Conformation of a tetradecapeptide epitope of myelin basic protein.

The peptide AcAla-Ser-Gln-Lys-Arg-Pro-Ser-Gln-Arg-His-Gly-Ser-Lys-Tyr, which comprises the first 14 residues of the acetylated N-terminus of myelin basic protein, is an epitopic site for two monoclonal antibodies to the human protein. The conformations of the tetradecapeptide in aqueous solutions were investigated employing high-resolution 1H- and 13C-NMR spectroscopy. Two-dimensional techniques were used to assign the spectra observed from both nuclei. Nuclear-Overhauser-effect data, amide proton temperature coefficients, 13C spin-lattice relaxation times, distance geometry calculations and dynamic simulated annealing provided evidence that the solution conformations of the tetradecapeptide included a nascent alpha-helix in the N-terminal segment, and a loop extending from Ser7 to Ser12 that bring His10 and Tyr14 into close proximity.

Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido[3H]GBR-12935.

A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido[3H]GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido[3H]GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of [3H]GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of less than or equal to 10 microM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido[3H]GBR-12935 binding to rat striatal membranes (r = 0.95, p less than 0.01). These data suggest that 3-azido[3H]GBR-12935, like other diphenylpiperazines such as [3H]GBR-12935 and [3H]GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido[3H]GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido[3H]GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine (but not other biogenic amines) and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido[3H]GBR-12935 is glycosylated.

Interactions of myelin basic protein with mixed dodecylphosphocholine/palmitoyllysophosphatidic acid micelles.

The interactions of myelin basic protein and peptides derived from it with detergent micelles of lysophosphatidylglycerol, lysophosphatidylserine, palmitoyllysophosphatidic acid, and sodium lauryl sulfate, and with mixed micelles of the neutral detergent dodecylphosphocholine and the negatively charged detergent palmitoyllysophosphatidic acid, were investigated by 1H NMR spectroscopy and circular dichroic spectropolarimetry. The results with single detergents suggested that there are discrete interaction sites in the protein molecule for neutral and anionic detergent micelles and that at least some of these sites are different for each type of detergent. The data on the binding of the protein and peptides to mixed detergent micelles suggested that intramolecular interactions in the intact protein and in one of the longer peptides limited the formation of helices and also that a balance between hydrophobic and ionic forces is achieved in the interactions of the peptides with the detergents. At high detergent/protein molar ratios, hydrophobic interactions appeared to be favored.

Human cytotoxic T-cell recognition of a synthetic peptide of myelin basic protein.

Previous studies with a panel of myelin basic protein (MBP)-specific human T-cell clones suggested a clustering of epitopes in the middle and at the C terminus of the molecule. The current study demonstrates that 19 of 40 clones recognize a synthetic peptide corresponding to residues 152 to 170 of the human MBP molecule and that 9 clones recognize a synthetic peptide corresponding to residues 86 to 105. Myelin basic protein-specific cytotoxic activity was restricted to the clones that recognized peptide 152-170, and this peptide served as a preferential cytotoxic T-cell target when attached to an autologous B-cell line. The specificity of MBP-directed cytotoxic activity appears to be much more restricted than the specificity demonstrated for proliferative activity.

Evidence for multiple human T cell recognition sites on myelin basic protein.

Myelin basic protein (BP)-specific T cell clones were used to study human T cell recognition sites on the BP molecule. Proliferation assays performed with a panel of xenogeneic BPs of known amino acid sequence and with large peptide fragments of human and guinea pig BPs demonstrated ten different patterns of reactivity. The data provide evidence for at least four different human T cell epitopes within the C-terminal half of the BP molecule, three within the N-terminal half, and three located within the central portion of the molecule. The results indicate that attempts to inhibit anti-BP responses in vivo in an antigen-specific manner will require the suppression of multiple T cell populations.

Conformation of a heptadecapeptide comprising the segment encephalitogenic in rhesus monkey.

The 17-residue peptide FKLGGRDSRSGSPMARR derived from myelin basic protein, containing an epitope encephalitogenic in rhesus monkey, has been studied in aqueous solution by high-resolution one- and two-dimensional carbon and proton nuclear magnetic resonance spectroscopy. The resonances of the spectra from both nuclei were assigned with the aid of two-dimensional correlated spectroscopy, pH and solvent titrations, and one-dimensional spin-decoupling techniques and by comparison of the spectra of the heptadecapeptide with those of a phosphorylated form of the peptide, the pentadecapeptide FKLGGRDSRSGSPMA, and the nonapeptide FKLGGRDSR. Amide proton temperature coefficients, coupling constants, 13C- spin-lattice relaxation times, and nuclear Overhauser effect data suggest the existence of three structured regions comprising residues 3-6, 7-12, and 12-14 in the solution conformations of the encephalitogenic heptadecapeptide.

