Robert F. Kibler

Experimental Allergic Encephalomyelitis in the Rat: Response to Encephalitogenic Proteins and Peptides

Lewis rats were used to determine the encephalitogenic activity of myelin basic protein of different species and of 45-residue fragments of basic protein. Basic protein from guinea pigs was more active than that from rats, and the fragments from the two species showed the same order of activity. Bovine basic protein was the least active of the intact proteins, and the respective fragment was inactive. Studies of serum-binding capacity did not support the hypothesis that blocking antibody played a role in this biological variation, whereas consideration of the amino acid sequences of the three fragments suggested that differences in primary structure, operating either at the sensitization or the effector phase of the immune response, could account for the variation.

THE MAJOR SITE OF GUINEA‐PIG MYELIN BASIC PROTEIN ENCEPHALITOGENIC IN LEWIS RATS12

In the Lewis rat, fragment 43–88 of the highly encephalitogenic guinea‐pig basic protein has been previously shown to retain the full activity of the parent protein. In the present studies this fragment was subjected to controlled chymotryptic digestion so that cleavage occurred only at tyrosine 67, generating two peptides, residues 43‐67 and residues 68‐88. When compared on an equimolar basis peptide 68‐88 had the same encephalitogenic activity as the intact fragment and induced the same degree of immunologically specific cell response as measured by the in vitro lymphocyte stimulation test. Peptide 68‐88 was further fragmented by selective tryptic cleavage at arginine 78 after blocking lysine 73 with citraconic anhydride. The two peptides, residues 68‐78 and residues 79‐88, were not encephalitogenic, indicating that residues adjacent to the point of cleavage contribute to the active site.

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid‐Sensitive Ca2+‐Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca2+, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X‐100 plus 4 mM EGTA, with or without further fractionation, showed that Ca2+‐dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE‐cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca2+ and phosphatidylserine; a protein kinase stimulated by Ca2+ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid‐sensitive Ca2+‐dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin‐sensitive species of Ca2+‐dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid‐sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N‐(6‐aminohexyl)‐5‐chloro‐l‐naphthalenesulfonamide (W–7).

Biological Activity and Synthesis of an Encephalitogenic Determinant

A 45-residue fragment of the basic protein of myelin is encephalitogenic in the rabbit and monkey but relatively inactive in the guinea pig. Synthetic peptides containing the sequence of a tryptic peptide of the fragment Thr-Thr-His-Tyr-Gly-Ser-Leu-Pro-Gln-Lys are moderately encephalitogenic.

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.

Encephalitogenic protein: structure.

Amino acid sequences of encephalitogenic proteins from bovine cord and rabbit brain are reported. The bovine protein contains 45 residues. The rabbit protein is identical except for two isopolar substitutions, a dipeptide and amino acid deletion. Analysis of this protein and a 140-residue myelin basic protein indicates that the smaller protein is a portion of the larger encephalitogen. The larger myelin protein contains at least two encephalitogenic sites.

Localization of 2',3'-cyclic nucleotide-3'-phosphohydrolase of rabbit brain by sedimentation in a continuous sucrose gradient.

Abstract— The enzyme 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase (CNP) has been assayed in fractions from a continuous sucrose density gradient zonal centrifugation of rabbit brain homogenates. Basic protein (BP) was also assayed by a radioimmunomethod. Fractions were examined by SDS‐polyacrylamide gel electrophoresis and by electron microscopy. These studies show that the major membrane fractions in the gradient differ greatly in the content of CNP and BP, and of high molecular weight proteins (HMW). The lightest membrane fractions contained numerous multilamellae, the highest content of BP and the lowest content of CNP and HMW, while the heaviest membrane fractions contained single membrane fragments and vesicles of unknown origin, the lowest content of BP and the highest content of CNP and HMW. The fraction containing the largest amount of membrane measured by turbidity, protein content, and water‐washed dry weight contained only half the CNP specific activity of a denser fraction in the gradient. CNP specific activity in the lightest fractions was insignificant compared to that of denser fractions. Thus, we conclude that this enzyme may be absent from the typical multilamellar myelin structures but present in the single‐membrane structures associated with myelin, such as the glial membrane and the paranodal segments of myelin adjacent to the axon. BP appears to occupy the opposite positions, highest in the multilamellae and lowest in the single‐membrane structures of myelin. These studies do not exclude the possibility that CNP may not be bound to myelin membranes, but rather to a membrane of different origin. Evidence that this enzyme is a myelin‐marker enzyme is circumstantial. Our evidence indicates the enzyme could be present either in a unique portion of myelin membranes or in another membrane structure.

