Proteolytic activation of receptor-bound anthrax protective antigen on macrophages promotes its internalization

Immunofluorescence and other methods have been used to probe the self-assem bly and internalization of the binary toxin, anthrax lethal toxin (LeTx), i n primary murine macrophages. Proteolytic activation of protective antigen (PA; 83 kDa, the B moiety of the toxin) by furin was the rate-limiting step in internalization of LeTx and promoted clearance of PA from the cell surf ace. A furin-resistant form of PA remained at the cell surface for at least 90 min. Oligomerization of receptor-bound PA63, the 63 kDa active fragment of PA, was manifested by its conversion to a pronase-resistant state, char acteristic of the heptameric prepore form in solution. That oligomerization of PA63 triggers toxin internalization is supported by the observation tha t PA20, the complementary 20 kDa fragment of PA, inhibited clearance of nic ked PA. The PA63 prepore, with or without lethal factor (LF), cleared slowl y from the cell surface. These studies show that proteolytic cleavage of PA , in addition to permitting oligomerization and LF binding, also promotes i nternalization of the protein. The relatively long period of activation and internalization of PA at the cell surface may reflect adaptation of this b inary toxin that maximizes self-assembly.