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.     

A new procedure for the isolation of the brain myelin basic protein in a lipid-bound form

Myelin basic protein has been isolated from bovine brain using the nonionic detergent n-octyl-polydisperse oligooxyethylene. The purified basic protein contains large amounts of heterogeneous lipids.     

Phosphatidylinositol-3 kinase p85 enhances expression from the myelin basic protein promoter in oligodendrocytes

Phosphatidylinositol-3 kinase (PI3K) is a family of enzymes that phosphorylates the D3 position of phosphoinositides in membranes which can then act as a second messenger and affect many essential cellular processes such as survival, proliferation and differentiation. Class IA PI3K is composed of two subunits: a regulatory subunit, p85, and a catalytic subunit, p110. The p85 subunit is composed of several adapter domains which, upon interaction with the appropriate molecules, transmit the signal to activate p110. We have used the spontaneously immortalized oligodendrocyte cell line, CG4, to examine the role of PI3K in maturation of the oligodendrocyte. We show that overexpression of the p85 subunit enhances expression of myelin basic protein (MBP) upon differentiation of CG4 cells and primary oligodendrocytes. In experiments in CG4 cells, neither cotransfection with the tumor suppressor PTEN, which dephosphorylates the D3 position of phosphoinositides, nor inhibition of PI3K activity with wortmannin mimics this effect. Further, we have shown that this effect is dependent on the coexpression of the two SH2 domains within p85. Thus, the p85-mediated enhancement of MBP promoter activity in oligodendrocytes appears to be independent of PI3K activity and dependent on the adapter functions of the p85 subunit's SH2 domains.     

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.     

Degradation of myelin basic protein by a membrane-associated metalloprotease: Neural distribution of the enzyme

A metalloprotease activity associated with myelin membrane preparations degrades myelin basic protein (MBP), generating a characteristic fragment designated peptide C (MBP 74-170). Using an immunoblotting assay, peptide C-generating activity was detected in mammalian, avian, reptilian, and amphibian brains. The activity was present in rat brain as early as postnatal day 1 and also in adult rat peripheral nerve. Immunohistochemistry with a monoclonal antibody to the purified enzyme revealed that the metalloprotease was present in oligodendrocytes of optic nerve, of both white and grey matter of spinal cord, and also in the cytoplasm of both myelinating and non-myelinating Schwann cells of peripheral nerve.Special issue dedicated to Dr. Alan N. Davison     

Role of phosphorylation in conformational adaptability of bovine myelin basic protein.

Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated form(s) of component 1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phosphorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in beta-structure(s).     

Analogous standard motifs in myelin basic protein and in MARCKS

Myelin basic protein (MBP) and myristoylated alanine-rich C-kinase substrate (MARCKS) are similar in terms of having extended conformations regulated by their environment (i.e., solubilised or lipid-associated), N-terminal modifications, a dual nature of interactions with lipids, binding to actin and Ca²⁺-calmodulin, and being substrates for different kinds of protein kinases. The further sequence similarities of segments of MBP with lipid effector regions of MARCKS, and numerous reports in the literature, support the thesis that some developmental isoform of MBP functions in signal transduction.     

Phosphorylation of myelin basic protein by glycogen phosphorylase kinase

The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases.     

Isolation and characterization of two novel peptide amides originating from myelin basic protein in bovine brain

During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-amide and Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Ser-Ala-amide. These 12 and 16 residues peptides had the primary structure identical to the N-terminal fragment of myelin basic protein (MBP). The peptides were therefore designated myelin peptide amide-12 (MPA-12) and-16 (MPA-16). Unlike other amidated peptides, MPA might be generated from MBP by hydroxyl radicals produced via a Fenton reaction in situ. However, this unique amidation seems to occur exclusively to MBP in a site specific manner in the brain.     

Antisense oligonucleotide to the 70-kDa heat shock cognate protein inhibits synthesis of myelin basic protein

Transfection of rat oligodendrocytes with an oligonucleotide sequence complementary to the mRNA encoding the initial ten amino acids of the rat 70-kDa heat shock cognate protein (HSC70) resulted in a rapid (within 24 h) and significant reduction in HSC70 synthesis (69% of control cells transfected with sense oligonucleotide). A further decrease to approximately 44% of controls was detected after 2 days. At that time, HSC70 protein content fell to approximately 49% of controls, and a significant reduction in the synthesis of myelin basic protein (MBP) was first detected (66% of controls). After 5 days, HSC70 synthesis returned to control levels. As HSC70 protein content recovered, so did the synthesis of MBP. Throughout the 5-day experimental period, only minor changes were detected in cell morphology, overall pattern of protein synthesis and the synthesis and content of proteolipid protein (PLP) and the pi isoenzyme of glutathione-S-transferase (pi). These data show that when HSC70 protein content is sufficiently reduced by antisense oligonucleotide, synthesis of MBP (but not PLP or pi) is correspondingly down-regulated, and provide evidence consistent with the role of HSC70 as a chaperone for MBP. Special issue dedicated to Dr. Marion E. Smith.     

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype. © 1996 John Wiley & Sons, Inc.     

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.     

Proteolysis of multiple myelin basic protein isoforms after neurotrauma: characterization by mass spectrometry

Neurotrauma, as in the case of traumatic brain injury, promotes protease over-activation characterized by the select fragmentation of brain proteins. The resulting polypeptides are indicators of biochemical processes, which can be used to study post-injury dynamics and may also be developed into biomarkers. To this end, we devised a novel mass spectrometry approach to characterize post-injury calpain proteolytic processing of myelin basic protein (MBP), a biomarker of brain injury that denotes white matter damage and recovery. Our approach exceeds conventional immunological assays in its deconvolution of multiple protein isoforms, its absolute quantification of proteolytic fragments and its polypeptide selectivity. We quantified and characterized post-injury proteolytic processing of all MBP isoforms identified in adult rat cortex. Further, the translation of calpain-cleaved MBP into CSF was verified following brain injury. We ascertained that the exon-6 sequence of MBP resulted in a characteristic shift in gel migration for intact and fragmented protein alike. We also found evidence for a second post-TBI cleavage event within exon-2 and for the dimerization of the post-TBI 4.3 kDa fragment. Ultimately, the novel methodology described here can be used to study MBP dynamics and other similar proteolytic events of relevance to brain injury and other CNS processes.     

Accumulation of galactosylsphingosine (psychosine) does not interfere with phosphorylation and methylation of myelin basic protein in the twitcher mouse

In attempts to elucidate mechanisms of demyelination in the twitcher mouse (Twi), phosphorylation and methylation of myelin basic protein (MBP) were examined in the brainstem and spinal cord of this species. Phosphorylation of MBP in isolated myelin by an endogenous kinase and an exogenous [³²P]ATP was not impaired and protein kinase C activity in the brain cytosol was not reduced. When the methylation of an arginine residue of MBP was examined in slices of the brainstem and spinal cord, using [³H]methionine as a donor of the methyl groups, no difference was found between Twi and the controls. Radioactivity of the [³H] methionine residue of MBP of Twi was also similar to that of the controls. Thus, accumulation of psychosine in Twi does not interfere with the activity of endogenous kinase, methylation of MBP, and the synthesis and transport of MBP into myelin membrane.     

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.     

Regulation of neuronal nitric oxide synthase by histone,protamine, and myelin basic protein

We examined the effects of endogenous basic proteins rich in the amino acidL-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca²⁺ gradient by a Ca²⁺ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influex of extracellular Ca²⁺ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca²⁺ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism ofL-arginine intoL-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.