PNS Myelin Research Articles

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

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

Myelin from Peripheral and Central Nervous System Is a Nonpermissive Substrate for Retinal Ganglion Cell Axons

In the CNS of mammals axonal regeneration is limited by inhibitory influences of the glial and extracellular environment. Myelin-associated inhibitors of neurite growth as well as some properties of so called "reactive astrocytes" which make the environment nonpermissive for axonal growth contribute to the inhibitory nature of the mammalian CNS. In contrast, the PNS is supportive of regeneration and Schwann cell surfaces and Schwann-cell-derived extracellular matrix provide suitable substrates for regenerating axons in vivo and in vitro. However, as the results presented here indicate, myelin derived from normal and axotomized sciatic nerves is a nonpermissive substrate for axonal regrowth. Addition of laminin to either CNS or PNS myelin or freezing of the myelin, however, allows reproducibly axonal growth. Membrane preparations from CNS or PNS tissue on the other hand allow axon outgrowth from retinal explants when adhesive substrates (e.g., polylysin) are available. This suggests that inhibitors of neurite growth are present in myelin from the CNS and PNS. Growth supportive substrates, which are present in large quantities after PNS but not after CNS injury, can overcome nonpermissive substrate properties.

Guillain-Barré syndrome: clinical and immunological aspects.

Immune-mediated PNS disorders comprise a significant segment of diseases of the nervous system. Studies on GBS as a prototype of these disorders and its experimental model EAN have helped to elucidate some of the mechanisms responsible for myelin injury in the PNS. These mechanisms, although partially understood have been useful in implementing therapies such as plasmapheresis and IVIG and various other immunomodulators. The question of whether an infectious agent such as a virus can directly damage the myelin sheath and/or Schwann cells or whether the agent triggers an immune response against self through antigenic mimicry remains unanswered. The association between C. jejuni infection and GBS has opened new areas of investigation in understanding the immunopathogenesis of the disease. Similar observations with other environmental factors may be made in the future, pointing to the possibility that GBS may not be caused by a single agent but could be the result of an immunological attack on the PNS myelin assembly by a variety of agents or factors. Regardless of the etiology, if the myelin injury is aggravated by product of the immune cells, such as various cytokines, neutralization of these factors could help lessen the burden of injury to the nerves. Future research in autoimmune disorders of the PNS needs to focus on identifying environmental factors that directly, or indirectly through antigen mimicry, damage the PNS myelin. In parallel, further understanding of the immunopathogenesis by dissecting the immunological phenomenon at the systemic and local levels, especially the role of cytokines, growth factors, and adhesion molecules will pave the way for more rational therapies, even if the causative factors are not known. Studies in laboratory animals have demonstrated the efficacy of selective immunotherapy through modulation of the trimolecular complex, i.e., T cell receptor, MHC/molecule, and antigen. Immunological tolerance, presumably through deletion of autoreactive clones, clonal anergy, or active suppression, has proven effective in animals. Other modes of immunotherapy such as nonspecific depletion of T or B cells or down-regulation of activated cells have also been shown to abolish or decrease the severity of experimental autoimmune neurological disorders, including EAN. These immunotherapeutic modalities may become applicable to human autoimmune neuropathies.

The number of Schmidt-Lanterman incisures is more than doubled in shiverer PNS myelin sheaths.

In the PNS, myelin basic protein (MBP) appears not to be essential for myelination, for in shiverer (shi) and mld mutant mice peripheral nerves, where MBP is not or only poorly expressed, myelination occurs normally. Only a few morphological abnormalities, i.e. reduction in axon calibre and myelin sheath thickness, and aberrant Schwann cell-axon contacts, have been reported. Here, we document a consistent difference between shi and wild type (wt) myelinated sciatic nerve fibres. The number of Schmidt-Lanterman incisures seen in longitudinally and transversely-sectioned sciatic nerves, or in teased fibres stained for the presence of F-actin, is dramatically increased in homozygous shi mice. With both methods, a twofold increase in Schmidt-Lanterman incisure number is seen in 15-day-old mice, the earliest time examined. The increase is slightly greater in nerve fibres from 30- and 90-day-old mice. The overproduction of Schmidt-Lanterman incisures in shi occurs in spite of the fact that the mean diameter of myelinated fibres in shi sciatic nerves is smaller than in wt sciatic nerves. These results lead us to suggest that the increase in Schmidt-Lanterman incisure density in shi compensates for a defect in Schwann cell-axon communication.

