Peripheral Nerve Myelin Research Articles

Suppression of Autoimmune Neuritis in IFN-γ Receptor-Deficient Mice

Experimental autoimmune neuritis (EAN) is an animal model of the human disease Guillain–Barré syndrome. In this autoimmune inflammatory disease, CD4+ T cells mediate demyelination in the peripheral nervous system (PNS). Infiltrating macrophages and T cells as well as cytokines like interferon (IFN)-γ are intimately involved in causing pathogenic effects. To investigate the role of IFN-γ in cell-mediated EAN, IFN-γ receptor-deficient mutant (IFN-γR−/−) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180–199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. IFN-γR−/− mice exhibited later onset of clinical disease. The disease was also less severe than in wild-type mice. Fewer IL-12-producing but more IL-4-producing cells were found in sciatic nerve sections from IFN-γR−/− mice than from wild-type mice on day 24 postimmunization, i.e., at the peak of clinical EAN. At the same time, IFN-γR−/− mice had less infiltration of inflammatory cells, including macrophages, CD4+ T cells, and monocytes, into sciatic nerve tissue and less demyelination. However, numbers of IFN-γ-secreting cells from the spleen were significantly augmented in the IFN-γR−/− mice, reflecting a failure of negative feedback circuits. The IFN-γR deficiency did not affect the production of anti-P0 peptide 180–199-specific antibodies. These results indicate that IFN-γ contributes to a susceptibility for EAN in C57BL/6 mice by promoting a Th1 cell-mediated immune response and suppressing a Th2 response.

Variability in immunohistochemistries of IgM M-proteins binding to sulfated glucuronyl paragloboside

Serum IgMs from 4 of 12 patients with polyneuropathy and IgM M-proteins that bind to sulfated glucuronyl paragloboside (SGPG) strongly immunostained the human peripheral nerve myelin (group A), whereas those from the other eight patients strongly immunostained the cytoplasm of the Schwann cells surrounding the myelin sheath with only weak staining of the myelin (group B). Strong immunostaining of peripheral myelin by IgMs from group A patients may be due to the strong cross-reactivities against P0 and peripheral myelin protein-22 (PMP-22), which are localized in compact myelin. Only three patients (all in group B) showed some response to the immunotherapies. Weak reactivities to P0 and and PMP-22 might indicate the possibility of improvement after the immunotherapies.

Contactin orchestrates assembly of the septate-like junctions at the paranode in myelinated peripheral nerve.

Rapid nerve impulse conduction depends on specialized membrane domains in myelinated nerve, the node of Ranvier, the paranode, and the myelinated internodal region. We report that GPI-linked contactin enables the formation of the paranodal septate-like axo-glial junctions in myelinated peripheral nerve. Contactin clusters at the paranodal axolemma during Schwann cell myelination. Ablation of contactin in mutant mice disrupts junctional attachment at the paranode and reduces nerve conduction velocity 3-fold. The mutation impedes intracellular transport and surface expression of Caspr and leaves NF155 on apposing paranodal myelin disengaged. The contactin mutation does not affect sodium channel clustering at the nodes of Ranvier but alters the location of the Shaker-type Kv1.1 and Kv1.2 potassium channels. Thus, contactin is a crucial part in the machinery that controls junctional attachment at the paranode and ultimately the physiology of myelinated nerve.

