Direct measurement of the energetics of association between myelin basic protein and phosphatidylserine vesicles

A newly designed high-sensitivity isothermal reaction calorimetry system has been used to investigate the thermodynamics of the association between myelin basic protein and phosphatidylserine vesicles. This instrument has allowed us to measure directly the energetics of the protein-lipid interaction under various conditions. Above the phospholipid phase transition temperature the enthalpy of association is highly exothermic amounting to -160 kcal/mol of protein. Below the phospholipid phase transition temperature the enthalpy of association is exothermic at protein/lipid ratios smaller than 1/50 and endothermic at higher protein/lipid ratios. These studies indicate that the association of myelin basic protein to phosphatidylserine vesicles consists of at least two stages involving different types of binding. The first stage, at low protein/lipid ratios, involves a strong exothermic association of the protein to the membrane and the second, at high protein/lipid ratios, a weaker association probably involving attachment of the protein to the membrane surface only. In the gel phase the second binding stage is endothermic and appears to be correlated with the formation of large vesicle aggregates. This vesicle aggregation is a reversible process dependent upon the physical state of the membrane. The isothermal titration studies have been complemented with high-sensitivity differential scanning calorimetry experiments. It is shown that the dependence of the phospholipid transition enthalpy on the protein/lipid molar ratio can be expressed in terms of the different protein-membrane association enthalpies in the gel and fluid phases of the membrane.     

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2′,3′-cyclic nucleotide-3′-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all of the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55 % and proteolipid protein for 46 % of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H. C., Burton, R. M., Fishman, M. A., Mitchell, R. F. and Prensky, A. L. (1972) J. Neurochem. 19, 2083–2089) exhibited a developmental “switch” between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain.

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2',3'-cyclic nucleotide-3'-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55% and proteolipid protein for 46% of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H.C., Burton, R. M., Fishman, M.A., Mitchell, R.F. and Prensky, A.L. (1972) J. Neurochem. 19, 2083-2089) exhibited a developmental "switch" between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.     

Protein kinase associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates

When highly purified myelin from rat sciatic nerve was incubated with [γ-³²P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and ³²P from [γ-³²P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg²⁺-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3′,5′-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing.From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

The role of charge microheterogeneity of human myelin basic protein in the formation of phosphatidylglycerol multilayers

Human myelin basic protein (HBP) was fractionated into its various charge isomers by chromatography on CM-52 columns at pH 10.6. Components 1,2,3 and "8" (C-1, C-2, C-3, and C-"8") were cleanly separated. Each component was combined with phosphatidylglycerol (PG) vesicles, at neutral pH at a concentration of 30% (w/w), protein/lipid. C-1, the most cationic of the components was the most effective at inducing the formation of multilayers when studied by liquid X-ray diffraction. C-3, which differs from C-1 by 2 positive charges was less effective than C-2. C-"8" was totally ineffective since the scattering pattern with this component was no different from that of the pure lipid. Thus a seemingly small change in net charge of the protein had a dramatic effect on the ability of the protein to organize the lipid into a crystalline, multilayer arrangement characteristic of compact myelin.     

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.     

Protein kinases associated with peripheral nerve myelin. 1. Phosphorylation of endogenous myelin proteins and exogenous substrates.

When highly purified myelin from rat sciatic nerve was incubated with [gamma-32P]ATP, protein components of the membrane were phosphorylated indicating the presence of both the substrate (receptor protein) and an endogenous kinase in the membrane. Polyacrylamide gel electrophoresis of the phosphorylated membrane proteins followed by scintillation counting of gel slices and autoradiography showed that the polypeptides of molecular weights 28000, 23000 and 19000 were phosphorylated, and 32P from [gamma-32P]ATP having been incorporated into serine residues of the substrate proteins. Phosphorylation of purified myelin was Mg2+-dependent, was optimal at pH 6.5 and was not stimulated by adenosine 3',5'-monophosphate. We found that proteins other than those in myelin, such as phosvitin, casein, protamine and histones, can also act as a substrate for the membrane associated kinase. Muscle protein kinase inhibitor had no effect on the endogenous phosphorylation of myelin proteins or on the phosphorylation of phosvitin by peripheral nerve myelin protein kinase. However, the phosphorylation of histone by peripheral nerve myelin protein kinase was inhibited by the protein kinase inhibitor. After washing the membrane with 150 mM KCl the protein kinase that utilizes histone as substrate was found in the supernatant. In contrast, the endogenous phosphorylation of membrane proteins or the phosphorylation of phosvitin by the membrane associated kinase was not affected by washing. From these findings we conclude that at least two protein kinase systems exist in purified peripheral nerve myelin. One system is not inhibited by muscle kinase inhibitor, is tightly bound to the membrane and utilizes as its receptor proteins either exogenous phosvitin or endogenous membrane proteins. The second system is inhibited by muscle kinase inhibitor, is removable from the membrane and utilizes histones as its receptor proteins.     

