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.     

Interaction of a purified hydrophobic protein from myelin with phospholipid membranes: studies on ultrastructure, phase transitions and permeability.

A purified protein fraction from the proteolipids of human brain myelin was recombined with different lipids either in aqueous buffer or in a chloroform-methanol-water (10:5:1, v/v/v) mixture. It was found that under both conditions it binds strongly to phospholipids irrespective of surface charge, the presence of cholesterol or double bonds on the fatty acyl chains. The buoyant density of the resulting lipoprotein membranes is intermediate to that of pure lipids, and proteins. The lipoproteins formed by either of these methods were observed by either freeze-fracture or negative stain electron-microscopy. The overall morphology was similar to that of pure phospholipids, showing large closed multilamellar vesicles. The presence of the protein was detected by the appearance of intramembrane particles in freeze-fracture. The addition of the N-2 protein generally increases the permeability vesicles to 22-Na-+ by 2-3 orders of magnitude depending on the concentration. The presence of calcium in the aqueous medium further increases the Na-+ efflux through negatively charged vesicles. Changes in lipid composition, surface charge, cholesterol, etc., have no appreciable influence on the effect of the protein. Differential scanning calorimetry indicates that the presence of small amounts of N-2 have no effect on the lipid phase transition from solid to liquid crystalline. As the amount of protein bound to the phospholipid increases, the enthalpy of the transition decreases, the main endothermic peak broadens, but there is no change on the midpoint temperature. Membranes containing 50% by weight of protein still show a transition with an enthalpy approximately one half that of the original lipid.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein: a possible role in myelin formation.

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, L-alpha-dimyristoyl phosphatidylcholine and DL-alpha-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15-18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associated species. A similar interaction was observed with diacyl phosphatidylserine vesicles. These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myelin, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.     

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).     

Interaction of lipid-bound myelin basic protein with actin filaments and calmodulin

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes (OLs) and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. MBP in solution has been shown by others to bind to both G- and F-actin, to bundle F-actin filaments, and to induce polymerization of G-actin. Here we show that MBP bound to acidic lipids can also bind to both G- and F-actin and cause their sedimentation together with MBP-lipid vesicles. Thus it can simultaneously utilize some of its basic residues to bind to the lipid bilayer and some to bind to actin. The amount of actin bound to the MBP-lipid vesicles decreased with increasing net negative surface charge of the lipid vesicles. It was also less for vesicles containing the lipid composition predicted for the cytosolic surface of myelin than for PC vesicles containing a similar amount of an acidic lipid. Calmodulin caused dissociation of actin from MBP and of the MBP-actin complex from the vesicles. However, it did not cause dissociation of bundles of actin filaments once these had formed as long as some MBP was still present. These results suggest that MBP could be a membrane actin-binding protein in OLs/myelin and its actin binding can be regulated by calmodulin and by the lipid composition of the membrane. Actin binding to MBP decreased the labeling of MBP by the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine (TID), indicating that it decreased the hydrophobic interactions of MBP with the bilayer. This change in interaction of MBP with the bilayer could then create a cytosol to membrane signal caused by changes in interaction of the cytoskeleton with the membrane.     

Mechanism of the interaction between myelin basic protein and the myelin membrane; the role of arginine methylation

The addition of solutions of bovine myelin basic protein to suspensions of unilamellar vesicles prepared from whole myelin suspensions results in the rapid equilibrium association of the vesicles into dimers, followed by time-dependent aggregation reactions. Other cationic proteins also induce the dimerization of the vesicles and equilibrium constants for dimer formation are obtained for bovine myelin basic protein, lysozyme, polyhistidine and myelin basic protein from carp, which differs from the bovine protein in that it contains no methylarginine residues. The bovine protein is more efficient at inducing dimer formation than the carp protein by approximately 0.93 kcal/mole; the carp protein is approximately as effective as the other cationic proteins examined. Complete methylation of the bovine MBP by AdoMet:MBP methyltransferase increases the interaction between MBP and the membrane by approximately 0.13 kcal/mole, consistent with the suggestion that a large portion of the free energy difference between the carp and bovine proteins arises from favorable interactions involving the methylarginine residues.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein. A possible role in myelin formation

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, l-α-dimyristoyl phosphatidylcholine and dl-α-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15–18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associating species.A similar interaction was observed with diacyl phosphatidylserine vesicles.These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myeling, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.     

