Enzymatic Regulation of Glycoprotein Synthesis in Peripheral Nervous System Myelin

The enzyme UDP-N-acetylglucosamine: dolichyl phosphate, N-acetylglucosamine-1-phosphate transferase initiates the synthesis of the oligosaccharide chain of complex-type glycoproteins. In view of the high content of glycoprotein in peripheral nerve myelin, the properties of this enzyme, its changes with age, and the effect of the specific inhibitor tunicamycin were investigated. The enzyme activity in rat peripheral nerve homogenate was completely dependent on the presence of exogenous dolichyl phosphate as well as Mg2+ and a detergent (Triton X-100) and was also greatly stimulated by a high salt concentration (0.4 M KCl) and AMP. The highest specific activity was present in the postmitochondrial membranes. The specific activity in postmitochondrial membranes in the presence of exogenous dolichyl phosphate reached a maximum at 17 days and remained relatively high throughout development, up to 2 years of age, but the activity was much lower when dolichyl phosphate was not added. This indicates that the enzyme level does not decrease with age, but that the content of the lipid cofactor may limit glycoprotein synthesis in vivo. Tunicamycin (5 micrograms) was injected intraneurally into 24-day-old rat sciatic nerve, and the enzyme was assayed from 1 to 24 days after injection. The specific activity of the transferase remained at low levels (5-40% of the level in control nerve) in most injected nerves assayed throughout this postinjection period. A protein previously identified as the unglycosylated P0 protein was synthesized in vitro by the tunicamycin-injected nerve and could be demonstrated to be incorporated into myelin in large amounts at 2 days and in small amounts at 6 days after injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological Nonidentity of 19K Protein and TP0 in Peripheral Nervous System Myelin

Peripheral nervous system myelin contains as the major structural protein a glycoprotein known as P0. Another glycoprotein present in smaller amounts, known as the 19K or X protein, has been previously identified as derived from P0 and identical with the main tryptic degradation product of P0 (TP0). Although both P0 and 19K protein incorporated fucose in vitro and stained on polyacrylamide gels with the periodic acid-Schiff stain for carbohydrate, only the P0 blotted to nitrocellulose paper showed immunoreactivity to an antibody to P0, whereas the 19K protein did not. Furthermore, when P0 was hydrolyzed with trypsin or elastase, the main degradation products reacted with P0 on immunoblots, whereas the 19K protein showed no immunoreactivity. From these studies and those of others, it may be concluded that the 19K protein shows some similarities to TP0, but probably has a different structure. P0 and 19K protein do not appear to be related as shown by lack of cross-immunoreactivity.     

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

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

Age-Related Changes of Ribonuclease Activities in Various Regions of the Rat Central Nervous System

Acid (pH 5.5), free, and latent alkaline (pH 7.4) RNases were assayed in homogenates of temporal cortex, hypothalamus, hippocampus, and cervicothoracic segments of spinal cord of rats at three different ages (5, 14, and 25 months old). Free alkaline RNase activity was lower (two- to fivefold) than the acid activity. Both free and inhibitor-bound alkaline RNases remained unchanged with age in all CNS regions examined. This result also indirectly indicates no change of RNase-inhibitor complex throughout aging. In contrast, the acid RNase activity showed a significant increase during aging in all tissues, with exception of the hypothalamus. Because this enzyme is localized mainly in the lysosomes, this result might be due to an increased lysosomal activity and/or to the release of hydrolases into the cytoplasm from these organelles, undergoing shrinkage and degeneration in aged animals.     

Glycoprotein Biosynthesis in Peripheral Nervous System Myelin: Effect of Tunicamycin

Abstract: The effect of an inhibitor of N -glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of ³H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P₀, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of ¹⁴Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P₀ peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with ³H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P₀ antiserum, was tentatively identified as a nonglycosylated P₀ protein that appears to be almost as well incorporated as P₀ into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P₀ is actually assembled into a site and configuration like that of P₀.     

The Glycoprotein Composition of Peripheral Nervous System Myelin Subfractions

Two fractions were isolated by continuous density gradient centrifugation from total particulate matter of rabbit sciatic nerves: a minor fraction, B, consisting of small-sized membrane fragments and a major fraction, C, of characteristic multilayered myelin figures, with maxima at 0.33 and 0.58 M-sucrose, respectively. In comparison with C, fraction B was enriched in CNPase and alkaline phosphatase activities and the P0, 23K and Z proteins, but was virtually devoid of basic protein. The glycoprotein composition of all fractions was examined with four fluorescein isothiocyanate-labelled lectins (WGA, Con A, RCA-60, U.E.). These revealed the presence of six glycoproteins in all fractions with similar lectin binding capacities and molecular weights ranging from 35,500 to 16,000, of which P0 was the predominant component. Material found on the heavy side of fraction C was characterized by the presence of a multitude of glycoproteins which bound variable proportions of the four different lectins, suggesting substantial variations in their carbohydrate moieties. Their absence from the central portion of fraction C points to a location other than that of compact PNS myelin.     

