Enzymes involved in the classic and non-classic processing of the neuroendocrine peptides.

摘要

Neuropeptides are synthesized as large precursors that undergo limited proteolysis as well as other post-translational modifications to gain full biological activity. In most cases, peptide precursors are processed at the dibasic or multi-basic sites, and such cleavage events are referred to as "classic cleavage". Prohormone Convertase-1 (PC1), along with prohormone convertase-2 (PC2) is believed to be responsible for the processing of many neuropeptide precursors at the "classic cleavage" sites. To explore the role that PC1 plays in neuropeptide processing in brain and pituitary, we used radioimmunoassays (RIA) as well as quantitative peptidomic methods and examined the changes in the levels of the multiple neuropeptide products in PC1 knockout (KO) mice. RIA analysis of ir-Met-Enk in brain extracts of these mice revealed an increase in the level of partially processed Met-Enk containing peptides suggesting that PC1 plays a physiological role in the processing of proenkephalin. Processing of the neuropeptide precursor VGF was also affected in PC1 KO mouse brains. In contrast, the processing of proSAAS was not altered in PC1 KO mouse brains. Quantitative peptidomics analysis revealed a number of changes in neuropeptide processing in PC1 KO mouse pituitary. Among them, the levels of oxytocin and chromogranin A and B derived peptides were dramatically decreased in the PC1 KO mice, while the levels of peptides derived from proopiomelanocortin and provasopressin were not altered. These results support the notion that PC1 plays a physiological role in the processing of multiple neuroendocrine peptide precursors and emphasizes the presence of a redundant system. In addition to processing at "classical" sites, the processing of peptide precursors also occurs at non-basic sites; the enzymes that are responsible for such cleavages have not been well explored. ECE-2 is a potential candidate for performing such cleavages since it exhibits a neuroendocrine distribution and an acidic pH optimum. In order to examine this possibility, we cloned, expressed and purified recombinant soluble ECE-2. We found that ECE-2 could generate a number of biologically active peptides from peptide intermediates by cleavages at "non-classic" sites, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. We also examined the subcellular localization of ECE-2 using immunocytostaining of AtT-20 cells and percoll-sucrose density fraction of bovine anterior pituitary. Finally, we examined the changes in the level of peptide processing in ECE-2 KO mouse pituitary by quantitative peptidomic method.