Progesterone stimulates Krox-20 gene expression in Schwann cells

The gene of the zinc finger transcription factor Krox-20 (Egr-2) is expressed in Schwann cells and plays an important role in myelination of peripheral nerves. We have shown that progesterone promotes myelination in the regenerating sciatic nerve and in cocultures of Schwann cells and sensory neurones. To determine whether progesterone regulates Krox-20 expression, we measured its effects on Krox-20 mRNA levels in the MSC80 mouse Schwann cell line by semi-quantitative RT-PCR. Although low levels of Krox-20 mRNA are detectable in MSC80 cells cultured in defined medium, treatment with 10 −6 M progesterone induces a rapid (15 min) and transient increase in the levels of Krox-20 mRNA. Lower doses of progesterone (10 −9, 10 −8 and 10 −7 M) are also effective in increasing Krox-20 mRNA. Other steroids including testosterone, dexamethasone, and estradiol are ineffective when added to the culture medium at 10 −6 M for 1 h. The induction of Krox-20 mRNA was also observed with the selective progesterone agonist Organon 2058 and was abolished by treating the MSC80 Schwann cells with the progesterone antagonist RU486, indicating that progesterone induces Krox-20 mRNA expression by binding to its intracellular receptor. The induction of Krox-20 by progesterone was also demonstrated in primary cultures of Schwann cells isolated from neonatal rat sciatic nerves, at the mRNA level by RT-PCR and at the protein level by immunohistochemistry. As Krox-20 is a necessary step for the initiation of myelin formation in peripheral nerves, its stimulation by progesterone suggests an important signalling function for this steroid in myelination.

EGR2 mutations in inherited neuropathies dominant-negatively inhibit myelin gene expression.

The identification of EGR2 mutations in patients with neuropathies and the phenotype Egr2/Krox20(-/-) have demonstrated that the Egr2 transcription factor is critical for peripheral nerve myelination. However, the mechanism by which these mutations cause disease remains unclear, as most patients present with disease in the heterozygous state, whereas Egr2(+/-) mice are phenotypically normal. To understand the effect of aberrant Egr2 activity on Schwann cell gene expression, we performed microarray expression profiling to identify genes regulated by Egr2 in Schwann cells. These include genes encoding myelin proteins and enzymes required for synthesis of normal myelin lipids. Using these newly identified targets, we have shown that neuropathy-associated EGR2 mutants dominant-negatively inhibit wild-type Egr2-mediated expression of essential myelin genes to levels sufficiently low to result in the abnormal myelination observed in these patients.

αβ1 Integrins Are Required For Normal Peripheral Nerve Development

Adhesion to basal lamina is required for Schwann cells (SC) to form normal myelin in peripheral nerves. In human Congenital Muscular Dystrophy (CMD), mutations in laminin 2 (α2 chain) cause a dysmyelinating peripheral neuropathy with slow nerve conduction velocities. The dy2J/dy2J mouse also carries a mutation in the laminin α2 chain, and is considered an animal model for CMD. The dysmyelination in the dy2J/dy2J mouse is dramatic in nerve roots, where SC fail to sort bundles of naked axons. In distal peripheral nerves, myelination can proceed, even if axon/SC interactions and nodes of Ranvier are abnormal. Which of the SC laminin receptors, integrins or dystroglycan mediate these functions is unclear because targeted inactivation of those genes results in embryonic death in mice prior to myelination. Several in vitro studies suggest β1 integrin as a candidate for a role in axon/SC interaction and myelination. To inactivate the β1 integrin gene (mIntb1) in SC only, we produced transgenic mice expressing Cre recombinase using the vector mP0TOT, that activates expression of genes in SC in parallel with P0 during development. By crossing mP0TOT(Cre) mice with mice carrying a mIntb1 flanked by loxP sites of recombination, we produced mice lacking β1 integrin specifically in SC. These mice have a severe dysmyelinating peripheral neuropathy, originating in embryonic nerve development. Although Schwann cells populate the length of forming nerves, and are present in numbers comparable to wildtype nerves, they are unable to penetrate bundles of naked axons in order to sort and ensheath them. In some nerve fascicles, SC can bypass the block, even in the absence of β1 integrin, and myelinate normally. These data show that αβ1 integrins are necessary for normal Schwann cell‐axon interactions, but not for myelination per se. The similarity of this phenotype to that of dy2J/dy2J mouse suggests that αβ1 integrins mediate some of the effects of altered laminin‐2 in impaired CMD peripheral nerves.

