Gladys E. Deibler

Differences between the two myelin basic proteins of the rat central nervous system. A deletion in the smaller protein.

Abstract Myelin of the rat central nervous system contains two highly basic proteins which differ in molecular size, amino acid composition, and encephalitogenic activity. The larger rat protein is very similar to the myelin basic proteins of beef and human in total polypeptide chain length, amino acid composition, encephalitogenic activity, and length of the polypeptide chain between the two methionyl residues. The length of polypeptide chain between the two methionyl residues of the smaller rat protein is considerably less than the corresponding segment of the larger. Both proteins contain 1 mole of tryptophan per mole of protein. The difference in amino acid compositions of the two rat proteins, together with the amino acid compositions of the tryptic peptides present in the larger rat protein but missing in the smaller indicate a deletion in the smaller protein corresponding to bovine and human residues 117–156 or 118–157. The new tryptophan-containing peptide created by the deletion has the composition (Phe, Ser, Trp, Gly 2 ) Arg. This deletion removes a major part of the peptide reported to be encephalitogenic in the guinea pig. Loss of the Gln-Lys portion of this latter peptide explains our observation that the smaller protein is much less encephalitogenic in the guinea pig than the larger.

Myelin basic proteins of the rat central nervous system Purification, encephalitogenic properties, and amino acid compositions

Abstract Rat myelin basic protein isolated from chloroform-methanol-pretreated spinal cords has been resolved into two components by electrophoresis in polyacrylamide gels at acid pH. The two components were separated by repeated fractionation on a Sephadex G-100 column and characterized by their ability to induce experimental allergic encephalomyelitis in guinea pigs and by their amino acid compositions. The larger component, which has the lower electrophoretic mobility, was found to be comparable to central nervous system myelin basic proteins of other mammalian species in encephalitogenic activity and amino acid composition. The smaller component, which comprised approximately three-fourths of the myelin basic protein extracted, was much less encephalitogenic than the larger, and its amino acid composition was somewhat different. Per 100 moles of total residues the smaller component is considerably richer in arginine and poorer in lysine than the larger component; however, both components contain approx. 23 moles of total basic residues and 14 moles of total acidic residues (including amides) per 100 moles of total residues. Per mole of protein the smaller component appears to have significantly fewer lysyl, glutamyl and/or glutaminyl, glycyl, alanyl, leucyl, tyrosyl, and phenylalanyl residues. It is suggested that the two components may be the products of two structurally related nonallelic genes; however, the possibility that the smaller component arise from the larger as the result of the action of a species-specific proteolytic enzyme system cannot be definitely excluded.

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.

THE OCCURRENCE OF TWO MYELIN BASIC PROTEINS IN THE CENTRAL NERVOUS SYSTEM OF RODENTS IN THE SUBORDERS MYOMORPHA AND SCIUROMORPHA

Extracts containing myelin basic proteins have been prepared from CNS tissue of representatives of the three suborders of Rodentia—Myomorpha, Hystricomorpha and Sciuro‐morpha. Analyses of the extracts by electrophoresis at low pH showed that one type (L) of myelin basic protein is present in the CNS of all of the rodents examined (rat, mouse, hamster, guinea pig, chinchilla, prairie dog, woodchuck and squirrel). This protein is comparable in molecular size and charge to the CNS myelin basic proteins found in several other mammalian orders. In the CNS of the myomorphs (rat, mouse, hamster) and sciuro‐morphs (prairie dog, woodchuck, squirrel) there is an additional type (S) of myelin basic protein of higher cathodic mobility and smaller molecular size. This additional protein is absent from the CNS of the hystricomorphs (guinea pig, chinchilla). These findings indicate that the presence of two myelin basic proteins originally reported in the CNS of the inbred rat is not an anomaly of inbreeding. These data further suggest that the presence of a single L‐type CNS myelin basic protein might be a general characteristic of hystricomorphs, while the presence of both L‐ and S‐type CNS myelin basic proteins might be a general characteristic of the myomorphs and sciuromorphs.