P2 Protein in Oligodendrocytes and Myelin of the Rabbit Central Nervous System

P2 protein, a myelin-specific protein, was detected immunocytochemically and biochemically in rabbit central nervous system (CNS) myelin. P2 protein was synthesized by rabbit oligodendrocytes and was present in varying amounts throughout the rabbit CNS. Comparison of P2 and myelin basic protein (MBP) stained sections revealed that P2 antiserum did not stain all myelin sheaths within the rabbit CNS. The proportion of myelin sheaths stained by P2 antiserum and the amount of P2 detected biochemically were greater in more caudal regions of the rabbit CNS. The highest concentration of P2 protein was found in rabbit spinal cord myelin, where P2 antiserum stained the majority of myelin sheaths. P2 protein was barely detectable biochemically in myelin isolated from frontal cortex, and in sections of frontal cortex only occasional myelin sheaths reacted with P2 antiserum. These results suggest the the regional variations in the amount of P2 protein are dut to regional differences in the number of myelin sheaths that contain P2 protein. P2 protein was detected immunocytochemically and biochemically in rabbit sciatic nerve myelin. Immunocytochemically, P2 antiserum only stained a portion of the myelin sheaths present. The myelin sheaths not reacting with P2 antiserum had small diameters and represented less than 10% of the total myelinated fibers.     

Bovine Peripheral Nervous System Myelin P2 Protein: Chemical and Immunological Characterization of the Cyanogen Bromide Peptides

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2-terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM-P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2-terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH2-terminal valine (Val) that was co-eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM-P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3-CN1-CN2(Val)-CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val). To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induced experimental allergic neuritis (EAN) in Lewis rats. The disease-inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.     

Release of Proteins from the Surface of Bovine Central Nervous System Myelin by Salts and Phospholipases

Incubation of bovine CNS myelin with phospholipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane. The myelin basic protein (MBP) was a major component of the dissolved protein. Comparable incubations with phospholipase C from Clostridium perfringens, phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP. However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9-12% of the total myelin protein, with MBP again as the predominant component. Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP. When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact. With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions. The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.     

Degradation of the P0, P1, and Pr, Proteins in Peripheral Nervous System Myelin by Plasmin: Implications Regarding the Role of Macrophages in Demyelinating Diseases

Activated macrophages secrete a variety of neutral proteinases, including plasminogen activator. Since macrophages are implicated in primary demyelination in the peripheral nervous system (PNS) in Guillain-Barré syndrome and experimental allergic neuritis, we have investigated the ability of plasmin and of conditioned media from cultured macrophages, in the presence of plasminogen, to degrade the proteins in bovine and rat PNS myelin. The results indicate that (a) the major glycoprotein P0 and the basic P1 and Pr proteins in PNS myelin are extremely sensitive to plasmin, perhaps more so than is the basic protein in CNS myelin; (b) the initial product of degradation of P0 by plasmin has a molecular weight higher than that of the "X" protein; (c) large degradation products of P0 are relatively insensitive to further degradation; and (d) the neuritogenic P2 protein in PNS myelin is quite resistant to the action of plasmin. Results similar to those with plasmin were obtained with conditioned media from macrophages and macrophage-like cell lines together with plasminogen activator, and the degradation of the PNS myelin proteins, Po and P1, under these conditions was inhibited by p-nitrophenylguanidinobenzoate, an inhibitor of plasmin and plasminogen activator. The results suggest that the macrophage plasminogen activator could participate in inflammatory demyelination in the PNS.     

Comparison of the Major Proteins of Shark Myelin with the Proteins of Higher Vertebrates

Abstract: On gel electrophoresis in dodecyl sulphate solutions shark CNS myelin showed four bands close in mobility to the proteolipid protein of bovine CNS myelin. They had apparent molecular weights of 21,000, 26,000, 27,000, and 31,500. Unlike bovine proteolipid protein, all of these shark proteins were shown to be glycosylated by staining gels with the periodate-Schiff reagent. Amino acid analyses of the polypeptides eluted from polyacrylamide gels indicated a high content of apolar amino acids and a composition approximating that of the P_o protein of bovine peripheral nervous system (PNS) myelin, rather than that of the CNS proteolipid protein. The shark poly-peptide of apparent molecular weight 31,500 was obtained by elution from dodecyl sulphate gels and antibodies raised against it in rabbits. By probing of electroblots with this antiserum the four shark CNS bands were shown to share common determinants with each other, with a major shark PNS protein and with sheep and chicken major PNS glycoproteins (P_o). The binding of antibody was unaffected by deglycosylation of the shark CNS polypeptides with anhydrous hydrogen fluoride. Together, these results appeared to establish that shark CNS myelin contains four proteins that are closely related to a major shark PNS protein and to the P_o protein of higher species.     

