A recombinant vaccinia virus vector was used to coexpress the two candidate mouse prohormone convertases, PC1 and PC2, together with mouse proopiomelanocortin (POMC) in the constitutively secreting cell line BSC-40 and in the endocrine tissue-derived cell lines PC12 and AtT-20, which exhibit regulated secretion. Monitoring of POMC processing demonstrated the distinct cleavage specificities of PCi and PC2, since in the cell lines analyzed (i) PCi cleaves POMC into corticotropin and fB-lipotropin, (ii) PC2 cleaves POMC into (3-endorphin, an N-terminally extended corticotropin containing the joining peptide, and either aMSH or desacetyl-aMSH, and (iii) PC2 cleaves POMC at the five pairs of basic residues analyzed, whereas PC1 cleaves two of them preferentially, suggesting that PC2 has a broader spectrum of activity than PC1. These data are consistent with our hypothesis on the physiological role of PCi and PC2 as distinct proprotein convertases acting alone or together to produce a set of tissuespecific maturation products in the brain and in peripheral tissues.Limited proteolysis of inactive precursors at pairs of basic residues or, less frequently, at single basic amino acids (1, 2) is a general mechanism by which the cell produces a variety of active proteins and peptides. Until recently, the molecular nature of the processing enzyme(s) was not identified except for the yeast pro-a mating factor precursor, where a subtilisin-like serine proteinase called KEX2 was proven to be the physiological convertase (3). In the last year, different groups obtained the complete cDNA structures of three distinct candidate mammalian convertases, furin (4), PC1 (5, 6), and PC2 (5, 7). The putative convertase function of these proteins was largely based on sequence homology with KEX2 (8, 9) and on their tissue and cellular distribution, especially for PC1 and PC2 (5, 6). Here we show that the expression of PC1 or PC2 by vaccinia virus (VV) recombinants results in the production of distinct proteolytic enzymes in three cell lines: the mouse pituitary corticotroph line AtT-20 and two that do not express these enzymes endogenously, the African green monkey kidney epithelial cell line BSC-40 and the rat pheochromocytoma cell line PC12. These proteinases cleave proopiomelanocortin (POMC) into the same set of distinct peptides known to be produced in vivo in the hypothalamus, adenohypophysis (AP), and pars intermedia of the pituitary, including adrenocorticotropin (ACTH), p-lipotropin (,8LPH), ,f-endorphin (f3End), and a-melanotropin (aMSH) or desacetyl-aMSH.MATERIALS AND METHODS Ws. Purified recombinant VVs using the full-length mouse (i) PC1 and mPC2 cDNA inserts (5, 6) (VV:mPC1 and VV:mPC2) were prepared as described (10). VV:POMC (mouse) was a gift of G. Thomas (Vollum Institute, Portland, OR).Northern Blot Analysis. Total RNA was prepared as described (11). The [a-32P]UTP-labeled cRNA probes were generated from cDNA sequences of mPC1 (6) and mPC2 (5) inserted in plasmid Rc/CMV vector (Invitrogen, San Diego) and linearized...

We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.

Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.

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