Major Peripheral Myelin Protein Research Articles

Inherited demyelinating peripheral neuropathies: relating myelin packing abnormalities to P0 molecular defects.

P0-glycoprotein, the major integral membrane protein of peripheral nerve myelin, is thought to mediate myelination and membrane interactions via its extracellular domain (P0-ED). Molecular modeling of P0-ED has suggested which of its amino acid side-chains may be involved in heterophilic and homophilic adhesions. We previously showed that some of these amino acids are the same ones that are substituted or deleted due to mutations in the human gene for P0 (MPZ), which correlate with certain cases of demyelinating motor and sensory peripheral neuropathies. In the current study, high magnification electron microscopy was used to examine the myelin membrane packing in sural nerve biopsies from patients with MPZ mutations. We found that there were distinguishable ultrastructural phenotypes that could be explained by the alterations in P0-ED. These phenotypes, which were not observed in a control nerve, included widening or irregularity of the extracellular apposition alone (delta Ser34; Arg69Cys), widening at both the extracellular and cytoplasmic appositions (Arg69His), the presence of focal bridges in the widened extracellular space (Arg69His), and a diminished (Arg69Cys) or absence (Arg69His) of staining of the double intraperiod line. Our study, which suggests that the altered P0 is incorporated into the myelin sheath, provides a unique basis for further molecular/ultrastructural correlations between P0-ED structure and myelination irregularities.

Inherited demyelinating peripheral neuropathies: Relating myelin packing abnormalities to P0 molecular defects

P0-glycoprotein, the major integral membrane protein of peripheral nerve myelin, is thought to mediate myelination and membrane interactions via its extracellular domain (P0-ED). Molecular modeling of P0-ED has suggested which of its amino acid side-chains may be involved in heterophilic and homophilic adhesions. We previously showed that some of these amino acids are the same ones that are substituted or deleted due to mutations in the human gene for P0 (MPZ), which correlate with certain cases of demyelinating motor and sensory peripheral neuropathies. In the current study, high magnification electron microscopy was used to examine the myelin membrane packing in sural nerve biopsies from patients with MPZ mutations. We found that there were distinguishable ultrastructural phenotypes that could be explained by the alterations in P0-ED. These phenotypes, which were not observed in a control nerve, included widening or irregularity of the extracellular apposition alone (delta Ser34; Arg69Cys), widening at both the extracellular and cytoplasmic appositions (Arg69His), the presence of focal bridges in the widened extracellular space (Arg69His), and a diminished (Arg69Cys) or absence (Arg69His) of staining of the double intraperiod line. Our study, which suggests that the altered P0 is incorporated into the myelin sheath, provides a unique basis for further molecular/ultrastructural correlations between P0-ED structure and myelination irregularities.

Membrane adhesion in peripheral myelin: good and bad wraps with protein P0

Recent molecular models and crystallographic analysis of the major protein of peripheral myelin have provided new insights into the molecular basis of membrane adhesion in myelin. These studies have proved useful in understanding the molecular basis of clinical phenotypes in certain demyelinating neuropathies.

Crystal Structure of the Extracellular Domain from P 0, the Major Structural Protein of Peripheral Nerve Myelin

P 0, the major protein of peripheral nerve myelin, mediates membrane adhesion in the spiral wraps of the myelin sheath. We have determined the crystal structure of the extracellular domain from P 0 (P 0ex) at 1.9 Å resolution. P 0ex is folded like a typical immunoglobulin variable-like domain; five residues at the C-terminus are disordered, suggesting a flexible linkage to the membrane. The requirements for crystallization of P 0ex are similar to those for maintaining the native extracellular spacing of adjacent myelin lamellae; thus, given the self-adhesive character of P 0ex, the crystal itself may reveal some of the natural interactions that occur between P 0 molecules in myelin. The structure leads to the suggestion that P 0 extracellular domains may emanate from the membrane surface as tetramers that link to tetramers on the opposing membrane surface, to result in the formation of networks of molecules. We report analytical ultracentrifugation data for P 0ex that support this idea.