Myelin basic protein is an endogenous inhibitor of the high-affinity cannabinoid binding site in brain.

Radioligand binding studies with the water-soluble cannabinoid [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) have revealed a saturable high-affinity site in brain that is specific for cannabinoids. To determine whether endogenous compounds of brain might act upon the site physiologically, we sought inhibitors in extracts of brain. An endogenous inhibitor has been purified to homogeneity by acid extraction of rat brain followed by adsorption to a reverse-phase matrix and gel filtration chromatography. The purified inhibitor has a subunit molecular mass of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of [3H]TMA binding by the purified inhibitor occurs with a Ki of about 4 nM in a noncompetitive manner. The molecular weight, abundance, and extraction properties are the same as a species of myelin basic protein (MBP). The MBPs of rat, rabbit, pig, and cow also inhibit [3H]TMA binding noncompetitively with similar potencies. The purified inhibitor comigrates with rat MBP-small form on SDS-PAGE, has a similar amino acid composition, and is recognized by antibody directed against MBP. Studies of fragments of rabbit MBP suggest that the determinants of affinity for the [3H]TMA site are contained primarily within the C-terminal half of the rabbit MBP. Synthetic polycationic peptides such as polylysine and polyarginine mimic the effects of MBP, suggesting that the high-affinity cannabinoid binding site recognizes large polycations. The identification of the endogenous inhibitor of [3H]TMA binding as MBP suggests that MBP interacts physiologically with the high-affinity cannabinoid site.

Electroimmunoblotting of myelin basic protein peptides: a novel approach to the rapid characterisation of antigenic specificities of monoclonal and polyclonal anti-MBP antibodies.

A rapid and sensitive method for the identification of antigenic determinants recognised by monoclonal and polyclonal antibodies directed against myelin basic protein (MBP) is described. By electroimmunoblotting a series of overlapping peptides covering the entire MBP molecule with monoclonal anti-MBP antibodies, the binding pattern of immunoreactive peptides can be rapidly determined and the reactive antigenic determinant identified. This procedure, which can be performed with both native and synthetic peptides, can also with appropriate modification, be applied to the analysis of naturally occurring or experimentally induced polyclonal anti-MBP autoantibodies.

Electroimmunoblotting of small peptides separated on urea-dodecyl sulphate (SUDS) gels. Application to myelin basic protein.

A method for the electroimmunoblotting and immunodetection of peptides of less than 50 amino acid residues is described. Excellent resolution of a mixture of myelin basic protein (MBP) peptides was achieved by electrophoresis in a polyacrylamide stacking, urea-dodecyl sulphate minislab gel. Following electrophoresis, the peptides were transferred to various matrices and probed with monoclonal and polyclonal antibodies. Variables such as transfer time, membrane type, fixation and the amount of peptide loaded on the gel have been optimized as a consequence native and synthetic peptides can now be visualized in gels and immunodetected on immobilizing matrices. This procedure is particularly suited to the analysis and identification of small MBP fragments arising in various neuropathological conditions as well as for the rapid characterization of antigenic determinants recognized by monoclonal and polyclonal anti-MBP antibodies.

Monoclonal antibodies reactive with myelin basic protein.

New monoclonal antibodies (MAbs) to myelin basic protein (BP) reveal epitopes to be in sequences 22-34, 75-82, 83-96, 118-131 and 125-131. Comparison of these results with those previously reported suggest that almost every sequence of about 10 amino acid residues may be sufficiently antigenic to make a single MAb but that certain regions are immunodominant, strong enough to make practically the same MAb repeatedly. One of these new MAbs (clone 3) has especially interesting reactivity, sharply limited to residues 75-82 in bovine and porcine BP: Lys-Ala-Gln-His-Gly-Arg-Pro. Whales presumably have the same sequence, since their BPs are fully reactive with clone 3 MAb, but all other species of BP, with known sequences of BP, have at least two changes in this sequence. Deletion of Lys75 (as in a tryptic peptide of porcine BP) reduces reactivity with the MAb about 10-fold, whereas substitution of Ala76 by Ser (as in all other species of BP) and either deletion of Gln77 (as in human, monkey and rabbit BP) or His78 (as in the guinea pig and rat BP) or substitution of Pro82 by Thr (as in human, monkey, rat and mouse BP) eliminates reactivity. We speculate that woodchuck and prairie dog BPs in this region closely resemble chicken BP, which has about 2% of the original reactivity. However, squirrel BP is unique, probably having only one of the changes in this region of BP, since it possesses 10-20 times the reactivity of chicken BP but still only 20-50% of the original reactivity with clone 3 MAb, a degree of reactivity not seen with any other species of BP.

