P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Developmental Expression of the P0 Glycoprotein and Basic Protein mRNAs in Normal and Trembler Mutant Mice

Mice affected by the autosomal dominant Trembler mutation exhibit a severe hypomyelinization of the PNS. Previous biochemical studies have shown that the accumulation of the major PNS myelin proteins, P0 and myelin basic protein (MBP), is strongly diminished in Trembler sciatic nerves during postnatal development. We performed Northern blots which showed that the size of mRNA species for P0 and MBP in normal and mutant mice are indistinguishable. Densitometric analysis of Northern blots showed that, in normal mice, the proportion of P0 mRNA increases up to the 12th day, then decreases slowly. At day 40, the proportion is 60% of the maximal value. In the mutant, the proportion of P0 mRNA increases up to the 12th day and then decreases much faster than in the control. At days 12 and 40, the P0 mRNA proportion measured in Trembler sciatic nerves represents only 40% and 7%, respectively, of the proportion measured in control littermates. The MBP mRNA proportion in the normal mice increases up to the 16th day, and then decreases to attain 45% of the maximum level at day 40. In the Trembler mouse, there is a maximum level at day 12, representing 25% of the normal level, but the MBP mRNA is barely detectable at days 8 or 40. Thus, these data seem to indicate that in the Trembler sciatic nerves, the proportions of P0 and MBP mRNAs are too small to allow the synthesis of normal levels of the corresponding proteins.     

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.     

Electron Microscopic Immunocytochemical Localization of Myelin Proteolipid Protein and Myelin Basic Protein to Oligodendrocytes in Rat Brain During Myelination

Electron microscopic immunocytochemical studies were carried out to localize myelin basic protein and myelin proteolipid protein during the active period of myelination in the developing rat brain using antisera to purified rat brain myelin proteolipid protein and large basic protein. The anti-large basic protein serum was shown by the immunoblot technique to cross-react with all five forms of basic protein present in the myelin of 8-day-old rat brain. Basic protein was localized diffusely in oligodendrocytes and their processes at very early stages in myelination. The immunostaining for basic protein was not specifically associated with any subcellular structures or organelles. The ultrastructural localization of basic protein suggests that it may be involved in fusion of the cytoplasmic faces of the oligodendrocyte processes during compaction of myelin. Immunoreactivity in the oligodendrocyte and myelin due to proteolipid protein appeared at a later stage of myelination than did that due to basic protein. Staining for proteolipid protein in the oligodendrocyte was restricted to the membranes of the rough endoplasmic reticulum, the Golgi apparatus, and apparent Golgi vesicles. The early, uncompacted periaxonal wrappings of oligodendrocyte processes were well stained with antiserum to large basic protein whereas staining for proteolipid protein was visible only after the compaction of myelin sheaths had begun. Our evidence indicates that basic protein and proteolipid protein are processed differently by the oligodendrocytes with regard to their subcellular localization and their time of appearance in the developing myelin sheath.     

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.     

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.     

Bovine Peripheral Nervous System Myelin P2 Protein: Chemical and Immunological Characterization of the Cyanogen Bromide Peptides

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2-terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM-P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2-terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH2-terminal valine (Val) that was co-eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM-P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3-CN1-CN2(Val)-CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val). To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induced experimental allergic neuritis (EAN) in Lewis rats. The disease-inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.     

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.     

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.     

Phospholipid-Sensitive Ca2+-Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.     

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.     

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.     

Spontaneous and Experimental Neuritis and the Distribution of the Myelin Protein P2 in the Nervous System

The P2 contents of nervous tissues from the human, rabbit, guinea pig, and Lewis rat were measured by radioimmunoassay. The ventral spinal roots contained more P2 than any other tissue. Human dorsal roots and peripheral nerves contained 41-65% of the amount in human ventral roots. Human olfactory and optic nerves and brain contained 1.1-2.7%, spinal cord, 2.8%, cranial nerve VIII, 11%, and cerebral grey matter, 0%. The relative amounts in the rabbit nervous system were similar except that the spinal cord contained 20% of the amount in the ventral roots. Qualitative estimates in the guinea pig showed that the spinal roots and peripheral nerves contained more P2 than the spinal cord, and that none was present in the brain. In the Lewis rat, P2 could be detected in the spinal roots and peripheral nerves but not in the CNS. The distribution of P2 in the human nervous system parallels the incidence and severity of lesions in acute polyradiculoneuritis. It also explains the absence of any lesions in the CNS when experimental allergic neuritis is induced in the Lewis rat.     

Cleavage of Rabbit Myelin Basic Protein by Thrombin

Rabbit myelin basic protein (BP) contains several Arg-X bonds with differing susceptibilities to thrombic cleavage as measured by the yields of the various cleavage products obtained under three different conditions. Under conditions where the thrombin-to-substrate ratio was very low (1 NIH unit/mg BP), the concentration of substrate was relatively low (4 mg BP/ml), and the incubation time was short (2 h), the rabbit BP was cleaved essentially completely and specifically at a single site, the Arg(95)-Thr(96) bond. The BPs of other species (beef, pig, guinea pig, rat) were similarly cleaved, no doubt because all have the same amino acid sequence in this region of the protein. Under conditions in which the enzyme-to-substrate ratio and the substrate concentration were higher (2 NIH units/mg BP, 8 mg BP/ml) and the incubation time was long (24 h), additional, partial cleavages occurred, principally at the Arg(43)-Phe(44) and Arg(128)-Ala(129) bonds, but with some cleavage at the Arg(31)-His(32) and Arg(63)-Thr(64) bonds as well. Under conditions in which all three variables were elevated (5 NIH units/mg peptide, 20 mg peptide/ml, 24 h), more extensive cleavage occurred at the above sites. In peptide (96-168), which we examined in detail, nearly complete cleavage of the Arg(128)-Ala(129) bond occurred, with partial cleavage at the unmethylated Arg(105)-Gly(106), Arg(111)-Phe(112), Arg(150)-Leu(151), and Arg(160)-Ser(161) bonds. The susceptibilities to cleavage of the Arg-X bonds in the BP can be explained with varying degrees of success in terms of the known specificity of thrombin. Cleavage of two of the bonds, Arg(128)-Ala(129) and Arg(160)-Ser(161), suggests the occurrence of a chain reversal or beta-turn in the sequence preceding the scissile bonds. Most cleavages of the BP with thrombin do not occur in the more hydrophobic regions; in particular, the hydrophobic region in the center of the molecule that includes the Phe-Phe(87-88) sequence is left intact.     

Expression and Localization of Myelin Basic Protein in Oligodendrocytes and Transfected Fibroblasts

Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Relationship of Myelin Basic Protein (Arginine) Methyltransferase to Myelination in Mouse Spinal Cord

Abstract: The relationship between the activity of myelin basic protein (arginine) methyltransferase and myelination in the mouse spinal cord has been examined. The activity of this methylase increases between 8 and 45 days postnatal age and correlates well with other parameters of myelination. A comparison of myelin basic protein methylase with histone methylase activity during development indicates that each is a distinct, specific enzyme activity. Together, these results are considered to establish myelin basic protein methylase as a myelination-related enzyme.