RESPONSE OF DIGESTIVE CYSTEINE PROTEINASES FROM THE COLORADO POTATO BEETLE (LEPTINOTARSA-DECEMLINEATA) AND THE BLACK VINE WEEVIL (OTIORYNCHUS-SULCATUS) TO A RECOMBINANT FORM OF HUMAN STEFIN-A

The effects of the cystatins, human stefin A (HSA) and oryzacystatin I (OCI) on digestive cysteine proteinases of the Colorado potato beetle (CPB), Leptinotarsa decemlineata, and the black vine weevil (BVW), Ot iorynchus sulcatus, were assessed using complementary inhibition assay s, cystatin-affinity chromatography, and recombinant forms of the two inhibitors. For both insects, either HSA and OCI used in excess (10 or 20 mu M) caused partial and stable inhibition of total proteolytic (a zocaseinase) activity, but unlike for OCI the HSA-mediated inhibitions were significantly increased when the inhibitor was used in large exc ess (100 mu M). As demonstrated by complementary inhibition assays, th is two-step inhibition of the insect proteases by HSA was due to the d ifferential inactivation of two distinct cysteine proteinase populatio ns in either insect extracts, the rapidly (strongly) inhibited populat ion corresponding to the OCI-sensitive fraction. After removing the cy statin-sensitive proteinases from CPB and BVW midgut extracts using OC I- (or HSA-) affinity chromatography, the effects of the insect ''non- target'' proteases on the structural integrity of the two cystatins we re assessed. While OCI remained essentially stable, HSA was subjected to hydrolysis without the accumulation of detectable stable intermedia tes, suggesting the presence of multiple exposed cleavage sires sensit ive to the action of the insect proteases on this cystatin. This appar ent susceptibility of HSA to proteolytic cleavage may partially explai n its low efficiency to inactivate insect OCI-insensitive cysteine pro teinases when not used in large excess. It could also have major impli cations when planning the use of cystatin-expressing transgenic plants for the control oi coleopteran pests. (C) 1996 Wiley-Liss, Inc.