Structure and bioactivity of the amino-terminal fragment of pro-opiomelanocortin

Abstract

The primary sequence of the amino-terminal or 16 K fragment (16 K) of pro-opiomelanocortin (POMC) is highly conserved throughout the mammals. This suggests an important biological role for this peptide. We have performed studies to determine the structure, biosynthetic origin and bioactivity of this pituitary peptide. A comprehensive study of all the biosynthetic derivatives of POMC in the neurointermediate lobe of the rat and mouse pituitary was undertaken. Inspection of the amino acid composition of these peptides indicated that cleavage at all available dibasic processing sites within POMC is essentially complete except for Arg 49-Lys 50 within the 1–74 16 K fragment (16 K 1–74). Only about 50% of 16 K 1–74 was found to be processed to give rise to the extreme amino-terminal 1 to 49 sequence (16 K 1–49) and the carboxyl-terminal 50 to 74 sequence (Lys 1 γ 3 melanotropin). Sufficient 16 K 1–77 and 16 K 1–49 was purified from bovine posterior pituitaries in order to determine if there are any structural features controlling the limited degree of processing of 16 K within the intermediate lobe. Both bovine 16 1–77 and 16K 1–49 were found to have cystine bridges linking cystine residues 2 and 24 and linking cystine residues 8 and 20. While 16K 1–77 was found to be O-glycosylated at threonine 45 and N-glycosylated as asparagine 65, 16 K 1–49 was found to have no carbohydrate content. Thus the presence of O-glycosylation at threonine 45 apparently inhibits cleavage at -Arg 49-Lys 50-. Lys 1γ 3 MSH 16 K 1–74 and 16 K 1–49 purified from rat neurointermediate pituitaries were tested for their ability to potentiate the action of corticotropin (ACTH) in an isolated rat adrenal cell bioassay. None of the 16 K-related peptides showed any intrinsic steroidogenic activity. Experiments were performed in which dispersed adrenal cells were incubated with serial dilutions of ACTH. Constant amounts of test peptides were added in concentrations ranging from 10 pM to 5 nM. Lys 1 γ 3 MSH potentiated the steroidogenic activity of ACTH by up to 2-fold with an ED 50 of approx 0.5 nM. 16 K 1–49 showed no ability to potentiate the action of ACTH. In contrast the most highly glycosylated form of 16 K 1–74 potentiated the action of ACTH by up to 6-fold.