Biosynthesis and maturation of the prohormone convertases PC3 and PC2
The prohormone convertases PC2 and PC3 are members of the highly conserved subtilisin-like serine protease superfamily. Targeted to the regulated secretory pathway of neuroendocrine cells they are involved in the proteolytic processing of a number of co-secreted polypeptides. To investigate the nature of their biosynthesis, maturation and possible interaction with the neuroendocrine polypeptide 7B2, a series of mutants were constructed and translated in a novel cell-free in vitro translation/translocation system derived from Xenopus eggs. It was shown that PC3 undergoes rapid (T1/2<10min), autocatalytic maturation, optimally at pH7, in a calcium-independent manner, from an 88kDa proform to an 80kDa mature form, in a proteolytic reaction occurring carboxy-terminal to RSKR83. This indicates the endoplasmic reticulum as the sub-cellular location for PC3 maturation in vivo. Conversely, PC2 matures optimally at pH5.5 in an autocatalytic reaction dependent on millimolar calcium. It was demonstrated that mutation of the PC2 unique oxyanion aspartate282 to asparagine increased its optimal processing pH requirements from acidic to neutral, in a reaction still dependent on millimolar calcium. This infers that the oxyanion aspartate of PC2 influences the pH at which the protease matures. In vitro the role of the neuroendocrine precursor 7B2 in PC2 biosynthesis was shown to be two-fold and pH-dependent. At acidic pH, pro-7B2 acted as an inhibitor of pro-PC2 maturation, whereas under neutral pH conditions pro-7B2 was seen to markedly enhance pro-PC2 maturation. Neither precursor nor mature 7B2 were shown to interact with the PC2 oxyanion mutant, pro- or mature PC3 in vitro. This is consistent with the proposed view that 7B2 specifically interacts with PC2 in the regulated secretory pathway and the association is dependent upon the nature of the PC2 oxyanion aspartate residue.