Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

Solid-Phase Immunoassay of PO Glycoprotein of Peripheral Nerve Myelin

To explore the immunological properties of PO protein, antibodies were elicited in rabbits against the purified chick PO protein. Peripheral nervous system protein was fractionated on sodium dodecyl sulfate-polyacrylamide slab gels and then transferred electrophoretically ("blotted") onto nitrocellulose sheets. The PO protein was detected by its capacity to bind its specific antibody present in the rabbit serum. The PO-specific antibody complex was then exposed to goat anti-rabbit immunoglobulin G (IgG) coupled to peroxidase or labeled with 125I. The resulting PO antigen-antibody "sandwich" was visualized and quantitated by densitometry of the colored peroxidase reaction product or by autoradiography and gamma-radiation counting of the 125I-IgG complex. The methods permitted quantitation of the PO protein in various nerve extracts. The limit of detection of the PO antigen was about 1 ng of protein. The antibody was specific for the PO glycoprotein in the peripheral nerve extracts. The PO proteins from various species, including human, were also detected by the antibody to chick PO protein. Preliminary experiments indicate the solid-phase immunoassay is a useful method for monitoring PO protein levels in small quantities of tissue extracts under various physiological and pathological conditions.     

Bony Fish Myelin: Evidence for Common Major Structural Glycoproteins in Central and Peripheral Myelin of Trout

Peripheral nervous system (PNS) myelin from the rainbow trout (Salmo gairdneri) banded at a density of 0.38 M sucrose. The main myelin proteins consisted of (1) two basic proteins, BPa and BPb (11,500 and 13,000 MW, similar to those of trout central nervous system (CNS) myelin proteins BP1 and BP2), and (2) two glycosylated components, IPb (24,400 MW) and IPc (26,200 MW). IPc comigrated with trout CNS myelin protein IP2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas trout CNS myelin protein IP1 had a lower molecular weight (23,000). Following two-dimensional separation, however, both IPb and IPc from PNS showed two components; the more acidic component of IPc comigrated with IP2 from CNS. PNS tissue autolysis led to the formation of IPa (20,000 MW), consisting of two components in isoelectric focusing of which again the more acidic one comigrated with the CNS autolysis product IP0. Limited enzymatic digestion of isolated IP proteins from PNS and CNS led to closely similar degradation patterns, being most pronounced in the case of IP2 and IPc. Immunoblotting revealed that all IP components from trout PNS and CNS myelins reacted with antibodies to trout IP1 (CNS) and bovine P0 protein (PNS) whereas antibodies to rat PLP (CNS) were entirely unreactive. All BP components from trout PNS and CNS myelins bound to antibodies against human myelin basic protein. On the basis of these studies trout PNS and CNS myelins contain at least one common IP glycoprotein, whereas other members of the IP myelin protein family appear closely related. In the CNS myelin of trout the IP components appear to replace PLP.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Metabolism of Phosphate and Sulfate Groups Modifying the P0 Protein of Peripheral Nervous System Myelin

We have examined the metabolism of phosphate and sulfate groups modifying the P0 protein, the major protein of peripheral nervous system myelin, using an in vitro incubation system. Incorporation of [3H]leucine into the P0 peptide backbone decreased approximately 25-fold between 10 and 90 days of age, a finding reflecting a decreased rate of myelin synthesis in the older animals. In contrast, incorporation of [32P]phosphate into P0 decreased only four- to fivefold, a result indicating that phosphate groups are metabolized independently of the peptide backbone. Developmental decreases in the incorporation of sulfate groups into P0 were similar to those seen for leucine, an observation suggesting that this modifying group is metabolized together with the peptide backbone as a single metabolic entity. The time course of labeling of P0 isolated from the starting homogenate and from myelin was also compared. Results are consistent with sulfation of P0 protein taking place before insertion of newly synthesized P0 into myelin. In contrast, incorporation of phosphate into P0 appears to involve both the newly synthesized pool and the preexisting pool of P0 in myelin. Presumably, entry of phosphate into P0 in myelin involves turnover of preexisting phosphate groups and rephosphorylation by myelin protein kinases. Developmental decreases in the specific activity of P0 phosphate groups in myelin are consistent with the presence of a small, rapidly turning-over pool of phosphorylated P0 (perhaps associated with the axon-myelin interface), which does not increase to the same extent as the marked increase in bulk myelin that occurs during development.     

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.     

A Rapid Method for Purification of Myelin Basic Protein

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.     

PO Protein and 2'',3''-Cyclic-Nucleotide 3''-Phosphodiesterase Activity in the Peripheral Nerve and Subcellular Fractions of the Trembler Mouse

To investigate the biochemical abnormalities of the Trembler mouse, the level of the PO protein (as % of total protein) and the activity of CNP was compared in the sciatic nerve and subcellular fractions of normal and mutant littermates. There was a significant decrease in both of these myelin markers in total nerve homgenates of the neurological mutant compared with the control animals. Immunoassay of the PO protein and polyacrylamide gel analysis of proteins indicated an accumulation of a protein with an apparent molecular weight of 67K in mutant nerve extracts. The mutant nerve also had relatively decreased levels of a protein of molecular weight about 41K that cross-reacted with antibody to PO protein. The Trembler mouse exhibited a larger percentage recovery of PO protein and CNP activity in subcellular fractions denser than the myelin sheath. Together these results are consistent with the theories that these denser components represent immature forms of myelin and that the Trembler mutant is characterized by hypomyelination.     

