Individual molecular species of phosphatidylcholine and phosphatidylethanolamine in myelin turn over at different rates.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of the myelin membrane exhibit heterogeneity with respect to metabolic turnover rate (Miller, S. L., Benjamins, J. A., and Morell, P. (1977) J. Biol. Chem. 252, 4025-4037). To test the hypothesis that this is due to differential turnover of individual molecular species (which differ in acyl chain composition), we have examined the relative turnover of individual molecular species of myelin PC and PE. Phospholipids were labeled by injection of [2-3H]glycerol into the brains of young rats. Myelin was isolated at 1, 15, and 30 days post-injection, lipids were extracted, and phospholipid classes were separated by thin-layer chromatography. The PC and PE fractions were hydrolyzed with phospholipase C, and the resulting diacylglycerols were dinitrobenzoylated and fractionated by reverse-phase high performance liquid chromatography. The distribution of radioactivity among individual molecular species was determined. The labeled molecular species of myelin PC were 16:0-16:0, 16:0-18:0, 16:0-18:1, and 18:0-18:1, with most of the label present in 16:0-18:1 and 18:0-18:1. Changes in distribution of label with time after injection indicated that 16:0-18:1 turned over more rapidly than 18:0-18:1. The labeled molecular species of myelin PE were 18:0-20:4, 18:1-18:1, 16:0-18:1, 18:0-18:2, and 18:0-18:1. As with myelin PC, 16:0-18:1 (and 18:1-18:1) turned over more rapidly than 18:0-18:1. The relative turnover of individual molecular species of PC in the microsomal fraction from forebrain was also examined. The molecular species profile was different from myelin PC, but again, 16:0-18:1 turned over more rapidly than the other molecular species. Thus, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Comparison of our results with previous studies of turnover of molecular classes of phospholipids indicates that in addition to polar head group composition (Miller et al., 1977), fatty acid composition is very important in determining the metabolic fate of a phospholipid.     

Age-Related Changes in the Ceramide Composition of the Major Gangliosides Present in Rat Brain Subcellular Fractions Enriched in Plasma Membranes of Neuronal and Myelin Origin

Abstract: Age-related changes of the ceramide composition of gangliosides were studied in the synaptosomal and myelin fractions from rat brain, carrying plasma membranes of neuronal and glial origin, respectively. The five major gangliosides (GM1, GD1 a, GD1 b, GT1 b, and GQ1 b) present in these fractions were separated and quantitated by normal-phase HPLC. Each ganglioside was then fractionated by reverse-phase HPLC into the molecular species carrying a single long-chain base (LCB). The largely preponderant LCBs in the synaptosomal and myelin fractions were the C18:1 and C20:1. The content of C20.1 LCB, generally low at 1 month, increased with age in all analyzed gangliosides and in all subcellular fractions and was greater in the "b series" than in the "a series" gangliosides. Remarkably, GM1 was the only ganglioside where the proportion of LCB 20:1 was higher in the synaptosomal fraction than in the myelin fraction. The fatty acid composition of the C18:1 or C20:1 LCB species of the different gangliosides in the synaptosomal and myelin fractions did not undergo appreciable changes with age. Stearic acid was largely predominant in all the gangliosides of the synaptosomal fraction, more in the C18:1 than in the C20:1 LCB species (80–90% vs. 60–70%). The gangliosides of the myelin fraction were characterized by a lower content of 18:0 and a much higher content of 16:0 and 18:1 fatty acids than those of the synaptosomal fraction. Thus, the ceramide composition is different in the gangliosides of neuronal and myelin origin and appears to be subjected to an age-related control.     

A comparative study on ceramide composition of cetacean brain gangliosides.

1. Ceramide composition and N-glycolylneuraminic acid content of gangliosides from gray and white matters and myelin of cerebrum and cerebellum were analyzed in eight species belonging to the suborder Odontoceti and two species to Mystacoceti. 2. The most characteristic feature was high contents of C20:0 (10-40%) and C24 species (5-40%). 3. Content of hydroxy fatty acid of C24 species was higher in cerebellum (5-20%) than cerebrum (0-3%). 4. Major component of long-chain base was dC18:1 (70-90%). 5. N-glycolylneuraminic acid was found in sperm whale, Dall's porpoise and killer whale (0.1-1.7%).     

