Increased Phosphorylation of Myelin Basic Protein During Hippocampal Long-Term Potentiation

Abstract: Hippocampal long-term potentiation (LTP) is a long-lasting and rapidly induced increase in synaptic strength. Previous experiments have determined that persistent activation of protein kinase C (PKC) contributes to the early maintenance phase of LTP (E-LTP). Using the back-phosphorylation method, we observed an increase in the phosphorylation of a 21-kDa PKC substrate, termed p21, 45 min after LTP was induced in the CA1 region of the hippocampus. p21 was found to have the same apparent molecular weight as the 18.5-kDa isoform of myelin basic protein (MBP) and was recognized by an antibody to MBP in western blotting and immunoprecipitation. Furthermore, p21 from control and potentiated hippocampal slices and purified MBP have identical phosphopeptide maps when back-phosphorylated and then digested with either endoproteinase Lys-C or endoproteinase Asp-N, suggesting that p21 and MBP are identical proteins. As there was no observed change in the amount of MBP in LTP, the increase in MBP phosphorylation during LTP cannot be explained by a change in the amount of protein. From these experiments, we conclude that the phosphorylation of the 18.5-kDa isoform of MBP is increased during E-LTP.     

Characterization of Basic Proteins from Goldfish Myelin

Abstract: Myelin basic protein (MBP) from common goldfish ( Carassius auratus ) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130–137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with ¹²⁵I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microhet-erogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis. Even from the limited sequence obtained, the sequence ATAST was found in goldfish, which is also present in human, rabbit, and guinea pig MBPs.     

Conservation Throughout Vertebrate Evolution of the Predicted β-Strands in Myelin Basic Protein

To identify functionally important parts of the 18.5-kDa myelin basic protein (MBP), the amino acid sequences from 10 species ranging from shark to human were aligned using the SEQHP computer program. The residues that are invariant or very conservatively substituted (Arg/Lys, Ser/Thr, Ile/Leu, Asp/Glu) among all 10 proteins were scored. Of the 72 conserved residues in the 170-residue human protein (42% conserved), 32 are found within the five beta-strands previously predicted (45 residues, 71% conserved), 23 within the small-loops region (42 residues, 55% conserved), but only 17 within the large-loops region (83 residues, 20% conserved). Of the 22 hydrophobic residues within the predicted beta-sheet of human MBP, 20 hydrophobic residues remain in the shark protein, 19 of them in the same positions. In contrast, there are 10 hydrophobic residues elsewhere in the human protein, but only 7 remain in the shark protein and only 1 of them is in the same position. The triprolyl sequence found in all mammalian MBPs and in the chicken MBP is not conserved in the shark protein. The four alternately spliced forms of mouse MBP can be accommodated by the beta-structural model, but not the 17-kDa human MBP, which lacks exon 5. These findings confirm the crucial role of the hydrophobic residues in the predicted beta-sheet for the structure and function of the protein. It seems likely that the conserved portions of the protein make an important contribution to the highly ordered lamellar structure of myelin.     

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca²⁺-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.     

Identification of the New Isoforms of Mouse Myelin Basic Protein: The Existence of Exon 5a

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.     

Expression of myelin protein genes in the developing brain

The major myelin proteins fall into two classes, the basic proteins and the proteolipid proteins. In mice, five forms of the myelin basic protein (MBP) have been identified with apparent molecular masses of 21.5 kD, 18.5 kD, 17 kD and 14 kD. The 17 kD MBP variant consists of two molecular forms with similar molecular masses but different amino acid sequences. Cell-free translation studies and analyses of MBP cDNAs have shown that each of the MBP variants is encoded by a separate mRNA of approximately 2 000 bp. The five mouse MBP mRNAs appear to be derived by alternative splicing of exons 2, 5, and 6 of the MBP gene. cDNAs encoding four forms of MBP have been isolated from a human fetal spinal cord library. The mRNAs corresponding to these cDNAs are probably derived by alternative splicing of exons 2 and 5 of the human MBP gene. Proteolipid protein (PLP) cDNAs have been isolated from several species and used to establish that the size of the major PLP mRNA is approximately 3 kb. Multiple size classes of the PLP mRNAs exist in mice and rats whereas the 3 kb mRNA is the predominant form in the developing human spinal cord. In normal mice, maximal expression of the PLP gene lags behind that of the MBP gene by several days. Studies on dysmyelinating mutants have determined some of the molecular defects with respect to these two classes of myelin proteins. For example, there is a deletion of a portion of the MBP gene in the shiverer mutant. In the quaking mutant, the expression of both classes of myelin proteins is significantly reduced prior to 3 weeks. However, after 3 weeks, MBP expression approaches normal levels but the newly synthesized protein fails to be incorporated into myelin. In the jimpy mutant, although the expression of both classes of proteins is reduced, PLP expression is most severely affected.     

