Mouse Myelin Basic Protein Research Articles

Herpes simplex virus-enhanced production of autoantibodies against myelin basic protein in mice

In 5 per cent of untreated and apparently healthy Swiss albino mice antibodies were demonstrable against myelin basic protein (MBP) prepared from human brain tissue. Associated with infection with herpes simplex virus (HSV) type 1, enhancement of the antibody formation against MBP was encountered. Thus of infected but symptom-free animals 15-25 per cent revealed anti-MBP antibodies. The latter type of antibodies appeared later than antibodies against HSV and were found predominantly in animals inoculated via the intraperitoneal route. No signs of demyelination in spinal cords or spinal nerve roots were observed in MBP antibody-positive HSV-immunized animals. Results reported may indicate triggering of an autoantibody formation against MBP and are discussed in relation to demyelinating disease.

Alternative splicing accounts for the four forms of myelin basic protein

We have isolated cDNA clones encoding the four different forms of mouse myelin basic protein (MBP) and have analyzed the structure of the MBP gene. The three larger forms of MBP differ from the smallest by the inclusion of either or both of two short amino acid sequences at positions 57 and 124 of the smallest protein. The mouse genome contains a single MBP gene comprised of seven exons. The two amino acid sequences present only in the larger MBPs are encoded by separate exons. Furthermore, all exons in the coding region begin or end in complete codons so that alternative splicing does not alter the reading frame. We conclude that the four forms of this myelin protein are encoded in separate mRNAs, each derived by a simple alternative splicing of the primary MBP gene transcript. Comparison of the amino acid sequence encoded by each exon with a recent model of the secondary structure of MBP suggests that each of the seven exons encodes one or two of the predicted structural motifs of the protein.

Encephalitogenic T cell clones specific for myelin basic protein. An unusual bias in antigen recognition.

Class II-restricted T cell clones specific for myelin basic protein (MBP) have been generated from PL/J and (PL/J X SJL/J)F1 [((PLSJ)F1] mice following sensitization to rat MBP. Of 17 T cell clones generated from (PLSJ)F1 mice, 5 are I-Au(A alpha uA beta u) restricted, one is restricted to I-As(A alpha sA beta s), 10 are restricted to hybrid I-A(u X s)F1 (A alpha sA beta u) determinants, and one clone is restricted to hybrid I-E(u X s) (E alpha uE beta s) molecules. Thus, of 16 I-A-restricted T cell clones generated from (PLSJ)F1 mice, only one is I-As-restricted, reflecting a lack of priming to MBP in association with I-As. T cell clones restricted to I-Au and to I-E (E alpha u E beta s) molecules recognize mouse (self) MBP. Furthermore, only the five T cell clones restricted to I-Au molecules recognize a determinant in common with mouse (self) MBP within the encephalitogenic N-terminal peptide. Three such I-Au restricted T cell clones, derived independently, cause paralysis in 100% of (PL/J X SJL/J)F1 mice tested. Acute, chronic unremitting, and chronic relapsing paralysis are all induced following injection of these clones. Administration of greater numbers of cloned T cells causes acute and fatal experimental allergic encephalomyelitis, while administration of lower numbers of cloned T cells is associated with chronic unremitting and relapsing paralysis. Paralysis induced with T cell clones shares many clinical, immunologic, and histologic aspects with human demyelinating diseases such as multiple sclerosis. Histopathology reveals perivascular lymphocytic infiltration, demyelination, and remyelination. These studies demonstrate the utility of T cell clones for analyzing the association between class II major histocompatibility complex molecules and disease susceptibility.

T-cell clones specific for myelin basic protein induce chronic relapsing paralysis and demyelination

Experimental allergic encephalomyelitis (EAE) serves as a model for autoimmune diseases mediated by T lymphocytes. Following sensitization to rat, mouse or guinea pig myelin basic protein (MBP) in complete Freund's adjuvant, inbred mouse strains PL/J (H-2u), SJL/J (H-2s) and (PL/J X SJL/J)F1((PLSJ)F1) develop EAE. Whereas sensitization to the N-terminal 37 amino-acid peptide of rat or guinea pig MBP [MBP(1-37)] induces EAE in PL/J mice, immunization to the C-terminal peptide (89-169) leads to EAE in SJL/J mice. The immune response to MBP in (PLSJ)F1 mice is not co-dominant; sensitization to the N-terminal peptide induces EAE, while sensitization to the C-terminal peptide does not. We have generated MBP-specific T-cell clones restricted to class II (Ia) antigens of the major histocompatibility complex (MHC) from PL/J and (PLSJ)F1 mice following sensitization to rat MBP. Two such I-Au-restricted T-cell clones that proliferate in response to the encephalitogenic N-terminal MBP peptide and recognize a shared determinant with mouse (self) MBP cause paralysis in 100% of (PLSJ)F1 mice tested. Paralysis is induced even when recipients are injected with as few as 1 X 10(5) cloned T cells. Relapsing paralysis followed in two-thirds of the recipients after recovery from acute paralysis, whereas one-third developed chronic persistent paralysis, a form of EAE not usually seen. Histopathology revealed intense perivascular inflammation, demyelination and remyelination within the central nervous system of paralysed mice. The experimental disease induced with these clones shares important features with human demyelinating diseases such as multiple sclerosis. This is the first demonstration that T-cell clones that respond to a defined self-antigen can induce clinical and histological autoimmune disease.

