Carboxy-terminal proteolytic processing at a consensus furin cleavage site is a prerequisite event for quail ZPC secretion

In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in granulosa cells is cleaved at a consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the effect of the proteolytic cleavage of proZPC on ZPC secretion by using a specific inhibitor of furin endoprotease and site-directed mutagenesis of the furin cleavage site. Western blot analysis demonstrated that the furin inhibitor efficiently blocked both the proteolytic cleavage of proZPC and the subsequent ZPC secretion. A site-directed mutant that possessed a mutated sequence for furin cleavage was not secreted from the cells. The immunocytochemical observations indicated that proZPC produced in the presence of a furin inhibitor or those produced by the site-directed mutant of the furin cleavage site had accumulated in the endoplasmic reticulum. These results indicate that proZPC is proteolytically cleaved at the consensus furin cleavage site with furin-like protease, and the failure of this cleavage results in its accumulation in the endoplasmic reticulum. Therefore, the C-terminal proteolytic processing of proZPC at the consensus furin cleavage site is a prerequisite event for quail ZPC secretion.