Differential racemization of aspartate and serine in human myelin basic protein

L-Aspartate and L-serine were found to undergo amino acid racemization in brain myelin basic protein (MBP) of aging humans. The observed racemization was different in each chromatographically purified MBP isoform. Pepsin digestion of MBP produced three peptides, each of which exhibited different degrees of racemization of the same amino acids. MBP isolated by the same method from rat and guinea pig brain showed little accumulation of D-amino acids. Total MBP isolated from SDS-polyacrylamide disc gel electrophoresis of total human myelin proteins (delipidated myelin) was racemized to the same extent as purified MBP, indicating that the racemization observed was not an artifact of the isolation procedure. Myelin proteolipid protein from the same gel was racemized approximately half as much as MBP. The age and environment of the aspartates and serines in myelin proteins may strongly affect their observed racemizations.     

Oligodendrocyte-specific expression of human immunodeficiency virus type 1 Nef in transgenic mice leads to vacuolar myelopathy and alters oligodendrocyte phenotype in vitro

Vacuolar myelopathy (VM) is a frequent central nervous system complication of human immunodeficiency virus type 1 (HIV-1) infection. We report here that transgenic (Tg) mice expressing even low levels of Nef in oligodendrocytes under the regulation of the myelin basic protein (MBP) promoter (MBP/HIV(Nef)) developed VM similar to the human disease in its appearance and topography. The spinal cords of these Tg mice showed lower levels of the myelin proteins MAG and CNPase and of the 21-kDa isoform of MBP prior to the development of vacuoles. In addition, Tg oligodendrocytes in primary in vitro cultures appeared morphologically more mature but, paradoxically, exhibited a less mature phenotype based on O4, O1, CNPase, and MBP staining. In particular, mature CNPase(+) MBP(+) Tg oligodendrocytes were less numerous than non-Tg oligodendrocytes. Therefore, Nef appears to affect the proper differentiation of oligodendrocytes. These data suggest that even low levels of Nef expression in human oligodendrocytes may be responsible for the development of VM in HIV-1-infected individuals.     

Developmental expression of the myelin gene MOBP in the rat nervous system

The myelin-associated/oligodendrocyte basic proteins (MOBPs) are recently discovered constituents of myelin and are small, cytoplasmic, and highly basic proteins exclusively expressed postnatally by oligodendrocytes. Due to a clustering of positively charged amino acids observed in the most abundant MOBP isoform similar to myelin basic protein (MBP) and P0, it was speculated that MOBP could function in myelin sheath compaction. The present report strongly supports this view. A direct comparison of MBP and proteolipid protein (PLP) gene expression with that of MOBP by in situ hybridization revealed a very similar regional distribution. It was found that MOBP expression was abundant in the rat CNS at postnatal day 15 (P 15) but is restricted to densely myelinated regions. In contrast to MBP and PLP, expression of MOBP was undetectable in the peripheral nervous system during the entire development. Interestingly, MOBP mRNA was localized in oligodendrocyte processes even at early postnatal stages and throughout development. MOBP showed a very specific timing of expression: in spinal cord and brain, MOBP gene expression occurred significantly later (2–3 days) than that of MBP and PLP, but slightly earlier than myelin oligodendrocyte glycoprotein gene expression. MOBP proteins appeared in spinal cord and brain stem also after MBP protein, suggesting that the MOBPs functionally act after the structural myelin proteins MBP and PLP. Our findings imply a function of MOBP during the late steps of myelin formation, presumably at the initiation of sheath compaction.     

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms

The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-II, although their functional expression is often less clear. Here, we investigated the role of exon-II-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-II-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage.     

Characterization of Myelin Basic Protein Charge Microheterogeneity in Developing Mouse Brain and in the Transgenic Shiverer Mutant