Endogenous phosphorylation of basic protein in myelin of varying degrees of compaction

Fractions containing myelin of varying degrees of compaction were prepared from human white matter. Protein kinase activity in these fractions was measured by using both endogenous and exogenous myelin basic protein (MBP) as substrates. In both cases, less compact myelin fractions possessed higher levels of protein kinase activity than the compact myelin fraction. In addition, the specific activity of phosphorylated basic protein was greater in the loosely compacted fractions than in compact multilamellar myelin. When basic protein in compact myelin or the myelin fractions was phosphorylated by the endogenous kinase, approximately 70% of the [32P]phosphate was incorporated at a single site, identified as Ser-102. The remaining 30% was found in three other minor sites. Electron microscopy of less compact myelin showed it was composed of fewer lamellae which correlated with a relative decrease in the proportion of cationic charge isomers (microheteromers) when MBP was subjected to gel electrophoresis at alkaline pH. The shift in charge microheterogeneity of basic protein to the less cationic isomers in the less compact myelin fractions correlated with an increase in protein kinase activity and a greater specific activity of phosphorylated basic protein.     

Shaping membrane interfaces in lipid vesicles mimicking the cytoplasmic leaflet of myelin through variation of cholesterol and myelin basic protein contents

Myelin basic protein (MBP) is an intrinsically disordered protein and in the central nervous system (CNS) mainly responsible for connecting the cytoplasmic surfaces of the multilamellar, compact myelin. Increased posttranslational modification of MBP is linked to both, the natural development (from adolescent to adult brains) of myelin, and features of multiple sclerosis. Here, we study how a combination of this intrinsically disordered myelin protein with varying the natural cholesterol content may alter the characteristics of myelin-like membranes and interactions between these membranes. Large unilamellar vesicles (LUVs) with a composition mimicking the cytoplasmic leaflet of myelin were chosen as the model system, in which different parameters contributing to the interactions between the lipid membrane and MBP were investigated. While we use cryo-transmission electron microscopy (TEM) for imaging, dynamic light scattering (DLS) and electrophoretic measurements through continuously-monitored phase-analysis light scattering (cmPALS) were used for a more global overview of particle size and charge, and electron paramagnetic resonance (EPR) spectroscopy was utilized for local behavior of lipids in the vesicles' membranes in aqueous solution. The cholesterol content was varied from 060 % in these LUVs and measurements were performed in the presence and absence of MBP. We find that the composition of the lipid layers is relevant to the interaction with MBP. Not only the size, the shape and the aggregation behavior of the vesicles depend on the cholesterol content, but also within each membrane, cholesterol's freedom of movement, its environmental polarity and its distribution were found to depend on the content using the EPR-active spin-labeled cholesterol (CSOSL). In addition, DLS and EPR measurements probing the transition temperatures of the lipid phases allow a correlation of specific behavior with the human body temperature of 37 °C. Overall, our results aid in understanding the importance of the native cholesterol content in the healthy myelin membrane, which serves as the basis for stable and optimum protein-bilayer interactions. Although studied in this specific myelin-like system, from a more general and materials science-oriented point of view, we could establish how membrane and vesicle properties depend on cholesterol and/or MBP content, which might be useful generally when specific membrane and vesicle characteristics are sought for.     

Accumulation of the four myelin basic proteins in mouse brain during development.

Myelin was isolated from the brains of mice 15, 20, 30, and 60 days after birth. The total amount of basic protein present in the isolated myelin was determined by radioimmunoassay. The 4 myelin basic proteins, with molecular weights of 21,500, 18,500, 17,000 and 14,000, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and their relative amounts were determined densitometrically. The absolute amount of each of the basic proteins was calculated from its relative amount on the gel and from the total amount of myelin basic protein in the sample as determined by radioimmunoassay. The results show that between 10 and 30 days after birth each protein accumulates at a characteristic rate so that the molar ratios among the 4 basic proteins are (in descending order according to their molecular weights) 1:5:2:10 during this period. Between 30 and 60 days after birth the 14 K and 18.5 K proteins continue to accumulate at reduced rates while the 21.5 K and 17 K proteins begin to disappear from the myelin membrane; 60 days after birth the molar ratios among the 4 basic proteins are 1:10:3.5:35. These developmental patterns of accumulation are discussed in relation to the possible role of each of the 4 myelin basic proteins in myelination.     

Metabolic turnover of myelin glycerophospholipids

The apparent half life for metabolic turnover of glycerophospholipids in the myelin sheath, as determined by measuring the rate of loss of label in a myelin glycerophospholipid following radioactive precursor injection, varies with the radioactive precursor used, age of animal, and time after injection during which metabolic turnover is studied. Experimental strategies for resolving apparent inconsistencies consequent to these variables are discussed. Illustrative data concerning turnover of phosphatidylcholine (PC) in myelin of rat brain are presented. PC of the myelin membrane exhibits heterogeneity with respect to metabolic turnover rates. There are at least two metabolic pools of PC in myelin, one with a half life of the order of days, and another with a half life of the order of weeks. To a significant extent biphasic turnover is due to differential turnover of individual molecular species (which differ in acyl chain composition). The two predominant molecular species of myelin PC turnover at very different rates (16:0, 18:1 PC turning over several times more rapidly than 18:0, 18:1 PC). Therefore, within the same membrane, individual molecular species of a phospholipid class are metabolized at different rates. Possible mechanisms for differential turnover of molecular species are discussed, as are other factors that may contribute to a multiphasic turnover of glycerophospholipids.Special issue dedicated to Dr. Marjorie Lees.     

