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After CNS demyelination, astrogliosis interferes with axonal regeneration and remyelination. We now provide evidence that myelin basic protein (MBP) can contribute to this observed astrocyte proliferation. We found that astrocytes grown in either serum-containing or serum-free medium proliferate in response to MBP. The mitogenic regions of MBP in both media were MBP(1-44), MBP(88-151) and MBP(152-167). The mitogenic effect of these individual peptides was potentiated by simultaneous treatment with microglia conditioned media (CM). MBP-induced proliferation was inhibited by suramin at concentrations known to block the fibroblast growth factor receptor (FGFR), whereas neither MBP(1-44), MBP(88-151) nor MBP(152-167) were affected. Cholera toxin B, that binds to ganglioside GM(1), inhibited the mitogenicity of MBP(1-44) and had no significant effect on the mitogenicity of MBP, MBP(88-151) or MBP(152-167). Treatment of astrocytes with MBP and MBP(152-167) caused a modest and transitory elevation of intracellular calcium, whereas treatment with MBP(1-44) resulted in a substantial and sustained increase in intracellular calcium. These results suggest that for cultured astrocytes 1) FGFR and extracellular calcium play a major role in MBP mitogenicity; 2) MBP(1-44), MBP(88-151) and MBP(152-167) are the mitogenic regions of MBP; 3) MBP(1-44) and MBP(152-167) interact with ganglioside GM(1) and FGFR, respectively; 4) Component(s) present in microglial CM potentiate the mitogenicity of MBP(1-44), MBP(88-151) and MBP(152-167). These data support the hypothesis that MBP related peptides in conjunction with microglial secreted factors may stimulate astrogliosis after demyelination in vivo., (Copyright 2000 Wiley-Liss, Inc.)

Myelin basic protein (MBP) and two peptides derived from MBP (MBP(1-44) and MBP(152-167)) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of 125I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of 125I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP(1-44)) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP(1-44) and MBP(152-167) associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.

In demyelinating diseases such as multiple sclerosis (MS), myelin membrane structure is destabilized as myelin proteins are lost. Calcium-activated neutral proteinase (calpain) is believed to participate in myelin protein degradation because known calpain substrates [myelin basic protein (MBP); myelin-associated glycoprotein] are degraded in this disease. In exploring the role of calpain in demyelinating diseases, we examined calpain expression in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS. Using double-immunofluorescence labeling to identify cells expressing calpain, we labeled rat spinal cord sections for calpain with a polyclonal millicalpain antibody and with mAbs for glial (GFAP, OX42, GalC) and inflammatory (CD2, ED2, interferon gamma) cell-specific markers. Calpain expression was increased in activated microglia (OX42) and infiltrating macrophages (ED2) compared with controls. Oligodendrocytes (galactocerebroside) and astrocytes (GFAP) had constitutive calpain expression in normal spinal cords whereas reactive astrocytes in spinal cords from animals with EAE exhibited markedly increased calpain levels compared with astrocytes in adjuvant controls. Oligodendrocytes in spinal cords from rats with EAE expressed increased calpain levels in some areas, but overall the increases in calpain expression were small. Most T cells in grade 4 EAE expressed low levels of calpain, but interferon gamma-positive cells demonstrated markedly increased calpain expression. These findings suggest that increased levels of calpain in activated glial and inflammatory cells in EAE may contribute to myelin destruction in demyelinating diseases such as MS.

Since calcium activated neutral proteinase (calpain) is present in the central nervous system (CNS) and degrades myelin proteins, this endopeptidase has been suggested to play a role in myelin destruction in demyelinating diseases such as multiple sclerosis (MS). In the present study, calpain immunocytochemical expression was examined in Lewis rats with acute experimental allergic encephalomyelitis (EAE), an animal model for MS and optic neuritis. To identify cells expressing calpain, we labeled rat optic nerve sections for calpain with a polyclonal myelin calpain antibody and with monoclonal antibodies for glial (GFAP, OX42) and inflammatory (CD2, ED2, ED1, IFN-gamma) cell-specific markers. The results showed increased calpain expression in microglia (OX42) and infiltrating macrophages (ED1,2) in EAE compared to normal controls. Astrocytes constitutively expressed calpain in controls and acute EAE. Reactive astrocytes in EAE located in or near inflammatory foci, exhibited markedly increased calpain expression. Most T cells in acute EAE showed low level calpain expression while activated IFN-gamma-producing lymphocytes in inflammatory foci exhibited elevated levels of calpain expression. Thus, our results demonstrate increased calpain expression (at transcriptional and/or translational levels) in a rat model of optic neuritis. A role for calpain in myelin destruction during optic neuritis may be relevant to the pathogenesis of this disorder., (Copyright 1998 Elsevier Science B.V.)

