A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P₀ protein and a basic protein of slightly larger mol wt. than the P₁ protein of rat myelin.     

Susceptibility of myelin proteins to a neutral endoproteinase: The degradation of myelin basic protein (MBP) and P2 protein by purified bovine brain multicatalytic proteinase complex (MPC)

Multicatalytic proteinase complex (MPC) was isolated from bovine brain and the susceptibility of myelin basic protein (MBP) and P₂ protein of bovine central and peripheral nervous system was examined. SDS-polyacrylamide electrophoretic analysis of purified MPC revealed protein bands of molecular weight ranging from 22–35 kDa. The enzyme is activated by SDS at a concentration less than 0.01%. Upon incubation with MPC, purified MBP and P₂ proteins were degraded into smaller fragments. There was a 57% and 100% loss of MBP at 2 and 6 hours of incubation. The P₂ protein which is not susceptible to any endogenous non-lysosomal enzyme thus far studied was digested into small peptide fragments only in the presence of SDS (0.01%) and not in its absence. These results indicate that MPC which is active at physiological conditions may have a role in the turnover of myelin proteins and in demyelinating diseases.     

Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane

Two major glycoproteins of bovine peripheral nerve myelin were isolated from the acid-insoluble residue of the myelin by a procedure involving delipidation with chloroform/methanol (2:1, v/v) and chromatography on Sephadex G-200 column with a buffer containing sodium dodecyl sulfate. The separation patterns of the proteins on the gel were affected considerably by the dodecyl sulfate concentration in the elution buffer. At above 2% dodecyl sulfate concentration in the elution buffer, the glycoproteins could be separated clearly on the gel and were purified. The purified proteins, the BR protein (mol. wt. 28 000) and the PAS-II protein (mol. wt. 13 000), were homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis. The NH₂-terminal amino acids of the BR and the PAS-II proteins were isoleucine and methionine, respectively. The BR protein contained glucosamine, mannose, galactose, fucose and sialic acids and the PAS-II protein contained glucosamine, mannose, galactose, fucose and glucose. Neither the BR protein nor the PAS-II were a glycosylated derivative of a basic protein of bovine peripheral nerve myelin, a deduction based on the results of amino acid analysis. The two major glycoproteins were observed commonly in the peripheral nerve myelin of cows, pigs, rabbits and guinea pigs, using dodecyl sulfate-polyacrylamide gel electrophoresis.     

Purification and partial characterization of two basic proteins from human peripheral nerve.

Two basic proteins, denoted P1 and P2 protein, were purified from human sciatic nerve. The isolation was achieved by the following steps: delipidation with chloroform/methanol mixtures, dry acetone and dry ether; acid extraction at pH 2; ion exchange chromatography on QAE-Sephadex A-25 and gel filtration on Sephadex G-100. P1, P2 proteins and the basic protein of the central nervous system have been shown to have different electrophoretic mobility, and each of the two peripheral basic proteins was shown to be homogeneous by disc electrophoresis. The molecular weight of P1 protein is around 14 100 and that of P2 protein is around 12 200, as determined by ultracentrifugal analysis. There was some difference in the amino acid composition of human P1 and P2 protein, and a marked difference between their composition and the composition of central basic protein and bovine peripheral P1 and P2 proteins which were described previously. When injected to animals, P1 protein induced only experimental allergic neuritis while P2 protein induced both mild experimental allergic neuritis and experimental allergic encephalomyelitis. Thus, the human P1 protein is similar to the bovine P1 protein and human P2 protein is similar to bovine P2 protein, concerning their electrophoretic mobilities, molecular weights and biological properties.     

COMPARATIVE STUDIES ON THE MYELIN PROTEINS OF BOVINE PERIPHERAL NERVE AND SPINAL CORD

Abstract— (1) Two myelin fractions of bovine peripheral nerve and spinal cord have been studied comparatively. Cholesterol as well as cerebroside content per mg of protein in the peripheral nerve myelin was less than that in the spinal cord myelin, while no significant difference in the total phospholipid content was noted.
(2) The basic proteins in myelin fractions were quantitatively estimated by disc gel electrophoresis. Around one-fourth of the total myelin protein in the bovine peripheral nerve was a basic protein with a mobility of 1.07 relative to lysozyme by Reisfeld's disc gel electrophoresis.
(3) The myelin proteins in the peripheral nerve were less completely solubilized than those of the spinal cord by treatment with deoxycholate as well as by Triton-salt solution. The protein fractions obtained from the peripheral nerve myelin by techniques similar to that for obtaining the proteolipids from the spinal cord myelin, contained different types of protein.
(4) 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the peripheral nerve myelin was only one tenth of that in the spinal cord myelin. The Triton-salt insoluble fraction showed remarkable high activity among subfractions of the spinal cord myelin.
(5) By immunological studies, it may be concluded that an antigenic substance for experimental allergic neuritis was localized in the peripheral nerve myelin, but not in its basic protein.     