Bovine P2 Protein: Sequence at the NH2-Terminal of the Protein

Sequence data from key fragments of the P2 protein established the order of cyanogen bromide (CNBr) peptides in the structure of the protein and the primary structure for approximately one-half of the molecule. Data were obtained from the three tryptic peptides of blocked NH2-terminal CNBr peptide (CN3), the large CNBr peptide of P2 protein (CN1), and a fragment obtained from P2 by cleavage at tryptophan with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine. This last fragment was found to contain an over-lapping sequence that proved the juxtaposition of CN1 and CN3 in P2 protein. Thus, based on this fact and the characteristics of the CNBr peptides, the P2 structure is composed of CNBr peptides in the order: CN3-CN1-CN2(Val)-CN2(Lys). A comparison was made between the partial sequence of P2 protein and the equivalent portion of the structure of bovine myelin basic protein. The structures of these two proteins were found to be distinctly different although certain similarities are found.     

人脑髓鞘碱性蛋白的分离纯化和鉴定

 本文介绍了从人脑中分离纯化髓鞘碱性蛋白的方法,人脑组织匀浆经甲醇—氯仿脱脂、酸提取、硫酸铵沉淀和羧甲基纤维素柱层析,得到了纯化的髓鞘碱性蛋白。该蛋白在SDS聚丙烯酰胺凝胶电泳中为单一带,分子量为21kD。在聚焦电泳中测得其等电点在pH10以上,氨基酸组成分析结果也与文献值接近。这为进一步研究人脑髓鞘碱性蛋白的抗原性创造了条件。     

Subcellular Fractionation of Rat Sciatic Nerve and Specific Localization of Ganglioside LM1 in Rat Nerve Myelin

Subcellular fractionation of rat sciatic nerve was developed to determine the specific localization of gangliosides in the nerve membrane fractions. Myelin, microsomal, and a plasma membrane-like fraction were isolated and purified by sucrose density gradient centrifugation. These subfractions were characterized by electron microscopy, marker enzyme assays, and their protein and lipid profile. In rat sciatic nerve myelin, 90 mol% of the total gangliosides were monosialogangliosides. LM1 (sialosyl-lactoneotetraosylceramide) (61 mol%) and GM3 (21%) were the major gangliosides of the rat nerve myelin. Two other neolacto series of gangliosides, viz., sialosyl-lactoneonorhexaosylceramide and sialosyl-lactoneooctaosylceramide, were also localized mostly in the myelin fraction. GM1 was only a minor (less than 2%) ganglioside in myelin. The ganglioside patterns of the microsomal and plasma membrane-like fractions were similar with minor quantitative differences and were entirely different from that of myelin. Monosialogangliosides were approximately 70-75 mol% of the total in these fractions. The major gangliosides of the microsomal and plasma membrane-like fractions were GM3 (approximately 40%) and GM1 (approximately 20%). LM1 in these fractions was minimal (less than approximately 5%). Significant amounts of GM3 with N-glycolylneuraminic acid (approximately 10%) and GM1b (4-14%) were also identified in the microsomal and plasma membrane-like fractions but not in myelin. These and the higher lactoneo series of gangliosides have not been previously reported to be present in the rat nervous system. Almost exclusive localization of LM1 in myelin in rat peripheral nervous system is consistent with our previous observation that deposition of LM1 in the nerve with age was very similar to that of myelin marker lipids cerebrosides and sulfatides.     

Changes in Some Myelin Protein Markers and in Cytoskeletal Components During Wallerian Degeneration of Mouse Sciatic Nerve

After transection of the mouse sciatic nerve, the sequence of events occurring in the distal degenerating segment was followed by the biochemical changes related to the cytoskeletal components and to the myelin protein markers. The components of the intermediate filaments and of the microtubules undergo early changes. Within 3 days, the neurofilament triplet and the peripherin disappear whereas many peptides bearing the antigenic determinant common to all classes of intermediate filaments accumulate. Several of them persist after 1 month. The tubulin pattern changes from a high level of microheterogeneity--reflecting mostly the axonal contribution--to a lower level displayed by the predominant Schwann cells. A decrease in the amount of the myelin markers is also observed. However, a month after transection, immunoreactive basic protein is still present in the degenerated segment homogenate.     