Treatment of cultured oligodendrocytes (OLGs) with micromolar quantities of myelin basic protein (MBP) caused a rapid, MBP-dose-dependent cell death. In contrast, a 72-hr incubation of OLGs with MBP peptides (1-44, 47-87, 88-151, or 152-167) at comparable concentrations had no effect on cell viability. MBP and MBP peptides (1-44 and 88-151) have been shown to interact with ganglioside GM1 (Tzeng et al.: J Neurochem Res: 42:758-767, 1995). This interaction has been reported to increase calcium influx. Therefore, using the fluorescent dye Indo-1 and an ACAS laser cytometer, we examined the level of intracellular calcium in OLGs after MBP treatment. MBP was shown to provoke a rapid, dramatic, and sustained rise of intracellular calcium in most OLGs. The levels of elevated intracellular calcium were sustained and did not return to baseline even after 10 min. This increase of intracellular calcium was suppressed in the presence of EGTA, indicating that the [Ca2+]i rise was due to the entry of extracellular calcium. Incubation of cultured OLGs with MBP peptides (1-44 or 88-151) caused a modest and transitory elevation of intracellular calcium ions in a lower percentage of OLGs. The potent OLG cytotoxicity of intact MBP and the loss of potency after proteolysis raise the possibility that MBP proteolysis during demyelination protects OLGs from death.

Previous studies have shown that myelin basic protein (MBP) is mitogenic for Schwann cells (SCs) in the presence of elevated intracellular cAMP. Two mitogenic regions of MBP have been identified: one mitogenic region within the first 44 residues of the aminoterminus (1-44) and the other mitogenic region within the terminal 15 residues of the carboxyl end of the molecule (152-167). Unlike the mitogenic effect of a myelin enriched fraction (MEF), the mitogenic effect of MBP was not reduced by the addition of the lysosomal inhibitor, ammonium chloride. These data indicate that MBP causes SC proliferation by direct interaction of MBP with a surface receptor. Using Scatchard analysis of the binding of MBP to SCs, we report that treatment with forskolin does not cause the upregulation of receptors for MBP. Moreover, MBP blocks the cross-linking of 125I-bFGF with two fibroblast growth factor (FGF) receptors having apparent molecular weights of 140 kDa and 120 kDa, respectively. Since neither TGF-beta nor PDGF-BB displaced cell surface bound 125I-MBP, we conclude that MBP binds to the FGF receptor rather than other growth factor receptors. Furthermore, only MBP interacted with ganglioside GM1, whereas MBP did not interact with this ganglioside. These results are consistent with the view that ganglioside GM1 mediates the mitogenic effects of MBP, while the FGF receptor mediates the mitogenic effect of MBP. Intracellular cAMP of SCs was transiently increased after the addition of macrophage conditioned medium, suggesting that macrophages may produce factors in vivo which can transiently elevate intracellular cAMP levels, allowing a wave of SC proliferation in response to MBP-related mitogens.

Myelin basic protein (MBP) occurs in multiple forms. Three of these isoforms from human MBP (HMBP) have been highly purified. HMBP, component 1 (18.5 kDa HMBP-1), was purified by ion-exchange chromatography at pH 10.6 in 2 M urea. During this ion-exchange chromatography, a fraction (Fraction 3), which contained HMBP component 3 (monophosphorylated or deamidated 18.5 kDa) and 17.2 kDa HMBP, was collected and further purified by fast protein liquid chromatography, which separated 17.2 kDa HMBP and HMBP component 3. When the latter was subjected to limited thrombic digestion, all of HMBP component 3 not phosphorylated at theonine 98 was cleaved. This digestion mixture was separated on Sephadex, and yielded pure component 3, monophosphorylated at theonine 98 (HMBP 3pT98), for which phosphate analysis yielded approximately 1 mole P/mole protein, and NMR showed only one phosphorylation site present. Circular dichroism (CD) studies were carried out on dilute solutions of HMBP-1 (18.5 kDa), 17.2 kDa HMBP, and HMBP3pT98 (phosphorylated 18.5 kDa). The CD spectrum of HMBP-1 was similar to that reported for rabbit MBP-1 and bovine MBP-1, but the spectra of 17.2 kDa HMBP and HMBP 3pT98 were distinctly different from HMBP-1. When analyzed by best-fit computations, 17.2 kDa HMBP showed about a 9% increase of ordered structure, and a greater increase, about 12%, was estimated for HMBP3pT98, attributable to beta-structure and beta turn.

