Myelin-Associated Glycoprotein and Related Glycoconjugates in Developing Cat Peripheral Nerve: A Correlative Biochemical and Morphometric Study

The expression and accumulation of the myelin-associated glycoprotein (MAG) and other glycoconjugates have been studied during myelination in the developing cat peripheral nervous system. The glycoconjugates studied have in common a similar carbohydrate determinant which is bound by many antibodies, including the mouse monoclonal antibody HNK-1, and human IgM paraproteins from patients with neuropathy. In addition to MAG, the reactive glycoconjugates include a 60-kilodalton (kD) glycoprotein and a group of 20-26 kD glycoproteins, as well as a group of recently identified acidic glycolipids, the major one of which is sulfate-3-glucuronyl paragloboside (SGPG). The accumulation of these glycoproteins and glycolipids is compared with the established myelin proteins P0, P1, and P2 and with morphometric indices of myelin volume and axonal perimeter. The study demonstrates that MAG appears and accumulates very early during myelination, being present at 15% of the maximum level prior to the appearance of P0, and at 80% of the maximum level when P0 is at 30% of its maximum level. In the adult, the level of MAG falls to 60% maximum. The 60 kD and 20-26 kD glycoproteins accumulate at the same time as or later than P0, suggesting that they are either compact myelin proteins or in membranes closely associated with compact myelin. SGPG accumulates with P0 early in myelination, but falls to 60% of maximum in the adult. By comparing biochemical and morphometric data, we demonstrate that P0 and other compact myelin proteins accumulate synchronously with the increase in myelin area. MAG accumulation, however, is closely related to changes in axonal perimeter, consistent with a predominant localization of MAG to the periaxonal membranes in the peripheral nervous system.     

Production and Characterization of Monoclonal Antibodies to Peripheral and Central Nervous System Myelin

Monoclonal antibodies against P0, myelin basic protein, or myelin-associated glycoprotein were generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with central and peripheral nervous system myelin proteins. The antibodies secreted were either IgG, IgM, or IgA. Clone C6B5 (iso-type IgM) secreted antibody(ies) that bound to both myelin basic protein and myelin-associated glycoprotein, although binding of antibody to myelin basic protein as detected by the immunoblot technique appeared to be much less than to the myelin-associated glycoprotein. Antibodies were characterized in solid-phase radioimmunoassay for their species cross-reaction, and histologically for the specificity of binding to myelin in central and peripheral nervous system tissues. These monoclonal reagents should prove valuable in studying CSF and myelin-producing cells, since in both cases the concentration of myelin proteins is low.     

Myelin protein zero/P0 phosphorylation and function require an adaptor protein linking it to RACK1 and PKC alpha

Point mutations in the cytoplasmic domain of myelin protein zero (P0; the major myelin protein in the peripheral nervous system) that alter a protein kinase Calpha (PKCalpha) substrate motif (198HRSTK201) or alter serines 199 and/or 204 eliminate P0-mediated adhesion. Mutation in the PKCalpha substrate motif (R198S) also causes a form of inherited peripheral neuropathy (Charcot Marie Tooth disease [CMT] 1B), indicating that PKCalpha-mediated phosphorylation of P0 is important for myelination. We have now identified a 65-kD adaptor protein that links P0 with the receptor for activated C kinase 1 (RACK1). The interaction of p65 with P0 maps to residues 179-197 within the cytoplasmic tail of P0. Mutations or deletions that abolish p65 binding reduce P0 phosphorylation and adhesion, which can be rescued by the substitution of serines 199 and 204 with glutamic acid. A mutation in the p65-binding sequence G184R occurs in two families with CMT, and mutation of this residue results in the loss of both p65 binding and adhesion function.     

The pH-dependent unfolding mechanism of P2 myelin protein: an experimental and computational study

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands beta-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.     