Chromosomal mapping of mouse myelin basic protein gene and structure and transcription of the partially deleted gene in shiverer mutant mice

The gene for mouse myelin basic protein (MBP) was mapped to chromosome 18 by hybridization of cloned MBP probes to DNA from hamster-mouse hybrid cell lines, showing it to be linked to the shiverer mutation which causes abnormal CNS myelination. Genomic blotting experiments show that in the mutants five of six exons which constitute the wild-type gene have been deleted. In shiverer brains the steady state level of transcripts that initiate correctly at the 5' end of the remaining exon 1 is reduced 16-fold. These RNAs are not spliced correctly and are not efficiently polyadenylated. It is proposed that the partial deletion of the MBP gene is an important part of the shiverer lesion.

Cloning and characterization of the myelin basic protein gene from mouse: one gene can encode both 14 kd and 18.5 kd MBPs by alternate use of exons

A mouse cosmid library was screened with a cDNA clone for rat small (M, 14,000) myelin basic protein (MBP). The entire MBP gene was isolated. Five exons were detected with the cDNA clone, which encode the Mr 14,000 MBP. A sixth exon, detected using a synthetic oligonucleotide, encodes the 41 amino acids specific to the Mr 18,500 form of MBP. Splicing together all six exons would give rise to Mr 18,500 MBP, while an mRNA omitting exon V would be translated to produce Mr 14,000 MBP. Further experiments indicate that there is probably a single MBP gene in the mouse genome, and that there is a single major 5' end for mouse MBP transcripts, 47 bp 5' of the initiator methionine codon.

Major histocompatibility complex-linked control of the murine immune response to myelin basic protein.

Recent experiments have shown that different regions of myelin basic protein (MBP) are encephalitogenic for different inbred strains of mice. It was therefore of interest to determine whether the immune response to MBP was MHC associated, and if so, what subregion controlled this response. Because PL/J and A/J mice were good responders to mouse MBP and C57Bl/10SN were not, B10.PL(73NS) and B10.A mice were immunized with mouse MBP under conditions designed to induce EAE. These strains were found to be highly susceptible. Intra-H-2 recombinant mice were then assessed for susceptibility. B10.A(4R) and B10.MBR were susceptible, whereas B10.A(5R) were resistant. Thus, EAE induced by purified MBP is under the control of the MHC, and the response maps to the I-A subregion. Production of IL 2 in vitro by T cells from MBP-primed mice in the presence of antigen and adherent cells was blocked by monoclonal antibody to the I-A, but not the I-E, subregion. When the specificity of the encephalitogenic response was tested, peptide 1-37 was active in B10.PL(73NS) and B10.A mice, whereas peptide 89-169 was active in A.SW, SWR, and B10.T(6R) strains. Serum from mice immunized with MBP peptides was assayed for antibody content. PL, B10.PL, and B10.A mice made a good antibody response to peptides 1-37 and 43-88 but were nonresponsive to peptide 89-169. SJL, A.SW, SWR, and B10.T(6R) mice responded well to peptide 89-169 but were poorly responsive to peptides 1-37 and 43-88.

Molecular Genetic Analysis of Myelin‐Deficient Mice: Shiverer Mutant Mice Show Deletion in Gene(s) Coding for Myelin Basic Protein

The gene expression of myelin basic proteins (MBPs) in shiverer mutant mice was investigated by the Northern and Southern hybridization techniques. In the control mice RNA molecules from the brains which were about 2,300 nucleotides in length were hybridized to cDNA of 1.8 kb encoding for a mouse MBP, but RNA from the brains of 3-week-old shiverer mutant mice contained no detectable amount of MBP transcripts hybridizing to this probe. Moreover the shiverer mutant mice lost several restriction fragments that hybridized to the same probe in the control mice when each of the five restriction enzymes, i.e., HindIII, PstI, PvuII, AccI, and StuI, was used. These data suggest that the shiverer mutation may correspond to the deletion of a large portion of MBP exon(s) in the gene, and this deletion causes inefficient transcription leading to the depletion of MBPs in the myelin and the dysmyelination observed in these mice.

