Specific Cleavage of the A1 Protein from Myelin with Cathepsin D

Abstract

Cathepsin D, purified from bovine and rabbit liver, hydrolyzed primarily one peptide bond in the A1 protein from bovine central nervous system myelin, the Phe-Phe bond between residues 42 and 43. Surprisingly, the other Phe-Phe linkage (residues 88 to 89) was not cleaved. In view of the high affinity of cathepsin D for the Phe-Phe bond, it was postulated that the resistant linkage was protected from enzymic attack either by the influence of surrounding amino acids or by conformational restrictions. The two peptides (Peptides CD1 and CD2), resulting from the action of bovine liver cathepsin D, were obtained in high yield by cellulose phosphate chromatography and shown to be homogeneous by gel electrophoresis at pH 4.5 and in sodium dodecyl sulfate. Peptide CD1 was identified as residues 1 to 42 and Peptide CD2 as residues 43 to 169 of the A1 protein sequence. The identity of Peptides CD1 and CD2 was established by end group analysis, by molecular weights obtained from sodium dodecyl sulfate gel electrophoresis, by amino acid analysis, and finally by peptide mapping of tryptic digests and analysis of each peptide. The amino acid analysis of each tryptic peptide was in excellent agreement with that expected from the position of the peptide on the peptide map. Peptide CD1, the 42-residue peptide, has special immunological significance since it contains a minor determinant responsible for allergic encephalomyelitis in the monkey. It also contains an antigenic determinant which displaces 80% of labeled A1 protein from rabbit antibody.