CNS myelin and oligodendrocytes of the Xenopus spinal cord--but not optic nerve--are nonpermissive for axon growth

In vitro assays reveal that myelin and oligodendrocytes of the Xenopus spinal cord (SC) are--unlike corresponding components of the optic nerve/tectum (OT)--nonpermissive substrates for regenerating retinal axons. The number of growth cones that crossed SC oligodendrocytes is low but increases significantly (four- to fivefold) in the presence of the antibody IN-1, in which case their numbers are similar to the number of growth cones (approximately 60%) that cross OT oligodendrocytes with or without IN-1. IN-1 neutralizes neurite growth inhibitors (NI) of rat CNS myelin, indicating that mammalian-like NI are associated with Xenopus SC myelin and oligodendrocytes but not with the OT. IN-1 immunocytochemistry on sections supports this view: SC myelin was stained with IN-1, whereas OT myelin and PNS myelin were not.

Macrophage recruitment in different models of nerve injury: lysozyme as a marker for active phagocytosis.

Macrophages play critical roles in both degenerative and regenerative processes following peripheral nerve injury. These include phagocytosis of debris, stimulation of Schwann cell dedifferentiation and proliferation, and salvage of myelin lipids for reutilization during regeneration. To better define the role of macrophages, we studied models of primary demyelination (tellurium intoxication) and secondary demyelination (nerve crush and cut). Sections of paraformaldehyde-fixed rat sciatic nerves at various stages of demyelination were stained with monoclonal antibody ED1, a standard macrophage marker, and a polyclonal antiserum specific for lysozyme (LYS). Near the peak of demyelination in all three models, LYS immunoreactivity colocalized with ED1 staining. Macrophages present in nerve after the period of maximal phagocytosis of myelin were much less immunoreactive for LYS. These results suggest LYS is a good marker for macrophages which are active in phagocytosis. Tellurium intoxication, which causes synchronous demyelination and subsequent remyelination of only about 25% of myelin internodes, recruited more macrophages (and induced more lysozyme expression) than either nerve crush or cut, which cause demyelination of all internodes distal to the injury site. This suggests that Schwann cells may recruit macrophages soon after metabolic insult and prior to actual demyelination. The final signal for macrophage recruitment is not directly related to the amount of damaged myelin. In the models listed above, steady state mRNA levels for apolipoprotein E (ApoE; possible mediator of cholesterol salvage), LYS, and P0 (major structural protein of PNS myelin), were analyzed by Northern blot analysis. LYS mRNA levels peaked sharply in all models, with a temporal pattern consistent with the expected presence of activated, phagocytic macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration

Following nerve injury, axons in the CNS do not normally regenerate. It has been shown that CNS myelin inhibits neurite outgrowth, though the nature of the molecules responsible for this effect are not known. Here, we demonstrate that the myelin-associated glycoprotein (MAG), a transmembrane protein of both CNS and PNS myelin, strongly inhibits neurite outgrowth from both developing cerebellar and adult dorsal root ganglion (DRG) neurons in vitro. This inhibition is reversed by an antiMAG antibody. In contrast, MAG promotes neurite outgrowth from newborn DRG neurons. These results suggest that MAG may be responsible, in part, for the lack of CNS nerve regeneration in vivo and may influence, both temporally and spatially, regeneration in the PNS.

Sulfate metabolism of rat P0 glycoprotein: some observations

It has been known for some time that P0, the major intrinsic protein in PNS myelin, contains sulfate. The position of sulfate has been described for beef PNS myelin, but rat PNS myelin differs somewhat from that of the beef, therefore an investigation of the location of sulfate in rat P0 was undertaken. Weanling rat nerves were incubated with [3H] amino acid mixture and [35S]O4, and purified myelin was prepared, and the proteins separated on polyacrylamide gels. The bulk of the [35S]O4 was incorporated into P0, but smaller peaks of sulfate label were found in the higher molecular weight proteins. With tunicamycin in the incubation mixture, sulfate incorporation was inhibited. Incubation of the labeled myelin mixture with endo F or glycanase resulted in total loss of sulfate label on P0, therefore all of the [35S]O4 was incorporated into the oligosaccharide chain, with none on the polypeptide. Castanospermine and deoxymannojirimycin inhibited [35S]O4 incorporation into P0, but no inhibition was exerted by swainsonine. These results indicate that sulfate resides in the core of the oligosaccharide chain, with none in the terminal region. Such a structure would correlate with the lack of an HNK-1 epitope, absent in the rat, but found in P0 of many species.

