Regulation of Myelination: Biosynthesis of the Major Myelin Glycoprotein by Schwann Cells in the Presence and Absence of Myelin Assembly

Schwann cell biosynthesis of the major myelin glycoprotein, P0, was investigated in the crush-injured adult rat sciatic nerve, where there is myelin assembly, and in the permanently transected nerve, where there is no myelin assembly. Endoneurial fractions from desheathed rat sciatic nerves distal to the crush were compared with similar fractions from the permanently transected nerves at 7, 14, 21, 28, and 35 days after injury. The Schwann cell expression of this asparagine-linked glycoprotein was evaluated after sodium dodecyl sulfate-pore gradient electrophoresis by Coomassie Blue and silver stain and by autoradiography after direct overlay of radioiodinated lectins [wheat germ agglutinin, gorse agglutinin, and concanavalin A (Con A)]. As evaluated by these parameters, the concentration of P0 after crush decreased and subsequently increased as a function of time after injury, corresponding to the events of demyelination and remyelination. After permanent transection, the P0 concentration decreased following the same time course found after crush. At subsequent time points, P0 could not be detected with Coomassie Blue stain, silver stain, or wheat germ agglutinin. Both gorse agglutinin and Con A, however, showed binding to P0. Radioactive precursor incorporation studies with [3H]fucose or [3H]-mannose into endoneurial slices at 35 days posttransection revealed active oligosaccharide processing of P0 glycoprotein by Schwann cells in this permanent transection model. Compared with other Schwann cell glycoproteins in the transected nerve, the highest level of incorporation of [3H]mannose was found in P0 which accounted for 42.7% of the incorporated label. In contrast, incorporation of [3H]mannose into endoneurial slices at 35 days after crush accounted for only 13.3% in P0. In addition, higher levels of Con A binding were observed in P0 in the transected nerve compared with the contralateral control or the crushed nerve. Both the [3H]fucose incorporation and gorse agglutinin binding to P0 in the transected nerve suggest posttranslational processing of this glycoprotein in the Golgi apparatus; however, the absence of wheat germ agglutinin binding, the high level of mannose incorporation, and the high level of binding by Con A imply that additional processing steps are required prior to its assembly into myelin.     

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.     

Catabolic Regulation of the Expression of the Major Myelin Glycoprotein by Schwann Cells in Culture

Previous studies have suggested that neonatal Schwann cell cultures deprived of axonal contact do not express components of the myelin membrane, including the major myelin glycoprotein, P0. In contrast, Schwann cells from permanently transected, adult nerve exhibit continued biosynthesis of P0 after culture, suggesting that the ability to express the myelin glycoprotein may depend on the degree of cellular differentiation. To examine further the ability of Schwann cell cultures to express P0 as a function of age, we have performed precursor incorporation studies on endoneurial explants from 4- to 12-day-old rat sciatic nerves after 5 days in culture. The data reveal that explants from 12-day-old animals synthesize detectable levels of this integral myelin protein when assayed by [3H]mannose incorporation, even though there is no apparent myelin assembly in the cultures. Pulse-chase analysis of cultures from 12-day-old rats demonstrates that [3H]mannose-labeled P0 is substantially degraded within 3 h. This catabolism largely can be prevented by the addition of swainsonine, ammonium chloride, or L-methionine methyl ester to the pulse-chase media. The former agent alters oligosaccharide processing whereas the latter two compounds inhibit lysosomal function. The P0 synthesized by the 12-day explant cultures following the addition of swainsonine is readily fucosylated, implying that the protein has progressed at least as far as the medial Golgi before its exit and subsequent catabolism. If cultures from 4-, 6-, and 8-day-old animals are analyzed for P0 biosynthesis by [3H]mannose incorporation in the presence of swainsonine, detectable levels of the glycoprotein are seen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Incorporation of P0 Protein into Liposomes: Demonstration of a Two-Domain Structure by Immunochemical and PAGE Analysis