Characterization of a novel monoclonal anti-myelin basic protein antibody: use in immunoblotting and immunohistochemical studies.

Monoclonal antibodies (MAbs) to the myelin basic protein (MBP) were produced in CAF1 (BALB/c x A/J) mice immunized with intact bovine MBP. A number of MAbs were obtained, one of which was characterized in detail with respect to its isotype, antigenic determinant on the MBP, the spectrum of antigens with which it reacted in mouse brain, and its immunohistochemical staining characteristics. This monoclonal, GB-1 (an IgG1), recognized an epitope within residues 30-51 of bovine MBP. It also reacted with a family of MBP-related proteins present in brain homogenates of mice from 7-35 days. Immunohistochemically, GB-1 stained myelinated fibers and oligodendrocytes in the rodent CNS. A second monoclonal (GB-2, and IgM) was partially characterized. It reacted with intact MBP when it was immobilized to plastic or nitrocellulose, but it was not found to be useful for immunoblots or immunohistochemistry.

Experimental allergic encephalomyelitis in rabbits. A major encephalitogenic determinant within residues 1-44 of myelin basic protein.

Experimental allergic encephalomyelitis could be induced in rabbits by injection in Freund's complete adjuvant of either peptide 1-44 or peptide 45-87 of rabbit myelin basic protein. In order to localize the encephalitogenic determinant present in peptide 1-44, several smaller derivative peptides were prepared and examined. Peptic peptide 15-44 and thrombic peptide 1-31 were as active as peptide 1-44, whereas peptic peptides 1-14 and 18-38 and BrCN peptide 22-44 were virtually inactive. Weak activity was shown by BrCN peptide 1-21. These results provide evidence that a major encephalitogenic determinant present in peptide 1-44 lies within sequence 15-31. The encephalitogenic activity of peptide 15-44 was essentially destroyed by oxidation of methionine-21 to methionine sulfoxide; methylation of Met-21, on the other hand, appeared to be relatively ineffective in eliminating the encephalitogenicity of peptide 1-44.

Evidence for specific polypeptide chain folding in myelin basic protein from reactions between fragments of the protein and monoclonal antibodies.

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)

NMR studies of myelin basic protein. XIII. Assignment of histidine residues in rabbit, bovine and porcine proteins.

Myelin basic protein from three species (rabbit, cow and pig) and peptides from enzymatic digests or cleavage of the proteins have been examined in aqueous solutions by proton nuclear magnetic resonance (NMR) at 400 MHz. The epsilon 1-CH and delta 2-CH resonances of all the histidine residues in the three proteins have been assigned and the pK values have been measured. The heterogeneity of chemical shifts among these resonances can be variously ascribed to persistent localized secondary structures and to effects arising from charged side-chains, particularly those of aspartic acid residues, and from side-chains of aromatic moieties.

Possible hydrophobic region in myelin basic protein consisting of an orthogonally packed beta-sheet.

Theoretical analysis was carried out to determine how the approximately 20% of beta-structure observed in the 18.5 kilodalton (kDa) myelin basic protein (MBP) could be organized into a relatively stable beta-sheet. The beta-sheet is presumed to consist of the five most hydrophobic segments of polypeptide chain, which have beta-structure potential. These correspond approximately to sequences 15-21, 37-45, 84-92, 106-112, and 148-154 (rabbit MBP sequence numbering) and constitute beta-strands a, b, c, d, and e, respectively. A number of constraints are imposed upon the sheet; e.g., it should have the same topology in all MBP forms (21.5, 18.5, 17, and 14 kDa); strand e should lie at the sheet edge; strands b, c, and d should be ordered sequentially; the sheet formed by strands a, b, c, and d should be antiparallel; a maximum of the nonpolar surface area should be removed from the aqueous milieu; and charged side chains should be solvent-accessible. On the basis of these constraints it is possible to propose six orthogonally packed beta-sheets having different topologies. If strand e is restricted to an antiparallel alignment, the number of different sheets is reduced to four. Each of these sheets can form a relatively compact hydrophobic globular region. Two of the strands (a and e) can undergo transitions to alpha-helix without disrupting the structure of the remaining sheet bcd or producing major topologic rearrangements of the polypeptide chain.