The degree of recycling of leucine derived from protein breakdown into the precursor pool for protein synthesis was measured in rat brain at different postnatal ages, and age-specific values were used in the calculation of regional (local) rates of cerebral leucine incorporation into protein (lCPSleu) in 44 brain regions and the brain as a whole. Early in development, a greater fraction of the precursor leucine pool is derived from protein breakdown, indicating that protein degradation is higher in young rats compared with adults. In whole brain and in most regions, values for lCPSleu were highest at 10 days and gradually decreased with age. By 60 days of age, values in cortex were approximately 60% of those at 10 days of age. In the paraventricular and supraoptic nuclei of the hypothalamus, however, lCPSleu increased during development, reaching peak values in adults. In white matter of the cerebellum and the cerebrum, peaks of lCPSleu were reached at 14 and 21 days, respectively, approximately at the times of maximum rates of myelination.

In order to determine the peptide bond specificity of calpain, human myelin basic protein (HMBP) was treated with purified calpain of bovine brain. Upon incubation, HMBP component I (HMBP-I) was degraded into several peptides as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Component I was more susceptible to degradation than components II and III. HMBP degradation products were separated by high performance liquid chromatography (HPLC) and the cleavage sites in HMBP molecules were determined by peptide sequence analysis and by N- and C-terminal analyses. The major cleavage site was found to be 94Val-95Thr with several minor cleavages at 49Arg-50Gly, 18Ala-19Ser, 23His-24Ala, 27Gly-28Phe, 59Asp-60Ser, 70Gly-71Ser, 97Arg-98Thr, 110Ser-111Leu, 145Asp-146Ala, and 156Leu-157Gly. These results indicate that calpain is involved in the limited proteolysis of human myelin basic protein and prolonged incubation causes further digestion of the large peptides.

The quantitative autoradiographic L-[1-14C] leucine method for the determination of regional rates of cerebral protein synthesis (lCPSleu) requires knowledge of the degree of recycling of leucine derived from protein degradation into the precursor pool for protein synthesis, which can be evaluated by measuring lambda i, the steady-state ratio of the leucine-specific activity in the precursor amino acid pool (tRNA-bound leucine) to that of the arterial plasma. To define the changes in lCPSleu during regeneration of the hypoglossal nerve, we examined the effects of axotomy on the value of lambda i. Because the concentration of tRNA-bound leucine in the hypoglossal nucleus is too low to measure, we measured the equivalent ratio for the total acid-soluble pool (psi i) and applied the linear relationship between lambda and psi found in the whole brain to calculate a value of lambda i in the ipsilateral and contralateral hypoglossal nuclei of 22 adult female rats 2, 18, 35, and 60 days after unilateral hypoglossal axotomy. Statistically significant but quantitatively inconsequential effects of axotomy on values of psi i and lambda i were found. Therefore, the mean value for lambda i (0.64) of the left and right hypoglossal nuclei in all 22 axotomized rats was used to calculate lCPSleu. In a separate group of 15 unilaterally axotomized rats, lCPSleu was determined by the autoradiographic technique; lCPSleu was increased on the axotomized side by 23% on day 2, 30% on day 18, and 13% on day 35. By postaxotomy day 60, lCPSleu had returned to normal.

Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N-methyl-D-aspartate (NMDA)-type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10-20 microM) of 2-amino-5-phosphonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 microM) of 2-amino-5-phosphonovaleric acid can protect cultures when added at much later times (30-60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 microns pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6-Diazo-5-oxo-L-norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.

The quantitative autoradiographic L-[1-14C]leucine method for the determination of regional rates of cerebral protein synthesis in vivo takes into account recycling of unlabeled leucine derived from protein degradation into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the apparent steady-state leucine specific activity in the precursor amino acid pool (tRNA-bound leucine) to that in the arterial plasma. In the whole brain of the conscious rat this ratio (lambda WB) equals 0.58. The equivalent ratio for leucine in the acid-soluble pool in whole brain (psi WB) is 0.49. A first-degree polynomial equation for lambda WB as a function of psi WB was fitted from paired determinations. To determine the degree of recycling in local regions of the brain, we have measured in individual brain regions (i) psi i and calculated lambda i assuming that the fitted equation also applies to these localized regions. Our results indicate that the degree of recycling into the precursor pool does vary regionally; lambda i in the individual regions varies from 0.62 in the hypoglossal nucleus to 0.50 in the globus pallidus. Local rates of protein synthesis were then determined by the autoradiographic technique with regional corrections for recycling of unlabeled leucine. Rates of leucine incorporation into protein averaged 6.1 nmol/g of tissue/min in the brain as a whole, with the rates in gray matter about twice those in white matter.

"Flooding" amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and "flooded" (10-28 mM) by comparing the steady-state specific activity of the tRNA-bound valine with that of the plasma valine. Under normal and "flooding" conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; "flooding" with a plasma level of 28 mM raised the brain acid-soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than "flooding" with a plasma level of 17 mM valine, which raised the brain acid-soluble level only to 2.3 mM. The results of these studies show that "flooding" amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.

