The association of myelin basic protein with itself and other proteins

Chromatographic studies were performed to measure myelin basic protein (MBP) interactions by covalently binding a number of different proteins to Sepharose and passing radioactive bovine MBP over these columns. Studies at a variety of pH values, ionic strengths and temperatures revealed that the bovine MBP could interact with itself as well as cytochrome c, lysozyme, and ovalbumin. Chromatographic profiles of elution volume vs. pH revealed that the interaction between MBP and these immobilized proteins was biphasic. The self-association of MBP was found to be strongest between pH 7.4 and 8.1 and at an elevated temperature. Titration of the amino acid residues responsible for the association of MBP with other proteins revealed apparent pKs ranging from 6.10 to 6.70. A pH dependence study at an elevated temperature shifted the apparent pK of the MBP interaction to a lower value with all the proteins except ovalbumin. After destroying 60% of the histidine residues in MBP by photooxidation and passing¹²⁵I-labeled photooxidized MBP over Sepharose columns containing immobilized protein, the second phase in binding was decreased significantly with immobilized cytochrome c, lysozyme, and MBP and to a smaller extent with ovalbumin. These results are consistent with the involvement of deprotonated histidine residues in the MBP-protein associations.     

Association of myelin basic protein with detergent micelles

Equilibrium measurements of the binding of central nervous system myelin basic protein to sodium dodecyl sulphate, sodium deoxycholate and lysophosphatidylcholine have been obtained by gel permeation chromatography and dialysis. This protein associates with large amounts of each of these surfactants: the apparent saturation weight ratios (surfactant/protein) being $\text{3.58 ± 0.12}\text{and}\text{2.30 ± 0.15}$ for dodecyl sulphate at ionic strengths 0.30 and 0.10, respectively, $\text{1.34 ± 0.10}$ for deoxycholate (at 0.12 ionic strength) and $\text{4.0 ± 0.5}$ for lysophosphatidylcholine. Binding to the ionic surfactants increases markedly close to their critical micelle concentrations. Sedimentation analysis shows that at 0.30 ionic strength in excess dodecyl sulphate the protein is monomeric. It becomes dimeric when the binding ratio falls below 1 at a free detergent concentration of approximately 0.25 mM: below this concentration much of the protein and detergent forms an insoluble complex. The amount of dodecyl sulphate bound at high concentrations and at both above-mentioned ionic strengths corresponds closely to that expected for interaction of a single polypeptide with two micelles. Variability of deoxycholate micelle size on interaction with other molecules precludes a similar analysis for this surfactant. Association was observed only with single micelles of lysophosphatidylcholine. The results provide strong evidence for dual lipid-binding sites on basic protein and indicate that lipid bilayer cross-linking by this protein may be effected by single molecules.     

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.     

An electronmicroscopic study of the distribution of myelin basic protein in multilayer dispersions of diacylphosphatidylserine

Although dispersions containing lipid and protein are widely used as model systems to explore the properties of biomembranes, the extent of mixing of the two components has generally not been determined. Here, the distribution of bovine myelin basic protein in dispersions with bovine brain L-alpha-diacylphosphatidylserine (PS) has been examined electronmicroscopically. Dispersions of PS were prepared by hydrating a known amount of dried lipid with buffer or with buffer containing an equal weight of myelin basic protein or lysozyme. The lipid-protein complexes were separated from unbound protein by centrifugation in 0-60% sucrose density gradients. In both systems only a few percent of the protein was unbound and the resultant recombinants, which gave single bands on the gradients, contained about 50% protein by weight. After removal of the sucrose by dialysis the dispersions were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated and embedded in epoxy resin. Thin sections cut from these blocks were incubated, after removal of osmium tetroxide, with antiserum raised in rabbits against human myelin basic protein. Excess antiserum was removed and the antigen-antibody complexes on the thin sections were labelled with 13 nm diameter colloidal gold particles stabilized with protein A. The distributions of these gold particles were examined under an electronmicroscope. Comparison of the labelling patterns for PS, PS-lysozyme and PS-basic protein demonstrated specific labelling in the last, and showed the gold particles to be uniformly dispersed. It was concluded that in these dispersions the protein and lipid were intimately mixed at the molecular level.     

Is myelin basic protein crystallizable?

Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized.     

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.     

