Characterization of the maturation of human pro-apolipoprotein A-I in an in vitro model

The reaction conditions and the protein structural features involved in the maturation of pro-apolipoprotein A-I (cleavage of pro-peptide) were invest igated in an in vitro model. ProapoA-I, mutants and wild type, were express ed in the PGEX/E. coli expression system as fusion proteins with glutathion e S-transferase (GST). Use of GST-proapoA-I and truncated forms of proapoA- I enabled quantitation of the amount of GST and apoA-T formed as a result o f cleavage following incubation with human serum. Deletion of the pro-pepti de (GST-apoA-I) resulted in complete inhibition of the reaction. Truncation of proapoA-I to residues 222, 150, 135, and 25 as well as substitution of residues -6, -5, and -4 with alanine did not affect the reaction. Substitut ion of residues -1, -2, 1, 3, and 4 with alanine either completely blocked or substantially inhibited cleavage of the pro-peptide. The reaction was in hibited by addition of EDTA, o-phenanthroline, dithiothreitol, and beta -me rcaptoethanol and to a lesser extent by p-chloromercuriphenylsulfonic acid, but not by leupeptin, N-ethylmaleimide, PMSF, pepstatin A, or trans-epoxys uccinyl-L-leucylamido(4-guanidino)butane. Calcium was essential for the act ivation of the cleavage enzyme, but it had a biphasic effect on the cleavag e, activating it at concentrations below 1.5 mM and inhibiting at concentra tions above 1.75 mM, Manganese alone was not essential for activation of th e enzyme nor did it modify the effect of low concentration of calcium. Howe ver, a high concentration of manganese partially reverted the inhibitory ef fect of a high calcium concentration. Thus, residues within -2 to +4 are in volved in forming the cleavage site for the maturation enzyme. The reaction of maturation is inhibited by metalloprotease inhibitors and is dependent upon calcium.