The ovary of the desert locust, Schistocerca gregaria, contains multiple inhibitors of serine proteases. Five serine protease inhibitors, designated SGPI-1^5 (Schistocerca gregaria protease inhibitors) were purified from methanolic extracts of mature ovaries and analyzed by mass spectrometry and amino acid sequencing. The revealed primary structures display amino acid similarities and are related to the serine protease inhibitors identified in the hemolymph of Locusta migratoria. All inhibitors show an in vitro inhibiting activity towards K K-chymotrypsin. In addition, SGPI-1 displays in vitro inhibiting activity towards trypsin, and SGPI-2 is a potent pancreatic elastase inhibitor. Differences in inhibitory specificities towards the locust endogenous serine proteases can be readily attributed to the amino acid sequence within the active region and also to amino acid residues beyond the P1-PP1 bond. A difference in one or two amino acid residues around the reactive sites results in considerable alteration of the inhibitory specificity. The temporal and spatial distribution of SGPI-1^5 was studied by RP-HPLC analysis. All inhibitors occur in hemolymph, ovaries, testes and fat body of adults but are absent in the gut. They are also present in larval hemolymph and fat body. An antibody raised against SGPI-2 shows positive immunostaining in the ovarian follicle cells.z 1998 Federation of European Biochemical Societies.

This study describes the cloning of two cDNAs encoding three serine-protease-inhibiting peptides, SGPI I, II and III, which were recently identified from ovarian extracts of the desert locust, Schistocerca gregaria. The first cDNA codes for the precursor polypeptides of SGPI I and SGPI II; the second encodes only a single inhibitor, SGPI III. Northern-blot analysis revealed an approximate length of 0.8 kb for SGPI-I/II mRNA and 0.6 kb for SGPI-III mRNA. The transcripts are present in several locust tissues, but they could not be detected in the midgut. The gene for SGPI-I/II is abundantly transcribed during all larval and adult stages, whereas SGPI-III mRNA is mainly present in adults. Northern-blot hybridization also revealed important changes in the SGPI-mRNA content during the molting cycle and during the adult reproductive cycle. Moreover, a differential hormonal control was observed in adult females which had been treated with precocene, juvenile hormone or ecdysone.

We have purified to homogeneity two forms of a new serine protease inhibitor specific for elastase/chymotrypsin from the ovary gland of the desert locust Schistocerca gregaria. This protein, greglin, has 83 amino acid residues and bears putative phosphorylation sites. Amino acid sequence alignments revealed no homology with pacifastin insect inhibitors and only a distant relationship with Kazal-type inhibitors. This was confirmed by computer-based structural studies. The most closely related homologue is a putative gene product from Ciona intestinalis with which it shares 38% sequence homology. Greglin is a fast-acting and tight binding inhibitor of human neutrophil elastase (k(ass)=1.2x10(7) M(-1) x s(-1), K(i)=3.6 nM) and subtilisin. It also binds neutrophil cathepsin G, pancreatic elastase and chymotrypsin with a lower affinity (26 nM< or =K(i)< or =153 nM), but does not inhibit neutrophil protease 3 or pancreatic trypsin. The capacity of greglin to inhibit neutrophil elastase was not significantly affected by exposure to acetonitrile, high temperature (90 degrees C), low or high pH (2.5-11.0), N-chlorosuccinimide-mediated oxidation or the proteolytic enzymes trypsin, papain and pseudolysin from Pseudomonas aeruginosa. Greglin efficiently inhibits the neutrophil elastase activity of sputum supernatants from cystic fibrosis patients. Its biological function in the locust ovary gland is currently unknown, but its physicochemical properties suggest that it can be used as a template to design a new generation of highly resistant elastase inhibitors for treating inflammatory diseases.