Immunomodulation of relapsing experimental allergic encephalomyelitis

Mice immunized to produce relapsing experimental allergic encephalomyelitis (R-EAE) were treated with immunomodulating agents known to affect acute monophasic experimental allergic encephalomyelitis. Pretreatment with either mouse spinal cord or myelin basic protein in incomplete Freund's adjuvant decreased the incidence of R-EAE from 77% to 28 and 31%, respectively. Single doses of cyclophosphamide (CY) at the time of immunization did not affect development of R-EAE. CY given repetitively in low doses decreased the incidence of R-EAE to 10%. Therefore, R-EAE can be altered by immunomodulation, but the patterns differ from those seen in acute EAE.

Characterization of mouse myelin basic protein messenger RNAs with a myelin basic protein cDNA clone.

Using a family of synthetic tetradecamer oligonucleotides as a primer for cDNA synthesis and a second family of tetradecamers as a hybridization probe, we have prepared and isolated a cDNA clone of mouse myelin basic protein (MBP). The clone, pNZ111, corresponds to the region of the mRNA that codes for an amino acid sequence present in all four major forms of MBP. The relative abundance of MBP mRNA, estimated by dot blot hybridization, increased with the age of the mouse to a maximum at 18 days, then decreased to about one-fourth of that amount at later ages. Mouse MBP mRNAs, selected by their ability to hybridize to the clone, translate into the four forms of myelin basic protein. In RNA blot analyses, pNZ111 hybridized to multiple species of mouse mRNA. The predominant hybridization is to a broad band of RNAs ranging in length from 2,350 to 2,100 bases. These mRNA species are extremely long, considering that the largest MBP could be encoded by approximately 600 bases. In addition to these, there are also minor bands that hybridize with pNZ111, including a band of 4,100 bases and smaller ones of 1,900, 1,500, and 1,200 bases.

Purification of mouse myelin basic proteins by immunoaffinity chromatography

The preparation of two immunosorbent columns specific for mouse myelin basic proteins (BPs) is described. The columns were prepared from either affinity purified mouse 14K-antibodies or bovine BP fragment (residues 1–115) antibodies. The column prepared from mouse 14K-antibodies bound 216–441 μg of mouse BPs within its working range at pH 4.5. The second column, prepared from antibodies specific for bovine fragment 1–115, bound 245–442 μg of mouse BPs under similar conditions. The ratios of 14K to 18.5 K proteins present in the immunopurified BPs reflected those in the samples originally applied onto the column. Therefore, both columns were equally effective for the small scale isolation of mouse BPs from whole brain. Because it is easier to prepare large quantities of bovine BP fragment 1–115 than the mouse 14K protein, it is less difficult to obtain antibodies against the bovine fragment. As a result, the use of immunosorbent columns containing immobilized bovine BP fragment 1–115-antibodies to purify mouse BPs represents a practical alternative to using immunosorbent columns prepared with mouse 14K-antibodies.

Encephalitogenic activity of the small form of mouse myelin basic protein in the SJL/J mouse.

Mouse myelin basic proteins (MBP) were prepared by ion-exchange chromatography of an acid extract of homogenized, delipidated mouse brain. The use of a linear salt gradient during ion-exchange chromatography gave three peaks, 1, 2, and 3, of MBP. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of these peaks showed that peaks 2 and 3 were a mixture of the large and small forms of mouse MBP, whereas peak 1 was solely the small form. Tryptic peptides of mouse and rat small form MBP were compared by high-performance liquid chromatography. The elution times and absorbances at 210 nm were identical for 16 of 21 peaks derived from small form MBP of the two species. Amino acid analysis showed that 10 corresponding peaks from the small form of mouse and rat MBP had identical amino acid composition. When tested for encephalitogenicity in SJL/J mice, both the mixture of large and small forms of mouse MBP and the small form mouse MBP were highly encephalitogenic.

Epitopes of peptide 43-88 of guinea pig myelin basic protein: localization with monoclonal antibodies.