Immunization with peptides is usually done with the aid of Freund's adjuvant. Using peptides derived from myelin basic protein, we show that aqueous solutions can be antigenic (encephalitogenic in this instance) in Lewis rats. The first procedure involved multiple doses of aqueous peptide, increased absorption into the lymphatic system from the peritoneal cavity in the postinflammatory state, and the use of pertussis vaccine. Three different peptides containing the major encephalitogenic site were active in this system, with the activity somewhat proportional to the size of the fragment. The second procedure, the direct delivery of peptide to lymph nodes by percutaneous inoculation, was equally successful and did not require the use of pertussis vaccine.

The induction of experimental allergic encephalomyelitis (EAE) with purified myelin basic protein (MBP) has, heretofore, required its incorporation in a water-in-oil emulsion or adsorption on particulate adjuvants. In the present work, the absorption of a saline solution of MBP from the peritoneal cavity into the mediastinal lymph nodes was increased by giving repeated inoculations or by pretreating rats with a peritoneal irritant. Under these conditions, the only adjuvant needed for production of EAE was aqueous pertussis vaccine which was injected separately a few hours or one day after the MBP. Pertussis vaccine was also necessary for production of EAE with intradermal injection of aqueous MBP. By injecting the aqueous MBP directly into pre-enlarged popliteal lymph nodes, it was possible to produce EAE without the pertussis vaccine. Thus, EAE can be induced in rats using MBP without the addition of Freund's adjuvant or pertussis vaccine.

Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated form(s) of component 1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phosphorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in beta-structure(s).

Myelin basic protein of rabbit brain (Mr = 18,200) was initially freed of the bulk of the nonphosphorylated species (mainly component 1) by Cm-cellulose chromatography at high pH. The remainder of the protein was subjected to peptic digestion at pH 6.00, which resulted in specific, essentially complete cleavage at several bonds (Phe-44--Phe-45, Phe-87--Phe-88, Leu-109--Ser-110, and Leu-151--Phe-152) and partial cleavage at the Tyr-14--Leu-15 bond. Gel filtration of the digest through Sephadex G-25 (fine) yielded three fractions, the first containing primarily peptides 1-44 and 45-87, the second peptides 15-44, 88-109, and 110-151, and the third peptides 1-14 and 152-168. Each fraction was chromatographed on Cm-cellulose at pH 8.2, and the resulting subfractions and partially purified peptides were analyzed for phosphoserine and phosphothreonine. Materials containing significant amounts of the phosphoamino acids were subsequently chromatographed on Cm-cellulose at pH 4.65, and the analyses for phosphoserine and phosphothreonine were repeated. The resulting purified peptic phosphopeptides were identified by amino acid analysis and tryptic peptide mapping. Comparison of the maps with those of the unphosphorylated counterparts located the tryptic phosphopeptides. These were recovered and their identities were established by amino acid analysis. In those cases where the phosphopeptide contained 2 Ser residues, the position of the phosphoserine was established by aminopeptidase M digestion. Five phosphorylation sites were found: Ser-7, Ser-56, Thr-96, Ser-113, and Ser-163. Only a small fraction of these sites was phosphorylated in the total basic protein, with values ranging from about 2 (ser-113) to 6% (Thr-96). With the possible exception of Ser-56, these sites are not the ones that have been reported to be phosphorylated in vitro by cyclic AMP-dependent protein kinase.

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.

A monoclonal antibody (immunoglobulin G1) has been produced that reacts against myelin basic protein present in or extracted from the brains of many mammals-with certain important exceptions. Because of known species differences in amino acid sequences of basic protein and of certain peptide fragments, the binding site for this particular antibody appeared likely to include residues 130 to 137. Confirmation of this hypothesis was obtained by amino acid composition of the major immunoreactive peptides produced by thermolysin digestion of human basic protein and isolated by high-performance liquid chromatography.

Rapid cleavage of bovine and guinea pig myelin basic proteins by pepsin at pH 6.0 is limited to the Phe-Phe bond in the middle of the molecule. In the rabbit protein, however, rapid cleavages occur elsewhere in addition to the Phe87-Phe88 bond in regions in which there are amino acid substitutions. Rapid cleavage occurs at the Leu151-Phe152 bond, at which Ile-151 has been replaced by Leu, the residue that actually contributes the scissile bond. Rapid cleavages occur at the Phe44-Phe45 and Leu109-Ser110 bonds, which in the bovine and guinea pig proteins are relatively resistant under the experimental conditions (pH 6.0). The increased susceptibility of these bonds in the rabbit protein appears to be related to the replacement of Gly-46 by Ser and the change in the sequence immediately NH2-terminal to Leu-109, from Leu-Ser to Thr-Val. These cleavages of the rabbit protein at the four very susceptible bonds have permitted us to isolate peptides (1-44), (45-87), (88-109), (110-151), and (152-168) in high yield. We have also isolated peptides (88-151), (1-14), and (15-44) in low yield; the latter two result from limited cleavage at the relatively resistant Tyr14-Leu15 bond. Peptide (88-109) has been chromatographically resolved into species differing in the degree of methylation of Arg-105; this resolution is thought to result from differences in hydrogen bonding ability of the guanidinium groups.

