Distribution of Myelin Basic Protein and P2 mRNAs in Rabbit Spinal Cord Oligodendrocytes

Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a "receptor" molecule, perhaps a membrane-anchored protein, for association with the cytoplasmic face of the myelin membrane.     

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.     

Phosphorylation of P0 Glycoprotein in Peripheral Nerve Myelin

The P0 protein in mammalian PNS myelin is known to undergo several posttranslational modifications, such as glycosylation, acylation, sulfation, and phosphorylation. Phosphorylation of purified P0 protein in vitro was studied comparatively using three enzymes, i.e., calcium/phospholipid-dependent protein kinase (protein kinase C), calcium/calmodulin-dependent protein kinase II (CaM kinase II), and the catalytic subunit of cyclic AMP-dependent protein kinase (A kinase). The phosphorylation of P0 protein by CaM kinase II was the greatest, followed by that by protein kinase C; phosphorylation by A kinase, however, was much lower. In order to identify phosphorylation sites, P0 protein was phosphorylated with [32P]ATP and each kinase and then digested with lysylendopeptidase. The resulting phosphopeptides were isolated by HPLC. Subsequent amino acid sequence analysis and comparison with the known sequence of P0 protein revealed that Ser181 and Ser204 were strongly phosphorylated by both protein kinase C and CaM kinase II. In addition, Ser214 was also phosphorylated by protein kinase C, but not by CaM kinase II. Because all of these sites are located in the cytoplasmic domain of P0 protein, phosphorylation may be important for maintenance of the major dense line of PNS myelin.     

Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

Age-Dependent Changes in the Oligosaccharide Structure of the Major Myelin Glycoprotein, P0

The single oligosaccharide moiety of the major myelin glycoprotein, P0, resides in an immunoglobulin-like domain that appears to participate in homophilic binding. The studies presented here indicate that the structure of the P0 oligosaccharide from rat nerve changes as a function of Schwann cell age. Examination of 5-day-old nerve revealed that P0 contained predominantly endo-beta-N-acetylglucosaminidase H (endo H)-resistant, complex-type oligosaccharide. In contrast, P0 from adult rats had mostly endo H-sensitive carbohydrate, indicating the presence of appreciable high-mannose and/or hybrid-type oligosaccharide on the glycoprotein. The endo H-sensitive and -resistant P0 of adult nerve could be readily phosphorylated by protein kinase C, as could the complex-type P0 from 5-day-old nerve. This suggests that the glycoprotein progresses to the plasma membrane and myelin regardless of the type of oligosaccharide chain. Analysis of 35SO4(2-)-labeled P0 showed that the sulfate group was found on both endo H-sensitive and -resistant oligosaccharide. The endo H-sensitive P0 carbohydrate from adult nerve appears to be primarily of the hybrid type, as evidenced by (a) the elution profile of [3H]mannose-labeled P0 glycopeptides from adult nerve during concanavalin A chromatography and (b) the inability of P0 from adult nerve to interact with Galanthus nivalis agglutinin. The observed age-dependent changes of P0 oligosaccharide may modify the binding properties of this myelin glycoprotein.     

Identification of the Palmitoylation Site in Rat Myelin P0 Glycoprotein

Abstract: P₀ glycoprotein, the major protein of PNS myelin, contains approximately 1 mol of covalently bound long-chain fatty acids. To determine the chemical nature of the fatty acid-protein linkage, P₀ was labeled in rat sciatic nerve slices with [³H]palmitic acid and subsequently treated with various reagents. The protein-bound palmi-tate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydrox-ylamine at pH 7.5. In addition, P₀ was deacylated by treatment with 10 m M NaBH₄ with the concomitant production of [³H]hexadecanol, indicating that the fatty acid is bound in a thioester linkage. This conclusion was supported further by the fact that deacylation with hydroxylamine generated free thiol groups, which were titrated with [¹⁴C]-iodoacetamide. To identify the cysteine residue involved in the thioester linkage, [¹⁴C]carboxyamidomethylated P₀was digested with trypsin and the resulting peptides analyzed by reversed-phase HPLC. Identification of the radioactive protein fragments by amino acid analysis and amino-terminal peptide sequencing revealed that Cys¹⁵³ in rat P₀ glycoprotein is the acylation site. The acylated cysteine is located at the junction of the putative transmem-brane and cytoplasmic domains. This residue is also present in the P₀ glycoprotein of other species, including human, bovine, mice, and chicken.     