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.     

SOME PROPERTIES OF A MAJOR STRUCTURAL GLYCOPROTEIN OF SCIATIC NERVE

The major protein of rat sciatic nerve is a glycoprotein which consists of a protein moiety attached to galactose, mannose and perhaps other sugars. On controlled tryptic digestion, it splits into a glycopeptide and a smaller fragment similar in molecular size to peripheral nerve basic proteins. Both the basic proteins of peripheral nervous system (PNS) myelin and the glycoprotein are antigenically active when administered to guinea pigs and produce sciatic nerve lesions similar to those described for experimental allergic neuritis. It is suggested from the amino acid analysis and its antigenic properties that the protein moiety of the glycoprotein may contain a sequence which is similar to the basic proteins of PNS myelin.     

The complete amino acid sequence of the rabbit P2 protein

P2 protein is a small basic protein (Mr = 14,820) found in peripheral nerve myelin and spinal cord myelin. There is now overwhelming evidence that P2 protein is the crucial antigen involved in the induction of experimental allergic neuritis, an autoimmune disease of the peripheral nervous system. The complete amino acid sequence of rabbit P2 protein was derived by sequence analysis of cyanogen bromide peptides and peptides obtained by proteolysis using Staphylococcus aureus V8 enzyme, trypsin, or clostripain. There are 131 amino acids and an excess of the basic amino acids lysine and arginine; histidine is absent. There are 3 highly hydrophobic regions in the P2 molecule. Probability analysis of the sequence predicts a high degree of beta structure, essentially in agreement with CD data.     

Proteolysis of peripheral nerve myelin in acute experimental allergic neuritis

Proteolysis of peripheral nerve myelin was studied in rats with experimental allergic neuritis (EAN). In vitro measurements using rat sciatic nerve homogenate and denatured bovine myelin as a substrate showed two myelin specific enzyme activities at pH 3.8 (inhibited by pepstatin) and pH 5.8 (inhibited by PMSF) in the normal rat and newly appearing activities at pH 2.8 (inhibited by pepstatin) and pH 5.0 (not characterized) in the EAN rat. In EAN the proteolytic activity was not restricted to myelin substrate but degraded total sciatic nerve protein as well. Endogenous sciatic nerve protease at pH 5.8 did not significantly change in activity during the course of disease. On the contrary, activity of acid protease at pH 2.8 corresponded well to the disease. Myelin degradation in EAN, therefore, appears to be mainly due to exogenous non-tissue protease.Abbreviations EAN experimental allergic neuritis - EDTA ethylenediaminetetraacetic acid - HBM hydroxymercuro benzoate - PLP proteolipid protein - PMSF phenylmethylsulfonyl fluoride - PNS peripheral nervous system - SDS sodium dodecylsulfate - TCA trichloroacetic acid This work is part of the M.D. thesis of R. B.     

GANGLIOSIDES OF PERIPHERAL NERVE MYELIN

—Gangliosides have been isolated from myelin obtained from three types of peripheral nerve: bovine spinal roots, bovine sciatic nerve and human sciatic nerve. Yields in most cases were 218–287 μg of lipid-bound sialic acid per g myelin, less than half that previously obtained from CNS myelin. Myelin accounted for approx 60% of total ganglioside present in whole spinal root. The human sample contained only N-acetylneuraminic acid but the two bovine preparations contained that as well as N-glycolylneuraminic acid; N-acetylglucosamine and N-acetylgalactosamine were both present in all three preparations. Sphingosine was the major long-chain base in each preparation while 4-eicosasphingenine (d20:1) comprised about 14% in the two bovine samples and 3% in the human sample. The major fatty acids in all preparations were 16:0, 18:0, 22:0, 24:0 and 24:1. Sialosylgalactosyl ceramide (G₇), a ganglioside characteristic of CNS myelin, was not detected in any of the PNS samples. The majority of gangliosides in bovine spinal root myelin were monosialo species, although the structures differed in some respects from those of CNS myelin. The molar concentration of lipid-bound sialic acid in PNS myelin is roughly equivalent to that of the P₁ basic protein.     

