Inactivation of the 7B2 inhibitory CT peptide depends on a functional furin cleavage site

The eukaryotic subtilisin prohormone convertase 2 (PC2) is known to require in vivo exposure to the neuroendocrine protein 7B2 in order to produce an enzymatically active species capable of proteolytic action on prohormone su bstrates. In the present study, we examined the role of the pentabasic site within 27-kDa 7B2 in this process. We prepared two His-tagged recombinant 7B2s by overexpression in bacteria: 7B2-Ser-Ser (SS), with an inactivating mutation in the CT peptide from Lys171-Lys172 (KK) to SS, rendering the CT peptide non-inhibitory; blockade-SS, a double mutant of both the CT peptide as well as of the pentabasic furin cleavage site. These purified proteins were used in a cell-free proPC2 activation assay. Both 7B2-SS as well as bl ockade-SS were able to facilitate the activation of proPC2 (as judged by ef ficient production of enzyme activity), suggesting that cleavage at the fur in site is not required for 7B2s lacking inhibitory CT peptides. Plasmids e ncoding proPC2 and various 7B2s were transiently transfected into human emb ryonic kidney (HEK293) cells and PC2 enzymatic activity and CT forms in eac h overnight conditioned medium were measured. Cells transfected with proPC2 and wild-type 7B2 secreted CT peptide cleavage products, but cells transfe cted with proPC2 and the blockade mutant overwhelmingly secreted intact, 27 -kDa, blockaded 7B2. Medium obtained from HEK293 cells transfected with pro PC2 and either wild-type 7B2, 7B2-SS, or blockade-SS exhibited PC2 activity , but medium from cells expressing the 7B2 blockade mutant did not. We conc lude that cleavage at the 7B2 furin consensus site is required to produce P C2 capable of efficient proteolytic inactivation of the CT peptide.