Preparation and characterization of unilamellar myelin vesicles

Myelin vesicles have been obtained from isolated rat brain myelin and shown by electron microscopy to consist of single bilayer membranes. The yield of the preparation is approximately 25% of the myelin proteins. The vesicles show a typical myelin protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contain activity for the myelin marker enzyme, 2',3'-cyclic nucleotide-3'-phosphohydrolase (CNPase). The preparation consists of both inside-out and right-side-out vesicles, and the proportion in each orientation varies from one preparation to another. The occurrence of two populations is demonstrated by the observation that hypotonically lysed vesicles compete to a greater extent than intact vesicles in a competitive enzyme-linked immunosorbent assay with myelin basic protein antiserum. In addition, only a portion of the CNPase activity of the vesicles is trypsin-sensitive and detectable in the absence of detergent; the remaining, trypsin-insensitive activity is present in detergent-disrupted membranes. Thus, there are vesicle populations in which myelin basic protein and CNPase are accessible and others in which they are inaccessible. A population of uniformly oriented right-side-out vesicles has been obtained by ConA-Agarose affinity column chromatography and elution of the bound fraction with methyl-alpha-D-mannopyranoside. In the absence of detergent, less than 10% of the total CNPase activity of these vesicles can be demonstrated, suggesting that the active site of CNPase is opposite to that of the ConA binding site and, therefore, appears to be on the cytoplasmic face of the myelin membrane.     

Reactions of fluorescent probes with normal and chemically modified myelin basic protein and proteolipid. Comparisons with myelin.

Basic (encephalitogenic) protein and water-soluble proteolipid apoprotein isolated from bovine brain myelin bind 8-anilino-1-naphthalenesulfonate and 2-p-toluidinylnaphthalene-6-sulfonate with resulting enhancement of dye fluorescence and a blue-shift of the emission spectrum. The dyes had a higher affinity and quantum yield, when bound to the proteolipid (Kans=2.3x10--6,=0.67) than to the basic protein (Kans=3.3x10--5,=0.40). From the efficiency of radiationless energy transfer from trytophan to bound ANS the intramolecular distances were calculated to be 17 and 27 A for the proteolipid and basic protein, respectively. Unlike myelin, incubation with proteolytic enzymes (e.g., Pronase and trypsin) abolished fluorescence enhancement of ANS or TNS by the extracted proteins. In contrast to myelin, the fluorescence of solutions of fluorescent probes plus proteolipid was reduced by Ca-2+,not affected by La-3+, local anesthetics, or polymyxin B, and only slightly increased by low pH or blockade of free carboxyl groups. The reactions of the basic protein were similar under these conditions except for a two- to threefold increase in dye binding in the presence of La-3+, or after blockade of carboxyl groups. N-Bromosuccinimide oxidation of tryptophan groups nearly abolished native protein fluorescence, but did not affect dye binding. However, alkylation of tryptophan groups of both proteins by 2-hydroxy (or methoxy)-5-nitrobenzyl bromide reduced the of bound ANS (excited at 380 nm) to 0.15 normal. The same effect was observed with human serum albumin. The fluorescence emission of ANS bound to myelin was not affected by alkylation of membrane tryptophan groups with the Koshland reagents, except for abolition of energy transfer from tryptophan to bound dye molecules. This suggests that dye binding to protein is negligible in the intact membrane. Proteolipid incorporated into lipid vesicles containing phosphatidylserine did not bind ANS or TNS unless Ca-2+, La-3+, polymyxin B, or local anesthetics were added to reduce the net negative surface potential of the lipid membranes. However, binding to protein in the lipid-protein vesicles remained less than for soluble protein. Basic protein or bovine serum albumin dye binding sites remained accessible after equilibration of these proteins with the same lipid vesicles. It is proposed that in the intact myelin membrane the proteolipid is probably strongly associated with specific anionic membrane lipids (i.e., phosphatidylserine), and most likely deeply embedded within the lipid hydrocarbon matrix of the myelin membrane. Also, in the intact myelin membrane the fluorescent probes are associated primarily, if not solely with the membrane lipids as indicated by the binding data. This is particularly the case for TNS where the total number of myelin binding sites is three to four times the potential protein binding sites.     