Complete amino acid sequence of PO protein in bovine peripheral nerve myelin

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.     

Fatty acid composition of phospholipids of myelin and synaptosomal proteolipid complexes from vertebrate brain.

1. Fatty acid composition of five main phospholipids of vertebrate brain myelin and synaptosomal proteolipids and membranes was studied. 2. Higher content of monoenoic and lower content of saturated and polyenoic fatty acids was found to be characteristic of phospholipids from myelin and myelin proteolipids as compared to phospholipids from synaptosomal proteolipids and membranes of vertebrates (from fishes to mammalians). Fatty acid composition of phospholipids of proteolipid complexes and of the membranes, from which they were isolated, were found to be similar in various species studied. 3. Microviscosity was found to be higher in myelin as compared to synaptosomal membranes of frog Rana temporaria and in rabbit Lepus cuniculus. It appears to be due to the difference in proteolipid content and in lipid composition of myelin and synaptosomal membranes.     

Detection of Multisulphated N-Linked Glycans in the L2/HNK-1 Carbohydrate Epitope Expressing Neural Adhesion Molecule P0

P0, the most abundant glycoprotein of PNS myelin, is a homophilic and heterophilic adhesion molecule. P0 is known to contain a glycoform population that expresses the L2/HNK-1 carbohydrate epitope found on other neural adhesion molecules, and to be functionally implicated centrally in neural cell adhesion and neurite outgrowth. This carbohydrate epitope has been characterized previously from glycolipid structures and contains a sulphated glucuronic acid residue. However, the L2/HNK-1 carbohydrate epitope has not been characterized in glycoproteins. Because P0 possesses only one glycosylation sequon, the number of P0 glycoforms is equal to the heterogeneity of the glycan species. Here we report that the carbohydrate analysis of L2/HNK-1-reactive P0 showed the presence of anionic structures containing sialic acid and sulphate in various combinations. At least one sulphate residue was present in 80% of the monosaccharide sequences, and 20% contained three sulphates. High-resolution P4 gel chromatography of the desialylated and desulphated oligosaccharides showed substantial heterogeneity of monosaccharide sequences. Sequential exoglycosidase digestions indicated that the majority of the structures were of the hybrid class, although the sulphated structures were found to be endoglycosidase H-resistant.     

A comparison of composition and fluidity of multiple sclerosis and normal myelin

Myelin was isolated from normal-appearing white matter from the brains of 11 multiple sclerosis (MS) patients, 11 normal individuals, and 1 patient with subacute sclerosing panencephalitis (SSPE). Ratios of protein, cholesterol, and phospholipid content, total fatty acid content, and total amino acid content were determined. The lipid bilayer fluidity of the myelin was measured using electron spin resonance and fluorescence polarization spectroscopy. The protein-to-phospholipid ratio was higher than normal, the cholesterol-to-protein ratio was lower than normal while the cholesterol-to-phospholipid ratio was normal in MS myelin. This suggested a relative increase of protein to lipid content in MS myelin. Of the fatty acid content of a total lipid extract, MS myelin had relatively more 16:0, less 18:3, and less 22:3, 24:1 than normal. The total amino acid content of MS myelin was altered in a way which suggested a decreased percentage of basic protein and an increased percentage of proteolipids. SSPE myelin had similar changes in amino acid content but not in protein-to-cholesterol or-phospholipid ratios or fatty acid content as MS myelin. There was no significant difference in myelin fluidity, however, between MS and normal myelin using fatty acid spin labels or fluorescent probes. A correlation was found between the fluidity and the cholesterol-to-phospholipid ratio but the MS and normal samples varied over a similar range. Although these alterations in lipid and protein composition had no effect on fluidity, they may nevertheless have serious consequences for myelin structure.     

Expression of Myelin Components in Mouse Schwann Cells in Culture

Mouse Schwann cells cultured in vitro are capable of expressing basal levels of the major myelin components P1, P2, P0, and galactocerebroside. Numerical counts of immunostained cultures indicated that between 22 and 40% of the cells are positive up to 21 days for all of the components indicated. Electrophoretic analysis of Schwann cells labeled with a 14C-amino acid mixture revealed the presence of proteins with relative mobilities identical to those of P0 and P1. Positive identification of the two proteins was indicated by immunoprecipitation of P1 and immunoblotting of P0. These data show that in the absence of neurites, Schwann cells in culture can express low levels of myelin characteristic components even in the absence of myelin assembly.     