Myelin basic protein-enhanced fusion of membranes

Myelin basic protein caused rapid aggregation of vesicles containing acidic phospholipids. Aggregation could be reversed by trypsin digestion of the myelin basic protein. Aggregated vesicles containing gel phase phospholipids or vesicles containing greater than 15 mol% lysolecithin underwent fusion. The extent of fusion was measured by irreversible changes in the light-scattering intensities or diffusion coefficients of the vesicles. Fusion was also measured by the fluorescence quenching which occurred when vesicles containing a covalently bound fluorophore, N-4-nitrobenzo-2-oxa-1,3-diazole, were fused with vesicles containing the covalently bound spin label, 4,4-dimethyl-oxazolidine-N-oxyl. The kinetics of fusion were first order in phospholipid and had half-times of 0.5–5 min depending on lysolecithin composition. This protein-enhanced membrane fusion may provide a valuable model system for studying some types of biological membrane fusions.     

An analysis of the regions of the myelin basic protein that bind to phosphatidylcholine

The interactions of phosphatidylcholine (PC) to regions of the myelin basic protein (MBP) was examined. In solid phase binding assays the nature of the binding of unilamellar vesicles of¹⁴C-labeled phosphatidylcholine to bovine 18.5 kDa MBP, its N- and C-terminal peptide fragments, photooxidized 18.5 kDa MBP and the mouse 14 kDa protein, with an internal deletion of residues 117–157, was studied. The data were analyzed by computer-generated Scatchard plots in which non-specific binding was eliminated. Non-cooperative, low affinity binding of PC vesicles to MBP was observed, and this binding found to be sensitive to pH and ionic changes. At an ionic strength of 0.1 and pH 7.4, the binding of PC to the 14 kDa mouse MBP exhibited a K_d similar to that obtained with both the N-terminal and photooxidized 18.5 kDa bovine MBP. The studies indicated that the sites of PC interaction with MBP are located in the N-terminal region of the protein. The C-terminal region appeared to modulate the strength of the interaction slightly. Under similar conditions, lysozyme did not bind PC liposomes, and histone bound them nonspecifically.     

Mechanisms of myelin basic protein and proteolipid protein targeting in oligodendrocytes (Review)

Summary

The segregation of proteins to specific cellular membranes is recognized as a common phenomenon. In oligodendrocytes of the central nervous system, localization of certain proteins to select regions of the plasma membrane gives rise to the myelin membrane. Whilst the fundamental structure and composition of myelin is well understood, less is known of the mechanisms by which the constituent proteins are specifically recruited to those regions of plasma membrane that are forming myelin. The two principal proteins of myelin, the myelin basic protein and proteolipid protein, differ greatly in character and sites of synthesis. The message for myelin basic protein is selectively translocated to the ends of the cell processes, where it is translated on free ribosomes and is incorporated directly into the membrane. Proteolipid protein synthesized at the rough endoplasmic reticulum, processed through the Golgi apparatus, and presumably transported via vesicles to the myelin membrane. This review examines the mechanisms by which these two proteins are targeted to the myelin membrane.     

Specificity of zinc binding to myelin basic protein

Z²⁺ appears to stabilize the myelin sheath but the mechanism of this effect is unknown. In a previous report we have shown that zinc binds to CNS myelin basic protein (MBP) in the presence of phosphate and this results in MBP aggregation. For this paper we used a solid phase zinc blotting assay to identify which myelin proteins bind zinc. MBP and a 58 kDa band were found to be the major targets of⁶⁵Zn binding. Moreover, using fluorescence, light scattering and electron microscopy we investigated the binding of zinc and other cations to purified MBP in solution. Among the cations tested for their ability to interfere with the binding of zinc, the most effective were cadmium, mercury and copper, but only cadmium and mercury increased the scattering intensity, whereas MBP aggregation was not inhibited by copper ions. Thus, the effect of zinc on the formation of MBP clusters seems to be specific.     