Two monoclonal antibodies, one raised by immunization with mouse myelin basic protein (MBP) and the second raised by immunization with peptide 68-88 of guinea pig MBP, were compared with respect to specificity. The former antibody (15.32) cross-reacted completely with rat, guinea pig, human, and bovine MBP. It also reacted with peptide 43-88 from each MBP. The latter antibody (22.17) was nonreactive with MBP, but cross-reacted with peptide 43-88 from rat, human, guinea pig, and bovine MBP. When tested with small peptides derived from peptide 43-88, antibody 22.17 reacted with an epitope in the C-terminal region. Antibody 15.32 reacted with an epitope in the N-terminal half of the peptide. The data show that 22.17 reacted with a unique epitope associated only with free peptide, whereas 15.32 recognized an epitope common to both peptide 43-88 and MBP.

Detection of autoimmune cells proliferating to myelin basic protein and selection of T cell lines that mediate experimental autoimmune encephalomyelitis (EAE) in mice.

A procedure is described for the detection of an in vitro proliferative response to the autologous mouse myelin basic protein in mice injected with mouse spinal cord homogenate (MSCH) or with myelin basic proteins (BP) of mouse (MBP) or rat (RBP) origin. The administration of MSCH, but not of MBP or RBP, in a suitable adjuvant could produce a reproducible clinical disease. Nevertheless, a proliferative response to the autologous MBP could not be detected after either inoculation. Only the removal of the adherent cell fraction from the immunized cell population and its replacement with fresh naive accessory cells could reveal a proliferative response to the autologous MBP and to the heterologous RBP. A heteroclitic cross-reactivity between MBP and RBP is demonstrated. The possibility of detecting an in vitro proliferative response to BP allowed the selection and propagation in vitro of cells specific to BP. T cell lines were established that specifically proliferated in response to BP and mediated EAE in normal mice. Intravenous inoculation of as few as 10(6) line cells was capable of producing clinical signs of EAE in normal recipients within 5 to 6 days. Thus, although an inoculation of MSCH was required for active induction of the disease, T cells specifically reactive against BP are sufficient for mediation of EAE in mice.

Interaction of the mouse and bovine myelin basic proteins and two cleavage fragments with anionic detergents

The binding of deoxycholate and dodecyl sulfate to the mouse and bovine myelin basic proteins and two peptide fragments, obtained by cleavage of the bovine basic protein at its single tryptophan residue, was examined. Complete equilibrium binding isotherms for both detergents were obtained by examining their binding to each of the polypeptides immobilized on agarose. The bulk of the binding of dodecyl sulfate was found to be highly cooperative, and at saturation all four polypeptides bound far more detergent than globular, water-soluble proteins. The sum of the dodecyl sulfate bound by each of the two bovine basic protein cleavage fragments was almost twice that bound by the intact protein at saturation, suggesting that cleavage of the bovine basic protein exposes sites for additional binding of dodecyl sulfate. At pH values below pH 8.0, an additional cooperative transition was observed below the critical micelle concentration of sodium dodecyl sulfate in the binding isotherms of all four polypeptides. The midpoint of this transition corresponded to an apparent p K of approximately 5.5; however, the destruction of 90% of the histidine residues in the bovine basic protein had no effect on this transition. At pH 9.2 and moderate ionic strength ( I = 0.1), the bulk of the binding of deoxycholate to the mouse and bovine basic proteins occurred at and above the critical micelle concentration of the detergent; and saturation values of deoxycholate binding to these two proteins were considerably higher than that reported for globular, water-soluble proteins. In marked contrast to the results with dodecyl sulfate, neither cleavage fragment was observed to bind deoxycholate. The results suggest that the higher ordered structure of the bovine basic protein may play an important role in the binding of anionic amphiphiles to the protein.

Administration of myelin basic protein-coupled spleen cells prevents experimental allergic encephalitis

Intravenous administration of mouse myelin basic protein covalently coupled with chromic chloride to syngeneic spleen cells (MBP-SC) prevents the subsequent induction of experimental allergic encephalitis (EAE). Whereas 1 in 28 mice receiving MBP-SC developed EAE after immunization with mouse spinal cord homogenate (MSCH) and adjuvants, 16 out of 25 mice receiving ovalbumin-coupled spleen cells (OA-SC) had EAE following encephalitogenic challenge. The effect of administration of antigen-coupled spleen cells on in vitro proliferation responses is shown.

Induction of experimental allergic encephalomyelitis in PL/J and (SJL/J x PL/J)F1 mice by myelin basic protein and its peptides: localization of a second encephalitogenic determinant.