Turnover rate of molecular species of sphingomyelin in rat brain

Turnover rate of individual molecular species of sphingomyelin of adult rat brain myelin and microsomal membranes was determined after an intracerebral injection of 100 Ci of [C³H₃]choline. Myelin and microsomal membrane sphingomyelins were isolated from the rest of the lipids. The individual molecular species of benzoylated sphingomyelin were separated and quantitated by reversed-phase high performance liquid chromatography. All individual major molecular species of microsomal and myelin sphingomyelin had maximum incorporation at 6 and 15 days, respectively, after the injection. The specific radioactivity of all the various molecular species of both myelin and microsomal sphingomyelin declined at a similar rate after reaching a maximum. There was no significant difference in the turnover rate of short chain (16:0, 18:0) and long chain (>22:0) fatty acid containing sphingomyelin. The average apparent turnover rate of myelin and microsomal sphingomyelin molecular species was about 14–16 days for the fast pool and about 45 days for the slow pool. It is concluded that individual molecular species of sphingomyelin of myelin and microsomal membranes turned over at a similar rate. Thus, turnover rate of sphingomyelin in myelin and microsomal membranes is not affected by the fatty acyl composition of the lipid.     

Immunocytological localization of the major peripheral nervous system glycoprotein P0 and the L2/HNK-1 and L3 carbohydrate structures in developing and adult mouse sciatic nerve

Immunocytological localization of the major glycoprotein of peripheral myelin P0 and its associated carbohydrate structures L2/HNK-1 and L3 was performed at the light- and electron-microscopic levels in mouse sciatic nerves at several developmental stages and in adulthood. P0 was first expressed on Schwann cells at the time that Schwann cells associated with axons on a 1:1 basis. P0 remains expressed at all times of myelin formation and in compact myelin. After cessation of myelination P0 is no longer detectable in the uncompacted parts of myelin, i.e., Schmidt-Lanterman incisures, paranodal loops, and outer and inner mesaxons. P0 is not detectable on basement membranes, interstitial collagens, and non-myelin-forming Schwann cells. The associated carbohydrate epitope L2 does not follow the expression of P0 at any developmental or adult stage. Until 21 days the L2 epitope is confined to nonmyelinated fibers. In sciatic nerves of mice older than 8 weeks, however, only a few nonmyelinated fibers remain L2-positive. L2 immunoreactivity is clearly seen in a subpopulation of compact myelin figures largely associated with motor fibers. The L3 epitope is never detectable on nonmyelinated fibers and becomes first visible when compact myelin is discerned. Unlike the L2 epitope L3 is present in most, if not all, compact myelin figures. These observations suggest that P0 may be involved in ensheathment of axons by Schwann cells at the decisive stages of initiation of myelination and later on, possibly in conjunction with the L3 carbohydrate structure, in maintenance of compact myelin. The appearance of the L2 carbohydrate epitopes in compact myelin of largely motor and fewer sensory nerve fibers at times when morphogenesis of myelin has ceased remains to be elucidated in functional terms.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

Binding of protein SCP to myelin, its identity with protein P2. A simple method of protein P2 isolation

Protein SCP is found in myelin of spinal cord and spinal roots. It is shown that its amount accounts for 12% of the total protein content in myelin of spinal roots and only for 2% in myelin of spinal cord. Almost all the studied protein is extracted from myelin with 0.1 M NaCl (80-90%). The absolute identity of protens SCP and P2 is established using the cross reaction immunodiffusion with monospecific antisera. It is shown that- N-terminal amino acid in protein SCP, like in protein P2 is blocked. On the basis of the data obtained a conclusion is made that protein P2 is not an integral protein of myelin. However, myelin is capable under conditions of a nonionic medium of binding protein which then may be easily extracted by increasing the medium ionic strength. This gave reasons to propose a method for protein P2 isolation from myelin using 0.15 M NaCl with the subsequent purification by means of Sephadex G-50 gelfiltration.     