Abstract: Myelin basic protein (MBP) is a highly heterogeneous family of membrane proteins consisting of several isoforms resulting from alternative splicing and charge isomers arising from posttranslational modifications. Although well characterized in the bovine and human species, those in the mouse are not. With the availability of a number of transgenic and knockout mice, the need to understand the chemical nature of the MBPs has become very important. To isolate and characterize the MBP species in murine brain, two methods were adapted for use with the small amounts of MBP available from mice. The first was a scaled-down version of the preparative CM-52 chromatographic system commonly used to isolate MBP charge isomers; the second was an alkaline-urea slab gel technique that required five times less material than the conventional tube gel system and, from these gels, western blots were readily obtained. Murine MBP was resolved into two populations of charge isomers: the 18.5- and 14-kDa isoforms. Isolation and characterization of these charge isomers or components permitted us to assign possible posttranslational modifications to some of them. Component 1 (C-1), the most cationic isomer, had a molecular weight of 14,140.38 ± 0.79. C-2 consisted of two 14-kDa species, 14,136.37 ± 0.74 and 14,204.45 ± 0.70. Two variants, 14,215.57 ± 0.94 and 18,413.57 ± 0.76, constituted C-3. C-4, C-5, and C-8 (the least cationic isomer) each consisted of both 14- and 18.5-kDa isoforms. During myelinogenesis, the 18.5-kDa isoform appeared first (day 4); the 14-kDa isoform appeared at day 16 and subsequently became the dominant isoform. The transgenic shiverer mutant synthesized mainly the 18.5-kDa isoform, but none of the 14-kDa isoform, similar to the 4-day-old mouse. We concluded that the trangenic shiverer was able to initiate myelinogenesis with the 18.5-kDa isoform, but was unable to complete myelinogenesis because of the absence of the 14-kDa isoform.     

Ganglioside-modulated protein phosphorylation in myelin

Gangliosides have profound effects on the phosphorylation of several proteins in myelin. Addition of polysialogangliosides to purified guinea pig brain myelin enhanced the endogenous phosphorylation of a 62-kDa phosphoprotein, but completely inhibited the phosphorylation of myelin basic protein (MBP) (18.5 kDa). The ganglioside-stimulated phosphorylation of the 62-kDa protein was dose-dependent and -specific. Asialo-GM1, ceramide trihexosides, N-acetylneuraminic acid, or colominic acid alone could not mimic this effect, suggesting that the activation process requires both the hydrophobic head group and the anionic character of the gangliosides. Studies on the time course of this reaction revealed that it was a rapid and reversible process and was affected only very slightly by Ca2+. Thus, the stimulatory effect of gangliosides may not involve Ca2+-gangliosides complexes or proteolysis, but may be mediated through an activation of a ganglioside-dependent protein kinase or due to substrate protein-glycolipid interaction. Modulation of the phosphorylation of MBP by gangliosides varies with the states of phosphorylation of this protein. Prior addition of ganglioside to myelin inhibited the phosphorylation of MBP. However, addition of gangliosides to myelin subsequent to maximal phosphorylation of MBP retarded the dephosphorylation of this protein. Phosphorylation of isolated MBP by protein kinase C was stimulated by gangliosides, provided phosphatidylserine was present. In contrast, the glycolipid inhibited the phosphorylation of a unique site catalyzed by cAMP-dependent protein kinase. This site was distinct from those phosphorylated by protein kinase C and was also sensitive to chymotryptic cleavage. Although the exact physiological significance of protein phosphorylation in myelin has yet to be established, gangliosides may play an important role in the modulation of this reversible post-translational modification mechanism.     

Structured functional domains of myelin basic protein: cross talk between actin polymerization and Ca(2+)-dependent calmodulin interaction

The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca(2+)-activated calmodulin (Ca(2+)-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22-K56), (S72-S107), and (S133-S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca(2+)-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca(2+)-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca(2+)-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca(2+)-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.     

Lateral self-assembly of 18.5-kDa myelin basic protein (MBP) charge component-C1 on membranes

Myelin basic protein (MBP), particularly the classic 18.5-kDa isoform, is a major structural protein of the myelin sheath of the central nervous system. It is an intrinsically disordered, peripheral membrane protein that shows structural polymorphism in combination with several overlapping interaction sites. Here, double electron-electron resonance (DEER) spectroscopy, in combination with a simplified, semi-quantitative analysis based on Monte Carlo simulations, is used to determine the distance distribution of murine 18.5-kDa MBP, unmodified charge component-C1, on large unilamellar vesicles of a lipid composition mimicking the cytoplasmic leaflet of myelin. Three singly spin-labeled MBP variants and a mixture of singly-labeled MBP variants are used. The MBPs, each bearing only one spin label, exhibit average intermolecular distances that are significantly shorter than the distances expected when assuming a random distribution at the employed lipid-to-protein ratios, indicating self-assembly on the membrane. The distribution of elliptical pervaded areas (hard ellipses) on a two-dimensional surface can serve as a model of the nonspecific self-assembly process. The corresponding pair correlation functions g(r) are determined from Monte Carlo simulations with variation of various parameters such as the ellipses' aspect ratios. Comparing the g(r) values with the DEER-derived distance distributions, the pervaded volume is best characterized by a nearly elliptical projection onto the membrane, with an aspect ratio of approximately 1.5, and with the longer semi-axis of approximately 1.4nm. The approach of using local information from DEER with low-resolution models derived from Monte Carlo simulations can be applied to study the lateral self-assembly properties of other protein complexes on membranes.     

