DIFFERENCES IN THE AUTOCATALYTIC CLEAVAGE OF PRO-PC2 AND PRO-PC3 CAN BE ATTRIBUTED TO SEQUENCES WITHIN THE PROPEPTIDE AND ASP(310) OF PRO-PC2

PC2 and PC3 are subtilisin-lilie proteases involved in the maturation of prohormones and proneuropeptides within neuroendocrine cells. They are synthesized as zymogens that undergo autocatalytic maturation with in the secretory pathway, Maturation of pro-PC2 is slow (t 1/2 > 8 h), exhibits a pH optimum of 5.5 and is dependent on calcium (K-0.5 2 mM) , while pro-PC3 maturation is relatively rapid (t 1/2 min), exhibits a neutral pH optimum and is not calcium dependent. These differences in the rates and optimal conditions for activation of the proteases may contribute to the diversity of products generated by these proteases i n different cell types. Although highly similar, there are two major d ifferences between pro-PC2 and pro-PC3: the presence of an aspartate a t position 310 in pro-PC2 compared with asparagine at the equivalent p osition in pro-PC3 land all other members of the subtilisin family), a nd the N-terminal propeptides, which exhibit low sequence identity (30 %). With a view to establishing the structural features that might be rc responsible for these differences in the maturation of pro-PC2 and pro-PC3, Asp(310) in pro-PC2 was mutated to Asn, and Asn(309) in pro- PC3 was mutated to Asp. Chimaeric proteins were also made consisting o f the pro-region of PC2 fused to the mature portion of PC3 and the pro -region of PC3 fused to the mature region of PC2, The wild-type and mu tant DNA constructs were then transcribed and translated in an in vitr o system capable of supporting maturation of pro-PC2 and pro-PC3. The results demonstrated that Asp(310) of pro-PC2 is responsible for the a cidic pH optimum for maturation. Thus changing Asp(310) to Asn shifted the pH optimum for maturation to pH 7.0. However? changing Asn(309) o f pro-PC3 to Asp had no effect on the optimum pH for maturation of pro -PC3. A chimaeric construct containing the propeptide of pro-PC2 attac hed to PC3 shifted the pH optimum for maturation from pH 7.0 to 6.0 an d slowed down the rate of maturation (t 1/2 > 8 h). When attached to P C2: the proregion of pro-PC3 had no effect on the optimum pH for matur ation (pH 5.5-6.0), but it did accelerate the rate of maturation (t 1/ 2 2 h). These results demonstrate that Asp(310) and the pro-region of pro-PC2 contribute to the acidic pH optimum and low rate of maturation of this zymogen relative to its closely related homologue PC3.