Clinical Phenotypes of Different MPZ (P 0) Mutations May Include Charcot–Marie–Tooth Type 1B, Dejerine–Sottas, and Congenital Hypomyelination

Hereditary demyelinating peripheral neuropathies consist of a heterogeneous group of genetic disorders that includes hereditary neuropathy with liability to pressure palsies (HNPP), Charcot–Marie–Tooth disease (CMT), Dejerine–Sottas syndrome (DSS), and congenital hypomyelination (CH). The clinical classification of these neuropathies into discrete categories can sometimes be difficult because there can be both clinical and pathologic variation and overlap between these disorders. We have identified five novel mutations in the myelin protein zero ( MPZ) gene, encoding the major structural protein (P 0) of peripheral nerve myelin, in patients with either CMT1B, DSS, or CH. This finding suggests that these disorders may not be distinct pathophysiologic entities, but rather represent a spectrum of related “myelinopathies” due to an underlying defect in myelination. Furthermore, we hypothesize the differences in clinical severity seen with mutations in MPZ are related to the type of mutation and its subsequent effect on protein function (i.e., loss of function versus dominant negative).

T cell antigenic and neuritogenic activity of recombinant human peripheral myelin P2 protein

The major neuritogenic protein of peripheral nerve myelin is the P2 protein. Human P2 is a candidate autoantigen in inflammatory demyelinating diseases of the peripheral nervous system. Since human P2 is not readily available, we produced full-length recombinant human P2 protein (rhP2) in Escherichia coli. RhP2 was recognized by neuritogenic rat T cell lines and induced experimental autoimmune neuritis in Lewis rats. Production of rhP2 allowed the generation of human T cell lines reactive to the autologous protein. Studies of human T cell autoreactivity as well as efforts to use hP2 as a tolerogen will be facilitated by the large-scale expression of rhP2.

Charcot-Marie-Tooth type 1B neuropathy: third mutation of serine 63 codon in the major peripheral myelin glycoprotein PO gene.

We report studies on two patients (a mother and her daughter) presenting with a Charcot-Marie-Tooth type 1 (CMT1) phenotype: low nerve conduction velocities of 13-15 m/s and an early onset at the age of walking. DNA analysis of the gene coding for the major peripheral myelin protein PO showed a new point mutation in exon 2, which resulted in substitution of a phenylalanine for serine at amino acid position 63 of PO. This is the third mutation reported at this codon, the two previously described leading to CMT1B (serine 63 deletion), or to Dejerine-Sottas disease (cysteine for serine 63 substitution), suggesting that different phenotypes can result from alteration of a single amino acid, depending on the type of the change involved.

Transforming growth factor-beta 1 and forskolin modulate gap junctional communication and cellular phenotype of cultured Schwann cells

Following peripheral nerve injury, Schwann cells undergo a series of cellular alterations that are thought to assist the regenerative process. Some of these changes are stimulated by the local release of cytokines and mitogenic factors. To test the hypothesis that cytokine regulation of gap junctional communication between cells helps to coordinate Schwann cell responses, cultured rat Schwann cells, from sciatic nerve, were utilized to study phasic changes induced by transforming growth factor-beta 1 (TGF beta 1), a cytokine released after nerve injury, or forskolin in combination with bovine pituitary extract (F-BPE), known for its mitogenic effects in vitro. In mitotically quiescent cultures, TGF beta 1 significantly decreased both electrical and dye coupling mediated by gap junctions. Single-channel analysis revealed that cultured Schwann cells expressed gap junctions with two distinct channel sizes of about 26 pS and 44 pS. TGF beta 1 treatment reduced coupling due to both populations of channels. Exposure to TGF beta 1 had a minimal effect on proliferation but significantly altered cellular morphology; cell bodies became flattened with multipolar processes within 72 hr. Additionally, immunolabeling for both low-affinity nerve growth factor receptor (L-NGFR) and glial fibrillary acidic protein (GFAP) were reduced, suggesting increased differentiation. In contrast, treatment with F-BPE significantly enhanced both electrical and dye coupling and stimulated Schwann cell proliferation. Additionally, cell bodies became more rounded with polarized, cytoplasmic processes contiguously aligned with adjacent cells. F-BPE reduced immunolabeling for L-NGFR but increased expression of both GFAP and the major peripheral myelin protein, P0. These data indicate that TGF beta 1 and/or F-BPE induce phenotypic changes in Schwann cells, including the coordinated regulation of proliferation and modulation of intercellular communication via gap junctions. Such mechanisms may underlie phasic responses that orchestrate recovery from nerve injury, indicating that Schwann cell gap junctions may be critical for peripheral nerve function.