Contact with myelin evokes a release of calcium from internal stores in neonatal rat oligodendrocytes in vitro

The response of neonatal rat oligodendrocytes to contact with myelin extracts prepared from the central and peripheral nervous system was examined. Contact with either CNS myelin or PNS myelin resulted in collapse of the fine structure of the leading edge of oligodendrocytes in vitro. The collapse of the fine structure of oligodendrocyte processes was preceded by a substantial (approximately fivefold) increase in intracellular free calcium concentration. The calcium concentration increase was due, at least in part, to a release of calcium from internal stores, since it persisted when extracellular calcium was removed by chelation with EGTA. The increase in calcium concentration and the coincident morphological change suggest that oligodendrocytes might be able to recognize and react to specific molecules on the surface of other oligodendrocytes.

Identification of the palmitoylation site in rat myelin P0 glycoprotein.

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 palmitate was released by incubation with the reducing agents dithiothreitol and 2-mercaptoethanol, and with 1 M hydroxylamine at pH 7.5. In addition, P0 was deacylated by treatment with 10 mM 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 P0 was 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 transmembrane and cytoplasmic domains. This residue is also present in the P0 glycoprotein of other species, including human, bovine, mice, and chicken.

Locus coeruleus neuron growth cones and spinal cord regeneration

One of the major impediments to successful recovery of function after a spinal cord injury is thought to be the reaction of the neuronal growth cone to inhibitory influences in the local environment in or around the site of the injury. The growth cones of locus coeruleus neurons studied in vitro collapsed upon contact with an extract of CNS myelin but did not collapse on contact with an extract of PNS myelin. Coincident with the collapse of the growth cone, was an increase in internal free calcium concentration that was predominantly the result of an influx of calcium through channels in the plasma membrane. Omega-conotoxin, which specifically blocks N-type voltage-gated calcium channels, blocked both the myelin-induced calcium influx and the coincident collapse of the growth cone.

Myelin P0‐Glycoprotein: Predicted Structure and Interactions of Extracellular Domain

Protein zero (P0), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P0 (P0-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 A, 50 A (native), and 90 A between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P0-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that beta-strand predictions for P0-ED are virtually superimposable with those of the VH domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P0-ED and a structure that has been solved crystallographically. The dimensions of P0-ED from the space-filling model, developed using PC-based molecular modeling software, were found to be 44 A x 25 A x 23 A. On the assumption that neither the shape nor the orientation of P0-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P0-ED from apposed membranes if the molecules were oriented so that the beta-strands were approximately perpendicular to the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular genetics of myelination: an update.

Recent molecular genetic studies have provided new insights into the structure and function of 2 of the major integral membrane proteins of myelin--the proteolipid protein (PLP) and protein zero (P0)--and have uncovered a third such protein--PMP22/gas3. The rumpshaker mouse has been shown to carry a point mutation in the PLP gene that uncouples a deleterious effect on CNS myelin assembly, which these mice exhibit, from oligodendrocyte degeneration and cell death, which they do not. The developmental importance of the P0 protein in PNS myelination has been dramatically demonstrated by the analysis of loss-of-function mutations engineered through the expression of antisense RNA and through the insertional inactivation of the P0 gene by homologous recombination in embryonic stem cells and the generation of P0-deficient mice. The cloned promoter of the P0 gene has been shown to drive quantitative, Schwann cell-specific expression of heterologous genes in transgenic mice. The PMP22/gas3 gene, previously cloned from fibroblast cell lines, has been found to encode an axonally regulated Schwann cell protein that is assembled into PNS myelin. Importantly, this gene appears to be the target of mutations that result in the Trembler alleles in mice, and in Charcot-Marie-Tooth disease Type 1a, the most common inherited peripheral neuropathy in humans.

Detection of multisulphated N-linked glycans in the L2/HNK-1 carbohydrate epitope expressing neural adhesion molecule P0.

