Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

Orientation of myelin proteolipid protein in the oligodendrocyte cell membrane

The orientation of proteins within a cell membrane can often be difficult to determine. A number of models have been proposed for the orientation of the myelin protein, proteolipid protein (PLP), each of which includes exposed domains on the intracellular and extracellular membrane faces. Immunolabeling experiments have localized the C-terminus and the region spanning amino acids 103–116 to the cytoplasmic face of the membrane, but no well characterized antibodies have been available that label extracellular PLP domains. In this report, we describe the generation and characterization of mouse monoclonal antibodies (mAb) against putative extramembrane domains. Three of the mAb, specific for PLP peptides 40–59, 178–191, or 215–232, immunostain live oligodendrocytes, indicating that these regions of the molecule are exposed on the external surface of the cell. In addition, we have used these mAb to study the time-course of incorporation of PLP into the oligodendrocyte membrane. These studies increase our knowledge of the orientation of PLP in the lipid bilayer and are relevant for understanding myelin function. Special issue dedicated to Dr. Marion E. Smith. Marion has filled many roles in my life (M. Lees): She has been a long time colleague, personal friend, meeting roommate, and traveling companion. Even our husbands have become good friends. Further, Marion’s scientific contributions in multiple aspects of neurochemistry have made her a role model for all scientists, and particularly for young women. It should be noted that all of the authors of this paper just happen to be women.     

A membrane receptor for gangliosides is associated with central nervous system myelin

A binding protein specific for major neuronal gangliosides was detected on rat brain membranes using a synthetic ganglioside-protein conjugate, 125I-(GT1b)4BSA (bovine serum albumin derivatized with 4 mol of ganglioside GT1b/mol of protein), as a radioligand. Specific binding of the ligand displayed marked regional variation within the brain, with white matter-enriched regions demonstrating the highest binding activity. Autoradiographic localization of 125I-(GT1b)4BSA binding to tissue sections revealed selective association with myelinated pathways throughout the brain. The ligand also bound preferentially to brain subcellular fractions enriched in myelin, even after removal of axolemma. In contrast, peripheral nerve myelin had little binding activity. The myelin-associated ganglioside receptor detected by 125I-(GT1b)4BSA binding appears to be a novel oligodendroglial membrane protein which preferentially recognizes neuronal gangliosides.     

Biochemical maturation of human central nervous system myelin.

—A developmental study of the lipid and protein composition of human CNS myelin was undertaken. The relative concentrations of the major lipid classes, cholesterol, glycolipids and phospholipids exhibited little change except for a modest decrease in the concentration of the phospholipids. In contrast to the total phospholipids, marked variations in the relative concentrations of individual phospholipids were found. Sphingomyelin increased over two-fold, and phosphatidyl choline decreased to almost half its original concentration. While the concentration of total myelin protein remained constant during maturation, variations in the concentrations of individual proteins were observed. Basic protein constituted 8·5 per cent of the total myelin proteins in the newborn brain and increased to about 30 per cent of the protein in the older ages. The concentrations of proteolipid protein and DM-20 seemed to increase with age, while the relative amounts of high molecular weight proteins decreased. The presence of myelin basic protein in newborn human brain was confirmed by electrophoretic studies involving several different polyacrylamide gel systems and by immunodiffusion experiments which showed a reaction of identity between a constituent present in the fraction containing the presumptive myelin basic protein and authentic myelin basic protein isolated from adult human brain.     

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta- galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.     

Glycoproteins associated with myelin in the central nervous system

Purified myelin fractions from the central nervous system contain one major myelin-associated glycoprotein and approximately 16 minor glycoproteins. While the genuine association of the major myelin-associated glycoprotein with the oligodendroglial myelin unit is demonstrated, the possibility exists that several of the minor glycoproteins have their origin in contaminating membranes not related to myelin. The major myelin-associated glycoprotein is probably not present in compacted myelin, but immunocytochemical and subfractionation studies indicate that it is confined to the periaxonal and paranodal region of the myelin sheath. In experimental demyelination and multiple sclerosis, the major glycoprotein is the first myelin constituent to be affected. Its localization on the membrane surface where myelin and axolemma are in close contact, and other indirect evidence indicate that the major glycoprotein, and possibly other myelin-associated glycoproteins, could play a role in the process of myelination and myelin maintenance.     