The amphiphilic nature of P0, the major glycoprotein of peripheral nerve myelin, has been suggested previously. In the present study, purified P0 from human peripheral nerve myelin was incorporated into an artificial lipid bilayer consisting of dimyristoyl lecithin and cholesterol. The liposomes were fractionated on a sucrose gradient. The continued expression of P0 antigenicity by the liposomes was shown by specific complement consumption with a multivalent antiserum against P0 or with an IgM monoclonal antibody. Both antibodies recognized P0 expressed on the surface of peripheral nerve myelin and the P0 liposomes. P0 liposomes and peripheral nerve myelin treated with trypsin lost the surface determinant that reacted with the monoclonal antibody. Analysis of the trypsin-treated liposomes and peripheral nerve myelin by polyacrylamide gel electrophoresis revealed molecular weights for this protein of 19,500 and 20,500, respectively. Similar treatment of the P0 in the fluid phase resulted in many smaller fragments. These results indicate that P0 consists of two domains, a hydrophilic domain accessible to trypsin digestion and a hydrophobic domain, which is potentially trypsin-sensitive, but shielded by the lipid bilayer. Binding studies with an anti-P0 monoclonal antibody and polyacrylamide gel analysis of the lipid-shielded P0 fragment in liposomes and peripheral nerve myelin suggest that the orientation of the protein in the liposome is similar to that in peripheral nerve myelin.     

EVIDENCE FOR THE CLOSE ASSOCIATION OF A GLYCOPROTEIN WITH MYELIN IN RAT BRAIN

Abstract— Myelin was purified from rats which had been injected intracerebrally with radioactive fucose in order to label specifically the glycoproteins. Myelin contained a small amount of fucose-labelled glycoproteins in comparison to that in other subcellular fractions, but polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed a unique pattern of radioactive glycoproteins dominated by a major peak. The same glycoprotein was not prominent in the other subcellular fractions which were examined. This major glycoprotein in the myelin fraction was also labelled after injection with [³H]glucosamine or N -[³H]acetylmannosamine. It was the most intensely staining myelin protein when gels were treated with periodic acid-Schiff reagents, an indication that, in terms of protein-bound carbohydrate, it is the major glycoprotein in the myelin fraction. The glycoprotein was present in myelin purified from rats ranging in age from 14 days to 14 months. Extensive recycling of the myelin through the purification procedures did not significantly reduce the amount of glycoprotein in the myelin. Double label experiments with [³H]fucose and [¹⁴C]fucose were used to compare glycoproteins in myelin purified from white and grey matter, respectively, and from mixed homogenates of myelinated and unmyelinated brain. The results obtained from these experiments suggested that the glycoprotein is closely associated with myelin and that it is not in an unrelated contaminating structure. Possible locations of the glycoprotein are discussed. They include the myelin membrane itself, the oligodendroglial plasma membrane, and the axolemma of myelinated axons.     

Evidence for the presence of major peripheral myelin glycoprotein P0 in mammalian spinal cord and a change of its glycosylation state during aging.

Glycoproteins, which react with Lens culinaris agglutinin, in the membrane preparation of various portions of brains and spinal cords, obtained from 9-week-old rats and 29-month-old rats, were comparatively analyzed by SDS-polyacrylamide gel electrophoresis. In contrast to the samples from brain, which showed similar staining patterns in the two different age groups, the glycoprotein patterns of spinal cords showed marked differences by the age of donors. The most prominent evidence is that a glycoprotein with an apparent molecular weight of 30 kDa (gp30) was detected in the aged rats, but not in the young adult rats. Based on the amino acid sequence data around the glycosylation site, the gp30 was identified as P0, which is a member of immunoglobulin superfamily and a major structural component of mammalian peripheral nerve myelin. This is the first report indicating that P0, which has been considered as a peripheral nerve-specific glycoprotein, occurs also in the spinal cord of mammals. In addition, nonglycosylated P0 molecule could be detected in the spinal cord of young adult rats by anti-P0 polyclonal antibody. These results indicate that the glycosylation state of the P0 molecule in the spinal cord changes during aging.     