FA2H is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin

Myelin in the mammalian nervous system has a high concentration of galactolipids [galactosylceramide (GalCer) and sulfatide] with 2-hydroxy fatty acids. We recently reported that fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is the major fatty acid 2-hydroxylase in the mouse brain. In this report, we show that FA2H also plays a major role in the formation of 2-hydroxy galactolipids in the peripheral nervous system. FA2H mRNA and FA2H activity in the neonatal rat sciatic nerve increased rapidly during developmental myelination. The contents of 2-hydroxy fatty acids were approximately 5% of total galactolipid fatty acids at 4 days of age and increased to 60% in GalCer and to 35% in sulfatides at 60 days of age. The chain length of galactolipid fatty acids also increased significantly during myelination. FA2H expression in cultured rat Schwann cells was highly increased in response to dibutyryl cyclic AMP, which stimulates Schwann cell differentiation and upregulates myelin genes, such as UDP-galactose:ceramide galactosyltransferase and protein zero. These observations indicate that FA2H is a myelination-associated gene. FA2H-directed RNA interference (RNAi) by short-hairpin RNA expression resulted in a reduction of cellular 2-hydroxy fatty acids and 2-hydroxy GalCer in D6P2T Schwannoma cells, providing direct evidence that FA2H-dependent fatty acid 2-hydroxylation is required for the formation of 2-hydroxy galactolipids in peripheral nerve myelin. Interestingly, FA2H-directed RNAi enhanced the migration of D6P2T cells, suggesting that, in addition to their structural role in myelin, 2-hydroxy lipids may greatly influence the migratory properties of Schwann cells.     

Production and Characterization of Monoclonal Antibodies to the Major Peripheral Myelin Glycoprotein P0

Several monoclonal antibodies were generated against the major glycoprotein P0 of human peripheral nervous system myelin. Antibodies were selected for their reactivity with P0 in Western blots. The antibodies were of the immunoglobulin G subclass and reacted with the glycopeptidase F-treated P0, indicating that the reactive epitope resides in the protein backbone. In fresh frozen and paraffin-embedded sections of central and peripheral nervous system of rat and human, P0 antibody 592 reacted with myelin sheaths of peripheral, but not central, nervous system.     

Isolation of an integral membrane glycoprotein by chloroform-methanol extraction and C3-reversed-phase high-performance liquid chromatography

Methodology is presented for the isolation of integral membrane proteins and applied to the purification of the major myelin glycoprotein, P0. This isolation scheme depends on the detergent solubilization of an isoosmotically extracted membrane fraction from sciatic nerve endoneurium, followed by the removal of lipids and detergent by chloroform/methanol extraction. The resulting membrane proteins are readily dissolved in acetic acid/water (1/1) and directly analyzed by reversed-phase high-performance liquid chromatography. The hydrophobic nature of the intrinsic membrane protein mixture results in strong binding to a C8 stationary phase, leading to poor resolution and yields. These problems can be eliminated by employing a C3 alkylsilane column, thereby allowing separation of the protein components and the isolation of P0. The purified P0 has an amino-terminal sequence that matches that predicted from nucleotide sequencing, and the glycoprotein contains the expected amount of sialic acid. This latter finding indicates that the isolation procedure is not detrimental to the complex-type oligosaccharide structure of P0 and should make the methodology readily applicable to the purification of other integral membrane proteins and glycoproteins.     

Proteolipid protein (PLP) of CNS myelin: positions of free, disulfide-bonded, and fatty acid thioester-linked cysteine residues and implications for the membrane topology of PLP.

Proteolipid protein (PLP), the major integral membrane protein of central nervous system myelin, contains 14 cysteine residues within its 276-residue polypeptide chain. We determined the state of all cysteine residues and localized four of them as free thiols at positions 24, 32, 34, and 168. Four cysteines are connected by disulfide bonds: Cys200-Cys219 and Cys183-Cys227. The remaining six cysteine residues at positions 5, 6, 9, 108, 138, and 140 are modified by long-chain fatty acids, mainly palmitic acid, in thioester linkage. The extreme hydrophobicity of PLP can therefore be explained by two structural features: a composition of approximately 50% apolar amino acid residues and a high degree of fatty acid acylation. A differential fluorescent-labeling technique was developed for the structural studies: the cysteine residues belonging to one of the three states were derivatized by N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) either directly (a), after thioester cleavage with hydroxylamine (b), or after disulfide cleavage with dithiothreitol (c). The protein was then proteolytically digested with thermolysin, and the labeled peptides were isolated by reversed-phase HPLC followed by sequence analysis. The results were further confirmed by determination of the fatty acid to protein stoichiometry. The structural data not only demand the revision of our concept of the membrane topology of PLP but will also promote more sophisticated studies on the mechanism of myelination and new functions of PLP.     