Morphological And Electrophysiological Signs Of Dysmyelination In Transgenic Mice Expressing CMT1B (MPZ DELSer34) or DSS (MPZ Ser34Cys) Mutations

Charcot‐Marie‐Tooth 1B neuropathy (CMT1B), Déjèrine‐Sottas syndrome (DSS) and Congenital Hypomyelination are each associated with mutations in MPZ, encoding P0 glycoprotein, the major structural protein of peripheral nerve myelin. To explore whether new abnormal functions of mutant MPZ can explain this phenotypic diversity, we expressed either of two MPZ mutations: DELSer34 (causing CMT1B) or Ser34Cys (causing DSS) in addition to the two normal endogeneous copies of Mpz in transgenic mice. We have shown previously that overexpression of wild type Mpz causes dose‐dependent dysmyelination. However, multiple lines of mice containing the MPZ mutants showed not only hypomyelination, but also abnormalities in myelin sheaths and onion bulbs that were never observed in Mpz overexpressor mice. To create a mouse model of CMT1B with no Mpz overexpression, one line of DELSer34 that expresses mutant Mpz at levels similar to one wildtype Mpz allele was crossed with heterozygous Mpz knock‐out mice, to obtain the genotype Mpzwt/−/MpzDELSer34. These mice manifest progressive peripheral neuropathy with hypomyelination, and onion bulbs that appear around 6 months of age. As a first step towards longitudinal studies of phenotype, we performed detailed neurophysiological analyses at 12 months of age. We found signs of demyelination, with statistically significant increases of both motor latency after proximal stimulation and F‐wave latencies, and decreases in both motor and mixed afferent nerve conduction velocities of sciatic nerve. The motor response was polyphasic and there was a trend towards reduced CMAP amplitudes. Thus, the clinical onset and progression, pathological features and neurophysiological findings provide a reasonable model of CMT1B. Longitudinal studies to correlate the onset and progression of morphological and electrophysiological abnormalities in these mice are ongoing.

Novel MPZ Mutation In A Sporadic CMT Patient

Mutations in the gene for the major structural protein component of peripheral nerve myelin, myelin protein zero (MPZ), are associated with some forms of hereditary neuropathies such as Charcot‐Marie‐Tooth disease type 1B (CMT1B), Dejerine‐Sottas syndrome (DSS) and congenital hypomyelinating neuropathy (CHN). The common pathological characteristics of these allelic disorders are severe demyelination and remyelination of peripheral nerves. Recently, MPZ mutations were also found in patients with the axonal form of CMT neuropathy (CMT2). We studied a patient with negative familiar history and clinical and electrophysiological features of Charcot‐Marie‐Tooth disease: distal muscle weakness and atrophy, foot deformities (pes cavus), and severely reduced nerve conduction velocities in the motor and sensory nerves. The sural nerve biopsy showed marked loss of myelinated fibers, few onion bulbs, and a high percentage of fibers showing excessive myelin outfoldings. DNA analysis excluded CMT1A duplication by Southern blot and by pulsed field gel electrophoresis methods. SSCP analysis of all six exons of MPZ revealed a shift band in exon 2 in the patient's DNA. No such difference was detected in normal controls. Direct sequencing disclosed a G → A transition at nucleotide position 181. This base substitution predicts the replacement of aspartic acid with asparagine at codon 61. A mutation at the same codon (but different amino acid replacement) was recently identified in a family with the axonal type of CMT, in which the disease was autosomal dominantly inherited. This finding provides further confirmation of the role of MPZ gene in peripheral neuropathies and suggests that MPZ coding region mutations may account for a considerable number of CMT cases which do not involve DNA duplication on 17p11.2‐p12. This research was partially supported by a MURST and an Ateneo grant to FA, by a Ministero della Sanità grant to PM. Our laboratory is a member of the European Charcot‐Marie‐Tooth Consortium co‐ordinated by Prof. Christine Van Broeckhoven.

