Encephalitogenic Basic Protein Research Articles

Suppression of experimental allergic encephalomyelitis by chemically modified encephalitogen

The results of enzymatic and chemical degradation studies of the basic myelin protein suggested that tryptophan is essential for encephalitogenic activity in the guinea pig. The object of the present investigation was to modify the protein through its tryptophan residue in such a way that it becomes non-encephalitogenic, yet suppresses experimental allergic encephalomyelitis (EAE). Accordingly, the single tryptophan residue of the protein was modified with specific reagents such as 2-hydroxy-5-nitrobenzylbromide (HNB-BR), 2-nitrophenylsulfenyl chloride (NPSCl) and performic acid. Injection of the modified proteins did not result in neurological disease, although some guinea pigs did exhibit histological changes in the brain. Starting 3 days after injection with the active inoculum, the treatment with the modified proteins was carried out for four weeks, using a total of 900 μg og the modified proteins. The guinea pigs treated with HNB and performic acid oxidized protein did not show neurological signs of the disease, but from the NPSCl group, two out of 10 developed EAE, although at a later data compared with those guinea pigs which were injected with the unmodified encephalitogen. The results not only confirm previous findings that intact tryptophan in the protein is a prime requirement for the production of EAE in guinea pigs, but also provide evidence that tryptophan is not essential for suppression of the disease. The conclusion we may draw from these studies is that the encephalitogenic basic myelin protein has two active sites: one which contains tryptophan required for the paralytic effect, the other without tryptophan which provides protection.

Localization of tumour specific antigen on external surface of plasma membrane.

FOLLOWING the adventitious finding that lymphocytes from patients with malignant (but not benign) neoplasia are sensitized to encephalitogenic basic protein of myelin (EF)1 and to a protein similarly extracted from a variety of tumours2, a logical step would be to determine the subcellular localization of the specific antigen(s) on or within the cancer cell and whether or not the antigen continues to be associated with human malignant (or transformed) cells long maintained in culture. If this were so, then such cultures might serve as a convenient and standard source for an antigen of considerable potential in the diagnosis—and perhaps also the treatment—of malignant disease. The allied question of the identity and uniqueness of the antigens extractable from different cell lines or tumours will be the subject of a separate communication.

Studies on protein extracted by chloroform/methanol and dilute acid from tissue membranes and particulate fractions

1. 1. Among the proteins extractable from tissue membranes and particulates by chloroform/methanol and dilute acid is one which appears common to all sources except the membrane of myelin. 2. 2. This protein has been designated AEMP (acid-extractable membrane protein). 3. 3. Polyacrylamide gel electrophoresis indicates that AEMP has a similar mobility to that of the encephalitogenic basic protein (EF) of myelin. 4. 4. The similarities and differences between EF and AEMP are discussed.

A myelin protein fraction extracted with thioethanol

A heterogeneous protein fraction has been extracted from myelin with properties different from the basic encephalitogenic protein or the Folch-Lees proteolipid protein. The major component of the protein fraction extracted from myelin with 0.014 M thioethanol migrated with an intermediate mobility between that of the encephalitogenic protein and that of the proteolipid protein on 7.5% polyacrylamide gel at acid pH. The amino acid composition of the fraction was different from both that of the encephalitogenic protein and that of the proteolipid protein. However, the amino acid composition was very similar to fractions of myelin obtained by Wolfgram and Eng et al. There were small amounts of glucose and galactose present, but no focose. The protein fraction was tested in guinea pigs for EAE activity, and it was shown to be encephalitogenic.

Purification of fully active human encephalitogenic basic protein on sulfoethyl-sephadex.