Modifications of myelin basic protein which occur during its isolation.

Abstract— Chromatography of myelin basic protein (BP) on carboxymethylcellulose gives a pattern of multiple components, of which three are major. Component 1 is considered the unmodified species of BP while component 2 has been found to be modified primarily by deamidation and component 3 by phosphorylation (Chouet al., 1976). 3 In contrast to BP prepared from tissue delipidated in the standard fashion in chloroform–methanol (CM powder), BP prepared from tissue delipidated first in acetone and then in chloroform–methanol (ACM powder) gave an elution pattern on carboxymethylcellulose characterized by a decrease in component 1 and an increase in the earlier eluting, less basic components. Studies with radiolabelled component 1 showed that this difference in elution patterns was due to the partial conversion of component 1 to less basic components during the extraction of ACM powder at neutral pH. The components derived from component 1 (D2, D3 and D4) were then isolated and subjected to tryptic peptide map analyses and determination of their carboxy‐terminal arginine content and content of phosphorus. None of the derived components contained phosphorus but tryptic peptide map analyses did show the presence of two minor peptides, T14M2 and T20M, previously found in component 2 from CM powder and considered to be the deamidation products of their parent peptides T14 and T20 (Chouet al., 1976). In addition components D3 and D4 were shown to have lost appreciable arginine from their carboxy‐termini. Since none of the efforts to reduce enzyme activity in vitro had any appreciable effect on components 2 and 3 it was concluded that phosphorylation probably occurs exclusively in vivo, that deamidation occurs both in vivo and in vitro and that loss of carboxy‐terminal arginine occurs exclusively in vitro.

Identity of myelin basic protein from multiple sclerosis and human control brains: discovery of a genetic variant.

Abstract— Myelin basic protein was isolated from the brains of 7 multiple sclerosis and 5 control patients. When acid extracts of the delipidated brains were chromatographed on carboxymethylcellulose at alkaline pH the elution profiles were the same for the two groups of patients. Component I, the most basic species of the protein, from 2 multiple sclerosis and one control brains was fragmented by limited pepsin digestion. Tryptic peptide maps were prepared from the three major products, fragment 1–38, 39–89 plus 45–89 and 90–170. The amino acid compositions of corresponding peptides were identical except for a 50:50 substitution of serine for glycine in tryptic peptide 44–49 from one (N.L.) of the 2 patients with multiple sclerosis. Peptide 44–49. isolated from intact component 1 from the other 6 multiple sclerosis and 5 control brains, did not show this substitution. In both multiple sclerosis and control basic proteins phosphorus was present only in fragment 90–170 of component 3 in the amount of 0.22 mol of phosphorus/mol of protein. These data suggest that there is no difference in either the amino acid sequence or in the modification of basic protein from control and multiple sclerosis patients. The amino acid substitution in patient N.L. represents the first example of a mutation in basic protein.

Antigenic regions for the humoral response to myelin basic protein.

Abstract The major encephalitogenic sites in myelin basic protein (BP) † differ among animals tested. In order to define further the antigenic features of BP and to explore the possibility that antigenic sites recognized for antibody production might also differ among species, the contribution of regions of BP to the total antigenic activity of the molecule was examined in guinea pigs and rabbits. Twenty-one guinea pigs, 13 Hartley and 8 strain 13, were given a series of immunizations with bovine or guinea pig BP administered with large amounts of Mycobacterium tuberculosis . Eleven New Zealand white rabbits were immunized with bovine or guniea pig BP alone or conjugated to albumin. Antisera were tested by double antibody radioimmunoassay for reactivity with BP and BP peptides. Antisera from all guinea pigs reacted with BP and peptide 89–169 but had little or no reactivity with peptides 1–36 and 43–88. All rabbit antisera reacted well with BP, peptide 89–169 and peptide 1–36. Reactivity of rabbit antisera with peptide 43–88 was variable. It was present, usually in low titers, in 5 animals. The pattern of reactivity of rabbit antisera to BP and BP peptides was established early during the course of immunization. Rabbit antisera reactive with BP peptide 43–88 showed limited, if any, activity toward peptide 79–88, thus providing additional information about the inaccessibility or conformational dependence of the antigenic site in the molecular region of residues 79–88.