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.     

Cell-free acylation of rat brain myelin proteolipid protein and DM-20.

Incubation of rat brain myelin with [3H]palmitic acid in the presence of ATP, CoA and MgCl2 or [14C]-palmitoyl-CoA in a cell-free system resulted in the selective labelling of 'PLP' [proteolipid protein; Folch & Lees (1951) J. Biol. Chem. 191, 807-817] and 'DM-20' [Agrawal, Burton, Fishman, Mitchell & Prensky (1972) J. Neurochem. 19, 2083-2089] which, after polyacrylamide-gel electrophoresis in SDS, were revealed by fluorography. These results provide evidence of the association of fatty acid-CoA ligase and acyltransferase in isolated myelin. Palmitic acid is covalently bound to PLP and DM-20, because 70 and 92% of the radioactivity was removed from proteolipid proteins after treatment with hydroxylamine and methanolic NaOH respectively. Incubation of myelin with [3H]palmitic acid in the absence of ATP, CoA, MgCl2, or all three, decreased incorporation of fatty acid into PLP to 3, 55, 18 and 2% respectively. The cell-free system exhibits specificity with respect to the chain length of the fatty acids, since myristic acid is incorporated into PLP at a lower rate when compared with palmitic and oleic acids. The acylation of PLP is an enzymic reaction, since (1) maximum incorporation of [3H]palmitic acid into PLP occurred at physiological temperatures and decreased with an increase in the temperature; (2) acylation of PLP with [3H]palmitic acid and [14C]palmitoyl-CoA was severely inhibited by SDS (0.05%); and (3) the incorporation of fatty acid and palmitoyl-CoA into PLP was substantially decreased by the process of freezing-thawing and freeze-drying of myelin. We have provided evidence that all of the enzymes required for acylation of PLP and DM-20 are present in isolated rat brain myelin. Acylation of PLP in a cell-free system with fatty acids and palmitoyl-CoA suggests that a presynthesized pool of non-acylated PLP and DM-20 is available for acylation.     

Fatty Acid Acylation of Rat Brain Myelin Proteolipid Protein In Vitro: Identification of the Lipid Donor

The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chloroform/methanol, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl-CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl-CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl-CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.     

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.     

Acylation of endogenous myelin proteolipid protein with different acyl-CoAs

Fatty acyltransferase activity that catalyzes the transfer of palmitic acid from palmitoyl-CoA to the endogenous myelin proteolipid protein has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the acylation of proteolipid protein was obtained in 0.1% Triton X-100, 2 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.5 and at 37 degrees C. Other detergents had little or no effect on the reaction whereas acylation was completely abolished by sodium dodecyl sulphate (0.1%). Pulse-chase experiments indicated that the reaction involves the net addition of fatty acid to the protein and not a rapid fatty acid exchange. The rate of acylation was linear up to 30 min, indicating that the concentration of endogenous protein acceptor was constant. Under these conditions and at short time periods, the enzyme activity versus acyl-CoA concentration showed a hyperbolic curve. The apparent Km and Vmax for palmitoyl-CoA was 41 microM and 115 pmol/mg protein/min. Similar values were obtained for stearoyl and oleoyl-CoA, whereas myristoyl-CoA showed a lower specificity for the enzyme. The acyl-CoA specificity was also studied in competition experiments using several saturated and unsaturated fatty acid-CoAs. The product of the reaction was identified as myelin proteolipid protein and the fatty acid was shown to be attached to the protein via an ester linkage. Limited proteolysis and peptide mapping showed that the same sites on the proteolipid protein were acylated when the reaction was carried out in isolated myelin preparations or in brain tissue slices, suggesting physiological importance for the in vitro acylation of endogenous myelin proteolipid protein.     

Decreased Fatty Acylation of Myelin Proteolipid Protein in the Twitcher Mouse

We examined chronological changes of myelin proteins of the brainstem and spinal cord of the twitcher mouse (15, 20, and 30 days old), a murine model of human globoid cell leukodystrophy caused by a genetic deficiency of galactosylceramidase I activity. The yield of myelin was normal until postnatal day 20, whereas galactosylsphingosine (psychosine) accumulated with age in myelin. The protein profiles of myelin and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase in the myelin remained normal throughout the experimental period. Fatty acylation of proteolipid protein (PLP) was examined in a cell-free system by incubation of myelin with [3H]palmitic acid, CoA, and ATP, and was normal at postnatal day 15, but decreased after postnatal day 20. Decreased fatty acylation of PLP was also observed in the twitcher mouse at postnatal day 20 when the isolated myelin was incubated with [14C]palmitoyl-CoA in the absence of ATP and CoA, or the slices of brainstem and spinal cord were incubated with [3H]palmitic acid. The activity of fatty acid:CoA ligase was reduced in myelin. These data suggest that decreased acylation of PLP in twitcher mouse myelin is probably due to reduced activities for both activation and transfer of fatty acid into PLP and that metabolic disturbance is present in myelin because acylation of PLP has been shown to occur in myelin membrane. Although psychosine (200 microM) inhibited only 17% of the acylation in vitro, it may be responsible for the reduced acylation of PLP in vivo.     