The activator of cerebroside sulphatase. Binding studies with enzyme and substrate demonstrating the detergent function of the activator protein.

1. Sulphatase A (cerebroside sulphatase) (EC 3.1.6.1.) and a 12-fold excess of its physiological activator protein were chromatographed together on Sephadex G-75. The elution buffer was the same as that used in the enzymic degradation of sulphatides. The two proteins were eluted in different peaks indicating that no stable complex formed. 2. Activator protein was incubated with sulphatides under conditions used favouring the sulphatase activity. Incubation solutions were then examined by electrophoresis on a polyacrylamide gel gradient. An one-to-one complex between activator and sulphatides was observed. Half maximal binding occurred with 2.5 nmol of sulphatides together with 1 or 2 nmol of activator in 100 micronl. 3. Cerebrosides as the enzymic degradation products of sulphatides, bind also to the activator protein. A ratio of one-to-one could possibly be obtained at high cerebroside concentrations. The binding to cerebrosides is less specific than that to sulphatides. A 7-fold excess of cerebrosides was necessary for half maximal binding. 4. In a mixture of sulphatides and cerebrosides the formation of the complex with the activator protein is partly inhibited. The total amount of bound lipids changed as the composition of the lipid mixture was varied. In a one-to-one mixture of the two lipids 60% of the total bound lipids are sulphatides and 40% are cerebrosides.     

Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain.

The synthesis and turnover of cerebrosides and phospholipids was followed in microsomal and myelin fractions of developing and adult rat brains after an intracerebral injection of [U-14C]serine. The kinetics of incorporation of radioactivity into microsomal and myelin cerebrosides indicate the possibility of a precursor-product relationship between cerebrosides of these membranes. The specific radioactivity of myelin cerebrosides was corrected for the deposition of newly formed cerebrosides in myelin. Multiphasic curves were obtained for the decline in specific radioactivity of myelin and microsomal cerebrosides, suggesting different cerebroside pools in these membranes. The half-life of the fast turning-over pool of cerebrosides of myelin was 7 and 22 days for the developing and adult rat brain respectively. The half-life of the slowly turning-over pool of myelin cerebrosides was about 145 days for both groups of animals. The half-life of the rapidly turning-over microsomal cerebrosides was calculated to be 20 and 40 h for the developing and adult animals respectively. The half-life of the intermediate and slowly turning-over microsomal cerebrosides was 11 and 60 days respectively, for both groups of animals. The amount of incorporation of radioactivity into microsomal cerebrosides from L-serine was greatly decreased in the adult animals, and greater amounts of the precursor were directed towards the synthesis of phosphatidylserine. In the developing animals, considerable amounts of cerebrosides were synthesized from L-serine, besides phosphatidylserine. The time-course of incorporation indicated that a precursor-product relationship exists between microsomal and myelin phosphatidylserine. The half-life of microsomal phosphatidylserine was calculated to be about 8 h for the fast turning-over pool in both groups of animals.     

Aliphatic aldehydes promote myelin basic protein-induced fusion of phospholipid vesicles

Myelin basic protein induces slow and limited fusion of phospholipid vesicles composed of a mixture of phosphatidylcholine and phosphatidylethanolamine. Addition of palmitoyl aldehyde to these vesicles dramatically increases their ability to fuse in the presence of myelin basic protein. Compared to aliphatic aldehydes, fatty acids are much less potent promoters of myelin basic protein-induced membrane fusion. The ability of aliphatic aldehydes to promote myelin basic protein-induced membrane fusion may be of relevance to myelin structure and function and, particularly, to the pathology of demyelinating diseases such as multiple sclerosis.     

Aliphatic aldehydes promote myelin basic protein-induced fusion of phospholipid vesicles

Myelin basic protein induces slow and limited fusion of phospholipid vesicles composed of a mixture of phosphatidylcholine and phosphatidylethanolamine. Addition of palmitoyl aldehyde to these vesicles dramatically increases their ability to fuse in the presence of myelin basic protein. Compared to aliphatic aldehydes, fatty acids are much less potent promoters of myelin basic protein-induced membrane fusion. The ability of aliphatic aldehydes to promote myelin basic protein-induced membrane fusion may be of relevance to myelin structure and function and, particularly, to the pathology of demyelinating diseases such as multiple sclerosis.     