Low-ultraviolet circular dichroism spectroscopy of sequential peptides 1-63, 64-95, 96-128, and 129-168 derived from myelin basic protein of rabbit.

Four sequential peptides (sequences 1-63, 64-95, 96-128, and 129-168) derived from rabbit myelin basic protein by thrombic cleavage were examined by low-ultraviolet circular dichroism spectroscopy in 0.5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH approximately 7.2) containing 0-92% trifluoroethanol (TFE). In the absence of the alcohol, all of the peptides contained a significant amount (17-29%) of beta-structure. In the presence of relatively low concentrations (up to 30%) of TFE, all of the peptides except 96-128 adopted considerable alpha-helix (16-33%). This involved a transition from the beta-structure in peptide 1-63 and transitions from the nonordered structure in peptides 1-63, 64-95, and 129-168. Furthermore, additional alpha-helix formed in peptide 1-63 between 30% and 92% TFE at the expense of nonordered structure, whereas the alpha-helix formation above 50% TFE in peptide 129-168 resulted largely from a beta-structure----alpha-helix transition. With the exception of the 129-168 peptide, approximately 65-100% of the maximum level of beta-structure persisted throughout the entire range of TFE concentration. In the case of peptide 129-168, however, most of the beta-structure was converted to alpha-helix and nonordered structure at 75% TFE. While the present results support our previous assignments of beta-structure- and alpha-helix-forming regions to specific amino acid sequences of the basic protein, they also demonstrate that the beta-structure----alpha-helix transitions evidenced at various concentrations of TFE were influenced to a considerable degree by the length of the peptide, presumably due to the presence or absence of interactions between noncontiguous portions of the myelin basic protein polypeptide chain.

Low-ultraviolet circular dichroism spectroscopy of oligopeptides 1-95 and 96-168 derived from myelin basic protein of rabbit.

Myelin basic protein (MBP) is a major protein constituent of the myelin sheath of the central nervous system, where it is believed to have functional alpha-helical segments. One element of the function of the protein might be "conformational adaptability" of specific regions of its amino acid sequence, since the purified protein appears to be largely devoid of ordered structure. To pursue this question, low-ultraviolet circular dichroism (CD) spectroscopy was conducted on the sequential thrombic peptides 1-95 and 96-168 of the protein in the presence of 0-92% trifluoroethanol (TFE), a solvent known to promote stable secondary structures in polypeptides. The series of CD spectra of the oligopeptides were subjected to a computerized best-fit analysis of four peptide conformations, the alpha-helix, beta-structure, beta-turn, and nonordered form. Agreement between experimental and best-fit composite spectra was achieved when standard CD curves of peptide conformations were derived from known theoretical spectra and experimental spectra of polypeptides. In dilute buffer alone, oligopeptides 1-95 and 96-168 evidence no alpha-helix but significant beta-structure (18% and 23%, respectively), as well as a predominant, extended nonordered conformation. However, the two parts of the protein differed in conformational adaptability. From 0% to 30% TFE, 96-168 exhibited concomitant transitions to 10% helix and 32% beta-structure from the nonordered form. In contrast, in 10-30% TFE, 1-95 underwent a transition to approximately 21% helix with partial loss of beta-structure as well as nonordered form; higher concentrations of TFE (40-75%) promoted additional transitions to both helix and beta-structure (totaling 33% and 25%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Cleavage of rabbit myelin basic protein by plasmin: isolation and identification of the major products.

Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion-exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy-terminal analyses with carboxypeptidases A, B, and Y and amino-terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89-Asn90, Lys133-Ser134, and Lys153-Leu154 bonds. Cleavages also occurred at the Arg31-His32, Lys53-Arg54, and Arg25-His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high-voltage electrophoresis-TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9-His10, Lys13-Tyr14, Lys103-Gly104, Lys137-Gly138, Lys140-Gly141, and Arg160-Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44-Phe45 bond. Instead it cleaves at the Arg31-His32 and Lys53-Arg54 bonds, thus preserving the entire hydrophobic sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe as well as short sequences to either side.

Conformation of two antigenic regions in myelin basic protein.

Four different regions of myelin basic protein from various species have been reported to be the antigenic sites (epitopes) for seven monoclonal antibodies evoked in rats or mice by guinea pig or monkey basic protein. The structures of the epitopes located in the amino-terminal region and in the eight-residue sequence including S-133, were examined by proton n. m. r at 400 MHz in aqueous solutions of peptides obtained by enzymatic cleavage of the rabbit protein. The data suggest conformational similarities between the two regions.