Increased susceptibility to degradation by trypsin and subtilisin of in vitro peroxidized myelin proteins

We examined the possibility that the peroxidative damage to central nervous system myelin produced by reactive oxygen species (ROS), could modify the susceptibility of its proteins to the proteolytic action of proteases such as trypsin and subtilisin. Purified myelin membranes obtained from adult rat brains were in vitro peroxidized by two non-enzymatic systems: Fe³⁺ plus ascorbic acid and Cu²⁺ plus hydrogen peroxide. Myelin proteins were severely affected by peroxidation. There was an increase in the amount of carbonyl groups (CO), accompanied by and enhanced susceptibility to degradation by trypsin and subtilisin of myelin basic proteins (MBP) and of the major proteolipid protein (PLP). The effect upon the degradation of myelin protein is a possible consequence of the appearance in the structure of myelin proteins of peroxidative modifications that contribute to the recognition by proteolytic enzymes. This hypothesis is supported by the fact that if peroxidation of myelin membranes is done in the presence of EDTA, both CO formation and increased sensitivity to enzymatic breakdown disappear. These results suggest that the appearance of abnormal post-translational modifications in the myelin membrane produced by peroxidation could constitute a putative mechanism of modulating the capacity of myelin proteins to be metabolized by proteases.     

Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin.

A full length cDNA of the major structural protein of peripheral myelin (P0 protein) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs (bp) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species.     

The P2 protein of bovine root myelin: partial chemical characterization.

The P2 protein of bovine root myelin has chemical characteristics which differentiate it from other myelin basic proteins. The tryptic peptide map of the bovine P2 protein is distinctly different from the map of either the rabbit PI protein or the bovine CNS myelin basic protein. The tryptic peptides of the P2 protein show no overlap in either map positions or amino acid content with the peptides of the CNS myelin basic protein. Analysis of the individual peptides in the P2 map accounted for all of the amino acids present in the analysis of the whole protein. The P2 protein has a blocked NH₂-terminus, lysine at its COOH-terminus and no hexosamine. CD studies revealed that the P2 protein has a very stable β-structure in aqueous solution at neutral or basic pH and retains much of this structure in acid and in 8 M urea. It is suggested that these structural properties are relevant to the dual role of the P2 protein as a membrane constituent and as an antigen.     

Effects of α-methylphenylalanine plus phenylalanine treatment during development on myelin in rat brain

The effects of hyperphenylalaninemia induced by treatment with -methylphenylalanine (MPA) plus phenylalanine (PHE) on body and brain weight, on myelin and synaptosome formation, and on the lipids and fatty acids of myelin were studied in rats. The administration of MPA (2.4 mol/g body wt) plus PHE (2.6 mol/g body wt) for 25 and 35 days beginning on the fifth postnatal day did not affect brain development. On doubling the dosage of PHE, body and brain weights and myelin yields were significantly lowered. The lipid composition of myelin from the brains of treated animals was largely unaffected; however, the concentration of sulfatides was significantly reduced. Unsaturated fatty acid levels in myelin from hyperphenylalaninemic rat brains were reduced while long-chain fatty acids were unaffected. We conclude that as in hyperphenylalaninemia induced by other methods, MPA+PHE treatment impairs body and brain growth, reduces myelin formation, and causes inhibition of fatty acid desaturation in the brain.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Expression of a P0-like glycoprotein in central nervous system myelin of amphibians (Ambystoma mexicanus,xenopus laevis and Rana catesbeiana)

• 1.1. The myelin protein profiles in the CNS and PNS of three species of amphibians were analyzed by biochemical and immunohistochemical methods.
• 2.2. The CNS myelin of the African clawed frog (Xenopus) and the Mexican salamander (axolotl) contained, in addition to proteolipid protein, a unique protein zero (P0)-like protein, whereas the adult bullfrog did not.
• 3.3. A strong expression of the P0-like protein in the bullfrog CNS myelin was found transiently at ontogenetically early phases including at the time of metamorphosis.
• 4.4. The CNS P0-like protein and the PNS P0 protein showed a difference in reactivity with lectins and anti-L2/HNK-1 antibodies, suggesting that the two proteins differ in some aspects of their carbohydrate structures.

     

Age-Related Changes of Myelin Proteins in the Rat Peripheral Nervous System

Age-related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS-PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS-PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.