Rapid screening of myelin genes in CMT1 patients by SSCP analysis: identification of new mutations and polymorphisms in the P0 gene.

Charcot-Marie-Tooth type 1 (CMT1) disease or hereditary motor and sensory neuropathy type I (HMSNI) is an autosomal dominant peripheral neuropathy. In most CMT1 families, the disease cosegregates with a 1.5-Mb duplication on chromosome 17p11.2 (CMT1A). A few patients have been found with mutations in the peripheral myelin protein 22 (PMP-22) gene located in the CMT1A region. In other families mutations have been identified in the major peripheral myelin protein P0 gene localized on chromosome 1q21-q23 (CMT1B). We performed a rapid mutation screening of the PMP-22 and P0 genes in non-duplicated CMT1 patients by single-strand conformation polymorphism analysis followed by direct polymerase chain reaction sequencing of genomic DNA. Six new single base changes in the P0 gene were observed: two missense mutations in, respectively, exons 2 and 3, two nonsense mutations in exon 4, and two silent mutations or polymorphisms in, respectively, exons 3 and 6.

Mutations in demyelinating peripheral neuropathies support molecular model of myelin P0-glycoprotein extracellular domain.

Homophilic interactions of the major integral membrane protein of peripheral nerve myelin, P0-glycoprotein, are thought to mediate membrane adhesion and compaction. Molecular modeling of its extracellular domain (P0-ED), based on its resemblance to an immunoglobulin variable domain and on X-ray diffraction measurements of inter-membrane spacings of myelin, has suggested which amino acid sidechains may be involved in the homophilic adhesion. Recently identified point-mutations in the human P0 gene result in amino acid substitutions in P0 protein and correlate with demyelinating motor and sensory neuropathies. The molecular model explains how these changes result in disrupted P0-P0 interactions; indicates how compensatory changes in amino acids, as occur in P0-ED of other species, preserve normal homophilic interactions; and predicts what other residue substitutions might underlie additional cases of demyelinating neuropathies.

P0 phosphorylation in nerves from normal and diabetic rats: role of protein kinase C and turnover of phosphate groups.

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P0 phosphate groups was studied in normal and experimentally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [32P]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P0, was increased 2-5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). This increase was blocked by the protein kinase inhibitors, H-7 and staurosporine. Both the basal and PDB-stimulated phosphorylation of P0 were significantly greater in segments of sciatic nerve from streptozotocin-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P0 and immunoblots of nerve proteins revealed a decrease in the content of the protein kinase C alpha-isoform. The adenylate cyclase activator, forskolin, had no effect on the PDB-stimulated phosphorylation of P0 in normal nerve but decreased phosphorylation in diabetic nerve. To measure turnover of P0 phosphate groups, nerves were incubated with 32P and incorporated label was then chased in radioactivity-free medium for up to 4 hours. P0 from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additional phosphate groups prelabeled in the presence of PDB disappeared after 2 hours of chase. P0 phosphate groups from diabetic nerve displayed similar turnover kinetics. When forskolin was added to the chase medium, the turnover of P0 phosphate moieties was accelerated in normal, but not in diabetic nerve.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion of the serine 34 codon from the major peripheral myelin protein P0 gene in Charcot–Marie–Tooth disease type 1B

Charcot-Marie-Tooth disease type 1B (CMT1B) is genetically linked to chromosome 1q21-23. The major peripheral myelin protein gene, P0, has been cloned and localized to the same chromosomal region. P0 is a 28 kDa glycoprotein involved in the compaction of the multilamellar myelin sheet and accounts for more than half of the peripheral myelin protein content. We checked whether P0 is altered in CMT1B, and show here that a 3 basepair deletion in exon 2 of the P0 gene is present in all affected individuals of a CMT1B family. The mutation results in the deletion of serine 34 in the extracellular domain of P0, suggesting that alterations of P0 cause CMT1B.

Charcot-Marie-Tooth neuropathy type 1B is associated with mutations of the myelin P0 gene.

