THE PROREGION OF PAPAYA PROTEINASE-IV INHIBITS COLORADO POTATO BEETLEDIGESTIVE CYSTEINE PROTEINASES

Three distinct digestive protease systems were induced in larvae of th e herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlin eata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases. As shown by gelatin/PAGE and complementary inhibition assays, a recombinant fo rm of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhib itor of cysteine proteinases, oryzacystatin I (OCT), In contrast with OCI, the inhibitory potency of the proregion was affected by an increa se of the temperature, suggesting a certain alteration of its structur al integrity by the insect non-target proteases, This apparent suscept ibility to proteolysis was confirmed by SDS-PAGE, after challenging th e proregion,vith the different insect extracts. As seen on gel, select ive inhibition of the insect aspartate proteinase, cathepsin D, with t he inhibitor pepstatin A preserved the activity of the proregion again st cysteine proteinases by preventing its hydrolysis, Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of 'companion' defense-related proteins from the action of insensitive pr oteases in target pests. (C) 1998 Federation of European Biochemical S ocieties.