Three insect peptides showing high sequence similarity and belonging to the same structural family incorporating a cysteine knot and a short three-stranded antiparallel ␤-sheet were studied. Their inhibitory effect on two serine proteases (bovine ␣-chymotrypsin and human leukocyte elastase) is reported. One of them, PMP-C, is a strong ␣-chymotrypsin inhibitor (K i ‫؍‬ 0.2 nM) and interacts with leukocyte elastase with a K i of 0.12 M. The other two peptides, PMP-D2 and HI, interact only weakly with ␣-chymotrypsin and do not inhibit leukocyte elastase. Synthetic variants of these peptides were prepared by solid-phase synthesis, and their action toward serine proteases was evaluated. This enabled us to locate the P1 residues within the reactive sites (Leu-30 for PMP-C and Arg-29 for PMP-D2 and HI), and, interestingly, variants of PMP-D2 and HI were converted into powerful inhibitors of both ␣-chymotrypsin and leukocyte elastase, the most potent elastase inhibitor obtained in this study having a K i of 3 nM.In the last decade, naturally occurring serine protease inhibitors (1) have been the focus of many studies, mainly for two reasons: first, the target proteins control functions in a variety of fundamental proteolytic processes in humans and mammals (blood clotting, digestion, inflammation, fibrinolysis), in invertebrates such as insects (immune system, digestion, protection against their predators) or worms (protection against their host), and plants (protection against insect attack); second, low molecular weight inhibitors of serine proteases have been attractive tools for studying the general aspects of protein conformation and protein-protein interactions (2). In the present study, we report the inhibitory properties of three homologous peptides (primary and tertiary structures) toward ␣-chymotrypsin, trypsin, and human leukocyte elastase.We have previously isolated two peptides, PMP-C and PMP-D2, from the brain and the fat body of the insect Locusta migratoria (3). These peptides are composed of 36 and 35 residues, respectively, and are cross-linked by three disulfide bonds. The Thr-9 of PMP-C has an uncommon O-glycosidic linkage to a single fucose moiety. There is 40% strict identity between PMP-C and PMP-D2 with conservation of the Cys positions (3). Moreover, they are located on the same peptidic precursor, and, by Northern blot analysis, it has been shown that the gene encoding this precursor is mainly transcribed in the fat body (4). In this paper, we describe the isolation and characterization of HI, a novel locust peptide.Because the isolation from insect extracts is time-consuming and yields only small amounts of peptides, we have prepared, at a reasonable scale (5-10 mg), by solid-phase synthesis, PMP-D2 (5) and PMP-C with and without the fucose moiety. 1Although they are small peptides with a high disulfide content, no sequence similarities could be found when comparing them with small toxins or small protease inhibitors. However, the milligram quantities of PMP-D2 obtained by solid-phase synthesi...

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