Human encephalitogenic basic protein (HEP) was obtained in high yield after chromatography on sulfoethyl-Sephadex C-50 (SE-Sephadex). The protein was shown to be homogeneous and was identical in physical properties with samples of human encephalitogenic protein obtained by other methods. It was also shown to induce typical clinical and histological experimental allergic encephalomyelitis (E.A.E.) in adult guinea pigs injected with 50 μg HEP in Freund's complete adjuvant (FCA). Gel-filtration radioimmunoassay was used to study immunogenicity, antigenicity, and antigenic cross-reactivity of HEP; seven of 11 guinea pigs injected with HEP in FCA had detectable serum antibody to 125I-HEP, indicating that the SE-Sephadex preparation was fully immunogenic and antigenic; HEP and another preparation of human encephalitogenic basic protein prepared by a different method were equally capable of inhibiting the binding between 125I-HEP and anti-HEP serum indicating that the two samples tested were antigenically identical. We believe that the new technique represents a significant technological advance because it provides a most effective and rapid method for the isolation of fully active encephalitogenic basic protein.

Proteins of peripheral nerve myelin in diabetic neuropathy

The proteins of human peripheral nerve myelin are similar to those of other mammalian peripheral nerves and different from those of the central nervous system. The most notable characteristics of the peripheral myelin are the lack of the encephalitogenic basic protein and the presence of the main protein between the major membrane protein and the group of basic proteins. The sedimentation properties of the diabetic peripheral myelin are different from those of normal myelin suggesting serious structural disintegration. The amount of the main protein is decreased and the basic proteins are also weaker. The intensity of the major membrane protein is increased. It is concluded that demyelination of the peripheral nerve in diabetic neuropathy is at least partly based on the inability of the Schwann cell to produce proteins for the formation of myelin.

Experimental allergic encephalomyelitis (E.A.E.): synthesis of the 7-11, 11-20, and 12-20 peptides of human encephalitogenic protein and inhibition of histological E.A.E. by the 12-20 peptide.

Peptides comprising the 7–11, 11–20, and 12–20 sequences of human encephalitogenic basic protein (HEP) were synthesized by the solid-phase procedure, purified by gel-filtration and high-voltage paper electrophoresis, and characterized by amino acid analysis, thin-layer chromatography, high-voltage paper electrophoresis, and paper chromatography.The peptides were studied for the capacity to induce E.A.E. when injected with Freund's complete adjuvant (FCA) into guinea pigs. The same peptides were also studied for the capacity to inhibit development of E.A.E. in guinea pigs injected with peptide/FCA 28 days before injection with encephalitogenic HEP in FCA. The results were as follows: (1) all three peptides induced mild clinical E.A.E. but the 12–20 peptide induced severe disease in five out of 40 guinea pigs; (2) none induced histological lesions typical of E.A.E.; (3) none induced antibody reactive with 125I-HEP; (4) none induced delayed sensitivity to the homologous peptide or HEP; (5) injection of the 12–20 peptide in FCA inhibited the development of histological E.A.E. in guinea pigs injected later with HEP/FCA, whereas the 7–11 and 11–20 sequences did not; (6) humoral immunity to injection of HEP/FCA was not prevented by prior injection of the synthetic peptides in FCA.

Probable Identity of Tissue Specific Historie with Encephalitogenic Protein

SOME years ago Tomasi and Kornguth isolated a basic protein from the brains of adult pigs1. Fluorescent immunohisto-chemical studies indicated that it was localized in the nuclei of neurones and spermatogonia and the suggestion was made that it was a tissue-specific histone2. The amino-acid composition of this brain protein is very similar to that of a basic encephalitogenic protein isolated from myelin of a variety of animal species3–12.

Molecular weight of encephalitogenic protein by electrophoresis in sodium dodecyl sulfate (SDS)-acrylamide gels.

SummarySDS-acrylamide electrophoresis studies indicate that the molecular weight of the basic encephalitogenic protein isolated from chloroform-methanol-extracted bovine brain is 22, 166 ± 577 daltons.

Specific binding of peroxidase-labeled myelin basic protein in allergic encephalomyelitis.

A conjugate of horseradish peroxidase and the encephalitogenic basic protein from myelin has been used to study the antigen reactivity of tissue in the autoimmune disease, experimental allergic encephalomyelitis. Control conjugates were also prepared of peroxidase and bovine serum albumin and of peroxidase and lysozyme, another basic protein. The basic protein from myelin conjugate was specifically bound by lymph node cells from rabbits immunized against the basic protein. Some of these cells appeared to be plasma cells. The conjugate was also specifically bound by occasional cells in the spinal-cord infiltrates of animals with early signs of allergic encephalomyelitis. These cells resembled large lymphocytes and plasma cells. There was no difference between the binding of basic protein of bovine and rabbit origin. The findings suggest the possibility that a local release of antibody within the target organ may play a role in the pathogenesis of allergic encephalomyelitis.