P0, the most abundant glycoprotein of PNS myelin, is a homophilic and heterophilic adhesion molecule. P0 is known to contain a glycoform population that expresses the L2/HNK-1 carbohydrate epitope found on other neural adhesion molecules, and to be functionally implicated centrally in neural cell adhesion and neurite outgrowth. This carbohydrate epitope has been characterized previously from glycolipid structures and contains a sulphated glucuronic acid residue. However, the L2/HNK-1 carbohydrate epitope has not been characterized in glycoproteins. Because P0 possesses only one glycosylation sequon, the number of P0 glycoforms is equal to the heterogeneity of the glycan species. Here we report that the carbohydrate analysis of L2/HNK-1-reactive P0 showed the presence of anionic structures containing sialic acid and sulphate in various combinations. At least one sulphate residue was present in 80% of the monosaccharide sequences, and 20% contained three sulphates. High-resolution P4 gel chromatography of the desialylated and desulphated oligosaccharides showed substantial heterogeneity of monosaccharide sequences. Sequential exoglycosidase digestions indicated that the majority of the structures were of the hybrid class, although the sulphated structures were found to be endoglycosidase H-resistant.

Tellurium‐Induced Alterations in 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase Gene Expression and Enzyme Activity: Differential Effects in Sciatic Nerve and Liver Suggest Tissue‐Specific Regulation of Cholesterol Synthesis

The demyelination of peripheral nerves that results from exposure of developing rats to tellurium is due to inhibition of squalene epoxidase, a step in cholesterol biosynthesis. In sciatic nerve, cholesterol synthesis is greatly depressed, whereas in liver, some compensatory mechanism maintains normal levels of cholesterol synthesis. This tissue specificity was further explored by examining, in various tissues, gene expression and enzyme activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Exposure to tellurium resulted in pronounced increases in both message levels and enzyme activity in liver, the expected result consequent to up-regulation of this enzyme in response to decreasing levels of intracellular sterols. In contrast to liver, levels of mRNA and enzyme activity in sciatic nerve were both decreased during the tellurium-induced demyelinating period. The temporal pattern of changes in 3-hydroxy-3-methylglutaryl-CoA reductase message levels in sciatic nerve seen following exposure to tellurium was similar to the down-regulation seen for mRNA specific for PNS myelin proteins. Possible mechanisms for differential control of cholesterol biosynthesis in sciatic nerve and liver are discussed.

A monoclonal antibody against HSV type 1 ribonucleotide reductase cross-reacts with the P 0 protein of peripheral nerve myelin

An epitope on peripheral nerve myelin was detècted by the use of a mouse monoclonal antibody directed against the 38 kDa subunit of herpes simplex virus (HSV) type 1 ribonucleotide reductase. Immunohistochemistry showed reactivity solely in PNS myelin. In nerve roots there was a sharp border in transitional zones to the negative CNS myelin. The immunoreactivity was found in rat, guinea pig, bovine and human peripheral nerves. Western blot analysis of peripheral nerve myelin as well as purified P 0 revealed a distinctly stained band corresponding to a molecular weight of approximately 29 kDa. The present finding of a shared antigenic determinant between HSV ribonucleotide reductase and peripheral nerve P 0 may be of pathogenetic relevance in virus induced demyelinating diseases in the peripheral nervous system.

Primary demyelination induced by exposure to tellurium alters mRNA levels for nerve growth factor receptor, SCIP, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, and myelin proteolipid protein in rat sciatic nerve

Weanling rats fed a diet containing tellurium develop a peripheral neuropathy characterized by a highly synchronous primary demyelination; this demyelination is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis. Expression of mRNA for the major structural proteins of PNS myelin, myelin basic protein and P 0, is coordinately down-regulated during the demyelinating phase and then up-regulated during the remyelinating phase (Toews et al., J. Neurosci. Res., 26 (1990) 501–507). We now report tellurium-induced alterations in gene expression for several proteins which are not major structural components of myelin in the peripheral nervous system. Expression of mRNA for nerve growth factor receptor in sciatic nerve was very low in control animals, but was markedly up-regulated after 3–5 days of exposure to tellurium, a time corresponding to the beginning of demyelination. Levels remained elevated during the subsequent period of remyelination. Expression of mRNA for SCIP (a presumptive transcription factor) was also up-regulated in sciatic nerve following tellurium exposure, with a time course similar to that for nerve growth factor receptor. When examined as a fraction of total RNA, steady-state mRNA levels for 2′,3′-cyclic nucleotide 3′-phosphodiesterase and the myelin proteolipid protein were decreased during the demyelinating phase; however, this decrease could be largely accounted for the increased levels of total RNA. When analyzed on a ‘per nerve’ basis, steady-state mRNA levels for these two proteins were actually increased about 2-fold by 9 days after beginning tellurium exposure. Differences in alterations of mRNA levels between these two ‘myelin-related’ proteins and the two major structural proteins of PNS myelin suggest that in the PNS, 2′,3′-cyclic nucleotide 3′-phosphodiesterase and the myelin proteolipid protein may play roles distinct from their functions in CNS myelin.