SJL/J, PL/J, and (SJL/J x PL/J)F1 mice were immunized with bovine, guinea pig, mouse, or rat myelin basic proteins (MBP) in adjuvant containing Mycobacterium tuberculosis H37Ra. Twenty-four and 72 hr later, Bordetella pertussis vaccine was given i.v. All MBP tested induced experimental allergic encephalomyelitis (EAE) in SJL/J and F1 mice; however, bovine MBP was inactive in PL/J mice. Each strain was immunized in a similar manner with peptic peptides, residues 1-37, 43-88, and 89-169 of guinea pig MBP. In contrast to the SJL/J strain, which has been shown to recognize a major encephalitogenic determinant in peptide 89-169, PL/J and F1 mice responded primarily to an encephalitogenic determinant within peptide 1-37. Analysis of antibody levels showed that SJL/J mice made no antibody to peptide 1-37, although anti-peptide 89-169 antibodies were consistently found. Conversely, PL/J mice responded well to peptide 1-37, but only an occasional animal made a significant response to peptide 89-169. (SJL/J x PL/J)F1 mice were more susceptible to EAE than either parental strain, as shown by the percentage of animals showing neurologic signs and by clinical severity.

Vitro synthesis of the four mouse myelin basic proteins: evidence for the lack of a metabolic relationship.

Abstract: Studies on the synthesis of the four immunologically related mouse myelin basic proteins (MBPs) were carried out to determine if these proteins were metabolically related. Two in vitro systems were used: (a) a homologous brain system consisting of free polysomes, pH 5 enzymes, and initiation factors; and (b) a reticulocyte lysate system directed with mRNA and supplemented with brain factors. Incorporation of [35S]methionine into the four MBPs (14K, 17K, 18.5K, and 21.5K) was detected by immunoprecipitation of the in vitro products of synthesis followed by sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. The four MBPs were identified by cross‐reactivity with purified anti‐MBP antibodies and their apparent molecular weights in SDS gels. Synthesis of all four proteins was detected in both systems soon after the incubations were begun. The kinetics of the labeling of the proteins showed no evidence of a precursor‐product relationship (i.e., 21.5K→ 18.5K; 17K → 14K) in either system. Inhibition studies with puromycin and “chase” experiments with unlabeled methionine demonstrated that neither system contained posttranslational “processing” activity. Thus, the 21.5K and 17K proteins were not being processed into the 18.5K and 14K MBPs by either . in vitro system. Detection of the synthesis of all four proteins in the reticulocyte system programmed with brain mRNA indicates that the four proteins are probably coded for by separate mRNAs. This conclusion was supported by studies using polyribosomes separated into different size classes, which suggest that the mRNAs for the four proteins may be translated on proteins of differing size class. It is proposed, therefore, that the four MBPs are the primary translation products of independent brain mRNAs and are not metabolically related.

Genetic control of antibody production to myelin basic protein in mice

Experimental allergic encephalomyelitis (EAE) is a disease produced by inoculation of myelin basic protein (BP) into susceptible animals. Data in this report link the production of anti-BP antibody to the murine H-2 histocompatibility background. H-2 k and H-2 a animals produce high levels of anti-BP antibodies as measuring during both primary and secondary immune responses. Strains with H-2 b,d,p,q,s haplotypes are poor responders after primary immunization; however some H-2 s and H-2 d animals showed an increase in antibody after boosting. The use of M. tuberculosis instead of M. butyricum resulted in greater antibody production in H-2 d strains. The response observed in 4 congenic pairs of mice support the association between antibody formation and the H-2 complex. Experiments with recombinant inbred strains indicate that responsible genes reside in the I-A region of the H-2 complex.

Cell-free synthesis of the myelin basic proteins in a wheat germ system programmed with brain messenger RNA.

Poly A(+) messenger RNA (mRNA) was isolated from the brains of 3-week-old mice and translated in a cell-free system derived from wheat germ. Maximal stimulation of the system by brain mRNA was observed at a relatively low K+ concentration (45 mM) and low mRNA concentration (1-10 microgram/ml). The translational system was dependent on an energy-generating system and stimulated by the addition of spermidine and transfer RNA. Under optimal conditions, incorporation was linear for almost 45 min, but the overall stimulation with brain mRNA was relatively low (about twofold). In spite of the low stimulation, analysis of the translation products indicated that in the presence of brain mRNA polypeptides which co-chromatographed and co-electrophoresed with the two mouse myelin basic proteins could be detected. In control experiments with liver poly A(+) mRNA, which stimulated the translational system to a greater extent than brain mRNA, no such polypeptides could be detected. In this system the ratio of synthesis of small myelin basic protein to large myelin basic protein was found to be about 4.0, which correlates well with that found in vivo.