Identification of the major sites of enzymic glycosylation of myelin basic protein

In the presence of porcine submaxillary N-acetylgalactosaminyltransferase and uridine diphospho-N-acetyl-D-galactosamine, approx. 1.2–1.5 mol of N-acetylgalactosamine were transfered per mol of myelin basic protein. Tritium-labelled N-acetylgalactosamine-labelled basic protein was digested with trypsin and the peptides were separated by HPLC and the radioactivity measured. Most of the radioactivity was associated with three peptide peaks (I, II and III) containing 17, 69 and 6% of the total radioactivity, respectively. The remaining radioactivity was distributed amongst several peptides, each containing less than 2.5% of the total radioactivity. Glycosylation of the basic proteins isolated from human, bovine and guine pig myelins showed that they were all equally good acceptors. In spite of differences in the peptide profiles of the basic proteins from different species, the distribution of radioactivity between the three peptide peaks was similar for all the species studies. The transfer of N-acetylgalactosamine to peptide II was much faster than to peptides I and III. The apparent K_m values of the three peptides were within a narrow range of 0.52–0.63 mM, whereas the V_max values were considerably different. The glycosylated peptide peaks (I, II and III) were separated by electrophoresis, the radioactivity measured, and amino acid compositions determined after hydrolysis. The major radioactive peptides of the human basic protein were identified with tryptic peptides containing the following sequences:

Full-size image (4K)

     

Conformation of bovine myelin basic protein purified with bound lipids

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having α-helix and β-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results. Received: 13 November 1998 / Accepted: 1 February 1999     

Primary structure of equine myelin basic protein by mass spectrometry

Equine myelin basic protein (MBP) has been isolated from spinal cord and shown to consist of a number of components (charge isomers) by alkaline-urea gel electrophoresis. Mass analyses of several of these components showed that each was posttranslationally modified and some have been identified. Component 1, the most cationic charge isomer, was sequenced by a combination of liquid chromatography and mass spectrometry of peptides obtained by proteolytic digestion. At 172 residues it is slightly larger than the bovine (169) and the human (170). A major difference between bovine and equine sequences was the replacement of AQGH (bovine residues 76-79) by SRDG (equine). A number of other replacements involving single amino acids were also found. Methylated arginine (residue 108 equine) was found as both the mono- and the dimethylated derivative and represents the first MS/MS evidence for this modification in any MBP.     

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and ³¹P, ¹³C, and ¹H nuclear magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5–, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and ¹³C- and ¹H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% -helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of -helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles. Correspondence to: G. L. Mendz     

Studies of posttranslational modifications in spiny dogfish myelin basic protein

The objective of this investigation was to determine whether nonmammalian myelin basic protein contained charge isomers resulting from extensive posttranslational modifications as seen in mammalian MBP. Four charge isomer components from dogfish MBP have been isolated. These forms arise by phosphorylation and deamidation modifications. Components C1, C2 and C3 have been characterized. We are currently characterizing component C8. Dogfish MBP is less cationic than mammalian MBP and has about 50% lower mobility on a basic pH gel electrophoresis relative to human and to bovine MBP. The mammalian component C1, which is unmodified, is modified in the dogfish by phosphorylation. The reduced electrophoretic mobility is largely attributable to the charge reduction resulting from phosphorylation in serine 72, 83, and 120 or 121 in C1, and C3. In component C2, two or three phosphate groups were distributed among residues 134, 138 and 139. It was found that dogfish amino acid residue 30 was a lysine residue and not a glutamate residue as reported in the literature.     

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.     

Fluorescence spectral resolution of myelin basic protein conformers in complexes with lysophosphatidylcholine

The structure of (Deibler) myelin basic protein in solution and in a lysolecithin lipid complex has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out using both static and time-resolved fluorescence techniques. Relative to the free protein, the lipid bound myelin basic protein showed a, twofold increase in fluorescence intensity and a marked blue-shift in the emission maximum wavelength. The multiexponential fluorescence decays and the decay associated spectra indicated that the protein exists in at least three different conformations both in buffer and in lipids. Fluorescence polarization and acrylamide quenching experiments showed that the tryptophan containing region of the protein is embedded in the lipid matrix. The binding of the protein to the lipid appears to be comparable with that predicted for the interaction of amphipathic helices with nonpolar lipids.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

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.     

Microstructural analysis of the effects of incorporation of myelin basic protein in phospholipid layers

We report an X-ray reflectivity study on the effects of adsorption of myelin basic protein (MBP) on Langmuir monolayers and on deposited Langmuir–Schaefer multilayers of the phospholipid dipalmitoyl phosphatidylglycerol (DPPG). We provide for the first time, direct microscopic evidence on the destructuring effects of MBP leading to plasticity of the DPPG layers supporting commonly accepted models of the stabilizing role of MBP in the myelin membrane. We also show how protein adsorption onto the layer is determined both by electrostatic and nonspecific hydrophobic interactions.