High Expression of the HNK‐1/L2 Carbohydrate Epitope in the Major Glycoproteins of Shark Myelin

The major 24- and 28-kDa glycoproteins in shark PNS and CNS myelin express high levels of the adhesion-associated HNK-1/L2 carbohydrate epitope. The 28-kDa protein, but not the 24-kDa protein, cross-reacts strongly with one of two anti-bovine P0 antisera not previously tested against fish myelin proteins. Shark PNS and CNS myelin also contains smaller amounts of high-molecular-weight HNK-1-positive proteins, including a prominent broad band in the 65-85-kDa range. Although myelin-associated glycoprotein (MAG) is well known to react with HNK-1 in some mammals, monoclonal and polyclonal anti-MAG antibodies did not react with the high-molecular-weight HNK-1-positive material in shark myelin, a result suggesting that it is not a MAG-like protein. The high expression of the HNK-1/L2 epitope in glycoproteins of shark myelin, including the major P0-related ones, suggests that this adhesion-related carbohydrate structure may have had an important role in the molecular evolution of the myelinating process.

Membrane flow within the myelin sheath in IDPN neuropathy

This report describes some aspects of beta,beta'-iminodipropionitrile (IDPN) neuropathy in rats as observed by ultrastructural methods and X-ray diffraction. Light microscopy shows gross swelling of the axons in proximal lumbar spinal roots 8 days after intraperitoneal injection of IDPN. Mean axon cross-sectional area and mean axon perimeter increased to 280% and 160% of their control values, respectively. At the same time, myelin membrane packing was not visibly disturbed. In addition, X-ray diffraction patterns, recorded under physiological conditions, demonstrate that the myelin lipid bilayer thickness and widths of the aqueous spaces between bilayers did not change. Related observations are made on posterior tibial nerve (PNS myelin) and ventral spinal cord (CNS myelin). The various observations together are interpreted in terms of a fluid myelin membrane. It is proposed that the myelin membrane flows during axon swelling even though normal membrane-membrane contacts are maintained within the sheath. Membrane flow and slippage between membranes are explained in terms of a molecular model of the myelin multilayer.

Effects of Inhibitors of Oligosaccharide Processing on P0Protein Synthesis and Incorporation into PNS Myelin

Four inhibitors of oligosaccharide processing were used to investigate their effects on the transport of PNS myelin glycoproteins through the secretory pathway, as well as to gain further insight into the structure of the oligosaccharide chains of the P0 and 19-kDa glycoproteins. Several different inhibitors of oligosaccharide processing were incubated with chopped peripheral nerves from young rats (21-24 days of age) and the uptake of 14C-amino acid and [3H]fucose or [3H]mannose was measured in P0 and the 19-kDa glycoprotein after separation of homogenate and myelin proteins on polyacrylamide gels. [3H]Mannose was not found as suitable as [3H]fucose as an oligosaccharide precursor because glucose used as an energy source profoundly inhibited the uptake of [3H]mannose. The substitution of pyruvate as an energy source, however, resulted in incomplete glycosylation, poor amino acid uptake, and truncated oligosaccharide chains. Endoglycosidase H cleaved approximately 50% of the P0 labeled with [3H]fucose and 14C-amino acid. The lower molecular weight protein resulting from endoglycosidase H cleavage contained approximately one-half the [3H]fucose label on the protein, whereas one-half remained on the oligosaccharide chain of the undegraded P0, indicating that at least one-half the P0 has a hybrid structure. Deoxynojirimycin, deoxymannojirimycin, and castanospermine inhibited incorporation of [3H]fucose into the oligosaccharide chains of P0 and the 19-kDa glycoprotein as predicted from their action in blocking various stages of trimming of high mannose structures before the addition of fucose. P0 synthesized in the presence of these inhibitors was cleaved to a greater extent by endoglycosidase H than the normal protein, indicating increased vulnerability to this enzyme with arrest of normal processing. Similar results were obtained for the 19-kDa glycoprotein. Both the incompletely processed P0 and the 19-kDa glycoprotein formed in the presence of these inhibitors appeared to be transported normally into myelin.