Expression of Myelin Proteolipid Protein and Basic Protein in Normal and Dysmyelinating Mutant Mice

Expression of myelin proteins was studied in the brains of 21-day-old normal mice and three dysmyelinating mutants-jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell-free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21-day-old quaking brain homogenates showed a reduction in the steady-state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21-day jimpy brain homogenates revealed no appreciable steady-state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.     

Expression of Myelin Basic Protein Genes in Several Dysmyelinating Mouse Mutants During Early Postnatal Brain Development

Northern blot and "dot" blot analyses using a myelin basic protein (MBP) specific cDNA probe and in vitro translation techniques were utilized to estimate the relative levels of myelin basic protein messenger RNA (mRNA) in the brains of C57BL/6J control mice, three dysmyelinating mutants (qk/qk, jp/Y, and shi/shi), and three heterozygote controls (qk/+, jp/+, and shi+) during early postnatal development. In general, the MBP mRNA levels measured directly by Northern blot and "dot" blot analyses correlated well with the indirect in vitro translation measurements. The Northern blots indicated that the size of MBP mRNAs in quaking and jimpy brain polysomes appeared to be similar to controls. Very low levels of MBP mRNAs were observed in shi/shi brain polyribosomes throughout early postnatal development. Compared to C57BL/6J controls, accumulation of MBP mRNAs in qk/qk and qk/+ brain polyribosomes was delayed by several days. That is, whereas MBP mRNA levels were below normal between 12 and 18 days, normal levels of message had accumulated in both qk/qk and qk/+ brain polyribosomes by 21 days. Furthermore, normal levels of MBP mRNAs were observed to be maintained until at least 27 days. MBP mRNA levels remained well below control levels in jp/Y brain polyribosomes throughout early postnatal development. The levels did, however, fluctuate slightly and peaked at 15 days in both jp/Y and jp/+ brains, 3 days earlier than in normal mice. Thus, it appears that jimpy and quaking mice exhibit developmental patterns of MBP expression different from each other and from C57BL/6J control mice.     

Developmental Expression of the Myelin Proteolipid Protein and Basic Protein mRNAs in Normal and Dysmyelinating Mutant Mice

Expression of the myelin proteolipid protein (PLP) was examined in the nuclei and polysomes of 12-27-day-old quaking, jimpy, and shiverer mouse brains and in 2-27-day-old normal brains and compared with expression of the myelin basic proteins (MBPs). Northern blots showed the presence of multiple mouse PLP RNAs, the developmental expression of which coincided with myelination. Two major mouse PLP RNAs, 3.5 and 2.6 kilobases in length, were observed in both cytoplasmic polyribosomes and nuclei, and, in addition, a larger 4.6-kilobase PLP RNA was observed in nuclei. Quantitative measurements with slot blot analyses showed that the levels of PLP and MBP RNAs peaked simultaneously at 18 days in nuclei but that maximal levels of PLP RNA lagged behind MBP RNA by several days in the polysomes. The developmental expression of both major classes of myelin protein mRNAs was affected in all three mutants. In shiverer brains, the levels of PLP mRNA in polysomes and nuclei were only 30-55% of control levels after 15 days. Thus, the deletion of a portion of the MBP gene appeared to have a major effect on the expression of the PLP gene in this mutant. In jimpy mice, where the mutation has been shown to involve the PLP gene, expression of MBP mRNA was also severely reduced, to less than 25% of control values. In quaking brains, the expression of each gene followed its own developmental course, different from each other and different from the normal mouse. The extent to which the expression of PLP and MBP was affected by the quaking mutation depended on the age at which it was examined.     

Abnormal Expression of the Myelin-Associated Glycoprotein in the Central Nervous System of Dysmyelinating Mutant Mice

Total cytoplasmic brain RNA was isolated at two different ages from three neurological mutant mice (qk/qk, jp/Y, and shi/shi) and their apparently normal littermates. This RNA was translated in vitro in a rabbit reticulocyte lysate system. Myelin-associated glycoprotein (MAG)-related polypeptides were immunoprecipitated from equal amounts of total translation products derived from mRNA of mutant animals, normal littermates, or control animals. The developmentally regulated synthesis of MAG polypeptides was compared among the mutants and normal animals. mRNA from qk/qk brains synthesized an overabundance of p67MAG (five- to sevenfold) which may be compensation for a decreased synthesis of p72MAG. mRNA from jp/Y brains synthesized less than 10% of normal amounts of both MAG polypeptides. The quantity of MAG synthesized by 15-day shi/shi brain mRNA was slightly decreased compared with normal brain mRNA but the quantity of MAG synthesized by adult shi/shi brain mRNA was normal. No apparent differences were detected in the sizes of the MAG polypeptides synthesized by any of the mutants studied. The data suggest that the genetic defect in qk/qk mutants directly or indirectly affects the coordinated developmental regulation of MAG polypeptide synthesis leading to an overabundance of the MAG polypeptide that is normally found in older animals. The jp/Y mutation appears to affect general myelin protein synthesis. Finally, shi/shi mutants may have a delayed synthesis of MAG. The data are discussed in the light of recent observations concerning the synthesis of myelin proteins and their proposed role in myelin assembly.     

