats

CLIP3 (cytoplasmic linker protein 3) is a 547 amino acid residue cytoplasmic protein that localises to Golgi stacks and tubulovesicular elements juxtaposed to Golgi cisternae. Composed of three Ank (ankyrin) repeats and two CAP-Gly (cytoskeleton-associated protein-glycine) domains, CLIP3 may function as a cytoplasmic linker protein that is involved in TGN–endosome dynamics. To define the expression and role of CLIP3 during peripheral nervous system degeneration and regeneration, we created an acute sciatic nerve injury (SNI) model in adult rats. Western blot analyses revealed prominent up-regulation of CLIP3 and PCNA (proliferating cell nuclear antigen) protein levels at 3?days after SNI. Immunohistochemistry displayed that the expression of CLIP3 was noticeably increased in the injured nerve. Immunofluorescence further revealed that the CLIP3 and PCNA proteins colocalised respectively with S100 in the cytoplasm of Schwann cells. The expression profile of the SC/neuron co-cultures demonstrated that CLIP3 and PCNA protein levels were markedly expressed during the early stage of myelination. These results suggest that CLIP3 is likely associated with the myelination of proliferating Schwann cells, and nerve tissue regeneration after peripheral nerve injury. CLIP3 and PCNA expression during early myelination may be related to the direct uptake and transport of lipids and cholesterol, which were derived from the degenerating myelin, by Schwann cells to prepare for the formation of myelin sheath-like structures around regenerated axons after SNI.     

Proteolipid protein and DM-20 are synthesized by Schwann cells,present in myelin membrane,but they are not fatty acylated

Proteolipid protein (PLP) and DM-20 were intensely labeled after immunoprecipitation of total cellular proteins and myelin proteins labeled with [³⁵S]methionine in nerve slices. These results provided evidence that PLP and DM-20 are incorporated into the myelin membrane following their synthesis in Schwann cells. In contrast, PLP and DM-20 were not fatty acylated after incubation of the nerve slices with [³H]palmitic acid, however, PO glycoprotein and 24kDa protein were heavily fatty acylated. The lack of fatty acylation of PLP and DM-20 in the peripheral nervous system suggests that fatty acyltransferase responsible for their acylation is absent or non-functional in the peripheral nervous system.     

Divalent cation-mediated interaction between cerebroside sulfate and cerebrosides: an investigation of the effect of structural variations of lipids by electrospray ionization mass spectrometry.

Divalent cations mediate a carbohydrate-carbohydrate association between the two major glycolipids, galactosylceramide (GalCer) and its sulfated form, cerebroside sulfate (CBS), of the myelin sheath. We have suggested that interaction between these glycolipids on apposed extracellular surfaces of myelin may be involved in the stability or function of this multilayered structure. A mutant mouse lacking galactolipids because of a disruption in the gene that encodes a galactosyltransferase forms myelin that initially appears relatively normal but is unstable. This myelin contains glucosylceramide (GlcCer) instead of GalCer. To better understand the role of GlcCer in myelin in this mutant, we have compared the ability of divalent cations to complex CBS (galactosyl form) with GlcCer or GalCer in methanol solution by using positive ion electrospray ionization mass spectrometry. Because both the alpha-hydroxylated fatty acid species (HFA) and the nonhydroxylated fatty acid species (NFA) of these lipids occur in myelin, we have also compared the HFA and NFA species. In addition to monomeric Ca2+ complexes of all three lipids and oligomeric Ca2+ complexes of both GalCer and GlcCer, Ca2+ also caused heterotypic complexation of CBS to both GalCer and GlcCer. The heterotypic complexes had the greatest stability of all oligomers formed and survived better at high declustering potentials. Complexes of CBS with GlcCer were less stable than those with GalCer. This was confirmed by using the free sugars and glycosides making up the carbohydrate headgroups of these lipids. HFA species of CBS and GalCer formed more stable complexes than NFA species, but hydroxylation of the fatty acid of GlcCer had no effect. The ability of GlcCer to also complex with CBS, albeit with lower stability, may allow GlcCer to partially compensate for the absence of GalCer in the mouse mutant.