Role Of Campylobacter Jejuni In Experimental Allergic Neuritis: A Morphological And Biochemical Study

Objective: The aim of the study was to evaluate if Campylobacter jejuni (C.j.) when used as an adjuvant would be able to produce a different form of Experimental Allergic Neuritis (EAN). We present here some preliminary results. Background: EAN is considered the in vivo model of Guillain‐Barrè Syndrome (GBS), which is often preceded by c.j. infection. EAN can be induced in Lewis rats by immunization with bovine peripheral nerve myelin in complete Freund's adjuvant (CFA), an emulsion formed by oil‐in‐water and dead mycobacteria. An adjuvant is usually necessary for the induction of EAN because it enhances the immunogenicity of the antigen. Clinically EAN is characterized by an acute monophasic course and progressive tail and limb weakness. The pathological finding is represented by marked demyelination affecting the roots and the sciatic nerve. Methods: 4 Lewis rats were immunized with an emulsion containing 2 mg of bovine peripheral myelin and C.j. strain Penner 0:41 in incomplete Freund's adjuvant (IFA). They were compared to 4 controls immunized with the same amount of peripheral myelin in CFA. The clinical course of the disease and the histological pattern of the roots and the sciatic nerve were examined. Anti‐peripheral myelin, anti‐C.jejuni and anti‐GM1 antibodies' reactivity was detected by an ELISA assay. A biochemical study was performed to test the role of cell‐ and humoral‐mediated responses. Results: The Lewis rats immunized with the C.j. as an adjuvant showed a delayed onset and a milder course of disease. Pathology in the roots was characterized by predominant demyelination, whereas the sciatic nerve presented very little signs of damage. Conclusion: This serotype of C.j. appears to be a less effective adjuvant in inducing EAN rather than Mycobacteria. Further studies are necessary to elucidate the pathogenetic mechanisms involved in GBS.

CD28–B7 costimulation: a critical role for initiation and development of experimental autoimmune neuritis in C57BL/6 mice

CD28 provides a critical costimulatory signal for antigen-specific T cell activation. Because CD28 is an important factor in the development of autoimmune diseases, we investigated its role in T cell-mediated experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. CD28-deficient mutant (CD28 −/−) C57BL/6 mice and corresponding wild-type mice were immunized with P0 peptide 180–199, a purified component of peripheral nerve myelin, and Freund’s complete adjuvant. As a result, all wild-type mice developed severe EAN, in contrast, none of the CD28 −/− mice manifested clinical signs of disease. Additionally, CD28 −/− mice had fewer IL-12 producing cells in sciatic nerve sections and fewer IFN-γ secreting splenic cells than wild-type mice on day 24 post immunization, i.e., at the peak of clinical EAN. At that time point, CD28 −/− mice had milder infiltration of such inflammatory cells as macrophages, CD4 + T cells and monocytes into sciatic nerve tissues and less demyelination than wild-type mice. Moreover, the CD28-deficiency led to reduced production of specific anti-P0 peptide 180–199 antibodies compared with wild-type mice. Evidently, CD28 is required for interaction with B7 to regulate the activation of T and B cells that initiates development of EAN.

P0 glycoprotein peptides 56–71 and 180–199 dose-dependently induce acute and chronic experimental autoimmune neuritis in Lewis rats associated with epitope spreading

Two synthetic peripheral nerve myelin P0 protein peptides, an immunodominant (amino acids 180–199) and a cryptic (amino acids 56–71) one, induced an acute or chronic course of experimental autoimmune neuritis (EAN) in Lewis rats, when given at low dose (50–100 μg/rat) or high dose (250 μg/rat), respectively. Corresponding to the different clinical course, pathological changes and immune responses were found: (1) Onset of clinical signs of P0 peptide 56–71 (P0 56–71) induced EAN was 1–3 days later than in P0 peptide 180–199 (P0 180–199) induced EAN at all immunizing doses, whereas the peak of the disease occurred at a similar time point post immunization (p.i.), i.e. at days 14–16 p.i. in P0 56–71 induced EAN and at day 16 p.i. in P0 180–199 induced EAN. (2) Intramolecular epitope spreading as assessed by delayed type hypersensitivity response occurred in P0 56–71 induced EAN at both low and high antigen doses and in P0 180–199 induced EAN at high antigen dose (250 μg/rat) only. (3) P0 180–199 stimulated higher levels of interferon-γ production in P0 180–199 induced EAN than in P0 56–71 induced EAN and vice versa. (4) Histopathologic evaluation revealed a similar grade of mononuclear cell infiltration in the sciatic nerves of both types of EAN, but more severe demyelination was found in P0 180–199 induced EAN compared to P0 56–71 induced EAN. The results support the hypothesis that high dose autoantigen immunization induces extensive determinant spreading and chronic course of autoimmune diseases.