Evolution of a neuroprotective function of central nervous system myelin

The central nervous system (CNS) of terrestrial vertebrates underwent a prominent molecular change when a tetraspan membrane protein, myelin proteolipid protein (PLP), replaced the type I integral membrane protein, P0, as the major protein of myelin. To investigate possible reasons for this molecular switch, we genetically engineered mice to express P0 instead of PLP in CNS myelin. In the absence of PLP, the ancestral P0 provided a periodicity to mouse compact CNS myelin that was identical to mouse PNS myelin, where P0 is the major structural protein today. The PLP-P0 shift resulted in reduced myelin internode length, degeneration of myelinated axons, severe neurological disability, and a 50% reduction in lifespan. Mice with equal amounts of P0 and PLP in CNS myelin had a normal lifespan and no axonal degeneration. These data support the hypothesis that the P0-PLP shift during vertebrate evolution provided a vital neuroprotective function to myelin-forming CNS glia.     

Divalent cations induce a compaction of intrinsically disordered myelin basic protein

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly-wrapped multilamellar structure around neurons. In mature myelin, the predominant splice isoform of classic MBP is 18.5 kDa. In solution, MBP is an extended, intrinsically disordered protein with a large effective protein surface for myriad interactions, and possesses transient and/or induced ordered secondary structure elements for molecular association or recognition. Here, we show by nanopore analysis that the divalent cations copper and zinc induce a compaction of the extended protein in vitro, suggestive of a tertiary conformation that may reflect its arrangement in myelin.     

Developmental regulation of myelin basic protein expression in mouse brain

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.     

The in vivo synthesis of myelin proteins in rabbit sciatic nerve

Rabbits were injected into the sciatic nerves with either ³⁵S-methionine, or ³H-fucose. After times ranging from 45 min to 15 days the nerves were removed and the total particulate material from the nerves fractionated to give seven subfractions with densities between 0.2 and 1.2 M sucrose. The patterns of radio-labelled proteins were examined by SDS-PAGE and quantitative fluorography. The results showed that the P₂ basic protein was metabolically far more active than either the major P₀ glycoprotein, or the basic protein BP. The P₂ protein also entered the myelin fractions more rapidly than either P₀, or BP components. The net synthesis of P₀ was slower than P₂ and BP and this intrinsic membrane protein remained associated with the denser membrane fractions (>0.7 M sucrose) for longer than the basic proteins prior to entering myelin. Newly synthesized high molecular weight proteins remained concentrated in the denser membrane fractions and turned over faster than the myelin proteins.

A low density myelin fraction (B) was detected in which both the P₂ protein and certain high molecular weight proteins became more rapidly labelled than in compact myelin. In this fraction the specific activity remained higher than that of compact myelin for up to five days after the injection of ³⁵S-methionine into the nerve.

The results indicate that the major PNS myelin proteins are incorporated into and turn over in the various compartments of the Schwann cell plasma membrane—myelin continuum at very different rates.     

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.     

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic protein

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca²⁺ in the micromolar range. Optimal activity is reached at a free Ca²⁺ concentration of about 2 μM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with ⁴⁵Ca²⁺, ⁸⁶Rb⁺, or ²²Na⁺, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80–90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb⁺ and Na⁺ was measured, when the vesicles were pretreated with ATP and Mg²⁺.     

A permeability change of myelin membrane vesicles towards cations is induced by MgATP but not by phosphorylation of myelin basic proteins

The existence of an endogenous protein kinase activity and protein phosphatase activity in myelin membrane from mammalian brain has now been well established. We found that under all conditions tested the myelin basic protein is almost the only substrate of the endogenous protein kinase in myelin of bovine brain. The protein kinase activity is stimulated by Ca2+ in the micromolar range. Optimal activity is reached at a free Ca2+ concentration of about 2 microM. Myelin membrane vesicles were prepared and then shown to be sealed by a light-scattering technique. After preloading with 45Ca2+, 86Rb+, or 22Na+, the self-diffusion (passive outflux) of these ions from myelin membrane vesicles was measured. Ionophores induced a rapid, concentration-dependent outflux of 80--90% of the cations, indicating that only a small fraction of the trapped ions was membrane bound. There was no difference in the diffusion rates of the three cations whether phosphorylated (about 1 mol phosphate per myelin basic protein) or non-phosphorylated vesicles were tested. In contrast, a small but significant decrease in permeability for Rb+ and Na+ was measured, when the vesicles were pretreated with ATP and Mg2+.     

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.