The Complete Amino Acid Sequence of Human P2 Protein

Abstract: The complete amino acid sequence of P2 protein from human peripheral nerve myelin was determined from nine staphylococcal protease peptides and four cyanogen bromide peptides. Human P2 protein is composed of 131 amino acids and has a molecular weight of 14,818. Compared to bovine P2 protein, there are replacements at nine positions (human↔bovine): 18(Asp↔Glu), 39(Thr↔Arg), 56(Thr↔Pro), 83(Ile↔Thr), 87(Gln↔Ala), 96(Arg↔Lys), 100(Lys↔Asn), 115 (Ala↔Val), and 121(Gly↔Asp).     

Novel Isoforms of Mouse Myelin Basic Protein Predominantly Expressed in Embryonic Stage

Abstract: Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well-known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.     

Impulse Conduction Regulates Myelin Basic Protein Phosphorylation in Rat Optic Nerve

The influence of action potential conduction in myelinated axons on the state of phosphorylation of myelin basic protein was studied in rat optic nerve incubated in vitro. For this purpose we used a technique that permits continuous recording of the responses of nerves to electrical stimulation together with the "back-phosphorylation" assay. Our results indicate that action potential conduction, but not electrical stimulation, increased the state of phosphorylation of myelin basic protein. The increment in basic protein phosphorylation was related to the number of impulses conducted, up to a maximal change which occurred after 12 X 10(3) impulses. Also, the effect of action potential conduction was reversible, since the state of myelin basic protein phosphorylation returned to control levels within 5 min of stopping stimulation. These findings raise the interesting possibility that myelin basic protein phosphorylation plays a role in some dynamic function of myelin, perhaps related to ion transport or to the process of recovery of ionic gradients.     

ADP-Ribosylation of Human Myelin Basic Protein

Abstract: When isolated myelin membranes were ADP-ribosylated by [³²P]NAD⁺ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein. As MBP contains several components that are ADP-ribosylated to different specific activities, the use of MBP, ADP-ribosylated in the natural membrane, to identify the sites involved would yield a mixture of peptides difficult to resolve. Therefore, to identify the sites ADP-ribosylated, an endoproteinase Lys-C digest of C-1 ADP-ribosylated by cholera toxin was prepared. Two radioactive peptides were isolated by reversed-phase HPLC. Amino acid and sequence analyses identified the radioactive peptides as residues 5–13 and 54–58 of the human sequence (sp. act., 0.89 and 0.62 nmol of ADP-ribose/nmol of peptide, respectively). The ADP-ribosylated residues were identified as Arg⁹ and Arg⁵⁴ by automated and manual Edman sequencing. Taken together with our previous observation that MBP binds GTP at a single site, these data suggest that MBP functions as part of a signal transduction system in myelin.     

Expression, Purification, and Encephalitogenicity of Recombinant Human Myelin Oligodendrocyte Glycoprotein

Abstract: Myelin oligodendrocyte glycoprotein (MOG), a putative autoantigen in multiple sclerosis (MS), is a quantitatively minor component of the CNS. In view of the difficulties associated with the purification of MOG from brain tissues, the extracellular domain of human MOG corresponding to the N-terminal 121 amino acids was expressed in Escherichia coli as a glutathione sulfotransferase fusion protein. The expressed protein was localized to inclusion bodies, and varying the growth parameters resulted in the solubilization of small amounts of GST-MOG that could be affinity purified on glutathione agarose columns. The fusion protein found in the inclusion bodies could be solubilized with urea. The solubilized fusion protein was cleaved with thrombin, and the extracellular domain was purified by CM Sephadex 50 chromatography to homogeneity. Injection of recombinant human MOG into different strains of mice resulted in the induction of an MS-like disease, characterized by severe neurological impairment and extensive CNS demyelinated lesions. Recombinant MOG produced in E. coli should prove to be useful as a highly purified biological reagent for immunological, pathological, functional, and structural studies.     

Myelin Basic Protein Is a Zinc-Binding Protein in Brain: Possible Role in Myelin Compaction

The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.     

Purification and Partial Structural and Functional Characterization of Mouse Myelin/Oligodendrocyte Glycoprotein

The myelin/oligodendrocyte glycoprotein (MOG) is found exclusively in the CNS, where it is localized on the surface of myelin and oligodendrocyte cytoplasmic membranes. The monoclonal antibody 8-18C5 identifies MOG. Several studies have shown that anti-MOG antibodies can induce demyelination, thus inferring an important role in myelin stability. In this study, we demonstrate that MOG consists of two polypeptides, with molecular masses of 26 and 28 kDa. This doublet becomes a single 25-kDa band after deglycosylation with trifluoromethanesulfonic acid or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, indicating that there are no or few O-linked sugars and that the doublet band represents differential glycosylation. Partial trypsin cleavage, which also gave a doublet band of lower molecular weight, confirmed this idea. MOG was purified by polyacrylamide gel electrophoresis, followed by electroelution. Three N-terminal sequences of eight to 26 amino acids were obtained. By western blot analysis, no binding was found between MOG and cerebellar soluble lectin. MOG does not seem to belong to the signal-transducing GTP-binding proteins. Reduced MOG concentrations were observed in jimpy and quaking dysmyelinating mutant mice, giving further support to its localization in compact myelin of the CNS.