Traditional reactive carbonyl scavengers do not prevent the carbonylation of brain proteins induced by acute glutathione depletion.

This study investigated the effect of reactive carbonyl species (RCS)-trapping agents on the formation of protein carbonyls during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH-depletor diethyl maleate in the absence or presence of chemically different RCS scavengers (hydralazine, methoxylamine, aminoguanidine, pyridoxamine, carnosine, taurine and z-histidine hydrazide). Despite their strong reactivity towards the most common RCS, none of the scavengers tested, with the exception of hydralazine, prevented protein carbonylation. These findings suggest that the majority of protein-associated carbonyl groups in this oxidative stress paradigm do not derive from stable lipid peroxidation products like malondialdehyde (MDA), acrolein and 4-hydroxynonenal (4-HNE). This conclusion was confirmed by the observation that the amount of MDA-, acrolein- and 4-HNE-protein adducts does not increase upon GSH depletion. Additional studies revealed that the efficacy of hydralazine at preventing carbonylation was due to its ability to reduce oxidative stress, most likely by inhibiting mitochondrial production of superoxide and/or by scavenging lipid free radicals.

Presence of alpha-globin mRNA and migration of bone marrow cells after sciatic nerve injury suggests their participation in the degeneration/regeneration process.

We have previously reported that in the distal stump of ligated sciatic nerves, there is a change in the distribution of myelin basic protein (MBP) and P0 protein immunoreactivities. These results agreed with the studies of myelin isolated from the distal stump of animals submitted to ligation of the sciatic nerve, showing a gradual increase in a 14 kDa band with an electrophoretic mobility similar to that of an MBP isoform, among other changes. This band, which was resolved into two bands of 14 and 15 kDa using a 16% gel, was found to contain a mixture of MBP fragments and peptides with great homology with alpha- and beta-globins. In agreement with these results, we have demonstrated that the mRNA of alpha-globin is present in the proximal and distal stumps of the ligated nerve. It is also detected at very low levels in Schwann cells isolated from normal nerves. These results could be due to the presence of alpha- and/or beta-globin arising from immature cells of the erythroid series. Also, they could be present in macrophages, which spontaneously migrate to the injured nerve to promote the degradation of myelin proteins. Cells isolated from normal adult rat bone marrow which were injected intraortically were found to migrate to the injured area. These cells could contribute to the remyelination of the damaged area participating in the removal of myelin debris, through their transdifferentiation into Schwann cells or through their fusion with preexisting Schwann cells in the distal stump of the injured sciatic nerve.

Nitric oxide reduces the palmitoylation of rat myelin proteolipid protein by an indirect mechanism.

Brain slices from 20-day-old rats were incubated with [3H]palmitate for 2 hours in the absence or presence of the NO-donors S-nitroso-N-acetyl-penicillamine (SNAP), ethyl-2-[hydroxyimino]-5-nitro-3-hexeneamide (NOR-3), 4-phenyl-3-furoxan carbonitrile (PFC) and sodium nitroprusside (SNP). Each of these drugs reduced the incorporation of [3H]palmitate into myelin proteolipid protein (PLP) in a concentration-dependent manner, SNP being the most active. The effect of SNAP was prevented by the NO-scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide). Furthermore, decayed-SNAP, sodium nitrite and N- nitrosopyrrolidine were inactive, suggesting that free NO and/or some of its direct oxidation products are the active molecular species. The amount of fatty acids bound to PLP and the rate of deacylation were unaffected by NO. Although NO diminished the number of thiols in brain and myelin proteins, with the formation of both nitrosothiols and disulfides, these changes did not parallel those in PLP acylation. In contrast, NO was effective at reducing the palmitoylation of brain and myelin lipids, and this effect along with that of PLP, was ascribed to a decrease in palmitoyl-CoA levels. The NO-induced reduction in acyl-CoA concentration was due to the decline in ATP levels, while the amount of [3H]palmitate incorporated into the tissue, the activity of palmitoyl-CoA ligase and palmitoyl-CoA hydrolase, and the concentration of CoASH were unaltered by the drugs. Experiments with endogenously-synthesized [18O]fatty acids confirmed that NO affects predominantly the ATP-dependent palmitoylation of PLP. In conclusion, the inhibitory action of NO on the fatty acylation of PLP is indirect and caused by energy depletion.

Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters.

Non-enzymatic thioesterification of specific cysteinyl peptides with fatty acyl-CoA has been previously demonstrated in both liposomes and aqueous medium. To identify the molecular basis for the differential reactivity of polypeptides in aqueous solutions, 26 synthetic cysteinyl peptides encompassing the palmitoylation sites of well known proteins (protein zero, proteolipid protein, beta-adrenergic receptor, p21(K-ras), transferrin receptor, CD-4 and SNAP-25) and six small thiol compounds were incubated separately with [3H]palmitoyl-CoA, [14C]acetyl-CoA and p-nitrophenyl thioacetate (NPTA). For each peptide, both the observed reaction rate constant at pH 7.5 and the pH-independent rate constant (k(2)) were calculated, and reactivity of the attacking sulfhydryl group was characterized using the Brønsted equation (log k(2)=beta(nuc) pK(a)+C). In general, peptides bearing basic and aromatic amino acid residues showed the lowest thiol pK(a)s, and consequently displayed the highest acylation rates. Reaction with palmitoyl-CoA was complicated to analyze because of the variable partition of peptides in the acyl chain donor/detergent micelles. In contrast, a linear Brønsted relationship was found for the reaction of the peptides with the water-soluble acetyl-CoA (beta(nuc)=0.59). A similar beta(nuc) value was obtained with the neutral NPTA, indicating that electronic effects other than those responsible for the acid-base properties of the thiol are less important. Thus, the concentration of the thiolate anion appears to be the major factor influencing the rate of the nucleophilic substitution reaction. These findings and the fact that the acylation sites in most proteins are surrounded by basic amino acids may partially explain the specificity of non-enzymatic palmitoylation regarding the acceptor sequences.

Chemical deacylation reduces the adhesive properties of proteolipid protein and leads to decompaction of the myelin sheath.

Myelin proteolipid protein (PLP) contains thioester-bound, long-chain fatty acids which are known to influence the structure of the molecule. To gain further insights into the role of this post-translational modification, we studied the effect that chemical deacylation of PLP had on the morphology of myelin and on the protein's ability to mediate the clustering of lipid vesicles. Incubation of rat optic nerves in isoosmotic solutions containing 100 mM hydroxylamine (HA) pH 7.4 led to deacylation of PLP and decompaction of myelin lamellae at the level of the intraperiod line. Incubation of nerves with milder nucleophilic agents (Tris and methylamine) or diluted HA, conditions that do not remove protein-bound fatty acids, caused no alterations in myelin structure. Other possible effects of HA which could have affected myelin compaction indirectly were ruled out. Incubation of optic nerves with 50 mM dithioerythritol (DTE) also led to the splitting of the myelin intraperiod line and this change again coincided with the removal of fatty acids. In addition, the apparently compacted CNS myelin in the PLP-less myelin-deficient rat, like that in tissue containing deacylated PLP, was readily decompacted upon incubation in isoosmotic buffers, suggesting that the function of PLP as a stabilizer of the interlamellar attachment is, at least in part, mediated by fatty acylation. Furthermore, in contrast to the native protein, PLP deacylated with either HA or DTE failed to induce the clustering of phosphatidylcholine/cholesterol vesicles in vitro. This phenomenon is not due to side-effects of the deacylation procedure since, upon partial repalmitoylation, the protein recovered most of its original vesicle-clustering activity. Collectively, these findings suggest that palmitoylation, by influencing the adhesive properties of PLP, is important for stabilizing the multilamellar structure of myelin.

Conserved fatty acid composition of proteolipid protein during brain development and in myelin subfractions.

Myelin proteolipid protein (PLP) is modified after translation by the attachment of long-chain fatty acids to several cysteine residues. In this study, the amount and pattern of fatty acids covalently bound to rat PLP were determined during brain development and in myelin subfractions. For this purpose, PLP was isolated by gel-filtration chromatography in organic solvents, subjected to alkaline methanolysis, and the released fatty acid methyl esters were analyzed by gas-liquid chromatography. At all ages examined, PLP had the same amount of covalently-bound fatty acids (3-4% w/w) and palmitate, oleate and stearate were always the major acyl chains. In contrast to myelin lipids, the fatty acid composition of PLP showed only minor changes between 15-days and 90-days of age. The amount and pattern of fatty acids bound to PLP prepared from three myelin subfractions were also indistinguishable. The conservation of a characteristic PLP-fatty acid make-up during brain development and in various myelin compartments suggests that this post-translational modification is essential for the normal functioning of the protein.