Quantitative Changes in Myelin Proteins in a Peripheral Neuropathy Caused by Tullidora (Karwinskia humboldtiana)

Peripheral nerve demyelination was induced in cats by oral administration of ether extracts of Tullidora (Karwinskia humboldtiana). Proteins from several hindlimb nerves, spinal roots, and dorsal columns of the spinal cord were subjected to slab gel electrophoresis and quantified by densitometry. In Tullidora-treated cats with severe motor disturbances, specific myelin proteins were reduced by at least 50% in motor nerves and less than 25% in cutaneous axons. There was a greater decrease of these proteins in the distal than in the cephalad segments of the sciatic nerve; no changes were detected either in the spinal roots or in the white matter of the spinal cord. Electron microscopy revealed intense demyelination in the motor nerves only. Both the density of the 100 A-thick neurofilaments and the relative proportion of a polypeptide with a molecular weight of 68,000 were considerably increased in the affected nerves. It is tentatively concluded that the active principles of Tullidora may enter the axons through the motor nerve terminals. The distal segments of the motor nerves would then be preferentially affected and demyelination could result from axonal damage.     

Immunochemical Characterization of Peripheral Nervous System Myelin 170,000-Mr Glycoprotein

A recently described 170,000-Mr glycoprotein, specific to peripheral nervous system (PNS) myelin, was purified from rat PNS myelin by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to immunize guinea pigs and rabbits. The resultant antisera proved specific for 170,000-Mr glycoprotein by enzyme-linked immunosorbent assay, by immunoprecipitation of the appropriate peptide from solubilized PNS myelin, and by immunoblot analysis of rat PNS myelin. The anti-rat 170,000-Mr glycoprotein antisera cross-reacted with proteins of similar molecular weight in human and bovine PNS myelin, but such proteins were not detected in human or rat CNS myelin or other rat tissues. The 170,000-Mr glycoprotein was also detected by this immunoblot procedure in recently isolated rat Schwann cells but not in those kept in culture for greater than or equal to 3 days. By indirect immunofluorescent microscopy, anti-rat 170,000-Mr glycoprotein antibody bound to rat PNS myelin sheaths but not to other rat tissues. Together, these studies indicate the 170,000-Mr glycoprotein is specific to PNS myelin of several species and that a neuronal influence may be required for its expression by Schwann cells.     

Progesterone Stimulates the Activity of the Promoters of Peripheral Myelin Protein-22 and Protein Zero Genes in Schwann Cells

Abstract: To understand better the mechanisms by which progesterone (PROG) promotes myelination in the PNS, cultured rat Schwann cells were transiently transfected with reporter constructs in which luciferase expression was controlled by the promoter region of either the peripheral myelin protein-22 (PMP22) or the protein zero (P₀) genes. PROG stimulated the P₀ promoter and promoter 1, but not promoter 2, of PMP22. The effect of PROG was specific, as estradiol and testosterone only weakly activated promoters. Dose-response curves for stimulation of both promoter constructs by PROG were biphasic. RU486, a PROG antagonist, did not abolish the effect of PROG, but stimulated promoter activities by itself. In the human carcinoma cell line T47D expressing high levels of PROG receptor, PROG did not stimulate the P₀ and PMP22 promoters, whereas the promoter region of the mouse mammary tumor virus was fully activated. Thus, the activation by PROG of promoter activity of two peripheral myelin protein genes is Schwann-cell specific.     

Relationship of ATP Turnover, Polyphosphoinositide Metabolism, and Protein Phosphorylation in Sciatic Nerve and Derived Peripheral Myelin Subfractions from Normal and Streptozotocin Diabetic Rats

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.     

Peripheral Nervous System Myelin and Schwann Cell Glycoproteins: Identification by Lectin Binding and Partial Purification of a Peripheral Nervous System Myelin-Specific 170,000 Molecular Weight Glycoprotein

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.     

Myelin Gangliosides of Human Peripheral Nervous System: An Enrichment of GM1 in the Motor Nerve Myelin Isolated from Cauda Equina

Myelins of the PNS were isolated from human motor and sensory nerves of cauda equina, and their ganglioside compositions were compared. The predominant ganglioside in the human PNS myelins, both from motor and sensory nerves, was LM1 (sialosylneolactotetraosylceramide). Sialosyl-nLc6Cer and disialosyl-nLc4Cer, GD3, GM3, and GD1b were detected as common components of the two nerve myelins. Furthermore, it was revealed that the motor nerve myelin contained GM1 (about 15% of total gangliosides), whereas sensory nerve myelin contained only a trace amount of GM1 (less than 5%), by TLC analyses together with TLC immunostaining using anti-GM1 antibody. As for the disialoganglioside fraction, the content of GD1a, as well as that of GM1, differed in motor and sensory nerves. Thus, the different contents of the ganglioseries gangliosides in human motor and sensory nerve myelins were demonstrated.