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.     

Morphometric Analysis of Normal, Mutant, and Transgenic CNS: Correlation of Myelin Basic Protein Expression to Myelinogenesis

The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoblot Identification of 13.5 Kilodalton Myelin Basic Protein in Goldfish Brain Myelin

Myelin isolated from goldfish brain shows a multilamellar structure with a major dense line and two intraperiod lines. Sodium dodecyl sulfate gel electrophoresis revealed that the protein profile of goldfish brain myelin is distinctly different from that of rat brain myelin. No protein migrating to the position of proteolipid protein or DM-20 was seen in goldfish myelin. Goldfish acclimated to 5 degrees, 15 degrees, and 30 degrees C showed no qualitative differences in myelin proteins. The 13.5 kD protein in goldfish brain myelin and brain homogenate was intensely immunostained with the antiserum to human basic protein by the immunoblot technique. In contrast, none of the proteins of goldfish myelin were immunostained with antiproteolipid protein serum; however, both proteolipid protein and DM-20 of rat brain myelin were immunostained. The significance of the synthesis of myelin proteins by astrocytes in the goldfish brain is discussed.     

Endogenous Basic Protein Phosphatases in the Brain Myelin

Direct treatment of brain myelin with freezing/thawing in 0.2 M 2-mercaptoethanol stimulated the endogenous myelin phosphatase activity manyfold when 32P-labeled phosphorylase a was used as a substrate, a result indicating that an endogenous myelin phosphatase is a latent protein phosphatase. When myelin was treated with Triton X-100, this endogenous latent phosphatase activity was further stimulated 2.5-fold. Diethylaminoethyl-cellulose and Sephadex G-200 chromatography of solubilized myelin revealed a pronounced peak of protein phosphatase activity stimulated by freezing/thawing in 0.2 M 2-mercaptoethanol and with a molecular weight of 350,000, which is characteristic of latent phosphatase 2, as previously reported. Moreover, endogenous phosphorylation of myelin basic protein (MBP) in brain myelin was completely reversed by a homogeneous preparation of exogenous latent phosphatase 2. By contrast, under the same conditions, endogenous phosphorylation of brain myelin was entirely unaffected by ATP X Mg-dependent phosphatase and latent phosphatase 1, although both enzymes are potent MBP phosphatases. Together, these findings clearly indicate that a high-molecular-weight latent phosphatase, termed latent phosphatase 2, is the most predominant phosphatase responsible for dephosphorylation of brain myelin.     

ADP-Ribosylation of Human Myelin Basic Protein

Abstract: When isolated myelin membranes were ADP-ribosylated by [³²P]NAD⁺ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg⁹ and Arg⁵⁴ by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.     

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.     

Rearrangement and Reactivation of the Myelin Basic Protein Locus in Myelin-Deficient (mld) Mouse Brain

Abstract: Myelin-deficient ( mld ) is a complex mutation affecting the myelin basic protein (MBP) locus of the mouse. It consists of duplication and partial inversion of the MBP gene and results in a dysfunctional MBP locus. The mutant phenotype is reversed, both in vivo and in vitro, in ∼5% of mld oligodendrocytes. One possible mechanism for the somatic reversion is recombination between homologous sequences of the duplicated gene copies to reconstitute a functional MBP locus. There are several possible recombination events that could reconstitute a functional MBP locus by DNA rearrangement. Two of these would result in reinversion and circularization of specific MBP gene sequences, respectively. In this work polymerase chain reaction analysis was used to detect both reinverted and circularized MBP gene sequences in mld mouse tissues, indicating that DNA rearrangement at the MBP locus does occur. Analysis of individually harvested cells showed that in revertant MBP-positive mld oligodendrocytes DNA rearrangement at the MBP locus was correlated with reactivation of the MBP gene. Fluctuation analysis showed that reactivation of the MBP locus is a stochastic event occurring with a frequency of ∼1.4 × 10⁻⁶ per cell per cell cycle during oligodendrocyte development. The frequency of rearrangement and reactivation of the MBP locus was comparable in double mutant ( mld/mld , scid/scid ) and single mutant ( mld/mld , + ^scid /+ ^scid ) mice, indicating that the scid factor is not required for MBP gene reactivation in mld . The significance of DNA rearrangement in mammalian development is discussed.     

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.     

Preparation and Properties of an Immunosorbent Column Specific for the Myelin Basic Protein

Abstract: An immunosorbent column specific for the myelin basic protein (BP) was prepared by coupling purified anti-BP antibodies to cyanogen bromide (BrCN)-activated Sepharose 4B. The BP-immunosorbent column bound BP between pH 4.5 and pH 6.8. In its working range the column bound approximately 400-475 μg of BP at pH 6.8 and 250 μg at pH 4.5 with recoveries of 72-77%. The BP-immunosorbent column could effectively separate BP from simple mixtures of BP and proteins of similar size and charge and from acid extracts of bovine brain. The results indicate that the BP-immunosorbent column can be used to isolate BP from a mixture of proteins and may be adapted for use in the small-scale purification of the myelin basic proteins involving a minimum number of steps.     

A Rapid Method for Purification of Myelin Basic Protein

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.