Two peptic fragments (residues 37-88 and 43-88) of guinea pig myelin basic protein which are capable of inducing experimental allergic encephalomyelitis in Lewis rats were cleaved to shorter fragments with alpha-protease (Crotalus atrox proteinase, EC 3.4.24.1) and thermolysin (EC 3.4.24.4). The fragments were isolated, purified, and identified by amino acid composition and NH2- and COOH-terminal residues. The time courses of the reactions, monitored by thin layer electrophoresis of the digests, showed that alpha-protease cleaves peptide (43-88) initially at the Pro(71)-Gln(72) bond, and that the product peptides are subsequently attacked at the Arg(63) -Thr(64), Ser(74)-Gln(75), Arg(78)-Ser(79), and Ser(76)-Gln(80) bonds. No significant cleavages occurred at the -Leu, -Val, and -Ala bonds. These results are in striking contrast to those obtained previously by others workers with other peptide substrates, where selective cleavage at hydrophobic residues occurred. Thermolysin was found to attack peptide (37-88) at the Phe(42)-Phe(43) bond very rapidly; the product peptides were subsequently attacked at the His(60)-Ala(61), Ser(38)-Ile(39)-Tyr(67)-Gly(68), and Pro(84)-Val(85) bonds. These cleavages are compatible with the known specificity of this enzyme. Several of the fragments prepared with these two enzymes, peptides (43-71), (61-88), (75-88), and (72-84) have been used in other studies to locate the encephalitogenic site in the parent peptic peptide.

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.

The myelin basic protein (BP) of pig brain was cleaved into its constituent tryptic peptides and the amino acid composition of each was determined. Those tryptic peptides that had not been sequenced previously were cleaved with dipeptidyl peptidases and the resulting dipeptides were trimethylsilated, separated by gas chromatography, and identified by mass spectrometry. Carboxypeptidases B and Y were used to establish the COOH-terminal sequences of some of the tryptic peptides; one tryptic peptide (sequence 76-92) was cleaved with thermolysin and the thermolytic peptides were analyzed. From the results of the present study together with those reported previously, it has been possible to determine the complete amino acid sequence of the protein. The protein consists of 172 residues and has a theoretical molecular weight of 18,604. Its amino acid sequence is identical with that reported for the homologous bovine protein with the following exceptions: Ser replaces (bovine) Ala2; His-Gly is inserted between Arg9 and Ser10; Ala replaces Ser45; His and Gly replace Gly76 and His77, respectively; Pro replaces Ser131 and Ser135; Ala is inserted between Gly142 and His143; and Gln replaces His143.

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.

Bovine and guinea pig myelin basic proteins were cleaved with pepsin at pH 3.0 or pH 6.0 (enzyme/substrate, 1:500, w/w), and the peptides were isolated and identified. At pH 3.0 cleavage of the bovine protein occurred principally at three sites: Phe-Phe (88-89), Phe-Phe (42-43), and Leu-Asp (36-37). Minor cleavages occurred at Leu-Ser (110-111), Phe-Ser (113-114), and Ile-Phe (152-153). A study of the time course of the hydrolysis showed that the reaction was biphasic; nearly all of the protein was cleaved at Phe-Phe (88-89) before significant cleavages at other sites occurred. At pH 6.0 cleavage of the bovine protein occurred almost exclusively at a single site, the Phe-Phe bond at position 88-89, resulting in bisection of the protein. Treatment of the guinea pig protein with pepsin under the same conditions resulted in the production of peptides which were identical with those of the bovine protein in chromatographic and electrophoretic properties and in N-terminal and C-terminal residues but which differed slightly in amino acid composition.