Periaxin Mutations Cause Recessive Dejerine-Sottas Neuropathy

The periaxin gene (PRX) encodes two PDZ-domain proteins, L- and S-periaxin, that are required for maintenance of peripheral nerve myelin. Prx(-/-) mice develop a severe demyelinating peripheral neuropathy, despite apparently normal initial formation of myelin sheaths. We hypothesized that mutations in PRX could cause human peripheral myelinopathies. In accordance with this, we identified three unrelated Dejerine-Sottas neuropathy patients with recessive PRX mutations-two with compound heterozygous nonsense and frameshift mutations, and one with a homozygous frameshift mutation. We mapped PRX to 19q13.13-13.2, a region recently associated with a severe autosomal recessive demyelinating neuropathy in a Lebanese family (Delague et al. 2000) and syntenic to the location of Prx on murine chromosome 7 (Gillespie et al. 1997).

No evidence of auditory dysfunction in guinea pigs immunized with myelin P0 protein

Recent data have focused on the peripheral nerve myelin glycoprotein P0 as a putative autoantigen involved in the autoimmune etiology of some cases of Meniere’s disease, idiopathic sensorineural hearing loss and sudden deafness. To determine whether antibodies to myelin P0 can alter cochlear function, 13 healthy guinea pigs were immunized with purified porcine myelin P0 while 10 controls were injected with saline water. The animals were then evaluated for evidence of evolving inner ear disease using immunological, electrophysiological and morphological methods. Twenty-six experimental ears were tested weekly with a brainstem auditory evoked potential technique for a period of 4 months and were compared to 20 control ears. Uniformly, all P0-sensitized guinea pigs showed antibodies to myelin protein P0 as evidenced by ELISA. Clinical signs of inflammatory demyelination were not discernible in P0-sensitized guinea pigs and all the animals were qualitatively normal. No significant increase of evoked potential thresholds was found in the P0-sensitized animals when compared to controls ( P>0.05). Peak latencies of waves I, II, III, IV and V and inter-peak latencies in P0-sensitized guinea pigs did not significantly differ from those of controls ( P>0.05). Histological sections of inner ear and peripheral nerves were free of disease in both groups. These findings indicate that the sole presence of antibodies to myelin P0 in the sera of guinea pigs or patients suspected of having autoimmune inner ear diseases is unlikely to elicit auditory abnormalities and that additional factors are necessary for the pathogenic development of these disorders.

ギランバレー症候群に自己免疫性溶血性貧血を併発した一症例

A 68-year-old man was admitted to our hospital with the chief complaint of progressive weakness of the limbs.Upon neurological examination, he demonstrated facial palsy, flaccid quadriplegia, and demon-strated no response to all deep tendon reflexes stimulation. Laboratory results showed abnormally elevated levels of anti-ganglioside GM1, asialo-GM1, GD1b, and GM3 antibodies. Stool culture for campylobacter species was negative.A diagnosis of a Guillain-Barre syndrome (GBS) was made and three plasma exchanges were administered. Following the treatment, muscle strength was gradually recovered. Progressive anemia of a mild degree was observed with an increase of unconjugated bilirubin. The direct (DAT) and indirect antiglobulin tests were positive, although the decrease of haptoglobin level was not so pronounced. Testing for Donath-Landsteiner antibody proved negative and cold hemagglutinin test was normal. DAT became negative concomitantly with the recovery from anemia and muscle weakness.The absorption of purified GM3 resulted in remarkably weak reactivity, indicating the affinity of anti-GM3 antibody with the erythrocyte membrane in this patient. Thus, the presence of anti-GM3 antibody, which has common reactivity with peripheral nerve myelin and erythrocyte membrane, may contribute to GBS becoming complicated with hemolytic anemia.

