IN-VITRO PROCESSING OF ANTHRAX TOXIN PROTECTIVE ANTIGEN BY RECOMBINANT PC1(SPC3) AND BOVINE INTERMEDIATE LOBE SECRETORY VESICLE MEMBRANES

Protective antigen (PA), an 83-kDa protein produced by Bacillus anthra cis, requires proteolytic activation at a tetrabasic site (RKKR(167)) before it can combine with either edema factor or lethal factor on the cell surface. The complex is then endocytosed and the target cell int oxicated, Previous work has demonstrated that furin, a ubiquitously di stributed, subtilisin-like protease, can perform this cleavage. In thi s study, another member of the furin family, PC1 (SPC3), was tested as a putative processing enzyme for PA, Recombinant PC1, partially purif ied from the medium of stably transfected L-cells, cleaved PA to a 63- kDa fragment (PA63) and a 20-kDa fragment (PA20). Amino-terminal seque nce analysis of the 63 kDa product demonstrated that cleavage occurred between Arg(167) and Ser(168). The pH optimum for in vitro PA cleavag e was 6.0 and the enzymatic activity was calcium-dependent. Medium fro m untransfected L-cells did not cleave PA, Site-directed mutagenesis o f the tetrabasic cleavage site revealed that PC1 preferred to cleave s equences containing basic residues at positions -1 and -4 relative to the wild-type cleavage site, demonstrating that PC1 can cleave substra tes at a monobasic residue site in vitro. Substrates having basic resi dues at the -1 and -2 positions were cleaved with approximately twofol d less efficiency than wild-type PA. Mutants of PA containing basic re sidues in positions -1 and either -2 or -4 of the cleavage site were p redicted to be substrates for PC1 and were more toxic to L-cells expre ssing PC1 than to untransfected L-cells, These results demonstrate tha t PA is cleaved by PC1 in vivo, Membranes from bovine intermediate lob e secretory vesicles which contain both prohormone convertases, PC1 an d PC2, also cleaved PA to PA63 with a pH optimum of 5.5, Immunodepleti on studies using antisera against PC1 and PC2 showed that these are th e enzymes primarily responsible for the cleavage of PA in the membrane preparation, Thus, both recombinant PC1 and a membrane preparation co ntaining endogenous PC1 can activate PA. (C) 1995 Academic Press, Inc.