The epitopes (antigenic sites) for seven monoclonal antibodies (MAbs) evoked in rats or mice by guinea pig or monkey myelin basic protein (BP) have been located in four different sequences of the BPs extracted from various species. Six of the MAbs were evoked by guinea pig BP. (1) One epitope, possibly a pair, is included within residues 1-14 of all BPs tested and reacts with two rat IgG MAbs. (2) A definite pair of overlapping epitopes includes the central Phe91-Phe92 sequence. One epitope is contained entirely within sequence 90-99 and reacts with a rat IgG MAb. The substitution of Ser in chicken BP for Thr97 destroys this epitope. The other epitope appears to include residues on the amino side of Phe44 and even of His32 and suggests some tertiary structure in BP. This epitope reacts with a mouse IgM MAb that does not recognize the chicken substitution. (3) The third epitope lies within residues 114-121, specifically including Trp118, and reacts with a rat IgG MAb. A cross-reacting epitope probably includes residues 44-45 in certain species (guinea pig and bovine but not rabbit). (4) Another pair of epitopes is located within residues 131-140 but is severely species-restricted. This region in guinea pig BP evoked a species-specific mouse IgM MAb. The same region in monkey BP evoked the seventh MAb, a mouse IgG, which reacts with human, chimpanzee, monkey, bovine, and rat-18.5 kDa BPs and to a lesser extent rabbit BP but not with guinea pig, pig, or chicken BPs. Some tertiary structure in guinea pig BP is also suggested by the reactivities with the IgM MAb. All of the MAbs react with myelin in histologic preparations, but the optimum method of preparation of the tissue varies with each.

A procedure for large scale isolation of myelin basic protein (BP) has been modified to insure BP preparations free of neutral proteinase activity. Fractions were monitored by electrophoretic analysis of BP solutions incubated under various conditions of temperature and pH. Maximum degradation of human BP prepared by the old batch procedure occurs at pH 7, approximately 47 degrees C. BP preparations obtained by the new procedure, as well as BP preparations purified by CM-cellulose chromatography, are stable under these conditions. The latter, however, do undergo significant breakdown at pH 9, 100 degrees C. The results suggest that the degradation observed under these conditions is non-enzymatic in nature.

This paper proposes a tentative amino acid sequence of guinea pig myelin basic protein obtained by comparison of peptide fragments of the guinea pig and bovine proteins. Analyses of the tryptic peptides confirmed the known sequence differences in the NH2-terminal half of the molecule and showed that in the COOH-terminal half of the guinea pig protein Ser131 was missing, Ala136 - His137 was deleted, Leu140 was replaced by Phe, and an extra Ala was inserted somewhere within sequence 142-151 (tryptic peptide T23 ). Sequence determination of guinea pig tryptic peptides corresponding to residues 130-134 ( T20 ), 135-138 ( T21 ), and 142-151 ( T23 ) of the bovine protein confirmed the above sequence changes and placed the extra Ala between Gly142 and His143 . The sequence of the region corresponding to bovine residues 130-143 is thus Ala-Asp-Tyr-Lys-Ser-Lys-Gly-Phe-Lys-Gly-Ala-His. No species differences were observed in the amino acid compositions of the remaining tryptic peptides obtained from the COOH-terminal half of the molecule. Based upon these results, the guinea pig basic protein contains 167 amino acid residues and has a molecular weight of 18,256.

Rabbit myelin basic protein (BP) contains several Arg-X bonds with differing susceptibilities to thrombic cleavage as measured by the yields of the various cleavage products obtained under three different conditions. Under conditions where the thrombin-to-substrate ratio was very low (1 NIH unit/mg BP), the concentration of substrate was relatively low (4 mg BP/ml), and the incubation time was short (2 h), the rabbit BP was cleaved essentially completely and specifically at a single site, the Arg(95)-Thr(96) bond. The BPs of other species (beef, pig, guinea pig, rat) were similarly cleaved, no doubt because all have the same amino acid sequence in this region of the protein. Under conditions in which the enzyme-to-substrate ratio and the substrate concentration were higher (2 NIH units/mg BP, 8 mg BP/ml) and the incubation time was long (24 h), additional, partial cleavages occurred, principally at the Arg(43)-Phe(44) and Arg(128)-Ala(129) bonds, but with some cleavage at the Arg(31)-His(32) and Arg(63)-Thr(64) bonds as well. Under conditions in which all three variables were elevated (5 NIH units/mg peptide, 20 mg peptide/ml, 24 h), more extensive cleavage occurred at the above sites. In peptide (96-168), which we examined in detail, nearly complete cleavage of the Arg(128)-Ala(129) bond occurred, with partial cleavage at the unmethylated Arg(105)-Gly(106), Arg(111)-Phe(112), Arg(150)-Leu(151), and Arg(160)-Ser(161) bonds. The susceptibilities to cleavage of the Arg-X bonds in the BP can be explained with varying degrees of success in terms of the known specificity of thrombin. Cleavage of two of the bonds, Arg(128)-Ala(129) and Arg(160)-Ser(161), suggests the occurrence of a chain reversal or beta-turn in the sequence preceding the scissile bonds. Most cleavages of the BP with thrombin do not occur in the more hydrophobic regions; in particular, the hydrophobic region in the center of the molecule that includes the Phe-Phe(87-88) sequence is left intact.