Effect of ATP depletion on the palmitoylation of myelin proteolipid protein in young and adult rats.

The present study was designed to determine whether the palmitoylation of the hydrophobic myelin proteolipid protein (PLP) is dependent on cellular energy. To this end, brain slices from 20- and 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various metabolic poisons. In adult rats, the inhibition of mitochondrial ATP production with KCN (5 mM), oligomycin (10 microM), or rotenone (10 microM) reduced the incorporation of [3H]palmitate into fatty acyl-CoA and glycerolipids by 50-60%, whereas the labeling of PLP was unaltered. Incubation in the presence of rotenone (10 microM) plus NaF (5 mM) abolished the synthesis of acyl-CoA and lipid palmitoylation, but the incorporation of [3H]palmitate into PLP was still not different from that in controls. In rapidly myelinating animals, the inhibition of both mitochondrial electron transport and glycolysis obliterated the palmitoylation of lipids but reduced that of PLP by only 40%. PLP acylation was reduced to a similar extent when slices were incubated for up to 3 h, indicating that exogenously added palmitate is incorporated into PLP by ATP-dependent and ATP-independent mechanisms. Determination of the number of PLP molecules modified by each of these reactions during development suggests that the ATP-dependent process is important during the formation and/or compaction of the myelin sheath, whereas the ATP-independent mechanism is likely to play a role in myelin maintenance, perhaps by participating in the periodic repair of thioester linkages between the fatty acids and the protein.

Fatty acid composition of myelin proteolipid protein during vertebrate evolution.

The hydrophobic myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids which are attached to intracellular cysteine residues via thioester linkages. To gain insight into the role of acylation in the structure and function of myelin PLP, the amount and pattern of acyl groups attached to the protein during vertebrate evolution was determined. PLP isolated from brain myelin of amphibians, reptiles, birds and several mammals was subjected to alkaline methanolysis and the released methyl esters were analyzed by gas-liquid chromatography. In all species studied, PLP contained approximately the same amount of covalently bound fatty acids (3% w/w), and palmitic, palmitoleic, oleic and stearic acids were always the major acyl groups. Although the relative proportions of these fatty acids changed during evolution, the changes did not necessarily follow the variations in the acyl chain composition of the myelin free fatty acid pool, suggesting fatty acid specificity. The phylogenetic conservation of acylation suggests that this post-translational modification is critical for PLP function.

Veratridine-induced depolarization reduces the palmitoylation of brain and myelin glycerolipids.

In this study, we have investigated the effect of neuronal depolarization on the palmitoylation of myelin lipids. For this purpose, brain slices from 60-day-old rats were incubated with [3H]palmitate for 1 h in the presence or absence of various drugs. Veratridine (100 microM) reduced the incorporation of [3H]palmitate into all brain glycerolipids by 40-50%, whereas the labeling of sphingolipids was unaffected. Similar results were obtained by using [3H]glycerol as a precursor, demonstrating that veratridine also causes a reduction in the de novo synthesis of glycerolipids. Both tetrodotoxin (1 microM) and ouabain (1 mM) prevented the effect of veratridine, indicating that it is mediated through the opening of voltage-gated sodium channels and involves the stimulation of the Na+/ K+ pump. Decreased levels of both ATP, due to activation of the Na+,K+-ATPase, and the precursor palmitoyl-CoA were found in the veratridine-treated slices, thus explaining the reduction in lipid synthesis. Neuronal depolarization also decreased the synthesis of lipids present in the myelin fraction. The relatively high specific radioactivity of myelin lipids and the results from both repeated purification experiments and mixing experiments ruled out the possibility that the radioactive lipids present in myelin could derive from contamination with other subcellular fraction(s). Because neither mature oligodendrocytes nor myelin is known to express voltage-dependent Na+ channels, it is conceivable that the effect of veratridine on myelin glycerolipid metabolism occurs by an indirect mechanism such as an increase in the extracellular [K+]. However, the presence of 60 mM KCl in the medium did not affect the acylation of either brain or myelin lipids. These results raise questions as to the absence of sodium channels in myelinating oligodendrocytes and/or myelin.