A Phorbol Ester-Sensitive Kinase Catalyzes the Phosphorylation of P0 Glycoprotein in Myelin

The proposed structural protein of peripheral nerve myelin, P0, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P0 is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P0 biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P0 phosphorylation. It is demonstrated that there is comparable P0 phosphorylation in both normal and crush-injured adult rat sciatic nerves, although the level of biosynthesis of P0 differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P0 biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P0 in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P0 phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P0 is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse-chase analysis. Addition of a biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate or 4 beta-phorbol 12,13-dibutyrate, substantially increases the extent of [32P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4 alpha-phorbol 12,13-didecanoate has no effect on P0 phosphorylation. Similarly, the addition of the cyclic AMP analog 8-bromo-cyclic AMP causes no appreciable changes in P0 labeling. These findings indicate that the phorbol ester-sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P0 within the myelin membrane.     

Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [³²P]PO₄³⁻ from [γ-³²P]ATP into protein constituents. Among these, P₀ glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P₀ protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two ³²P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P₀ sequence revealed, besides a Lys¹⁷² to Arg substitution, that in the first peptide, two serine residues (Ser¹⁷⁶ and Ser¹⁸¹) were phosphorylated, Ser¹⁷⁶ appearing to be modified subsequently to Ser¹⁸¹. In the second peptide, Ser¹⁹⁷, Ser¹⁹⁹, and Ser²⁰⁴ were phosphorylated. All these serines are clustered in the C-terminal region of P₀ protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P₀. We found that, in vivo, Ser¹⁸¹ and Ser¹⁷⁶ are not phosphorylated, whereas Ser¹⁹⁷, Ser¹⁹⁹, Ser²⁰⁴, Ser²⁰⁸, and Ser²¹⁴ are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.     

Myelin P0 Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated

Abstract: P₀, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys¹⁵³. To gain insights into the mechanism of P₀ acylation, the in vitro palmitoylation of both P₀ and a synthetic Cys¹⁵³-containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P₀ with [³H]palmitoyl-CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P₀ was not affected by boiling the isolated P₀ for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonezymatic nature of the reaction. After chemical deacylation, P₀ was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [¹⁴C]palmitoyl-CoA, the synthetic peptide encompassing the natural P₀ acylation site (I¹⁵⁰RYCWLRR¹⁵⁷) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37°C, peptide palmitoylation followed a second-order reaction (k₂ = 246 ± 6 M^?1 min^?1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl-CoA. The activation energy calculated from the Arrhenius plot is ～2 kcal/mol and much lower than that of enzyme-catalyzed transacylations. Finally, two other P₀ peptides (V¹²¹PTRYG¹²⁶ and K¹⁰⁹TSQVTL¹¹⁵) as well as various unrelated thiol-containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.     

Formation of a Disulfide Bond in the Immunoglobulin Domain of the Myelin P0 Protein Is Essential for Its Adhesion

Abstract: It is widely accepted, although never demonstrated, that the formation of a disulfide bond in the majority of immunoglobulin (Ig)-like domains stabilizes their final conformation and thus is essential to their functioning as adhesion/recognition molecules. The myelin P₀ protein, which has been shown directly to behave as a homophilic adhesion molecule, contains a single Ig-like domain, stabilized by a putative Cys²¹-Cys⁹⁸ disulfide bond. To test if this bond is indeed necessary to the adhesive function of P₀, the nucleotides in the P₀ cDNA coding for Cys²¹ were altered to code for an alanine. The mutated P₀ cDNA was transfected into Chinese hamster ovary cells, expression of the mutated P₀ protein was characterized, and the adhesiveness of Cys²¹-mutated P₀-expressing cells and that of cells expressing equivalent surface amounts of the unmutated protein were compared. It was found, as we previously reported, that incubation of a single cell suspension of the unmutated P₀-expressing cells resulted in the rapid formation of large aggregates. In contrast, after a similar incubation the cells expressing the Cys²¹-mutated P₀ were still mostly single cells, a result indistinguishable from that observed with the control transfected cells. This suggests that the P₀ protein, when mutated at Cys²¹, does not behave as a homophilic adhesion molecule, which in turn implies that the formation of an Ig domain disulfide bond is essential to the functioning of this molecule.     