EXPERIMENTAL ALLERGIC NEURITIS IN THE SJL/J MOUSE: INDUCTION OF SEVERE AND REPRODUCIBLE DISEASE WITH BOVINE PERIPHERAL NERVE MYELIN AND PERTUSSIS TOXIN WITH OR WITHOUT INTERLEUKIN‐12

We report a reproducible model of experimental allergic neuritis (EAN) with severe clinical signs and consistent pathological features in mice. Pertussis toxin (PT) in the presence or absence of murine recombinant interleukin‐12 (mrIL‐12) was used as an adjuvant with bovine peripheral nerve myelin (BPNM) to induce clinical EAN in SJL/J mice. After immunization with a combination of BPNM in complete Freund's adjuvant (CFA) and PT, mice developed severe consistent signs of EAN. The additional treatment of immunized mice with mrIL‐12 prolonged the course of EAN characterized by earlier clinical signs of the disease and delayed the recovery stage. Mice injected with BPNM and CFA without PT developed mild clinical signs. Histological examination of the caudae equinae and the sciatic nerves taken from mice with clinical signs of EAN during the recovery stage revealed severe demyelination, remyelination and remnants of mononuclear cell infiltration. Moderate to severe EAN can be induced in SJL/J mice by the injection of a combination of BPNM in CFA and PT. This model can provide a better understanding of mechanism of demyelination in infiltrating peripheral neuropathy.

Epitope-tagged P(0) glycoprotein causes Charcot-Marie-Tooth-like neuropathy in transgenic mice.

In peripheral nerve myelin, the intraperiod line results from compaction of the extracellular space due to homophilic adhesion between extracellular domains (ECD) of the protein zero (P(0)) glycoprotein. Point mutations in this region of P(0) cause human hereditary demyelinating neuropathies such as Charcot-Marie-Tooth. We describe transgenic mice expressing a full-length P(0) modified in the ECD with a myc epitope tag. The presence of the myc sequence caused a dysmyelinating peripheral neuropathy similar to two distinct subtypes of Charcot-Marie-Tooth, with hypomyelination, altered intraperiod lines, and tomacula (thickened myelin). The tagged protein was incorporated into myelin and was associated with the morphological abnormalities. In vivo and in vitro experiments showed that P(0)myc retained partial adhesive function, and suggested that the transgene inhibits P(0)-mediated adhesion in a dominant-negative fashion. These mice suggest new mechanisms underlying both the pathogenesis of P(0) ECD mutants and the normal interactions of P(0) in the myelin sheath.

Electrophysiological features of inherited demyelinating neuropathies: A reappraisal in the era of molecular diagnosis.

The observation that inherited demyelinating neuropathies have uniform conduction slowing and that acquired disorders have nonuniform or multifocal slowing was made prior to the identification of mutations in myelin-specific genes which cause many of the inherited disorders involving peripheral nerve myelin. It is now clear that the electrophysiological aspects of these disorders are more complex than previously realized. Specifically, certain mutations appear to induce nonuniform slowing of conduction which resemble the findings in acquired demyelinating neuropathies. It is clinically important to recognize the different electrodiagnostic patterns of the various inherited demyelinating neuropathies. In addition, an understanding of the relationship between mutations of specific genes and their associated neurophysiological findings is likely to facilitate understanding of the role of these myelin proteins in peripheral nerve function and of how abnormalities in myelin proteins lead to neuropathy. We therefore review the current information on the electrophysiological features of the inherited demyelinating neuropathies in hopes of clarifying their electrodiagnostic features and to shed light on the physiological consequences of the different genetic mutations.