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.     

Structural and Functional Characterization of Human Peripheral Nervous System Myelin Protein P2

The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 Å. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces.     

Proteolipid protein and DM-20 are synthesized by Schwann cells,present in myelin membrane,but they are not fatty acylated

Proteolipid protein (PLP) and DM-20 were intensely labeled after immunoprecipitation of total cellular proteins and myelin proteins labeled with [³⁵S]methionine in nerve slices. These results provided evidence that PLP and DM-20 are incorporated into the myelin membrane following their synthesis in Schwann cells. In contrast, PLP and DM-20 were not fatty acylated after incubation of the nerve slices with [³H]palmitic acid, however, PO glycoprotein and 24kDa protein were heavily fatty acylated. The lack of fatty acylation of PLP and DM-20 in the peripheral nervous system suggests that fatty acyltransferase responsible for their acylation is absent or non-functional in the peripheral nervous system.     

Conformational changes induced by ionic strength and pH in two bovine myelin basic proteins.

The structures of two biologically different myelin proteins, A1 from the central nervous system and P2 from the peripheral nervous system, were investigated. Both proteins were isolated from nerve tissues. Conformational changes in the homogeneous proteins were examined in aqueous solutions by means of circular dichroism measurements. The secondary structures of both proteins proved to be very stable between pH 2.5 and pH 11.7. Unlike the P2 protein, the A1 protein is stable up to pH 13 without detectable conformational changes. The stereochemistry of the polypeptide chains of both proteins is markedly different in the presence of urea. While the value of theta222 for the A1 protein changes linearly with increasing urea concentration, a sigmoidal curve was obtained for the P2 protein. The observed differences in the dichroic properties of the basic myelin proteins A1 and P2 indicate the possibility of further structure - function correlations.     

The three-dimensional structure of P2 myelin protein.

The three-dimensional structure of P2 protein from peripheral nervous system myelin has been determined at 2.7 A resolution by X-ray crystallography. The single isomorphous replacement/anomalous map was interpreted using skeletonized electron density on a computer graphics system. An atomic model was built using fragment fitting. The structure forms a compact 10-stranded up-and-down beta-barrel which encapsulates residual electron density that we interpret as a fatty acid molecule. This beta-barrel shows some similarity to, but is different from, the retinol binding protein family of structures. The relationship of the P2 structure to a family of cytoplasmic, lipid binding proteins is described.     

HISTOCHEMISTRY OF MYELIN—XII ANIONIC STAINING OF MYELIN BASIC PROTEINS FOR HISTOLOGY, ELECTROPHORESIS AND ELECTRON MICROSCOPY

Abstract— Phosphotungstic acid haematoxylin, trypan blue and amidoblack techniques have been developed as anionic dye methods for staining myelin basic proteins. All methods displayed central and peripheral nervous system myelin in histochemical prepa rations and stained brain basic proteins in electrophoretic polyacrylamide gels: phosphotungstic acid haematoxylin appeared to be the most selective of these techniques. Electron photomicrographs of peripheral nerve stained by phosphotungstic acid haematoxylin showed that the major part of myelin basic protein is located in the period dense line. The basic proteins stained by phosphotungstic acid haematoxylin showed an early loss in rat sciatic nerve undergoing Wallerian degeneration and had completely disappeared from the centre of 20 plaques of multiple sclerosis.     

Myelin tetraspan family proteins but no non-tetraspan family proteins are present in the ascidian (Ciona intestinalis) genome

Several of the proteins used to form and maintain myelin sheaths in the central nervous system (CNS) and the peripheral nervous system (PNS) are shared among different vertebrate classes. These proteins include one-to-several alternatively spliced myelin basic protein (MBP) isoforms in all sheaths, proteolipid protein (PLP) and DM20 (except in amphibians) in tetrapod CNS sheaths, and one or two protein zero (P0) isoforms in fish CNS and in all vertebrate PNS sheaths. Several other proteins, including 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin and lymphocyte protein (MAL), plasmolipin, and peripheral myelin protein 22 (PMP22; prominent in PNS myelin), are localized to myelin and myelin-associated membranes, though class distributions are less well studied. Databases with known and identified sequences of these proteins from cartilaginous and teleost fishes, amphibians, reptiles, birds, and mammals were prepared and used to search for potential homologs in the basal vertebrate, Ciona intestinalis. Homologs of lipophilin proteins, MAL/plasmolipin, and PMP22 were identified in the Ciona genome. In contrast, no MBP, P0, or CNP homologs were found. These studies provide a framework for understanding how myelin proteins were recruited during evolution and how structural adaptations enabled them to play key roles in myelination.     

