7B2 can function as an extracellular chaperone for proPC2 activation: uncoupling of solubilization from zymogen capacitation

Abstract

Prohormone convertase 2 (PC2), a protease belonging to the prohormone convertase family, requires early interaction with the neuroendocrine protein 7B2 for production of an active enzyme molecule. However, the biochemical mechanism underlying the 7B2-mediated capacitation process is still enigmatic. We previously found that proPC2 expressed in CHO cells lacking 7B2 (CHO/PC2 cells) is secreted as an inactivatable species. We have here investigated the role of extracellular 7B2 on the solubilization and acquisition of enzyme activity. We found that addition of exogenous recombinant 7B2 to CHO/PC2 cells can prevent proPC2 aggregation and restore enzyme activity. Interestingly, when 7B2 was added to the conditioned medium in the absence of cells, an anti-aggregant activity was observed, but the PC2 enzyme activity could not be rescued. Velocity sedimentation experiments showed that addition of extracellular 7B2 increased the amounts of several different PC2 species: two soluble activatable proPC2 species (the intact zymogen and a zymogen with a partially cleaved propeptide) as well as a soluble, inactive 66 kDa PC2 form. Our results suggest that extracellularly added 7B2 possesses chaperone-like activities to block partially unfolded proPC2 forms from aggregating and to refold the solubilized proPC2 species. Since acquisition of activity could be uncoupled from aggregation, either a separate capacitation process occurs on the cell surface and/or in local microenvironments close to cell membranes, resulting in the production of a zymogen competent for activation; or proPC2 released into medium lacking 7B2 becomes conformationally incompetent for activation even though soluble.