Biosynthesis of Neutral Glucocerebroside Homologues in the Absence of Myelin Assembly After Nerve Transection

The biosynthesis of myelin-associated glycolipids during various stages of myelination was studied by in vitro incorporation of [3H]Gal, [3H]Glc, or [35S]sulfate into the endoneurium of rat sciatic nerve. In the normal adult nerve, where the level of myelin assembly is substantially reduced and Schwann cells are principally involved in maintaining the existing myelin membrane, [3H]Gal was primarily incorporated into monogalactosyl diacylglycerol (MGDG) and the galactocerebrosides (GalCe) with lower levels of incorporation into the sulfatides. Such incorporation was enhanced 35 days after crush injury of the adult rat sciatic nerve, which is characterized by active myelin assembly. In contrast, at 35 days after permanent nerve transection where there is no axonal regeneration or myelin assembly, the incorporation of [3H]Gal or [3H]Glc into GalCe was nearly undetected whereas the incorporation of [3H]Gal into MGDG was completely inhibited. Instead, the 3H-labeled glycolipids in transected nerve were identified as the glucocerebrosides (GlcCe) and oligohexosylceramide derivatives with tetrahexosylceramide being a major product. In contrast, [35S]sulfate was incorporated into endoneurial sulfatides in the transected nerve, which suggests that endogenous GalCe rather than newly synthesized GalCe served as the substrate for the sulfotransferase reaction. The GlcCe homologues are not considered as constituents of the myelin membrane but are likely plasma membrane components synthesized in the absence of myelin assembly. It is likely that the cells responsible for GlcCe biosynthesis are Schwann cells, since they comprise 90% of the total endoneurial cell area in the distal nerve segment at 35 days after transection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Myelination: Schwann Cell Transition from a Myelin-Maintaining State to a Quiescent State After Permanent Nerve Transection

Permanent nerve transection of the adult rat sciatic nerve forces Schwann cells in the distal nerve segment from a myelin-maintaining to a quiescent state. This transition was followed by serial morphometric evaluation of the percentage fascicular area having myelin (myelin percent of area) in transverse sections of the distal nerve segment and revealed a rapid decline from a normal value of 36.6% to 3.2% by 14 days for the sciatic nerve to less than 1.0% throughout the remaining time course (up to 105 days). No evidence of axonal reentry into the distal nerve segment or new myelin formation was observed at times under 70 days. In some of the distal nerve segments at 70, 90, and 105 days, new myelinated fibers were observed that usually consisted of only a few myelinated fibers at the periphery and in the worst case amounted to 1.6% (myelin percent of area). Radioactive precursor incorporation of [3H]mannose into endoneurial slices at 4 and 7 days after transection revealed two species of the major myelin glycoprotein, P0, with Mr of 28,500 and 27,700. By 14 days after nerve transection, only the 27,700 Mr species remained. Incorporation of [3H]mannose into the 27,700 Mr species increased progressively to 35 days after transection and then began to decline at 70 and 105 days. Alterations in the oligosaccharide structure of this down-regulated myelin glycoprotein accounted for the progressive increase in mannose incorporation. Lectin affinity chromatography of pronase-digested P0 glycopeptides on concanavalin A-Sepharose revealed that the 28,500 Mr species of P0 had the complex-type oligosaccharide as the predominant oligosaccharide structure (92%). In contrast, the high mannose-type oligosaccharide was the predominate structure for the 27,700 Mr form, which increased to 70% of the total radioactivity by 35 days after nerve transection. Since the biosynthesis of the complex-type oligosaccharide chains on glycoproteins involves high mannose-type intermediates, the mechanism of down-regulation in the biosynthesis of this major myelin glycoprotein, therefore, results in a biosynthetic switch from the complex-type oligosaccharide structure as an end product to the predominantly high mannose-type oligosaccharide structure as a biosynthetic intermediate. This biosynthetic switch occurs gradually between 7 and 14 days after nerve transection and likely reflects a decreased rate of processing through the Golgi apparatus. It remains to be determined if the high mannose-type oligosaccharide chain on P0 can undergo additional processing steps in this permanent nerve transection model.     