A Novel Locus for Autosomal Recessive Peripheral Neuropathy in the EGR2 Region on 10q23

During our studies of Romany (Gypsy) families with hereditary motor and sensory neuropathy-Lom, we have identified a large kindred with two independently segregating autosomal recessive neuropathies. The novel disorder, named "hereditary motor and sensory neuropathy-Russe" (HMSNR), presented as a severe disabling form of Charcot-Marie-Tooth disease with prominent sensory loss, moderately reduced motor nerve conduction velocity, and a high threshold for electrical nerve stimulation. A genome scan in two branches of the large kindred detected linkage to the 10q22-q23 region containing the early growth response 2 gene (EGR2), a transcription factor with a key role in peripheral nerve myelination. The results of sequence analysis and the detection of an intragenic polymorphism allowed us to exclude EGR2 as the HMSNR gene. Further analysis done using linkage and recombination mapping refined the position of the HMSNR gene to a small interval on 10q23.2, flanked by markers D10S581 and D10S1742, telomeric to EGR2. In this interval, a conserved seven-marker haplotype is shared by all disease chromosomes, suggesting a single founder mutation. The homozygosity region is contained in bacterial-artificial-chromosome contig 1570 of the Sanger Centre physical map and has an estimated physical size of approximately 500 kb.

Structure of a major oligosaccharide of PASII/PMP22 glycoprotein in bovine peripheral nerve myelin.

The amino acid sequence of the glycopeptide obtained from bovine PASII/PMP22 protein in the PNS myelin was determined to be Gln-Asn-Cys-Ser-Thr, where the asparagine was glycosylated. To eliminate all the contaminated P(o) glycopeptides from the PASII/PMP22 glycopeptide preparation, we used a fluorescent probe, N-[2-(2-pyridylamino)ethyl]maleimide, which reacts with the cysteine of the PASII/PMP22 glycopeptides. The labeled PASII/PMP22 glycopeptides were isolated by HPLC and were digested further with glycopeptidase A. The resultant oligosaccharides were conjugated with 2-aminopyridine (PA) as a fluorescent tag. One major PA-oligosaccharide, OPPE1, was purified by HPLC. The structure of OPPE1 was elucidated by fast atom bombardment mass spectrometry and (1)H-NMR studies and comparing the derivatives of PA-OPPE1 and PA-oligosaccharides of gamma-globulin on HPLC. The structure, SO(4)-3GlcAbeta1-3Galbeta1-4GlcNAcbeta1-2Manalpha1+ ++-6(GlcNAcbeta1-4) (GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1- 4(Fucalpha1-6)GlcNAc- PA, was identical to the pyridylaminated form of the major oligosaccharide D8 of bovine P(o) previously reported.

Evidence for expression of some microtubule-associated protein 1B in neurons as a plasma membrane glycoprotein.

Microtubule-associated protein (MAP) 1B is a high-molecular-weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for axonal growth. Various biochemical and immunocytochemical results are reported, indicating that a significant fraction of MAP1B is expressed as an integral membrane glycoprotein in vesicles and the plasma membrane of neurons. MAP1B is present in microsomal fractions isolated from developing rat brain and fractionates across a sucrose gradient in a manner similar to synaptophysin, a well-known vesicular and plasma membrane protein. MAP1B is also in axolemma-enriched fractions (AEFs) isolated from myelinated axons of rat brain. MAP1B in AEFs and membrane fractions from cultured dorsal root ganglion neurons (DRGNs) remains membrane-associated following high-salt washes and contains sialic acid. Furthermore, MAP1B in intact DRGNs is readily degraded by extracellular trypsin and is labeled by the cell surface probe sulfosuccinimidobiotin. Immunocytochemical examination of DRGNs shows that MAP1B is concentrated in vesicle-rich varicosities along the length of axons. Myelinated peripheral nerves immunostained for MAP1B show an enrichment at the axonal plasma membrane. These observations demonstrate that some of the MAP1B in developing neurons is an integral plasma membrane glycoprotein.