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.     

Myelin-Associated Glycoprotein and Other Proteins in Trembler Mice

The myelin-associated glycoprotein (MAG) and other myelin proteins were quantitated in homogenates of whole sciatic nerve from adult and 20-day-old Trember mice. In the nerves of adult mice, the concentration of MAG was increased from 1.1 ng/micrograms of total protein in the controls to 1.4 ng/micrograms protein in the Tremblers. By contrast, the concentrations of P0 glycoprotein and myelin basic proteins were reduced to 27% and 20% of control levels, respectively. Immunoblots demonstrated that P2 was also greatly reduced in the Trembler nerves. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was 65% of the control level. Immunoblot analysis showed that MAG had a higher than normal apparent Mr in the sciatic nerves of the Trembler mice, but its apparent Mr was normal in the brains of these mutants. In 20-day-old Tremblers, the P0 and myelin basic protein were reduced slightly less to about 40% of the level in the nerves of age-matched controls. CNP and MAG levels were not significantly different from those in controls, and MAG exhibited a shift toward higher apparent Mr similar to that in the adults. The maintenance of high MAG levels despite the severe deficit of myelin, as reflected by the decrease of the major myelin proteins, is consistent with the immunocytochemical localization of MAG in periaxonal Schwann cell membranes, Schmidt-Lantermann incisures, lateral loops, and the outer mesaxon and its absence from compact myelin. The abnormal form of MAG in the peripheral nervous system (PNS) of the Trembler mice may contribute to the pathology in this mutant.     

Hydrodynamic and circular dichroic analysis of mammary-derived growth inhibitor (MDGI)

The mammary-derived growth inhibitor exists in solution as a monomeric molecule with a molar mass of 14,500 +/- 400 g/mol. The largest diameter and the height of the polypeptide chain were estimated to be 3.75 +/- 0.25 nm and 2.01 +/- 0.13 nm respectively. This is in good agreement with the structurally related bovine peripheral myelin P2 protein (about 70% amino acid sequence homology). CD measurements have revealed MDGI to be a protein with about 50% beta structure and less than 20% alpha helix similarly as in fatty acid-binding proteins. Removal of endogenous long-chain fatty acid by lipidex or storage in the frozen state lead to a destabilization of the active MDGI conformation which is accompanied by a loss of its activity with regard to growth inhibition of Ehrlich Ascites cells.     

Opalin, a transmembrane sialylglycoprotein located in the central nervous system myelin paranodal loop membrane

In contrast to compact myelin, the series of paranodal loops located in the outermost lateral region of myelin is non-compact; the intracellular space is filled by a continuous channel of cytoplasm, the extracellular surfaces between neighboring loops keep a definite distance, but the loop membranes have junctional specializations. Although the proteins that form compact myelin have been well studied, the protein components of paranodal loop membranes are not fully understood. This report describes the biochemical characterization and expression of Opalin as a novel membrane protein in paranodal loops. Mouse Opalin is composed of a short N-terminal extracellular domain (amino acid residues 1-30), a transmembrane domain (residues 31-53), and a long C-terminal intracellular domain (residues 54-143). Opalin is enriched in myelin of the central nervous system, but not that of the peripheral nervous system of mice. Enzymatic deglycosylation showed that myelin Opalin contained N- and O-glycans, and that the O-glycans, at least, had negatively charged sialic acids. We identified two N-glycan sites at Asn-6 and Asn-12 and an O-glycan site at Thr-14 in the extracellular domain. Site-directed mutations at the glycan sites impaired the cell surface localization of Opalin. In addition to the somata and processes of oligodendrocytes, Opalin immunoreactivity was observed in myelinated axons in a spiral fashion, and was concentrated in the paranodal loop region. Immunogold electron microscopy demonstrated that Opalin was localized at particular sites in the paranodal loop membrane. These results suggest a role for highly sialylglycosylated Opalin in an intermembranous function of the myelin paranodal loops in the central nervous system.     

Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.