Myelin Proteolipid Protein Contains Thioester-Linked Fatty Acids

Myelin proteolipid protein (PLP) is known to contain long-chain, covalently bound fatty acids. Previous studies, including our own, have suggested the occurrence of an oxyester type of linkage between fatty acids and PLP. However, we found that protein-SH groups are required in the acylation reaction, suggesting the possible presence of thioesters. In the present study, we have examined the nature of the acyl-PLP linkages by determining whether free thiol groups are generated on removal of fatty acids. Incubation of reduced and carboxyamidomethylated proteolipid apoprotein (RCM-APL) with 0.2 M hydroxylamine and [14C]iodoacetamide at pH 7.5 and 37 degrees C resulted in the release of fatty acids and the concomitant labeling of newly formed thiol groups. Incubation with Tris or methylamine at pH 7.5 failed to remove fatty acids and generate free -SH groups. The possibility that on treatment buried thiol groups became exposed was essentially excluded because (1) similar results were obtained in 2-chloroethanol, a solvent in which acylated and deacylated PLP have the same conformation, and (2) small PLP peptides were labeled only in the presence of hydroxylamine. On incubation with [14C]methylamine at pH 9.0, RCM-APL was not labeled, thus excluding the occurrence of intramolecular thiol esters. On the other hand, fatty acids were released as radioactive N-methyl fatty acylamide, indicating the presence of intermolecular thioesters between fatty acids and protein. These results demonstrate that a large proportion of fatty acids covalently bound to PLP are liked to -SH groups.     

Cysteine-108 is an acylation site in myelin proteolipid protein.

Myelin proteolipid protein (PLP) contains covalently bound long-chain fatty acids. A large proportion of these acyl moieties are bound in thioester linkages, as demonstrated by alkylation of newly formed SH groups upon deacylation. To identify the Cys residue(s) involved in the thioester linkage(s), reduced and carboxyamidomethylated proteolipid protein was labeled with [14C]iodoacetamide upon deacylation with neutral hydroxylamine. The labeled protein was digested with trypsin or pepsin, and peptides analyzed by RP-HPLC. Identification of the isolated radioactive peptides by amino acid analysis, peptide sequencing and/or fast-atom bombardment-mass spectrometry revealed that Cys108 in the bovine PLP sequence is an acylated site. The sequence surrounding the palmitoylation site in the myelin PLP is strikingly similar to that found in rhodopsin. Furthermore, as in rhodopsin and other members of the G protein-coupled receptor family, this Cys residue is located within a hydrophilic, basic, and possibly cytoplasmic, domain.     

Myelin proteolipid protein is not esterified at the site of synthesis

Brain slices prepared from 20-day old rats were incubated with [³H]palmitic acid to study its incorporation into myelin proteins. After separation by SDS-PAGE, most of the label was found to be associated with the major proteolipid protein (PLP) and with the intermediate protein (I). The radioactivity measured in PLP at short incubation times was shown to be due to palmitic acid bound to the protein by ester linkages. Time-course incorporation of [³H]palmitic acid into PLP of fraction SN₄ (a myelin like membrane) and of purified myelin showed that the former was poorly labeled and no relationship of the type ‘precursor-product’ between these fractions could be detected. Incorporation of the fatty acid into PLP was not affected by inhibition of the synthesis or transport of myelin PLP with cycloheximide or colchicine, indicating that the pool of PLP that can be acylated must be larger than the extramyelin pool. Addition of unlabeled palmitic acid to the incubation medium, 30 min after the addition of [³H]palmitate, stopped the appearance of label in myelin PLP almost immediately, indicating that there is no significant extramyelin pool of PLP destined for transport into myelin. The results presented in this paper strongly suggest that esterification of PLP takes place in the myelin membrane or at a site very close to it.     

Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP(104-117) and PLP(139-151)) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.     

Simultaneously entry of palmitic acid into proteolipid protein in different myelin subfractions of rat brain

Rats of 20-days of age were injected intracranially with radioactive palmitic acid to study its incorporation into proteolipid protein (PLP) of myelin and myelin subfractions. At short times (120 min), the radioactivity present in PLP was shown to be due to palmitic acid bound to the protein by ester linkages. The specific radioactivity of palmitic acid labeled PLP was identical in all the myelin subfractions except the myelin-like fraction, in which it was lower, suggesting that the entry of the fatty acid into PLP of the different subfractions occurs simultaneously.Experiments using time staggered injections of ¹⁴C- and ³H-labeled palmitic acid also showed that entry of the fatty acid into PLP of the various subfractions was simultaneous. These results seem to indicate that the acylation of PLP occurs in the myelin membrane and that synthesis and transport of this protein are events unrelated to the acylation process.     