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis.

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca2+. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.     

Immune lysis of reconstituted myelin basic protein--lipid vesicles and myelin vesicles

Complement-mediated lysis of reconstituted lipid-myelin basic protein (BP) vesicles and myelin vesicles due to antibody raised against BP and isolated myelin is measured by determination of the amount of a water-soluble spin label, tempocholine chloride, released from the vesicles. The response is shown to be antigen-specific, antibody-dependent, and complement mediated. The relative response to different anti-BP antibody samples is similar to that determined by radioimmunoassay procedures. In contrast to immunoassays with BP in aqueous solution, this method measures immune recognition of the protein in either a synthetic or a natural membranous environment. This is important because this protein has been shown to have a different conformation when bound to lipid bilayers than in aqueous solution and its conformation depends on lipid composition. It is also a more rapid method because no separation of spin label still trapped in the vesicles and that released due to immune lysis is required. In synthetic membranes consisting of sphingomyelin, cholesterol, and an acidic lipid, either phosphatidylglycerol, phosphatidic acid, or phosphatidylserine, the response was greatest when the acidic lipid was phosphatidic acid. The response did not depend significantly on the antigen concentration expressed as molar ratio of BP to sphingomyelin, over the range 0.15:600 to 2:600, although it decreased at molar ratios less than 0.15:600. The antigen density required for immune lysis of vesicles containing this protein antigen is similar to that reported elsewhere for lipid antigens, although the time required for maximal lysis was greater. Both anti-BP and anti-myelin antibodies caused a greater specific complement-mediated response with synthetic vesicles than with myelin vesicles, which may be due to the different lipid and/or protein composition of myelin. Response was also obtained with the myelin vesicles, however, indicating that some determinants of BP can be recognized on the surface of the bilayer in isolated myelin by anti-BP.     

A calorimetric study of the thermotropic behaviour of mixtures of brain cerebrosides with other brain lipids

We have used a computer-controlled differential scanning calorimeter to determine the phases present in mixtures of the brain galactocerebrosides with other representative brain lipids. There are two types of brain galactocerebroside, those which possess an alpha-hydroxy substituent on the acyl chain (HFA) and those that do not (NFA). In the liquid crystalline state both cerebrosides were miscible with all the lipids studied, but in the gel state they were immiscible with cholesterol and the brain phosphatidylcholines. However, cholesterol mixtures in which the cholesterol mole fraction exceeded one third formed homogeneous metastable gel states on cooling from above the melting point of the cerebroside. Relaxation to the stable two phase state took place slowly over several hours. The solubilities of the galactocerebrosides in the other main brain sphingolipid, sphingomyelin, were much higher. Only in the case of the NFA galactocerebroside and at low mole fractions of sphingomyelin was immiscibility detected. Ternary mixtures of the two cerebrosides with sphingomyelin/cholesterol and phosphatidylcholine/cholesterol (PC/Chol) showed different miscibility characteristics. On cooling from 80 degrees C all mixtures formed homogeneous gel states. However, on standing the cerebrosides separated into discrete gel phases in all mixtures but one, that in which HFA galactocerebrosides were mixed with sphingomyelin and cholesterol. The cerebroside in the mixture with the composition closest to that of myelin, HFA/PC/Chol, melted at 38 degrees C. On scanning guinea pig CNS myelin which had been equilibrated at 5 degrees C a transition was detected with Tmax 33 degrees C. On the basis of comparison with the HFA/PC/Chol mixture we propose that the transition in myelin at this temperature is due to the melting of a galactocerebroside gel phase.     

Localization of sites for ionic interaction with lipid in the C-terminal third of the bovine myelin basic protein.

