INHIBITION OF INTRACELLULAR PROTEOLYTIC PROCESSING OF SOLUBLE PROPROTEINS BY AN ENGINEERED ALPHA(2)-MACROGLOBURIN CONTAINING A FURIN RECOGNITION SEQUENCE IN THE BAIT REGION

The bait region of the general protease inhibitor alpha(2)-macroglobul in (alpha(2)M) was mutated by introducing a recognition sequence of fu rin. This did not interfere with folding, S-ester formation or tetrame rization of the mutant recombinant alpha(2)M (r alpha(2)M) Mutant r al pha(2)M inhibited furin in vitro, by a similar mechanism to that used by plasma alpha(2)M to inhibit high-molecularmass proteases. The mutan t alpha(2)M was intracellularly active in COS-1 cells in inhibiting th e endogenous processing of the soluble substrates for furin (von Wille brand factor, transforming growth factor beta 1 and a soluble form of the envelope glycoprotein gp160 from HIV-1) but not the membrane-bound form of gp160. The intracellular activity of mutant alpha(2)M strongl y indicated that alpha(2)M attains its native conformation, and thus t hat the unusual internal S-ester is formed, before alpha(2)M passes th rough the cleavage compartment(s). Our results show for the first time that modu lation of the bait region of alpha(2)M allows the creation of an inhibitor against membrane-bound proteases. It can be expected t hat the use of alpha(2)M-bait mutants will become important as a techn ique for the study of various proteolytic processes and for the identi fication of the proteases involved.