Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes.

Abstract

Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrineprecursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursorprocessing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processingdeficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg-sites flanking corticotropin. The -Lys-Arg-within 13-lipotropin was less efficiently cleaved to release f-endorphin.Expression of PC2 together with PC3 resulted in efficient conversion of i-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of j3-lipotropin to y-lipotropin and 8-endorphin in the regulated secretory pathway. Finally, the processing selectivities ofPC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffmn cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.

Malignant Gastric Carcinoid Causing Ectopic ACTH Syndrome: Discrepancy of Plasma ACTH Levels Measured by Different Immunoradiometric Assays

“…In contrast, aberrant processing of POMC, such as unprocessed POMC, and/or enhanced processing of ACTH into a-MSH and CLIP, by non-pituitary tumors, leads to increased proportion of circulating levels of HMW form ACTH and ACTH-related fragments in patients with ectopic ACTH syndrome [20,21].…”

“…In contrast to high ACTH levels determined by Allegro ® assay kit, those determined by Yuka ® assay kit in the same patient's plasma samples were almost normal. Two-site IRMA kit by Allegro ® employs 125 I-labeled antibody against Nterminal fragment (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) and antibody against Cterminal fragment(34-39) of ACTH, while that by Yuka ® employs 125 I-labeled antibody against Nterminal fragment (20)(21)(22)(23) and antibody against Cterminal fragment(35-39) of ACTH, respectively. These data strongly suggest that various circulating forms of ACTH molecule in patient's plasma could be differentially recognized by these two different IRMA kits.…”

“…A 39 amino-acid residue ACTH is synthesized from a larger precursor molecule, termed POMC, via specific processing by PC 1/3 in a tissue-specific fashion [20][21][22]; in the normal anterior pituitary, POMC is processed to b-LPH and pro-ACTH, which is further cleaved in to an N-terminal peptide containing g-MSH (N-POC), joining peptide, and ACTH. In contrast, aberrant processing of POMC, such as unprocessed POMC, and/or enhanced processing of ACTH into a-MSH and CLIP, by non-pituitary tumors, leads to increased proportion of circulating levels of HMW form ACTH and ACTH-related fragments in patients with ectopic ACTH syndrome [20,21].…”

Sorting and processing of secretory proteins

“…These convertases have thus been well conserved throughout evolution. In keeping with their proposed role in proprotein conversion, these enzymes correctly cleave precursor proteins at paired basic residues [196,197,[221][222][223][224]. Kex2, furin and PC6B have hydrophobic transmembrane domains, while PCI, PC2, PC4, PC5, PC6A and PACE 4 do not.…”

“…The cleavage specificity of PCI and PC2 was first demonstrated by their co-transfection with mouse POMC and POMC mutants into the constitutively secreting BSC-40 cells and cell lines derived from endocrine tissue, namely PC12 and AtT20 cells, using the vaccinia expression system [223,224]. PC2 efficiently produced a-MSH and ,-endorphin-(1-3 1), while PCI generated ACTH and ,i-LPH.…”