A conformation change induced in the basic encephalitogen by lipids

Using ORD/CD we have studied the conformation of the basic encephalitogenic protein isolated from normal human myelin. Using the curve fitting method we have found this protein to be essentially random. When sodium dodecyl sulphate was added to a solution of the protein the random curve obtained by ORD was altered to an α-helical type. Addition of sodium dodecyl sulphate, sodium oleate or triphosphoinostide to the protein, all had the effect of changing the CD curve from random to α-helical type. The most effective agent was sodium dodecyl sulphate since it produced the largest rotation. Positively charged lipids, cetyltrimethylammonium bromide and lysolecithin did not affect the random CD curve at pH 2.0. However, at pH 5.0 where lysolecithin is a zwitterion, the CD curve appeared to be α-helical.

Protein composition of multiple sclerosis myelin.

Myelin was purified from control and multiple sclerosis (MS) white matter. Purity of samples was checked with electron microscopy and biochemical analyses, and protein composition of myelin samples was evaluated with horizontal gel electrophoresis. Results showed a typical and consistent pattern in electropherograms with major protein constituents and some minor ones. All other proteins were similar in control and MS myelin samples except the encephalitogenic (basic) protein which showed decreased values in MS myelin. The role of encephalitogenic protein in the pathogenesis of MS is discussed on the basis of these findings.

The vulnerability of immature rabbits to experimental allergic neuritis: A light and electron microscope study

The vulnerability of immature rabbits to experimental allergic neuritis (EAN) was investigated. The incidence, severity and duration of EAN signs are described; histological changes occuring in dorsal roots and peroneal nervws were observed by light and electron microscopy. Unequivocal signs of EAN occured only in rabbits older than 4 weeks at the time of antigen challenge. Immature EAN rabbits differ in the course of the disease from adults in (a) lower incidence, (b) a greater recovery rate. The time of onset of signs and the duration of signs were not significantly different from the adult group. The results are interpreted in terms of the development of immunological competence, and relative rates of myelic synthesis and myelin degradation. As in the adult, EAN in immature rabbits is characteristically a segmental demyelinating, axon-sparing condition. Demyelination is largely mediated by cells of exogenous origin, though evidence for a contribution by circulating antibodies and degradation of myelin by the Schwann cell is discussed. The type of demyelination represented by EAN is contrasted with that occuring after diptheritic intoxication. The vulnerability of spinal roots to demyelination in EAN is interpreted in terms of the greater permeability of neural capillaries in roots compared with peripheral nerves. The functional significance of the splitting of myelin at the intraperiod line is considered to be related to the putative distribution of encephalitogenic basic protein. A hypothesis is proposed to account for the sparing of the Schwann cell plasma membrane in an autoimmune response which selectively degrades myelin.

Amino acid sequence of the encephalitogenic basic protein from human myelin.

Myelin from the central nervous system contains an unusual basic protein, which can induce experimental autoimmune encephalomyelitis. The basic protein from human brain was digested with trypsin and other enzymes and the sequence of the 170 amino acids was determined. The localization of the encephalitogenic determinants was described. Possible roles for the protein in the structure and function of myelin are discussed.

Isolation and partial characterization of methylated arginines from the encephalitogenic basic protein of myelin.

Two methylated derivatives of arginine were isolated from the encephalitogenic protein of myelin from the central nervous system. Evidence is presented for the proposed structures, omega-NN'-dimethylarginine and omega-N-monomethylarginine. In the encephalitogenic protein from human brain the proportion 1:6:10 for arginine:monomethylarginine:dimethylarginine residues was found to occur at position 107. Possible roles for the methylated arginine in conformational changes and altered ion-exchange behaviour are discussed.

An encephalitogenic basic protein from human peripheral nerve.

A basic protein isolated from human peripheral nerve was found to produce allergic encephalomyelitis in guinea pigs with very little neuritis. Immunologic cross-reactivity was found between the periph

Suppression of experimental allergic encephalomyelitis by a synthetic polypeptide.