Metalloendoprotease Cleavage of 18.2- and 14.1-Kilodalton Basic Proteins Dissociating from Rodent Myelin Membranes Generates 10.0- and 5.9-Kilodalton G-Terminal Fragments

Rat and guinea pig myelin membranes were incubated at physiological ionic strength with millimolar concentrations of Ca2+/Mg2+ ions (37 degrees C; pH 7.4). After 1-3 h, electrophoresis of the membranes revealed loss of 50% of 18.2- and 14.1-kilodalton (kDa) forms of myelin basic protein (MBP). Concomitantly, peptides representing 25% of the original membrane-associated MBP were detected in incubation media. Roughly equal amounts of MBP fragments with molecular masses of 10.0 and 8.4 kDa were found in media from guinea pig myelin incubations. Media from rat myelin experiments contained a major 8.4-kDa and minor 10.0- and 5.9-kDa MBP peptides. Kinetic studies implied that proteolysis occurred subsequent to MBP dissociation from the membranes. Immunoblotting studies indicated that both the 18.2- and 14.1-kDa forms of MBP were cleaved near residue 73 to produce a 10.0- and 5.9-kDa C-terminal fragment, respectively. Degradation of MBP in myelin membranes was partially inhibited by only 5-20% using leupeptin (20 microM) but up to 50% by dithiothreitol mM), phenylmethylsulphonyl fluoride (1 mM), and phosphoramidon (50 microM) but up to 50% by dithiothreitol (DDT, 10 mM). Only DDT and 1,10-phenanthroline substantially blocked the formation of the characteristic 10.0-and 5.9-kDa C-terminal fragments. This suggests that MBP, dissociating from myelin membrane preparations, is cleaved near residue 73 by a metalloendoprotease distinct from N-ethylmaleimide/leupeptin-sensitive calpains and phosphoramidon-sensitive endopeptidase 24.11.     

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.     

The Lateral Membrane Organization and Dynamics of Myelin Proteins PLP and MBP Are Dictated by Distinct Galactolipids and the Extracellular Matrix

In the central nervous system, lipid-protein interactions are pivotal for myelin maintenance, as these interactions regulate protein transport to the myelin membrane as well as the molecular organization within the sheath. To improve our understanding of the fundamental properties of myelin, we focused here on the lateral membrane organization and dynamics of peripheral membrane protein 18.5-kDa myelin basic protein (MBP) and transmembrane protein proteolipid protein (PLP) as a function of the typical myelin lipids galactosylceramide (GalC), and sulfatide, and exogenous factors such as the extracellular matrix proteins laminin-2 and fibronectin, employing an oligodendrocyte cell line, selectively expressing the desired galactolipids. The dynamics of MBP were monitored by z-scan point fluorescence correlation spectroscopy (FCS) and raster image correlation spectroscopy (RICS), while PLP dynamics in living cells were investigated by circular scanning FCS. The data revealed that on an inert substrate the diffusion rate of 18.5-kDa MBP increased in GalC-expressing cells, while the diffusion coefficient of PLP was decreased in sulfatide-containing cells. Similarly, when cells were grown on myelination-promoting laminin-2, the lateral diffusion coefficient of PLP was decreased in sulfatide-containing cells. In contrast, PLP''s diffusion rate increased substantially when these cells were grown on myelination-inhibiting fibronectin. Additional biochemical analyses revealed that the observed differences in lateral diffusion coefficients of both proteins can be explained by differences in their biophysical, i.e., galactolipid environment, specifically with regard to their association with lipid rafts. Given the persistence of pathological fibronectin aggregates in multiple sclerosis lesions, this fundamental insight into the nature and dynamics of lipid-protein interactions will be instrumental in developing myelin regenerative strategies.     

Marburg's Variant of Multiple Sclerosis Correlates with a Less Compact Structure of Myelin Basic Protein

Multiple sclerosis (MS) is an autoimmune disease in which the myelin sheath of the central nervous system is degraded, and the 18.5 kDa isoform of myelin basic protein (MBP) is reduced in cationicity. In a unique case of acute, fulminating MS (Marburg's variant), MBP is considerably less cationic than MBP from both normal, and chronic MS-afflicted individuals. This electron microscopical study has identified that,in vitro,the less cationic Marburg MBP isomer forms a more extended protein-lipid complex than MBP from healthy or chronic MS-afflicted individuals. This correlation implies that chemical modifications to MBPin vivocontribute directly to the structural instability of myelin, and subsequent autoantigenic presentation of this protein, observedin vivoin MS.