The myelin basic protein from bovine brain tissue was purified and the two peptides obtained by cleavage of the polypeptide chain at the single tryptophan residue were isolated. The interaction of these peptides and the intact basic protein with complex lipids was investigated by following the solubilization of lipid-protein complexes into chloroform in a biphasic solvent system. The C-terminal peptide fragment (residues 117-170) and the intact basic protein both formed chloroform-soluble complexes with acidic lipids, but not with neutral complex lipids. The N-terminal fragment (residues 1-115) did not form chloroform-soluble complexes with either acidic or neutral complex lipids. The molar ratio of lipid to protein that caused a 50% loss of protein from the upper phase to the lower chloroform phase was the same for the intact basic protein as for the smaller C-terminal peptide fragment. Phosphatidylserine and phosphatidylinositol were approximately twice as efficient as sulphatide at causing protein redistribution to the chloroform phase. The results are interpreted as indicating that the sites for ionic interactions between lipid and charged groups on the basic protein of myelin are located in the C-terminal region of the protein molecule.     

Membrane instability induced by purified myelin components. Its possible relevance to experimental allergic encephalomyelitis

The fusogenic properties of purified myelin components in a system employing chicken erythrocytes were studied. Sulphatides, myelin basic protein and the apoprotein of Folch-Lees proteolipid were capable of individually inducing membrane fusion in the presence of Ca²⁺. By contrast, cerebrosides or a mixture of sulphatides and myelin basic protein (molar ratio 19 : 1) did not show such effect. The fusogenic ability of sulphatide was correlated to its behaviour in mixed monolayers with phospholipids at the air-water interface. Mixed films of sulphatides with phosphatidylcholine or sphingomyelin but not with phosphatidylethanolamine showed reductions of molecular packing and surface potential similar to those found for other fusogenic compounds. The effects of myelin components described could be of importance in the membrane instability and vesicular disruption of myelin occurring in demyelinative disorders.     

Lipid and basic protein interaction in myelin

1. Purified myelin labelled with [(3)H]myo-inositol or [1-(14)C]acetate was incubated with trypsin or acetylated trypsin at 37 degrees C, pH8.0 for 30min. 2. After incubation and centrifugation analysis of the myelin pellet showed marked digestion of basic protein on polyacrylamide-gel electrophoresis. Proteolipid and Wolfgram proteins remained unchanged. 3. A loss of 15% of total protein and loss of all classes of lipids was also found. Most significant lipid losses were phosphoinositides, phosphatidylserine and sulphatide. 4. A low-density material containing more phospholipid than cholesterol and galactolipid was isolated from the supernatant obtained after centrifugation of trypsin-treated myelin. 5. Interaction of sulphatide and myelin basic protein was shown to take place in a biphasic system. Basic protein does not form any complex either with cerebroside or cholesterol in the same solvent system. 6. The release of acidic lipids from myelin suggests that they may be linked to basic protein by ionic forces and the neutral lipids may be by lipid-lipid interactions. 7. The relevance of these studies as a model of brain degeneration is discussed.     

Cytoplasmic domain of human myelin protein zero likely folded as beta-structure in compact myelin

Myelin protein zero (P0 or P0 glycoprotein), the major integral membrane protein in peripheral nervous system myelin, plays a key role in myelin membrane compaction and stability. While the structure of P0 extracellular domain was determined by crystallography, the paucity of any structural data on the highly positive-charged P0 cytoplasmic domain (P0-cyt) has greatly limited our understanding of the mechanism of P0 function. Here, using circular dichroism and intrinsic fluorescence spectroscopy, we attempted to elucidate the structure of human P0-cyt (hP0-cyt) in membrane mimetic environments composed of detergents or lipid vesicles. We found that the secondary structure of P0-cyt was polymorphic—at the lipid/protein ratio corresponding to that of mature peripheral myelin (~50:1), hP0-cyt mainly adopted a β-conformation, whereas when the proportion of lipid increased, the structure underwent a β→α transition. By contrast, the secondary structure of the major isoform of myelin basic protein, another myelin protein with a very large positive charge, remained unchanged across a wide range of lipid/protein ratios. We propose that when hP0-cyt is bound at sufficient concentration to lamellar lipid bilayers such as myelin, it folds into a β-conformation; before this threshold lipid/protein ratio is reached, the domain is α-helical. We suggest that the cytoplasmic apposition (major dense line) in compact myelin may be stabilized via the hydrogen-bonding of β-strands formed as a result of local P0-P0 aggregation.