MUTATIONS WITHIN THE PROPEPTIDE, THE PRIMARY CLEAVAGE SITE OR THE CATALYTIC SITE, OR DELETION OF C-TERMINAL SEQUENCES, PREVENTS SECRETION OF PROPC2 FROM TRANSFECTED COS-7 CELLS

PC2 is a neuroendocrine endoprotease involved in the processing of pro hormones and proneuropeptides. PC2 is synthesized as a proenzyme which undergoes proteolytic maturation within the cellular secretory appara tus. Cleavage occurs at specific sites to remove the N-terminal propep tide. The aim of the present study was to investigate structural requi rements for the transfer of proPC2 through the secretory pathway. A se ries of mutant proPC2 constructs were transfected into COS-7 cells and the fate of the expressed proteins followed by pulse-chase analysis a nd immunocytochemistry. Human PC2 was secreted relatively slowly, and appeared in the medium primarily as proPC2 (75 kDa), together with muc h lower amounts of a processed intermediate (71 kDa) and mature PC2 (6 8 kDa). Mutations within the primary processing site or the catalytic triad caused the protein to accumulate intracellularly, whereas deleti on of part of the propeptide, the P-domain or the C-terminal regions a lso prevented secretion. Immunocytochemistry showed that wild-type hPC 2 was localized mainly in the Golgi, whereas two representative mutant s showed a distribution typical of proteins resident in the endoplasmi c reticulum. The results suggest that proenzyme processing is not esse ntial for secretion of PC2, but peptides containing mutations that aff ect the ability of the propeptide (and cleavage sites) to fold within the catalytic pocket are not transferred beyond the early stages of th e secretory pathway. C-terminal sequences may be involved in stabilizi ng such conformations.