STRUCTURE OF TERMINAL REGIONS OF ENCEPHALITOGENIC BASIC PROTEIN FROM BOVINE MYELIN

Treatment of the bovine encephalitogen (A1 protein) with cyanogen bromide resulted in complete cleavage of the two methionyl-alanine linkages present in the protein, giving rise to three peptides (CB1, CB2, and CB3). Although the reaction mixture retained full encephalitogenic activity, located exclusively in peptide CB2, the antibody-combining activity was lost. Neither peptide CB1 nor CB2 was active in the passive hemagglutination inhibition test; and peptide CB2 was negative in the Ouchterlony test. It was concluded that encephalitogenic site of the A1 protein does not coincide with the site involved in interaction with antibody. Peptide CB1 contains N-acetylalanine, and thus occupies the N-terminal region of the A1 protein. The amino acid sequence of peptide CB1 was determined from tryptic, chymotryptic, and peptic peptides. The complete sequence is: N-Ac-Ala-Ser-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Ser-Ala-Ser-Thr-Hser-OH. Thus, the first methionine residue of the A1 protein is located at position No. 20 from the N-terminal acetylalanine. The internal peptide (CB2), containing approximately 141 amino acids, has an N-terminal alanine residue and contains the only tryptophan found in the original protein. The amino acid sequence of peptide CB3, H-Ala-Arg-Arg-OH, is identical with the C-terminal sequence obtained for the A1 protein, and reveals the location of the second methionine residue at position No. 4 from the C-terminal arginine. The C-terminal region was further clarified by treating the A1 protein and peptide CB2 with carboxypeptidase. The sequence was found to be: -His-Phe-Met-Ala-Arg-Arg-OH. The C-terminal sequence of -Met-Ala-Arg-Arg-OH was also established for encephalitogenic A1 proteins derived from human, monkey, dog, and guinea pig brain. © 1969.