Proteolytic profile of recombinant pro-opiomelanocortin in embryonal carcinoma P19 cells: conversion to beta-lipotropin and secretion are inhibited following incubation with canavanine.
A variety of proteins and peptides are produced through limited proteolysis of precursors at paired basic residues. This proteolytic bioactivation is carried out by subtilisin-like proteases, called convertases. The mRNAs of several convertases are expressed during prenatal life as well as in P19 embryonal carcinoma cells, which are a model of the totipotent cells of the embryo before and at the time of implantation. To determine whether converting activities accompany convertase mRNA expression in the early embryo, we transferred the gene of pro-opiomelanocortin (POMC) into P19 cells, by lipofection, and searched for the presence of mature peptides by high-performance liquid chromatography and radioimmunoassay techniques. In P19 cells, POMC, a precursor of several endocrine peptides, is mainly processed to beta-lipotropin rather than to beta-endorphin, both peptides having been identified by their immunoreactivity, polarity, and molecular size. These results indicate that converting capacities appear early in the embryo and that they are more similar to the activity of furin and of convertase PC1 than that of convertase PC2 in their cleavage selectivity of POMC sites. Efficiency of POMC processing can reach 50%, suggesting that convertases, with other proteases, can have an important role in ontogenesis. As for other peptide precursors in endocrine cells, the conversion of POMC in P19 cells was inhibited by the biosynthetic replacement of its arginine residues by the analog canavanine. However, the incorporation of canavanine into P19 cells also inhibited peptide secretion, suggesting that inhibition of conversion in these cells as well as in endocrine cells could indirectly result from the impairment of intracellular traffic and not only from a direct inhibition of the converting activity.
Bolduc
D
D
Département de chimie et de biochimie, Université du Québec, Montréal, Canada.
Cadet
N
N
Sayasith
K
K
Paquin
J
J
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002172:Canavanine / Q000494:pharmacology*; D018236:Carcinoma, Embryonal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D006868:Hydrolysis; D051379:Mice; D011333:Pro-Opiomelanocortin / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism*; D013552:Swine; D014407:Tumor Cells, Cultured; D008083:beta-Lipotropin / Q000378:metabolism*
D011994:Recombinant Proteins; D002172:Canavanine; D011333:Pro-Opiomelanocortin; D008083:beta-Lipotropin; D001120:Arginine; D010450:Endopeptidases