P0, a major structural protein of peripheral myelin, is a homophilic adhesion molecule and maps to chromosome 1q22-q23, in the region of the locus for Charcot-Marie-Tooth neuropathy type 1B (CMT1B). We have investigated P0 as a candidate gene in two pedigrees with CMT1B and found point mutations which are completely linked with the disease (Z = 5.5, theta = 0). The mutations, glutamate substitution for lysine 96 or aspartate 90, are located in the extracellular domain, which plays a significant role in myelin membrane adhesion. Individuals with CMT1B are heterozygous for the normal allele and the mutant allele. Our results indicate that P0 is a gene responsible for CMT1B.

Repression of the myelin P 0 gene by the POU transcription factor SCIP

SCIP is a POU domain transcription factor expressed by Schwann cells, the myelin-forming glial cells of the peripheral nervous system. In this study, we investigate SCIP regulation of gene encoding P 0, the major structural protein of peripheral myelin. We find that SCIP represses transcription of this gene through the joint action of the SCIP POU domain and an amino terminal domain that acts cell specifically. Maximal repression is DNA-binding-dependent, and analysis of the P 0 promoter reveals the presence of multiple SCIP binding sites. Suprisingly, none of these sites in their native positions dramatically affect P 0 promoter activity or its repression by SCIP, although they mediate repression when moved closer to the P 0 transcription start site. We proposed that repression occurs through a quenching mechanism mediated by the SCIP POU and amino terminal domains acting in concert with other nuclear proteins, including a Schwann cell-specific adapter.

Production and characterization of monoclonal antibodies to the extracellular domain of P0

Seven monoclonal antibodies were raised against the immunoglobulin-like extracellular domain of P0 (P0-ED), the major protein of peripheral nervous system myelin. Mice were immunized with purified recombinant rat P0-ED. After fusion, 7 clones (P01-P07) recognizing either recombinant, rat, mouse, or human P0-ED were selected by ELISA and were characterized by Western blot, immunohistochemistry, and a competition assay. Antibodies belonged to the IgG or IgM class, and P04-P07, reacted with P0 in fresh-frozen and paraffin-embedded sections of human or rat peripheral nerve, but not with myelin proteins of the central nervous system of either species. Epitope specificity of the antibodies was determined by a competition enzyme-linked immunosorbent assay (ELISA) and a direct ELISA using short synthetic peptides spanning the entire extracellular domain of P0. These assays showed that P01 and P02 exhibiting the same reaction pattern in Western blot and immunohistochemistry reacted with different distant epitopes of P0. Furthermore, the monoclonal antibodies P05 and P06 recognized 2 different epitopes in close proximity within the neuritogenic extracellular sequence of P0. This panel of monoclonal antibodies, each binding to a different epitope of the extracellular domain of P0, will be useful for in vitro and in vivo studies designed to explore the role of P0 during myelination and in demyelinating diseases of the peripheral nervous system.

The major peripheral myelin protein zero gene: structure and localization in the cluster of Fc gamma receptor genes on human chromosome 1q21.3-q23.

We have characterized the human gene encoding the major peripheral myelin protein zero (P0) and assigned it, by in situ hybridization, to the q21.3-q23 region of human chromosome 1. This region is known to contain a cluster of interspersed genes coding for the related human leukocyte receptors of the Fc portion of the immunoglobulin G (Fc gamma RI, II, III). This colocalization was refined by the finding of a yeast artificial chromosome (YAC) of the Centre d'Etude du Polymorphisme Humain (CEPH) library, hybridizing to the P0 and Fc gamma RIIA genes, demonstrating their physical linkage. These data may have important implications in demyelinating diseases studies like Charcot-Marie-Tooth disease type 1B (CMT1B).

Mouse P0 gene disruption leads to hypomyelination, abnormal expression of recognition molecules, and degeneration of myelin and axons

We have used homologous recombination in embryonic stem cells to generate mice carrying a mutation in the gene encoding P 0, an immunoglobulin-related recognition molecule and the major protein of peripheral nervous system myelin. These mice are deficient in normal motor coordination and exhibit tremors and occasional convulsions. Axons in their peripheral nerves are severely hypomyelinated and a subset of myelin-like figures and axons degenerate. The mutation leads to an abnormal regulation of some, but not all, molecules involved in myelination. These results demonstrate that P 0 is essential for the normal spiraling, compaction, and maintenance of the peripheral myelin sheath and the continued integrity of associated axons. They further suggest that this protein conveys a signal that regulates Schwann cell gene expression.