Palmitoylation of proteolipid protein from rat brain myelin using endogenously generated 18O-fatty acids.

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains covalently bound fatty acids, predominantly palmitic acid. This study adapts a stable isotope technique (Kuwae, T., Schmid, P. C., Johnson, S. B., and Schmid, H. O. (1990) J. Biol. Chem. 265, 5002-5007) to quantitatively determine the minimal proportion of PLP molecules which undergo palmitoylation. In these experiments, brain white matter slices from 20-day-old rats were incubated for up to 6 h in a physiological buffer containing 50% H218O. The uptake of 18O into the carbonyl groups of fatty acids derived from PLP, phospholipids, and the free fatty acid pool was measured by gas-liquid chromatography/mass spectrometry of the respective methyl esters. Palmitic acid derived from PLP acquired increasing amounts of 18O, ending with 2.9% 18O enrichment after 6 h of incubation. 18O incorporation into myelin free palmitic acid also increased over the course of the incubation (67.2% 18O enrichment). After correcting for the specific activity of the 18O-enriched free palmitic acid pool, 7.6% of the PLP molecules were found to acquire palmitic acid in 6 h. This value is not only too large to be the result of the palmitoylation of newly synthesized PLP molecules, it was also unchanged upon the inhibition of protein synthesis with cycloheximide. 18O enrichment in less actively myelinating 60-day-old rats was significantly reduced. In conclusion, our experiments suggest that a substantial proportion of PLP molecules acquire palmitic acid via an acylation/deacylation cycle and that this profile changes during development.

The mechanism and functional roles of protein palmitoylation in the nervous system.

Palmitoylation refers to the covalent attachment of long-chain fatty acids, mostly palmitic acid, to the side chain of cysteine residues of proteins. In recent years, a considerable number of functionally relevant nervous system proteins including ion channels, neurotransmitter receptors, signal transduction components and cell-adhesion molecules have been found to be palmitoylated. However, the lack of a generalized and unambiguous role for the fatty acids in these proteins has questioned the importance of palmitoylation in the functioning of the nervous system. Last year, it was found that the deficiency of palmitoyl-protein thioesterase (PPT), one of the enzymes responsible for the removal of palmitate from proteins, is the underlying cause of infantile neuronal ceroid lipofuscinosis (INCL). This finding not only constitutes a step forward in elucidating the pathogenesis of INCL, but it also provides new impetus in the search for the function(s) of protein palmitoylation. This work succinctly outlines the molecular mechanisms involved in dynamic acylation and the potential biological role(s) of this modification, and it constitutes an introduction to those presentations in this issue which specifically deal with the pathogenic mechanisms of INCL.

Palmitoylation of myelin P0 protein is independent of its synthesis and parallels that of phospholipids.

To determine whether the acyl chains modifying P0, the major protein of PNS myelin, turn over independently of the protein backbone, sciatic nerve slices from 10 to 65 day-old rats were incubated with a mixture of [3H]palmitic acid and [14C]amino acids, and proteins were analyzed by electrophoresis. Incorporation of [14C]amino acids into nerve P0 decreased approximately 10-fold between 10 and 65 days of age. In contrast, palmitoylation of P0, although maximal at 10 days-of age, decreased only 3-4-fold during the same period. In the same experiments, the incorporation of [3H]palmitate into the nerve and into various lipids classes diminished by a comparable extent (2.5-fold). Thus, if corrected by the uptake of the tritiated precursor, palmitoylation of P0 remains nearly constant throughout development, and it is therefore independent of protein synthesis. Preincubation of nerve slices with cycloheximide for one hour reduced the incorporation of [3H]palmitate into both P0 and phospholipids in a concentration-dependent manner. At 10 microM cycloheximide, palmitoylation of P0 was unaffected while its synthesis was still repressed, indicating that these events are uncoupled. The effect of cycloheximide on fatty acid uptake can be attributed to inhibition of the palmitoyl-CoA : lysophosphatidylcholine acyltransferase activity. Neither the distribution of palmitate between albumin and lipid membranes nor the activities of other lipid-metabolizing enzymes were affected by the inhibitor. In conclusion, these results indicate that P0 palmitoylation occurs mainly on the preexisting molecules, and it therefore constitutes a dynamic event.