Forty myelin basic protein (BP)-reactive T-cell clones were isolated from a patient with multiple sclerosis and used to identify human T-cell recognition sites on the BP molecule. At least three sites have been identified: one in the N-terminal half of the molecule (residues 1-97), one in the C-terminal (residues 98-170), and one which spans residues 97-98. The clones exhibited a marked preference for the C-terminal half of the molecule. No cross-reactivity with measles virus was detected. These clones will be useful for both the further delineation of the human T-cell recognition sites on BP and the generation of anticlonotypic monoclonal antibodies.

The anatomical distribution of P2 protein was studied in human autopsy tissue. Spinal cord (SC) and peripheral nerve (PN) were stained by the peroxidase-antiperoxidase method with antisera to bovine P2, glial fibrillary acidic protein, and myelin basic protein (BP). P2 antiserum did not stain all of the myelin in the PN. The staining was randomly distributed and discontinuous along a given myelinated axon. P2 antiserum also stained SC myelin in a pattern similar to the PN. Only a fraction of the sheaths stained, in contrast to BP antiserum that stained all myelin sheaths in both the SC and PN. P2-positive myelin was distributed throughout the SC white matter, including an occasional myelinated fiber in the SC grey matter. P2 and BP antisera did not stain regions of demyelination in a case of idiopathic polyneuritis, while adjacent myelinated PN stained normally. Absorption of the P2 antiserum with P2, bovine PN or bovine SC (carefully dissected to eliminate PN contamination) nullified the specific staining in both the PN and SC; however, absorption with BP or hemispheric myelin did not eliminate P2 staining. The P2 antiserum formed a single immunodiffusion line with pure P2 and acid extracts of bovine SC and PN myelin, but not with an acid extract of bovine hemispheric myelin. Electrophoresis of defatted bovine SC produced a distinct band corresponding to P2. Therefore, three lines of evidence, immunocytochemical, immunodiffusion and electrophoretic, suggest that P2 is present in PN and SC but not in hemispheric myelin.

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)

Human myelin basic protein was subjected to ion-exchange chromatography at high pH to separate the differently charged components. Polyacrylamide gel electrophoretic patterns of the fractions showed that the less basic fractions 3, 4, and 5 contained significant amounts of a protein somewhat smaller than the more common 18.5-kDa form. Fraction 3 consisted of approximately equal amounts of this smaller polypeptide and component 3, the 18.5-kDa form found in other mammalian myelin basic protein preparations. The two proteins in fraction 3 were separated by fast protein liquid chromatography. Both have blocked N termini and identical C termini (-Met-Ala-Arg-Arg). When the tryptic digests of the two proteins were fractionated by HPLC, the elution profiles were similar, except that four peaks found in the chromatogram of the larger protein were missing from the chromatogram of the smaller one. In addition, an extra peak was found in the elution pattern of the latter chromatogram. Amino acid analysis of the individual tryptic peptides indicated that the smaller protein lacked residues 106-116 (-Gly-Arg-Gly-Leu-Ser-Leu-Ser-Arg-Phe-Ser-Trp-). The deleted portion corresponds exactly to the amino acid sequence encoded by exon 5 of the mouse basic protein gene. This new form of myelin basic protein has a molecular weight of 17,200, calculated from its amino acid composition.

Treatment of rabbit myelin basic protein component 1 with pepsin (enzyme:substrate, 1:500 w/w) in 0.5 M-ammonium formate (pH 6.00) for 15-20 min at room temperature resulted in limited cleavage of the protein. The resulting fragments were isolated by ion-exchange chromatography and gel filtration and identified by amino acid and COOH-terminal analyses and by tryptic peptide mapping. All of the possible products resulting from incomplete cleavages at the highly susceptible Phe44-Phe45, Phe87-Phe88, Leu109-Ser110, and Leu151-Phe152 bonds were isolated: peptides (1-151), (1-109), (1-87), (45-168), (45-151), (45-109), (88-168), (88-151), and (110-168). Of these, peptides (1-151), (1-87), and (88-151) were recovered in the greatest yield (0.14-0.19 mol per mol of starting protein). Relatively low yields (0.04 mol/mol starting protein) were obtained for peptides (1-109) and (110-168), indicating that the Leu109-Ser110 bond is somewhat more resistant to peptic cleavage than are the Phe-Phe and Leu-Phe bonds. Smaller fragments of the basic protein were also recovered: peptides (1-44), (1-28), (45-87), (88-109), (110-151), and (152-168). Many of the individual peptides could be readily identified in electrophoretograms of the total peptic digest. The relative electrophoretic mobilities of the above-mentioned peptides, together with the previously isolated peptides (1-14) and (15-44), were determined in 15% (w/w) polyacrylamide slab gels containing 1 M-acetic acid and 8 M-urea.