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

A full length cDNA of the major structural protein of peripheral myelin (P0 protein) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs (bp) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species.

Correction of Altered Metabolic Activities in Sciatic Nerves of Streptozocin-Induced Diabetic Rats: Effect of Ganglioside Treatment

The effect of ganglioside administration to nondiabetic and streptozocin-induced diabetic rats on sciatic nerve Na(+)-K(+)-ATPase, polyphosphoinositide (PPI) turnover, and protein phosphorylation was investigated. Gangliosides were injected (10 mg/kg body wt i.p.) for 10 or 30 days beginning 20 days after induction of diabetes. Na(+)-K(+)-ATPase activity was reduced nearly 50% in diabetic nerve and was restored to normal by both ganglioside treatments. The elevated levels of fructose and sorbitol and depressed content of myoinositol in diabetic nerve were unaffected by 30 days of ganglioside treatment, indicating that the restoration of Na(+)-K(+)-ATPase activity is not dependent on normal concentrations of these compounds. In the same nerves, 32P incorporation into phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate increased 73-76 and 39-53%, respectively, in diabetic compared with nondiabetic tissue. Ganglioside administration abolished the elevated labeling of PPIs after 30 days but was ineffective after only 10 days. Neither ganglioside regimen was able to reverse enhanced phosphorylation of the major peripheral nerve myelin protein P0. The finding that gangliosides can more quickly correct the effects of diabetes on Na(+)-K(+)-ATPase activity than on PPI turnover suggests that the mechanisms underlying these two phenomena are not closely related and are distinct from the sequence of events responsible for altered myelin protein phosphorylation.

Reconstitution into liposomes of highly purified PO glycoprotein from avian peripheral nerve myelin

To explore the adhesive, possibly fusogenic properties of the major protein of peripheral nerve myelin (PO protein), three methods were employed for the reconstitution of this glycoprotein into liposomes of various lipid compositions. The most successful technique for the preparation of proteoliposomes was a detergent solubilization that employed Bio-Beads for the removal of Triton X-100 from the PO protein – lipid – detergent mixture. This method yielded a similar level of PO protein relative to lipids in the liposomes to that found in myelin. The protein to lipid ratio of the reconstituted proteoliposomes was only 1.4-fold lower than the initial protein to lipid ratio employed to prepare the liposomes. In contrast, the solvent evaporation and reverse-phase evaporation methods provided liposomes with 30 times lower protein to lipid ratio when compared with PO protein to lipid ratio initially used for the preparation of proteoliposomes. The incorporation of PO protein was dependent on the lipid composition of the vesicles and on the degree of purification of the protein. Proteolytic enzyme treatment showed that proteoliposomes containing the highly purified form of PO protein were partially or completely sensitive to trypsin treatment, whereas in liposomes containing a partially purified form of PO protein, a 22.4-kilodalton fragment was completely protected from trypsin digestion, indicating a transmembrane orientation of the partially purified polypeptide. Endoglycosidase F treatment of the liposomes established that all of the oligosaccharide moieties were removed from the liposome-associated protein, indicating the oligosaccharide moiety was exposed to the outside surface of the liposomes. Taken together, the results of limited proteolysis and endoglycosidase treatments did not provide strong evidence for the transmembrane orientation of highly purified PO in the liposomes, but were consistent with a surface of partial hairpin configuration, where the extracellular domains are facing the outside milieu. Electron microscopic examinations revealed highly convoluted, elongated shapes arid extensive fusion of PO liposomes containing lipids capable of adopting the hexagonal (H11) phase. Proteoliposomes prepared with the simple lipid composition of dipalmitoyl phosphatidylcholine -cholesterol (10:1 molar ratio) yielded stable, unilamellar liposomes of 80 nm average diameter, that encapsulated water-soluble drugs efficiently into the intravesicular compartment.Key words: liposomes, PO glycoprotein, myelin, peripheral nerve.