Proteolysis of 125I-labelled tetanus toxin by subcellular fractions from an Epstein-Barr-virus-transformed B lymphoblastoid cell line, JY, was investigated. Fractions enriched in lysosomes and plasma membranes cleaved the toxin molecule at several sites, with a pH optimum of 5.5. N-Terminal sequence analysis of Mr-81,000, -45,000 and -35,000 proteolytic fragments indicated cleavage of the Asp-460-Leu-461, Asp-872-Glu-873 and Ile-1013-Thr-1014 peptide bonds, all sites located within the heavy chain of the toxin molecule. Additional sites near the C-terminus of the heavy chain, giving rise to low-Mr peptides, were implicated. The toxin light chain was more resistant to proteolysis. A similar pattern of fragmentation was observed with tetanus toxin biosynthetically radiolabelled with 14C-labelled amino acids, showing that the proteolysis was not an artifact caused by iodination. The proteolytic activity was inhibited by the serine proteinase inhibitor di-isopropyl phosphorofluoridate, thiol-blocking proteinase inhibitors N-ethylmaleimide and iodoacetamide, and by EDTA. These results represent a preliminary characterization of the processing in vitro of tetanus toxin by an antigen-presenting cell line.