Myelin P0 glycoprotein and a synthetic peptide containing the palmitoylation site are both autoacylated.

P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153-containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl-CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonenzymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl-CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37 degrees C, peptide palmitoylation followed a second-order reaction (k2 = 246 +/- 6 M-1 min-1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl-CoA. The activation energy calculated from the Arrhenius plot is approximately 2 kcal/mol and much lower than that of enzyme-catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol-containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.

Overview: protein palmitoylation in the nervous system: current views and unsolved problems.

Palmitoylation refers to a dynamic post-translational modification of proteins involving the covalent attachment of long-chain fatty acids to the side chains of cysteine, threonine or serine residues. In recent years, palmitoylation has been identified as a widespread modification of both viral and cellular proteins. Because of its dynamic nature, protein palmitoylation, like phosphorylation, appears to have a crucial role in the functioning of the nervous system. Several important questions regarding the post-translational acylation of cysteine residues in proteins are briefly discussed: (a) What are the molecular mechanisms involved in dynamic acylation? (b) What are the determinants of the fatty acid specificity and the structural requirements of the acceptor proteins? (c) What are the physiological signals regulating this type of protein modification, and (d) What is the biological role(s) of this reaction with respect to the functioning of specific nervous system proteins? We also present the current experimental obstacles that have to be overcome to fully understand the biology of this dynamic modification.

Identification of the palmitoylation site in rat myelin P0 glycoprotein.

P0 glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P0 was labeled in rat sciatic nerve slices with [3H]palmitic acid and subsequently treated with various reagents. The protein-bound palmitate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydroxylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 mM NaBH4 with the concomitant production of [3H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [14C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [14C]carboxyamidomethylated P0 was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys153 in rat P0 glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmembrane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken.

Proteolipid protein from the peripheral nervous system also contains covalently bound fatty acids.

Proteolipid protein, the major protein component of central nervous system (CNS) myelin, is known to contain approximately 2% by weight of covalently bound fatty acids. Recently, this protein was also found, though at a lower concentration, in the peripheral nervous system (PNS). The present study was undertaken to determine whether PNS proteolipid protein, like its CNS counterpart, was fatty acylated. Myelin PLP and DM-20, the two proteins that constitute the proteolipid protein fraction, were isolated from bovine sciatic nerves by chromatography on a methylated Sephadex G-100 column. The identity of the isolated proteins was determined by (a) SDS-PAGE, (b) Western blot analysis, and (c) amino acid analysis, and fractions containing only PLP and DM-20 were subjected to acid-methanolysis. Gas-liquid chromatography of the released methyl esters revealed the presence of fatty acids (2.9% w/w) distributed as approximately 47% palmitic, 22% stearic, and 19% oleic acid. Similar results were obtained for bovine white matter proteolipid protein when isolated and analyzed in parallel with the peripheral nerve protein. These data unequivocally demonstrate that both PNS and CNS proteolipid protein are equally acylated.

Characterization of proteolipid protein fatty acylesterase from rat brain myelin.