AbstractThree random basic copolymers of amino acids were tested for their effect on experimental allergic encephalomyelitis (EAE). One of these copolymers denoted as Cop 1, composed of alanine, glutamic acid, lysine and tyrosine, with a molecular weight of 23 000, showed a marked suppressive effect on the disease. The intravenous administration of Cop 1 in physiological saline, as late as 5 days following the challenge with the disease‐inducing dose of the basic encephalitogenic protein, reduced the clinical incidence of EAE from 64% in the control group to 22%; the histological lesions were also decreased both in prevalence and in severity. The suppressive effect on the disease attained by the synthetic copolymer is of the same order of magnitude as that previously reported for the basic encephalitogen.The effect of the copolymers appears to be specific, since neither an acidic amino acid copolymer, nor unrelated basic proteins, had any protective action. On the other hand, a second batch of Cop 1 showed activity identical to that of the first batch. The potential applicability of this non‐encephalitogenic and non‐immunosuppressive material is discussed.

Localization of the Active Site through Chemical Modification of the Encephalitogenic Protein

Abstract The encephalitogenic basic protein of the myelin contains 168 amino acid residues with a calculated molecular weight of 18,200. Among these residues, there are 2 methionines and 1 tryptophan. In order to locate and identify the number of encephalitogenic determinants of this protein, chemical degradation of the methionine and modifications of the tryptophan by various specific reagents were carried out. Cleavage of the polypeptide chain at the methionyl residues was effected with cyanogen bromide. Three peptides were produced. Peptide C-1 (20 amino acid residues) was derived from the amino-terminal part of the protein, Peptide C-2 (143 residues) from the middle part, and Peptide C-3 (3 residues) from the carboxyl terminus. Only Peptide C-2 is active at the same level as the parent protein. These results are in agreement with our previous report that part of the amino-terminal segment of the protein can be removed without affecting the biological activity. The tryptophan residue was modified by the following reagents: 2-hydroxy-5-nitrobenzyl bromide, oxidation by performic acid, alkylation with 2-nitrophenylsulfenyl chloride, and formylation with formic acid saturated with HCl gas. The modified proteins, except the formylated one, were not encephalitogenic in guinea pigs. The animals did not show any neurological sign during the assay period of 30 days. The same compounds were tested with rabbits and the rabbits developed typical experimental allergic encephalomyelitis. It is concluded that there are at least two encephalitogenic determinants. One contains tryptophan, a residue essential for activity in guinea pigs, and the other is active in rabbits without the tryptophan residue. The results suggest that the encephalitogenic determinant is species-specific.

Amino acid sequence of the basic protein of the myelin membrane.

The amino acid sequences of the encephalitogenic basic protein, A1, from bovine and human myelin are similar, differing by only 11 residues. The sequence reveals that while basic residues are spread randomly over most of the polypeptide chain, several regions (8-10 residues) exist that are nonpolar in character. The bovine protein has 170 residues with molecular weight 18,400. The human protein, which has an additional His-Gly sequence, contains 172 residues. The major encephalitogenic determinant (tryptophan region) of the bovine protein differs from the human only by a lysine to arginine substitution. The structural features of the A1 protein are discussed, with special reference to its role in stabilization of the myelin membrane, and its relation to multiple sclerosis.

Immunologic Properties of the Main Encephalitogenic Peptide from the Basic Protein of Human Myelin

Abstract The encephalitogenic basic protein from human brain was digested with pepsin and encephalitogenic peptides were isolated. Two overlapping peptides containing 15 and 22 amino acids had similar encephalitogenicity as the parent protein. Both peptides induced delayed hypersensitivity to the protein but no detectable circulating antibody in guinea pigs. There was a striking correlation between the occurrence of experimental autoimmune encephalomyelitis and low yields of peritoneal exudate cells. The main encephalitogenic determinant resides within the sequence Ser-Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-. We postulate that the integrity of this region of the basic protein of myelin is vital to the normal functioning of the central nervous system. These small peptides should be of value in investigating the role of delayed hypersensitivity in autoimmune disease.