Experimental allergic encephalomyelitis. Isolation of basic proteins and polypeptides from central nervous tissue

1. Basic protein (mol.wt. 16500) and polypeptides (mol.wt. 3500) were isolated from bovine spinal cord by a procedure involving defatting, acid extraction of the defatted material and repeated chromatography on Sephadex G-50. Similar fractions were isolated from guinea-pig brain. 2. These fractions produced experimental allergic encephalomyelitis in guinea pigs. 3. The polypeptides appeared to be derived from a basic protein of myelin as a result of the action of an acid proteinase during extraction with acid. Similar proteolysis might also occur in the isolation of other biologically active polypeptides from acetone-dried powders of nervous tissue. The activity of the acid proteinase was lowered by defatting with chloroform-methanol. 4. Peptides from tryptic digests of encephalitogenic polypeptides and protein were also encephalitogenic, which suggests that the encephalitogenic determinant may be quite a short sequence of amino acids. 5. These encephalitogenic polypeptides are further examples of antigens of low molecular weight.     

A biochemical comparison of Xenopus laevis and mammalian myelin from the central and peripheral nervous systems.

Myelin purified from the central nervous system of Xenopus laevis contained the same major lipid and protein components as human myelin. However, some minor differences in the myelin proteins were noted. The Xenopus basic protein had a higher apparent mol wt. on sodium dodecyl sulfate gels than the corresponding mammalian protein. The absolute specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in the Xenopus myelin was considerably higher than in mammals. There were differences in the high mol wt. proteins, and the glycoproteins in Xenopus myelin were more heterogeneous than those in mammals. Peripheral myelin from Xenopus sciatic nerve was compared with that from the rat. The lipids in the two types of myelin were similar. There was a major glycoprotein in the Xenopus myelin corresponding to the P0 protein and a basic protein of slightly larger mol wt. than the P1 protein of rat myelin.     

The P2 protein of bovine root myelin: partial chemical characterization.

The P2 protein of bovine root myelin has chemical characteristics which differentiate it from other myelin basic proteins. The tryptic peptide map of the bovine P2 protein is distinctly different from the map of either the rabbit PI protein or the bovine CNS myelin basic protein. The tryptic peptides of the P2 protein show no overlap in either map positions or amino acid content with the peptides of the CNS myelin basic protein. Analysis of the individual peptides in the P2 map accounted for all of the amino acids present in the analysis of the whole protein. The P2 protein has a blocked NH₂-terminus, lysine at its COOH-terminus and no hexosamine. CD studies revealed that the P2 protein has a very stable β-structure in aqueous solution at neutral or basic pH and retains much of this structure in acid and in 8 M urea. It is suggested that these structural properties are relevant to the dual role of the P2 protein as a membrane constituent and as an antigen.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

A permanent rat T cell line that mediates experimental allergic neuritis in the Lewis rat in vivo

A rat T cell line of the "helper" phenotype (W3/25-positive, OX 8-negative) has been derived from Lewis rats inoculated with P2 protein isolated from bovine PNS myelin. The line LiP2/A is exquisitely specific for P2 protein, exhibiting no reactivity to bovine basic protein or to PPD. In addition to responding strongly to the intact P2 protein, the line cells show some response to a synthetic peptide containing the neuritogenic amino acid sequence of P2 protein (SP-B, residues 66-78). Intravenous inoculation of naive rats with as few as 10(4) activated LiP2/A cells leads to the onset of mild clinical signs of experimental allergic neuritis. Higher doses of cells lead to more severe clinical disease. Histologic examination of clinically ill animals confirmed the disease as EAN. The pathologic lesions were confined to the PNS and spared the central nervous system. The lesions consisted of marked perivascular cuffs and infiltrates of inflammatory cells associated with marked degenerative changes--demyelination and some axonal degeneration.     

The binding of calmodulin to myelin basic protein and histone H2B.

1. A calmodulin-binding protein of apparent mol.wt. 19 000 has been purified from chicken gizzard. Similar proteins have been isolated from bovine uterus, rabbit skeletal muscle and rabbit liver. 2. These proteins migrated as an equimolar complex with bovine brain calmodulin on electroporesis on polyacrylamide gels in the presence of Ca2+ and 6M-urea. The complex was dissociated in the presence of EGTA. 2. The chicken gizzard calmodulin-binding protein has been shown to be identical with chicken erythrocyte histone H2B on the basis of partial amino acid sequence determination. 4. The calmodulin-binding proteins of apparent mol.wt. 22 000 isolated previously from bovine brain [Grand & Perry (1979) Biochem. J. 183, 285-295] has been shown, on the basis of partial amino-acid-sequence determination, to be identical with myelin basic protein. 5. The activation of bovine brain phosphodiesterase by calmodulin is inhibited by excess bovine uterus calmodulin-binding protein (histone H2B). 6. The phosphorylation of myelin basic protein by phosphorylase kinase is partially inhibited, whereas the phosphorylation of uterus calmodulin-binding protein (histone H2B) is unaffected by calmodulin or troponin C. 7. The subcellular distribution of myelin basic protein and calmodulin suggests that the two proteins do not exist as a complex in vivo.     