A protein fatty acylesterase activity that catalyzes the removal of fatty acid from exogenous proteolipid protein (PLP) has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the deacylation of PLP was obtained in 0.5% Triton X-100, 1 mM dithiothreitol at pH 7.0 and at 37 degrees C. Other detergents (octyl beta-D-glucoside, Nonidet P-40, and Tween 20) have little or no effect, whereas deacylation was completely abolished by 0.1% sodium dodecyl sulfate or boiling the membrane fraction for 5 min prior to incubation. Under optimal conditions, the rate of deacylation was linear up to 20 min, and the apparent Km for bovine [3H]palmitoyl-PLP was 18 microM. The myelin-associated PLP fatty acylesterase has no apparent requirements for divalent cations (Ca2+, Mg2+, Mn2+), and chelators such as EDTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, and 1,10-phenantroline have little or no effect on enzyme activity. Sulfhydryl and histidine residues are needed for full enzyme activity, whereas the "active serine"-directed inhibitor phenylmethylsulfonyl fluoride has no effect. The myelin-associated protein fatty acylesterase was present throughout brain development and in all myelin subfractions, in agreement with the dynamic metabolism of PLP-bound fatty acids. Enzyme activity was also present in sciatic nerve, brain cortex, and heart whereas liver was devoid of activity. Several esterases, including phospholipase A2, glyoxalase II, and acetylcholinesterase, did not remove fatty acid from PLP. Myelin basic protein, palmitoyl-CoA hydrolase, and myelin-associated nonspecific esterase were also ruled out as the PLP fatty acylesterase. Thus, all data seem to indicate that this enzyme is different from esterases of the lipid metabolism. Finally, stimulation of protein phosphorylation with Ca2+, but not with cyclic-AMP, inhibited PLP deacylation, suggesting that the myelin-associated protein fatty acylesterase activity is regulated by endogenous Ca(2+)-dependent protein kinases.

Rapid metabolism of fatty acids covalently bound to myelin proteolipid protein.

Proteolipid protein (PLP), the major protein of central nervous system myelin, contains approximately 2 mol of covalently bound fatty acids. In this study, the in vivo turnover rate of the acyl chains bound to PLP was determined in 40-day-old rats after a single intracranial injection of [3H]palmitic acid. The apparent half-life of total fatty acids bound to PLP was approximately 7 days. After correction for acyl chain interconversion, the half-life of palmitate bound to PLP was only 3 days. This turnover rate is much more rapid than that of the protein moiety calculated under the same experimental conditions (t1/2 = 1 month). Additional evidence for the dynamic metabolism of acyl groups was provided by experiments in brain tissue slices which showed that acylation of PLP occurs in adult animals as well as during active myelination. Acylation of endogenous PLP in purified myelin and its subfractions was also studied during rat brain development using either [3H]palmitoyl-CoA or [3H]palmitic acid plus ATP and CoA. Labeling of endogenous PLP with [3H]palmitoyl-CoA was observed as early as 10 days postnatal and continued at the same rate throughout development. When [3H]palmitic acid was used as precursor in the presence of both ATP and CoA, esterification of myelin PLP occurred rapidly in adult animals, indicating that both nonacylated PLP and acyl-CoA ligase are present in myelin. Finally, pulse-chase experiments in a cell-free system showed that PLP-bound fatty acids turn over with a half-life shorter than 10 min. These observations are consistent with the concept that acylation of myelin PLP is a dynamic process involved mainly in myelin maintenance and function.

Fatty acid composition of human myelin proteolipid protein in peroxisomal disorders.

Myelin proteolipid protein (PLP) is an acylated protein which contains approximately 2 mol of ester-bound fatty acids. In this study, the amount and composition of fatty acids covalently bound to human myelin PLP were determined during development and in peroxisomal disorders. Palmitic, oleic, and stearic acids accounted for most of the PLP acyl chains. However, in contrast to PLP in other species, human PLP contains relatively more very long chain fatty acids (VLCFA). The fatty acid composition remained essentially unchanged between 1 day and 74 years of age. The total amount of fatty acid bound to PLP was not altered in any of the pathological cases examined. However, in the peroxisomal disorder adrenoleukodystrophy, the proportions of saturated and, to a lesser extent, monounsaturated VLCFA bound to PLP were increased at the expense of oleic acid. Smaller, but significant, changes were observed in adrenomyeloneuropathy. The reduction in the levels of oleic acid was also observed in two other peroxisomal disorders, the cerebrohepatorenal (Zellweger) syndrome and neonatal adrenoleukodystrophy, as well as in the lysosomal disorder Krabbe globoid cell leukodystrophy. However, in these disorders, the decrease in oleic acid occurred at the expense of stearic acid, and not VLCFA. The results indicate that, although a characteristic PLP fatty acid pattern is normally maintained, changes in the acyl chain pool can ultimately be reflected in the fatty acid composition of the protein. The altered PLP-acyl chain pattern in peroxisomal disorders may contribute to the pathophysiology of these devastating disorders.