PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin

Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of ³H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P₀, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of ¹⁴Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P₀ peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with ³H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P₀ antiserum, was tentatively identified as a nonglycosylated P₀ protein that appears to be almost as well incorporated as P₀ into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P₀ is actually assembled into a site and configuration like that of P₀.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

Progesterone Stimulates the Activity of the Promoters of Peripheral Myelin Protein-22 and Protein Zero Genes in Schwann Cells

Abstract: To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P₀) genes. PROG stimulated the P₀ promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P₀ and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.     

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.     

Developmental regulation of insulin receptor gene in sciatic nerves and role of insulin on glycoprotein P0 in the Schwann cells

In view of the observations that Schwann cells contain insulin receptors, in the present study, we have investigated the developmental regulation of insulin receptor gene in the sciatic nerves of different postnatal age group rats. We have also investigated the role of insulin in the expression of the major PNS myelin glycoprotein P zero (P0) in normal as well as high glucose conditions in primary rat Schwann cells. The expression of insulin receptor gene in sciatic nerves appeared to be differentially regulated. The steady-state levels of insulin receptor mRNA increased remarkably during development and after postnatal day 10, when the peak of myelin structural gene (P0) expression occur and slowly increased further until at least postnatal day 90 in parallel with the growth of the myelin sheath. By employing immunofluorescence and RT-PCR, we observed significant increase in the P0 protein and mRNA levels in Schwann cells in response to the insulin than in insulin deprived counterparts. The presence of insulin in the high glucose medium ameliorated the altered protein and mRNA of P0 in Schwann cells compared to the insulin deprived counterparts. These studies demonstrate the importance of insulin and its receptor as possible regulatory factors in the PNS and also emphasizes their novel therapeutic applications in demyelinating diseases, especially in diabetic poly-neuropathy.     

The Second Messenger, Cyclic AMP, Is Not Sufficient for Myelin Gene Induction in the Peripheral Nervous System

Abstract: The adenylyl cyclase-cyclic AMP (cAMP) second messenger pathway has been proposed to regulate myelin gene expression; however, a clear correlation between endogenous cAMP levels and myelin-specific mRNA levels has never been demonstrated during the induction or maintenance of differentiation by the myelinating Schwann cell. Endogenous cAMP levels decreased to 8–10% of normal nerve by 3 days after crush or permanent transection injury of adult rat sciatic nerve. Whereas levels remained low after transection injury, cAMP levels reached only 27% of the normal values by 35 days after crush injury. Because P₀ mRNA levels were 60% of normal levels by 14 days and 100% by 21 days after crush injury, cAMP increased only well after P₀ gene induction. cAMP, therefore, does not appear to trigger myelin gene induction but may be involved in myelin assembly or maintenance. Forskolin, an activator of adenylyl cyclase, increased endoneurial cAMP levels only in the normal nerve, and in the crushed nerve beginning at 16 days after injury, but at no time in the transected nerve. Only by treating transected nerve with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of cAMP phosphodiesterases, in combination with forskolin was it possible to increase cAMP levels. No induction of myelin genes, however, was observed with short- or long-term treatment with IBMX and forskolin in the transected nerve. A three-fold increase in phosphodiesterase activity was observed at 35 days after both injuries, and a nonmyelinated nerve was shown to have even higher activity. These experiments, therefore, suggest an important role for phosphodiesterase in the inactivation of this second messenger-dependent stimuli when Schwann cells are non-myelinating, such as after sciatic nerve injury or in the nonmyelinated nerve, which again implies that cAMP may be required for the maintenance of the myelin sheath.     

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.     

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.     

Basic proteins of rodent peripheral nerve myelin: immunochemical identification of the 21.5K, 18.5K, 17K, 14K, and P2 proteins

Abstract: Proteins in peripheral nervous system and central nervous system myelin and homogenates of sciatic nerve and brain from young and adult mice and rats were characterized with affinity-purified anti-P₂ and anti-myelin basic protein sera after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose sheets. Using this method we have identified a component of rodent peripheral nervous system myelin as P₂ protein. Peripheral nervous system myelin also showed the presence of four basic proteins in addition to P₂ protein. These were found to be analogous to the 14, 17, 18.5, and 21.5K species found in the central nervous system myelin. A number of high-molecular-weight proteins were also detected with anti-myelin basic protein serum in peripheral nervous system, as well as central nervous system myelin. In addition, we report the presence of a high-molecular-weight P₂ cross-reactive protein in rodent brain stem homogenates, but not in central nervous system myelin.     

Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell–cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P₁ phosphorylated and P₀ unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.     

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.     

The in vivo synthesis of myelin proteins in rabbit sciatic nerve

Rabbits were injected into the sciatic nerves with either ³⁵S-methionine, or ³H-fucose. After times ranging from 45 min to 15 days the nerves were removed and the total particulate material from the nerves fractionated to give seven subfractions with densities between 0.2 and 1.2 M sucrose. The patterns of radio-labelled proteins were examined by SDS-PAGE and quantitative fluorography. The results showed that the P₂ basic protein was metabolically far more active than either the major P₀ glycoprotein, or the basic protein BP. The P₂ protein also entered the myelin fractions more rapidly than either P₀, or BP components. The net synthesis of P₀ was slower than P₂ and BP and this intrinsic membrane protein remained associated with the denser membrane fractions (>0.7 M sucrose) for longer than the basic proteins prior to entering myelin. Newly synthesized high molecular weight proteins remained concentrated in the denser membrane fractions and turned over faster than the myelin proteins.

A low density myelin fraction (B) was detected in which both the P₂ protein and certain high molecular weight proteins became more rapidly labelled than in compact myelin. In this fraction the specific activity remained higher than that of compact myelin for up to five days after the injection of ³⁵S-methionine into the nerve.

The results indicate that the major PNS myelin proteins are incorporated into and turn over in the various compartments of the Schwann cell plasma membrane—myelin continuum at very different rates.     

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.     

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.     

Hemichannels Formed by Connexin 43 Play an Important Role in the Release of Prostaglandin E2 by Osteocytes in Response to Mechanical Strain

Osteocytes embedded in the matrix of bone are mechanosensory cells that translate strain into signals and regulate bone remodeling. Our previous studies using osteocyte-like MLO-Y4 cells have shown that fluid flow shear stress (FFSS) increases connexin (Cx) 43 protein expression, prostaglandin E₂ (PGE₂) release, and intercellular coupling, and PGE₂ is an essential mediator between FFSS and gap junctions. However, the role of Cx43 in the release of PGE₂ in response to FFSS is unknown. Here, the FFSS-loaded MLO-Y4 cells with no or few intercellular channels released significantly more PGE₂ per cell than those cells at higher densities. Antisense Cx43 oligonucleotides and 18 β-glycyrrhetinic acid, a specific gap junction and hemichannel blocker, significantly reduced PGE₂ release by FFSS at all cell densities tested, especially cells at the lowest density without gap junctions. FFSS, fluid flow-conditioned medium, and PGE₂ increased the activity of dye uptake. Moreover, FFSS induced Cx43 to migrate to the surface of the cell; this surface expressed Cx43 developed resistance to Triton-X-100 solublization. Our results suggest that hemichannels formed by Cx43, instead of intercellular channels, are likely to play a predominant role in the release of intracellular PGE₂ in response to FFSS.