In vivo acylation of proteolipid protein and DM-20 in myelin and myelin subfractions of developing rat brain: Immunoblot identification of acylated PLP and DM-20

The acylation of proteolipid protein (PLP) was examined in myelin and myelin subfractions from rat brain during the active period of myelination. Proteolipid protein and DM-20 in myelin and myelin subfractions were readily acylated in developing rat brain 22 hours after intracerebral injection of [3H]palmitic acid. No differences in the relative specific activity of PLP in myelin from 9-, 15-, and 30-day-old rat brains was observed; however, the relative specific activity of PLP in the heavy myelin subfraction tended to be higher than that in the light myelin subfraction. The acylation of PLP was confirmed by fluorography of immuno-stained cellulose nitrate sheets, clearly establishing that the acylated protein is in fact the oligodendroglial cell- and myelin-specific protein, PLP. Since PLP is acylated in the 9-day-old animal, when little compact myelin is present, it is possible that the acylation of PLP is a prerequisite for the incorporation of this protein into the myelin membrane.     

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.     

Myelin P0 Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated

Abstract: 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 nonezymatic 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°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 ～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.     

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.     

Effect of cycloheximide on palmitylation of PO protein of the peripheral nervous system myelin.

Incubation of rat sciatic nerve slices with Krebs-Ringer bicarbonate buffer containing [3H]palmitic acid resulted in the acylation of the PO glycoprotein and a 24 kDa protein of the peripheral nerve myelin. Radioactivity was removed from PO after treating PO with hydroxylamine (83%) and methanolic KOH (97%). These results provided evidence that the radioactivity incorporated into PO was not due to the metabolic conversion of [3H]palmitic acid into amino acids or sugars. PO was more heavily labelled in the homogenate than in the myelin membrane in 8-day-old rat nerve between 5 min and 2 h of incubation. These results suggested that PO may be primarily acylated in the cell body. Incubation of purified myelin with [1-14C]palmitoyl-CoA resulted in the non-enzymic acylation of PO. This provided evidence of the absence of fatty acyltransferase from the purified peripheral nerve myelin. Glycosylation of PO has been shown to occur in the Golgi complex, and monensin inhibited glycosylation of PO in the homogenate and myelin by 53 and 61% respectively. These results suggest that the processing of PO in the Golgi complex and the assembly of PO into myelin is impaired by monensin. However, fatty acylation of PO was unimpaired by monensin, suggesting that the addition of fatty acids may not occur in the Golgi complex. There was a progressive decrease in the acylation of PO between 5 min (28%) and 2 h (61%) in the presence of cycloheximide, as the pool of previously synthesized PO was gradually depleted. These results also provide evidence that palmitylation of PO is not coupled to protein synthesis, and acylation of this protein probably occurs in the early subcompartment of the Golgi complex, which appears to be insensitive to monensin.     

An ordered reaction mechanism for bacterial toxin acylation by the specialized acyltransferase HlyC: formation of a ternary complex with acylACP and protoxin substrates

The 110 kDa haemolysin protoxin (proHlyA) is activated in the Escherichia coli cytosol by acyl carrier protein-dependent fatty acylation of two internal lysine residues, directed by the co-synthesized protein HlyC. Using an in vitro maturation reaction containing purified protoxin peptides and acylACP, we show unambiguously that HlyC possesses an apparently unique acyltransferase activity fully described by Michaelis-Menten analysis. The Vmax of HlyC at saturating levels of both substrates was approximately 115 nmol acyl group min-1 mg-1 with KMacylACP of 260 nM and KMproHlyA of 27 nM, kinetic parameters sufficient to explain why in vivo HlyC is required at a concentration equimolar to proHlyA. HlyC bound the fatty acyl group from acylACP to generate an acylated HlyC intermediate that was depleted in the presence of proHlyA, but enriched in the presence of proHlyA derivatives lacking acylation target sites. HlyC was also able to bind in vivo 4'-phosphopantetheine. Substitution of conserved amino acids that could act as putative covalent attachment sites did not prevent binding of the fatty acyl or 4'-phosphopantetheine groups. These data and substrate variation analyses suggest that the unique acylation reaction does not involve covalent attachment of fatty acid to the acyltransferase, but rather that it proceeds via a sequential ordered Bi-Bi reaction mechanism, requiring the formation of a non-covalent ternary acylACP-HlyC-proHlyA complex.     

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.     

In vitro acylation of myelin PLP and DM-20 in the quaking mouse brain

Both proteolipid proteins (PLP) and DM-20 were found to be present by the immunoblot technique in myelin isolated from quaking mouse brain; however, the relative concentration of these proteins in myelin from quaking brain was substantially reduced when compared to the control. Brain slices from littermate control and quaking mice were incubated with [³H]palmitic acid to determine the incorporation of fatty acid into myelin proteolipid proteins. Fluorography of gels containing myelin proteins from control and quaking mice brain revealed that both PLP and DM-20 were acylated. The incorporation of [³H]palmitic acid into quaking myelin PLP and DM-20 was reduced by 75% and 20% respectively of those in control brain. The significance of differential acylation of quaking myelin PLP and DM-20 is discussed with respect to availability of non-acylated pools of proteolipid proteins and the activities of acylating enzymes.     

Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein

Abstract: 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 palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P₀ was deacylated by treatment with 10 m M 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 P₀was 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 transmem-brane and cytoplasmic domains. This residue is also present in the P₀ glycoprotein of other species, including human, bovine, mice, and chicken.