A quantitative immunohistochemical study of the endoneurium in the rat dorsal and ventral spinal roots

The dorsal and ventral spinal roots contain different types of axons. The endoneurial extracellular matrix (ECM) among them is produced by Schwann cells and fibroblasts under the control of the axons. Chondroitin sulfate proteoglycan, fibronectin, tenascin-C, and thrombospondin are common components of the endoneurial ECM involved in the normal function as well as regeneration of the peripheral nerve. The present paper demonstrates a comparison of immunofluorescence staining for chondroitin sulfate proteoglycan, fibronectin, tenascin-C, and thrombospondin in the endoneurium of the rat dorsal and ventral spinal roots. Sections through the dorsal and ventral roots were cut simultaneously and adhered to the same microscopic slide. They were incubated simultaneously and the intensity of immunofluorescence staining was assessed by computer-assisted image analysis using interactive segmentation of digitized pictures to select the areas of measurement. The measurement of the immunofluorescence brightness revealed that the endoneurium of the dorsal roots was immunostained for the studied molecules at a higher intensity than in the ventral roots. The results suggest quantitative differences of the endoneurial content of the spinal dorsal and ventral roots probably corresponding to the presence of various types of axons. On the other hand, the different concentration of ECM molecules in the endoneurium of dorsal and ventral roots might be related to the formation of extrinsic conditions differently supporting regeneration of afferent and motor axons after their injury.     

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.     

Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction.

Protein zero (P0), an integral membrane glycoprotein synthesized by Schwann cells, is the major glycoprotein of peripheral nerve myelin. The predicted disposition of P0 with respect to the membrane bilayer postulates the existence of extracellular and intracellular domains, that mediate compaction of the myelin lamellae. We used in vitro translations programmed with sciatic nerve mRNA and cells transfected with a P0 cDNA construct to study the biosynthesis and topology of P0 in the bilayer. The behavior of P0 at the cell surface, when expressed under physiological conditions, was also examined. We have verified the topological predictions of an earlier model, derived from analysis of a P0 cDNA, and provide evidence that the extracellular domain of P0 mediates homotypically cell-cell interactions in the transfectants.     

The role of macrophages in demyelinating peripheral nervous system of mice heterozygously deficient in p0

Mice heterozygously deficient in the p0 gene (P0(+/-)) are animal models for some forms of inherited neuropathies. They display a progressive demyelinating phenotype in motor nerves, accompanied by mild infiltration of lymphocytes and increase in macrophages. We have shown previously that the T lymphocytes are instrumental in the demyelination process. This study addresses the functional role of the macrophage in this monogenic myelin disorder.In motor nerves of P0(+/)- mice, the number of macrophages in demyelinated peripheral nerves was increased by a factor of five when compared with motor nerves of wild-type mice. Immunoelectron microscopy, using a specific marker for mouse macrophages, displayed macrophages not only in the endoneurium of the myelin mutants, but also within endoneurial tubes, suggesting an active role in demyelination. To elucidate the roles of the macrophages, we crossbred the myelin mutants with a spontaneous mouse mutant deficient in macrophage colony-stimulating factor (M-CSF), hence displaying impaired macrophage activation. In the P0-deficient double mutants also deficient in M-CSF, the numbers of macrophages were not elevated in the demyelinating motor nerves and demyelination was less severe. These findings demonstrate an active role of macrophages during pathogenesis of inherited demyelination with putative impact on future treatment strategies.     

External labeling of galactose in surface membrane glycoproteins of the intact myelin sheath.