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.     

Bovine P2 Protein: Sequence at the NH2-Terminal of the Protein

Sequence data from key fragments of the P2 protein established the order of cyanogen bromide (CNBr) peptides in the structure of the protein and the primary structure for approximately one-half of the molecule. Data were obtained from the three tryptic peptides of blocked NH2-terminal CNBr peptide (CN3), the large CNBr peptide of P2 protein (CN1), and a fragment obtained from P2 by cleavage at tryptophan with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. This last fragment was found to contain an over-lapping sequence that proved the juxtaposition of CN1 and CN3 in P2 protein. Thus, based on this fact and the characteristics of the CNBr peptides, the P2 structure is composed of CNBr peptides in the order: CN3-CN1-CN2(Val)-CN2(Lys). A comparison was made between the partial sequence of P2 protein and the equivalent portion of the structure of bovine myelin basic protein. The structures of these two proteins were found to be distinctly different although certain similarities are found.     

Partial Structure and Mapping of the Human Myelin P2 Protein Gene

Abstract: The myelin P₂ protein, a 14,800-Da cytosolic protein found primarily in peripheral nerves, belongs to a family of fatty acid binding proteins. Although it is similar in amino acid sequence and tertiary structure to fatty acid binding proteins found in the liver, adipocytes, and intestine, its expression is limited to the nervous system. It is detected only in myelin-producing cells of the central and peripheral nervous systems, i.e., the oligodendrocytes and Schwann cells, respectively. As part of a program to understand the regulation of expression of this gene, to determine its function in myelin-producing cells, and to study its role in peripheral nerve disease, we have isolated and characterized overlapping human genomic clones encoding the P₂ protein. We report here on the partial structure of this gene, and on its localization within the genome. By using a panel of human-hamster somatic cell hybrids and by in situ hybridization, we have mapped the human P₂ gene to segment q21 on the long arm of chromosome 8. This result identifies the myelin P₂ gene as a candidate gene for autosomal recessive Charcot-Marie-Tooth disease type 4A.     

Structure of Bovine P2 Basic Protein: Sequence of a Carboxyterminal Segment That Is a Neuritogen in Rabbits

The sequence of the carboxyterminal 18 amino acids released by cyanogen bromide digestion of the bovine P2 protein was determined. It has several interesting structural and immunological properties. It contains the only two half-cystines in the molecule that have the capacity to form an intrachain disulfide bond. Using the rabbit as a test animal, this carboxyterminal peptide was capable of producing experimental allergic neuritis. The sequence of this peptide is Val-Val-Glu-Cys-Lys-Met-Lys-Asp-Val-Val-Cys-Thr-Arg-Ile-Tyr-Glu-Lys-Val.     

Cell-Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice

Total polyribosomes were isolated from the brains of 16-20 day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shil/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 +/- 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and less than 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an "assembly" or "synthesis" defect.     

Conformations of P2 Protein of Peripheral Nerve Myelin by Nuclear Magnetic Resonance Spectroscopy

High-resolution 1H NMR spectra of P2 protein from bovine peripheral nerve myelin indicate that the protein contains a high degree of tertiary structure in aqueous solution. Denaturation of the protein in urea solutions is a multi-step process. Binding of lysophosphatidylcholine micelles to the protein causes a conformational change and a broadening of NMR peaks from side chains of aromatic amino acid and methionine residues, with much less effect on upfield methyl resonances.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.