The C-terminal half of the bovine myelin basic protein, peptide 89-169, was treated with BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine], and the products were isolated by repeated gel filtration through Sephadex G-50. They consisted of uncleaved peptide 89-169 in which approximately 30% of the tyrosine had been monobrominated and the tryptophan converted to oxindolealanine, peptide 116-169 modified by partial bromination (30%) of the tyrosine, and two chromatographic forms of peptide 89-115. The major form contained the lactone of dioxindolealanine at the C terminus; the minor form contained the uncyclized oxidation product. Each form of peptide 89-115 was resolved into several components by electrophoresis in polyacrylamide gels (10%, w/w) containing 1 M acetic acid and 8 M urea. The presence of three of these components could be explained by partial deamidation of Asn-91 and Gln-102. Studies on the oxidation of tryptophan-containing model peptides by BNPS-skatole indicated that the reaction can also include partial bromination of the dioxindole and its lactone and partial cleavage at the amino peptide bond of the tryptophan.

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.

In order to determine whether bovine, rabbit, and monkey myelin basic proteins (BPs) have the sequence Gly-His or His-Gly at positions corresponding to bovine sequence 76-77, we isolated the tryptic peptides encompassing the sequence in question in these proteins and cleaved them into dipeptides with dipeptidyl aminopeptidase I (EC 3.4.14.1). Analysis by gas chromatography/mass spectrometry of the dipeptides released showed that in no case did His follow Gly or Gly precede His. The identification of peptides Ala-Gln and His-Gly (bovine BP) and Ser-His and Gly-Arg (rabbit and monkey BPs) established the His-Gly sequence. A similar sequence analysis of tryptic peptide (80-91) of human BP confirmed the sequence Thr-Gln-Asp-Glu-Asn-Pro (80-85).

The epitopes (antigenic determinants) evoked by guinea pig or monkey myelin basic protein (BP) for 7 monoclonal antibodies (MAb) have been localized to 4 different sequences of the BPs extracted from various species. The first pair of epitopes is included within residues 1-14 and reacts with 2 rat IgG MAbs having slightly different specificities. The second pair of epitopes overlap within the sequence 86-100, requiring the Phe(91)-Phe(92) residues. One includes only residues 90-100 and reacts with a rat IgG MAb, the other is longer and reacts with a mouse IgM MAb. The third epitope lies within residues 114-124 (specifically including the single Trp) and reacts with a rat IgG MAb. A cross-reacting epitope in the amino half of the molecule is present in certain species (human, bovine and guinea pig). The fourth pair of epitopes includes residues 131-140 and is severely species-restricted. A mouse IgM MAb reacts practically only with guinea pig BP. Another IgG MAb reacts with human, chimpanzee, monkey, bovine and rat-18.5 kDa BPs, to a much lesser extent with rabbit and rat-14 kDa BPs and not with guinea pig, pig or chicken BPs. A cross-reacting epitope in the amino half of the molecule may be present in the more reactive species.

A quantitative autoradiographic method for the determination of local rates of protein synthesis in brain in vivo is being developed. The method employs L-[1-14C]leucine as the radiolabeled tracer. A comprehensive model has been designed that takes into account intracellular and extracellular spaces, intracellular compartmentation of leucine, and the possibility of recycling of unlabeled leucine derived from steady-state degradation of protein into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the steady-state precursor pool distribution space for labeled leucine to that of unlabeled leucine. The values obtained were 0.58 in whole brain and 0.47 in liver. These results indicate that there is significant recycling of unlabeled amino acids derived from steady-state protein degradation in both tissues. Any method for the determination of rates of cerebral protein synthesis in vivo with labeled tracers that depends on estimation of precursor pool specific activity in tissue from measurements in plasma must take this recycling into account.

A reversed-phase high-performance liquid chromatography (HPLC) system was developed to obtain individual tryptic peptides of myelin basic protein (BP). Because of the similar charge and hydrophobicity of some of the tryptic peptides of the whole protein, several of these were not clearly separated by a single HPLC system. Therefore, the BP was first cleaved specifically between residues 97 and 98 with thrombin, and the two resulting fragments were separated by ion-exchange chromatography. When the thrombic fragments were digested with trypsin separately and subjected to HPLC, all of the peptides were satisfactorily separated. Elution times of all of the tryptic peptides of human BP were established. Differences among homologous peptides, derived from different mammalian BPs, were readily detected from their elution patterns inasmuch as a change in a single amino acid residue was usually sufficient to cause a shift in the retention time of the peptide. An amino acid difference detected by a peak shift could be confirmed by amino acid analysis. The technique has been used to isolate short peptides of rabbit, monkey, porcine, bovine, and human BP for sequence analysis.