The molecular organization of surface galactose residues in glycoproteins of the intact myelin sheath was investigated using the enzymatic membrane probe, galactose oxidase. Rat spinal cords treated under physiological conditions with this nonpermanent probe were labeled specifically in galactose residues by reduction with tritiated sodium borohydride. The enzymatically modified proteins from isolated myelin were analyzed electrophoretically and their specific radioactivities determined. Results indicated tritium label associated with a surprising variety of high molecular weight proteins. The most extensively labeled peak corresponded to the major myelin glycoprotein as indicated by the coincidence of tritium label with that of [14C]fucose used as an internal marker for the glycoproteins. The radioactivity associated with this protein was 1.1 to 2.7 times higher after treatment with galactose oxidase when compared to reduction in the absence of the enzyme and 1.4 to 4.8 times higher when oxidized and reduced after prior treatment with neuraminidase. The results suggest a complex heterogeneity of minor glycoproteins associated with isolated myelin. It is concluded that from this complexity of glycoproteins, a major glycoprotein is at least partially localized on the external surface of either the intact myelin sheath or the closely associated oligodendroglial plasma membrane. Such a localization of this glycoprotein and the probable localization of the other glycoproteins enhances their potential role in specific interactions in the process of mpyelination or myelin maintenance.     

Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin

The myelin sheath is a multilayered membrane, unique to the nervous system, which functions as an insulator to increase greatly the velocity of axonal impulse conduction. We have used the techniques of differential screening and hybrid selection to identify a cDNA clone encoding the Schwann cell glycoprotein P0, the major structural protein of the peripheral myelin sheath. The sequence of this protein, deduced from the nucleotide sequence of the cloned cDNA, indicates that P0 is an integral membrane protein containing a single membrane-spanning region, a large hydrophobic extracellular domain, and a smaller basic intracellular domain. The structure of the protein suggests that each of these domains plays an essential role in generating the highly ordered structure of the myelin sheath. Furthermore, we find that the induction of P0 mRNA coincides with the initiation of myelin formation, and we propose a model in which the glycoprotein serves as a molecular guidepost for this process.     

Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane.

The effect of ionic strength on the proteolysis by trypsin of the major membrane-penetrating protein (polypeptide 3) in the erythrocyte membrane was studied. Both the intracellular and extracellular regions of the protein are susceptible to trypsin proteolysis under hypo-osmotic conditions, whereas under iso-osmotic conditions the extracellular region of the protein is resistant to trypsin, and the intracellular region yields only two cleavage products with trypsin. Studies of the fragments obtained from polypeptide 3 by trypsin digestion under iso-osmotic conditions of 'ghosts' radioiodinated with lactoperoxidase confirmed our earlier conclusions that the polypeptide chain of polypeptide 3 traverses the membrane twice. Ionic-strength-dependent changes were also observed in the incorporation of iodine by lactoperoxidase into the individual extracellular tyrosine sites of the protein. These results show that polypeptide 3 undergoes ionic-strength-dependent changes in structure.     

Complete amino acid sequence of PO protein in bovine peripheral nerve myelin

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.     

A LOW MOLECULAR W eight GLYCOPROTEIN ASSOCIATED WITH ISOLATED MYELIN: DISTINCTION FROM MYELIN PROTEOLIPID PROTEIN

Abstract— A low molecular weight glycoprotein has been demonstrated in myelin isolated from immature rat brains. Both short term and long term fucose incorporation studies have identified this protein in the proteolipid protein region of a sodium dodecyl sulfate, polyacrylamide gel. A 1.7-2.1 fold increase in radioactive fucose in this glycoprotein relative to the major myelin glycoprotein was seen after long term fucose incorporation (21 days) compared to short term incorporation (18–22 h). The demonstration that this fucose-labelled protein is distinguishable from that of proteolipid protein was achieved by a variety of independent techniques. One technique involved a comparison of ether-ethanol extracted, freshly isolated, myelin with myelin extracted with chloroform-methanol. Treatment of isolated myelin with chloroform-methanol results in the solubilization of the proteolopid protein and its subsequent absence on gel electrophoresis while, in contrast, an enhancement of fucose label was observed in the same region of the polyacrylamide gel. Another procedure involved the electrophoretic separation of the radioactive fucose peak from that of proteolipid protein by employing a continuous, phosphate buffered, gel system. Finally carbohydrate analysis by gas-liquid chromatography of a partially purified proteolipid protein fraction did not reveal significant amounts of carbohydrates which are characteristic of glycoproteins. The identification of this minor glycoprotein comigrating with proteolipid protein indicates, therefore, a greater complexity associated with the purified myelin membrane than has been previously demonstrated.     

