Structural Elements That Direct Specific Processing of Different Mammalian Subtilisin-like Prohormone Convertases

Abstract

PC1 and PC2 are two important subtilisin-like prohormone convertases (PC) that undergo differential endoproteolytic processing steps and sequentially mediate proopiomelanocortin (POMC) processing. To investigate the structural elements directing the processing of different PCs, we constructed a series of mutant and chimeric PC proteins and expressed them in cell lines with different patterns of expression of endogenous PCs: AtT-20, hEK293, and hLoVo cells. The COOH-terminally truncated PC1 underwent efficient proregion cleavage and rapid secretion in all three cell lines, while proregion cleavage and secretion were completely blocked in an active-site mutant of PC1. The truncated PC1 produced dramatic changes in POMC processing in AtT-20 cells. PC2 with the potential oxyanion hole Asp residue changed to Asn was processed and altered several aspects of POMC processing in a manner similar to that of wild-type PC2. PC1 protein with its proregion substituted with that of furin was cleaved after its proregion, producing active PC1 enzyme. A similar furin/ PC2 fusion protein underwent proregion cleavage at low efficiency. By contrast, when the proregions of PC1 and PC2 were substituted with one another, both fusion proteins failed to cleave the foreign prosequences, were unable to undergo oligosaccharide maturation, and remained in the ER. Although inactive PC mutants could theoretically function as dominant negatives, none interfered with the processing of endogenous active PCs or with POMC processing. We conclude that the COOHterminal of PC1 plays an important role in the routing or storage of PC1, the proregions of these PC proteins are replaceable in a molecule-specific manner, removal of proregion is essential for routing and for endoproteolytic activity, and the role of the potential oxyanion hole in PC2 is still unclear.Virtually all known mammalian bioactive peptides are synthesized first as precursor proteins in which product peptides are flanked by cleavage sites, most often paired basic amino acids. In different neuroendocrine tissues, depending on the availability of different endoproteases, precursors are cleaved into tissue-specific sets of final products. Proopiomelanocortin (POMC) 1 is the common precursor of several important peptide hormones, including ACTH, ␣-MSH, and ␤-endorphin. In anterior pituitary corticotropes, major POMC-derived peptides include ACTH and ␤-lipotropin, whereas in intermediate pituitary melanotropes, ACTH is further processed into ␣-MSH, and ␤-lipotropin is processed to ␤-endorphin(1-31) and ␤-endorphin(1-27) (1, 2).In the last few years, a group of subtilisin-like serine endoproteases that cleave specifically at sequences containing paired basic amino acids have been identified in mammalian tissues (3-9). The group includes PC1 (prohormone convertase 1, also called PC3), PC2, PC4, PC5/6, furin, and PACE4. PC1 and PC2 are predominantly detected in neuroendocrine tissues. Using as a model the AtT-20 mouse corticotrope tumor cell line, which has very high levels of endogenous P...

Two Activation States of the Prohormone Convertase PC1 in the Secretory Pathway

“…Following the cleavage of its profragment in the endoplasmic reticulum, mPC1 undergoes an additional cleavage in the secretory granules that results in the removal of its C-terminal tail (11)(12)(13). In this study, we have investigated the role of the C-terminal tail of mPC1 on prorenin processing.…”

“…The C-terminal cleavage occurs at the dibasic Arg-Arg 617-618 site (15), possibly by an autocatalytic event (15,16). The Cterminal tail has been proposed to play a role in sorting of PC1 to the regulated secretory pathway (17), but its exact role on PC1 activity is still poorly understood.…”

“…Following its synthesis as a 753-amino acid zymogen (9, 10), PC1 undergoes an autocatalytic intramolecular processing of its N-terminal profragment in the endoplasmic reticulum, resulting in an 87-kDa enzyme (11,12). This 87-kDa form is targeted to the regulated secretory pathway where it is further shortened by removal of 135 amino acids of its C-terminal tail leading to a 66-kDa form (11)(12)(13)(14). The C-terminal cleavage occurs at the dibasic Arg-Arg 617-618 site (15), possibly by an autocatalytic event (15,16).…”

Processing and trafficking of a prohormone convertase 2 active site mutant

“…This result stands in contrast to data obtained for other convertases, which indicate that catalytic triad mutants of human furin (furD46A, furH87A, and furS261A) expressed in transfected COS-1 cells are retained in the ER and are unable to undergo autocatalytic maturation [9,10]. Similarly, when PC1-S382A was expressed in HEK293 cells, no processed and secreted enzyme was detected, suggesting ER retention similar to furin [11]. Our observation that the PC2-S383A mutant was secreted as effectively as wild type enzyme from CHO-K1 cells suggests that for PC2, possibly alone among convertases, the serine to alanine active site mutation does not affect the ability of the zymogen to fold correctly.…”

“…Interestingly, catalytic triad mutants of human furin (furD46A, furH87A, and furS261A) do not undergo autocatalytic maturation, but instead accumulate in the ER [9,10]; similar results were found for a catalytic site mutant of PC1, PC1-S382A [11]. In this paper we have investigated the fate of a catalytic site mutant of proPC2 and show that in contrast to other convertases, this active site mutant is apparently folded correctly, as evidenced by its ability to efficiently traverse the secretory pathway.…”