Molecular mimicry and the autoimmune response to the peripheral nerve myelin PO glycoprotein

In the Lewis rat immunisation with the myelin PO glycoprotein can induce an inflammatory demyelinating disease of the peripheral nervous system, experimental allergic neuritis (EAN), which has many clinical and histopathological parallels with the human disease the Guillain-Barre syndrome. In view of the reported association of GBS with a number of infectious agents we have investigated whether molecular mimicry may occur between microbial antigens and the PO protein that could possibly trigger a similar pathogenic autoimmune response in man. A computer search of the available protein sequence data bases identified several absolute sequence homologies between PO and viral proteins that involve five or more consecutive amino acid residues. Four of these sequence homologies involved viral pathogens previously associated with the Guillain-Barre syndrome, namely Epstein-Barr virus (EBV), cytomegalovirus (CMV), Varicella zoster virus (VZV) and human immunodeficiency virus I (HIV I). Although, sequence homologies were also found between viral peptides and the neuritogenic determinants of PO, residues 56–71 and 180–199, these homologies proved incapable of eliciting EAN in the Lewis rat. These observations are discussed with reference to the role that molecular mimicry between T cell epitopes on pathogen derived antigens and the PO protein may play in the pathogenesis of the Guillain-Barre syndrome.Abbreviations EAN Experimental allergic neuritis - EAE experimental allergic encephalomyelitis - PNS peripheral nervous system - CNS central nervous system - MBP myelin basic protein - GBS Guillain Barre syndrome - CFA complete Freund's Adjuvant - LPC lysophosphatidyl choline - VZV Varicella zoster virus - CMV cytomegalovirus - EBV Epstein Barr virus - HIV I human immunodeficiency virus I Special issue dedicated to Dr. Alan N. Davison     

ELECTROPHORETIC STUDIES OF BASIC PROTEINS CAPABLE OF INDUCING EXPERIMENTAL ALLERGIC NEURITIS

Abstract— A basic protein fraction capable of inducing experimental allergic neuritis was isolated from peripheral nervous tissues of ox and rabbit. The electrophoretic mobility was lower than that of lysozyme at pH 4 , 2 and 8 , 2. The yield was approximately 0 , 1 g per cent of fresh tissue weight.     

A RADIOIMMUNOASSAY FOR MYELIN BASIC PROTEIN AND ITS USE FOR QUANTITATIVE MEASUREMENTS

—A specific radioimmunoassay (RIA) for myelin basic protein is described which is sensitive to 10⁻⁹ g of basic protein. The amount of basic protein detected in isolated myelin by the RIA and by SDS-gel electrophoresis and spectrophotometric quantitation agree to within experimental error. In contrast to isolated myelin, the major portion of the basic protein in fresh tissue is not accessible to its antibody. It is shielded from its antibody in a complex which is disrupted by heat, organic solvents, and various detergents. Maximum antibody binding was obtained with tissue heated to 100°C for 10 min. It is possible to calculate that the RIA quantitatively detects basic protein in boiled tissue. Boiled adult rat brain contains approximately 2·5 μg of basic protein/mg wet wt of cerebral cortex. The antibody to basic protein has no capacity to bind non-neural tissues.     

Effect of inorganic lead exposure on myelination in the rat

The effect of defined lead burdens on myelination of the central and peripheral nervous systems was studied in neonatal Long-Evans rats. Pups were exposed to inorganic lead (100 or 400 mg Pb as lead acetate/kg body wt/day by gastric intubation) from day 2 following birth to 30 days of age. Accumulation of myelin in forebrain was not affected by the 100-mg dosage, but at the 400 mg/kg dosage level, myelin accumulation was reduced by approximately 42% on a per gram forebrain basis relative to vehicle-intubated animals. The deficit was over 50% on a per forebrain basis, since there was also a slight reduction in brain weight. This lead effect was observed at both 15 and 30 days of age. Accumulation of myelin in optic nerve (determined on the basis of proteolipid protein concentration) was also reduced by 30% relative to controls by this dosage level. However, myelination in sciatic nerve (determined on the basis of P₀ protein concentration) was not affected by this exposure regimen. Myelin deficits were greater than could be accounted for by undernutrition arising secondary to lead exposure and were not due to a developmental delay in the onset of myelination.