Opalin, a transmembrane sialylglycoprotein located in the central nervous system myelin paranodal loop membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.     

Autoacylation of myelin proteolipid protein with acyl coenzyme A

Rat brain myelin proteolipid protein (PLP) is known to contain long chain, covalently bound fatty acids. In the course of characterizing the mechanism of acylation, we found that the isolated PLP, in the absence of any membrane fraction, was esterified after incubation with [3H]palmitoyl coenzyme A (CoA). This observation demonstrated that the protein acts as both an acylating enzyme and an acceptor. Thus, acylation occurs by an autocatalytic process. The possibility of a separate acyltransferase that copurifies with PLP was essentially excluded by adding brain subcellular fractions to the reaction mixtures and by changing the isolation procedure. After deacylation, the protein was acylated at a 4-fold greater rate, suggesting that the original sites were reacylated. The palmitoyl-CoA concentration followed Michaelis kinetics, confirming that spontaneous acylation was not occurring. Pulse-chase experiments indicated that the reaction entails net addition of acyl groups. Although fatty acids are bound via an O-ester linkage, free SH groups are required in the reaction. Denaturation of the protein by sodium dodecyl sulfate or heat inhibits the reaction, whereas cerulenin has little or no effect. PO, the major protein in peripheral nerve myelin, is also an acylated protein, but it was not labeled upon incubation of either peripheral myelin or the isolated protein with [3H]palmitoyl-CoA, demonstrating that it is acylated by a different route. Several synthetic peptides derived from PLP sequences with sites known to be acylated in vivo as well as a series of deacylated PLP tryptic peptides were not labeled, indicating that integrity of the protein is required for acylation. Limited proteolysis and peptide mapping showed that the same sites are acylated in vitro or in vivo, suggesting that the autocatalytic acylation reaction is physiological.     

Studies on autoimmune encephalomyelitis in the guinea pig--III. A comparative study of two autoantigens of central nervous system myelin.

Abstract— A new CNS myelin autoantigen(s) (referred to as M2), different from the encephalitogenic basic protein (BP), can be detected with guinea-pig demyelinating and complement fixing (CF) sera raised against guinea pig CNS tissue or myelin (Lebar et al., 1976). M2 and BP were present in mouse, rat, rabbit, bovine and human CNS tissues when tested with guinea-pig homologous specific antisera; they were not present in non-CNS tissues. Both autoantigens were also detected in newborn guinea-pig myelin and myelin-like fractions. The CF activity of myelin with demyelinating (anti-M2) sera was not altered by trypsin; however, absorption experiments showed that M2 was partly trypsin sensitive. Both antibodies against the trypsin sensitive and the trypsin resistant determinants of M2 were demyelinating. Both determinants of M2 were preselit in mouse, rat, rabbit, bovine‘and human CNS tissues and in guinea-pig newborn myelin. CF BP activity of myelin was partially or even totally abolished by trypsin, but the persistent encephali-togenicity of trypsin-treated myelin could be attributed to non-CF encephalitogenic peptides from BP. In accordance with recent work our results tend to support an inner localization of BP in myelin; M2, on the other hand, would be a surface antigen(s).     

Localization of five somatostatin receptors in the rat central nervous system using subtype-specific antibodies.

The cloning of five members of the somatostatin receptor family, sst1-sst5, as well as two isoforms of the somatostatin receptor 2, sst2A and sst2B, enabled us to generate specific anti-peptide antisera against unique sequences in the carboxyl-terminal tail of each somatostatin receptor subtype. We used these antibodies in multicolor immunofluorescent studies aimed to examine the regional and subcellular distribution of somatostatin receptors in adult rat brain. Several findings are notable: The cloned sst1 receptor is primarily localized to axons, and therefore most likely functions in a presynaptic manner. The cloned sst2 receptor isoforms exhibit strikingly different distributions, however, both sst2A and sst2B are confined to the plasma membrane of neuronal somata and dendrites, and therefore most likely function in a postsynaptic manner. The cloned sst3 receptor appears to be excluded from 'classical' pre- or postsynaptic sites but is selectively targeted to neuronal cilia. The cloned sst4 receptor is preferentially distributed to distal dendrites, and therefore most likely functions postsynaptically. The cloned sst5 receptor was not detectable in the adult rat brain, however, prominent sst5 expression was found in the pituitary. Furthermore, sst1-containing axons either co-contained somatostatin or were closely apposed by somatostatin-positive terminals in a regional-specific manner. Neuronal somata and dendrites containing either sst2A, sst2B or sst4 were found to exist in close proximity, although not necessarily synaptically linked, to somatostatin-positive terminals. Together, in the central nervous system the effects of somatostatin are mediated by several different receptor proteins which are distributed with considerable regional overlap. However, there appears to be a high degree of specialization among somatostatin receptor subtypes with regard to their subcellular targeting. This subtype-selective targeting may be the underlying principal of organization that allows somatostatinergic modulation of neuronal activity via both pre- and postsynaptic mechanisms.     