Damage to Neurons in Culture Following Medium Change: Role of Glutamine and Extracellular Generation of Glutamate

Abstract— 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 μM) of 2‐amino‐5‐phos‐phonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 μM) 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 μrn 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.

Cleavage of Rabbit Myelin Basic Protein by Pepsin

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 Tyr14Leu15 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.

Peptide specificities of myelin basic protein-reactive human T-cell clones.

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.

COMPARATIVE STUDIES OF GUINEA PIG AND BOVINE MYELIN BASIC PROTEINS. PARTIAL CHARACTERIZATION OF CHEMICALLY DERIVED FRAGMENTS AND THEIR ENCEPHALITOGENIC ACTIVITIES IN LEWIS RATS

Guinea pig and bovine myelin basic proteins were chemically cleaved at the carboxyl peptide bonds of methionyl and tryptophanyl residues to yield several fragments. Comparison of the bovine fragment consisting of the first 20 residues of the protein with the corresponding guinea pig fragment showed that the latter differs in containing histidine and glycine (one residue of each), an additional threonyl residue, and one fewer alanyl residues. Comparison of the bovine fragment consisting of the C‐terminal 54 residues of the protein (residues 117‐170) with the corresponding guinea pig fragment showed that the latter differs in containing one fewer histidyl and leucyl residues and an additional phenylalanyl residue. Tests of encephalitogenic activity in Lewis rats showed that these two fragments from both species were much less active, on a molar basis, than the uncleaved protein. On the other hand, examination of the bovine fragments consisting of residues 1‐116 and 21‐116 and the corresponding fragments obtained from the guinea pig protein revealed activity at least as high as that of the respective uncleaved proteins.

PARTIAL CHARACTERIZATION OF BASIC PROTEINS OF CHICKEN, TURTLE AND FROG CENTRAL NERVOUS SYSTEM MYELIN

Myelin basic proteins were isolated from CNS tissues of chicken, turtle and frog and compared with the corresponding protein of bovine origin. At acid pH all four proteins had comparable mobilities in polyacrylamide gels. Upon electrophoresis at alkaline pH the submammalian proteins, like the bovine protein, were separated into multiple components. The components of the chicken and frog proteins had exceptionally high and low mobilities, respectively, while those of the turtle protein had mobilities comparable to those of the bovine protein. The chicken and turtle proteins were similar to the bovine protein in amino acid composition except for containing considerably more serine and valine and having higher proportions of histidine to lysine. The frog protein differed further in having an unusually high content of tyrosine (approx 9 mol/mol protein), an unusually high arginine: glycine ratio (1.09) and practically no methylated arginine (0‐0.036 mol/mol protein). Like those of mammalian origin, the submammalian proteins each contained a single tryptophan and two methionines. Arginine, serine and glycine together accounted for approximately 40 per cent of the residues in each protein. The chicken and turfle proteins each contained roughly equal amounts of NG‐monomethyl‐ and NG, NG‐dimethylarginine, the two derivatives together comprising 0.5‐0.6 mol/mol protein. No NG, NG‐dimethylarginine was detected in any of the proteins examined. The microheterogeneity observed in the chicken and turtle proteins upon electrophoresis at alkaline pH was reproduced upon alkaline pH chromatography on carboxymethylcellulose. Chromatographic fractions of the chicken protein which differed electrophoretically at alkaline pH had virtualy identical amino acid compositions and apparent molecular weights and all contained comparable amounts of both NG‐monomethyl‐ and NG, NG‐dimethylarginine. Treatment of the submammalian proteins with BNPS‐skatole yielded two fragments comparable in size, charge and staining characteristics to those similarly produced from the bovine protein (residues 1‐116 and 117‐170). Fragments produced from the frog protein by treatment with BrCN were comparable in size and charge to those similarly produced from the bovine protein; those produced from the chicken and turtle proteins were much different. In immunodiffusion studies the submammalian and bovine proteins showed reactions of identity when tested against rabbit anti‐chicken basic protein serum.

Cleavage of rabbit myelin basic protein by thrombin.

Abstract: 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 β‐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.