Cytoplasmic domain of human myelin protein zero likely folded as beta-structure in compact myelin

Myelin protein zero (P0 or P0 glycoprotein), the major integral membrane protein in peripheral nervous system myelin, plays a key role in myelin membrane compaction and stability. While the structure of P0 extracellular domain was determined by crystallography, the paucity of any structural data on the highly positive-charged P0 cytoplasmic domain (P0-cyt) has greatly limited our understanding of the mechanism of P0 function. Here, using circular dichroism and intrinsic fluorescence spectroscopy, we attempted to elucidate the structure of human P0-cyt (hP0-cyt) in membrane mimetic environments composed of detergents or lipid vesicles. We found that the secondary structure of P0-cyt was polymorphic—at the lipid/protein ratio corresponding to that of mature peripheral myelin (~50:1), hP0-cyt mainly adopted a β-conformation, whereas when the proportion of lipid increased, the structure underwent a β→α transition. By contrast, the secondary structure of the major isoform of myelin basic protein, another myelin protein with a very large positive charge, remained unchanged across a wide range of lipid/protein ratios. We propose that when hP0-cyt is bound at sufficient concentration to lamellar lipid bilayers such as myelin, it folds into a β-conformation; before this threshold lipid/protein ratio is reached, the domain is α-helical. We suggest that the cytoplasmic apposition (major dense line) in compact myelin may be stabilized via the hydrogen-bonding of β-strands formed as a result of local P0-P0 aggregation.     

Posttranslational Protein Modification: Biosynthetic Control Mechanisms in the Glycosylation of the Major Myelin Glycoprotein by Schwann Cells

The posttranslational processing of the asparagine-linked oligosaccharide chain of the major myelin glycoprotein (P0) by Schwann cells was evaluated in the permanently transected, adult rat sciatic nerve, where there is no myelin assembly, and in the crush injured nerve, where there is myelin assembly. Pronase digestion of acrylamide gel slices containing the in vitro labeled [3H]mannose and [3H]fucose P0 after electrophoresis permitted analysis of the glycopeptides by lectin affinity and gel filtration chromatography. The concanavalin A-Separose profile of the [3H]mannose P0 glycopeptides from the transected nerve revealed the high-mannose-type oligosaccharide as the predominant species (72.9%), whereas the normally expressed P0 glycoprotein that is assembled into the myelin membrane in the crushed nerve contains 82.9-91.9% of the [3H]mannose radioactivity as the complex-type oligosaccharide chain. Electrophoretic analysis of immune precipitates verified the [3H]mannose as being incorporated into P0 for both the transected and crushed nerve. The high-mannose-type glycopeptides of the transected nerve isolated from the concanavalin A-Sepharose column were hydrolyzed by endo-beta-N-acetylglucosaminidase H, and the oligosaccharides were separated on Biogel P4. Man8GlcNAc and Man7GlcNAc were the predominant species with radioactivity ratios of 12.5/7.2/1.4/1.0 for the Man8, Man7, Man6, and Man5 oligosaccharides, respectively. Jack bean alpha-D-mannosidase gave the expected yields of free Man and ManGlcNAc from these high-mannose-type oligosaccharides. The data support the notion that at least two alpha-1,2-mannosidases are responsible for converting Man9GlcNAc2 to Man5GlcNAc2. The present experiments suggest distinct roles for each mannosidase and that the second mannosidase (I-B) may be an important rate-limiting step in the processing of this glycoprotein with the resulting accumulation of Man8GlcNAc2 and Man7GlcNAc2 intermediates. Pulse chase experiments, however, demonstrated further processing of this high-mannose-type oligosaccharide in the transected nerve. The [3H]mannose P0 glycoprotein with Mr of 27,700 having the predominant high-mannose-type oligosaccharide shifted its Mr to 28,500 with subsequent chase. This band at 28,500 was shown to have the complex-type oligosaccharide chain and to contain fucose attached to the core asparagine-linked GlcNAc residue. The extent of oligosaccharide processing of this down-regulated glycoprotein remains to be determined.