1H-nuclear magnetic resonance study of the association of the basic protein of central nervous system myelin with lysophosphatidylcholine

High-resolution 270 MHZ 1H-nuclear magnetic resonance spectroscopy has been used to follow the interaction of myristoyllysophosphatidylcholine with bovine myelin basic protein. At lipid/protein ratios up to 30:1 it proved possible to follow changes in the spectra of both the protein and the lipid. Lysophosphatidylcholine induced several changes in the protein spectrum. Foremost amongst these changes were downfield shifts of histidine C2 protons, and upfield shifts and broadening of the phenylalanine aromatic proteins. Several other resonances assigned to nonpolar amino acid side chains also broadened. But even at a lipid/protein molar ratio of 30:1 the majority of the protein appeared to remain in a loosely coiled conformation. In the presence of the protein the lipid acyl chain peaks were moved upfield and broadened, whereas the resonances associated with the head-group protons were unaffected. These changes were consistent with partial immobilization of the acyl chain of lysophosphatidylcholine on binding to the basic protein, with hydrophobic interactions providing the predominant attraction between this lipid and the basic protein.     

In vitro synthesis of myelin proteolipid protein from rat brain using a total homogenate system

In vitro synthesis of myelin proteolipid protein (PLP) was explored at different ages using rat brain total homogenates, incubated for 30 min with [³H]glycine. Total proteolipids, extracted from the incubated samples, were separated by SDSPAGE and the radioactivity was measured in the band corresponding to myelin PLP. The incorporation into PLP in relation to the incorporation into brain total proteins increased from 0.04% at 10 days of age to 0.63% at 20 days, and declined slowly thereafter. Time course experiments were carried out using brain homogenates obtained from rats of 20 days of age (i.e. at the period of maximal synthesis of PLP). Labeled PLP molecules were already found at 2.5 min of incubation and the incorporation of the label into this protein, relative to the incorporation into total proteins, did not vary throughout the entire incubation time (30 min). Pulsechase experiments using a similar system and adding cycloheximide at different incubation times showed that the appearance of label into mature PLP was immediately blocked by the inhibitor of protein synthesis. These data suggest that PLP is synthesized as such and not as a pre-protein which is subsequently processed to render mature PLP.     

Galactosphingolipids and axono-glial interaction in myelin of the central nervous system

The myelin of central and peripheral nervous system of UDP-galactose-ceramide galactosyltransferase deficient mice (cgt ^-/-) is completely depleted of its major lipid constituents, galactocerebrosides and sulfatides. The deficiency of these glycolipids affects the biophysical properties of the myelin sheath and causes the loss of the rapid saltatory conduction velocity of myelinated axons. With the onset of myelination, null mutant cgt ^-/- mice develop fatal neurological defects. CNS and PNS analysis of cgt ^-/- mice revealed (1) hypomyelination of axons of the spinal cord and optic nerves, but no apoptosis of oligodendrocytes, (2) redundant myelin in younger mice leading to vacuolated nerve fibers in cgt ^-/- mice, (3) the occurrence of multiple myelinated CNS axons, and (4) severely distorted lateral loops in CNS paranodes. The loss of saltatory conduction is not associated with a randomization of voltage-gated sodium channels in the axolemma of PNS fibers. We conclude that cerebrosides (GalC) and sulfatides (sGalC) play a major role in CNS axono-glial interaction. A close axono-glial contact is not a prerequisite for the spiraling and compaction process of myelin. Axonal sodium channels remain clustered at the nodes of Ranvier independent of the change in the physical properties of myelin membrane devoid of galactosphingolipids. Increased intracellular concentrations of free ceramides do not trigger apoptosis of oligodendrocytes.