A gut-specific chitinase gene was cloned from the mulberry longicorn beetle, Apriona germari. The A. germari chitinase (AgChi) gene spans 2894 bp and consists of five introns and six exons coding for 390 amino acid residues. AgChi possesses the chitinase family 18 active site signature and three N-glycosylation sites. Southern blot analysis of genomic DNA suggests that AgChi is a single copy gene. The AgChi cDNA was expressed as a 46-kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AgChi showed activity in a chitinase enzyme assay. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-linked glycosylation, revealed that AgChi is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity. Northern and Western blot analyses showed that AgChi was specifically expressed in the gut; AgChi was expressed in three gut regions, indicating that the gut is the prime site for AgChi synthesis in A. germari larvae.
A serine protease gene was cloned from the bumblebee, Bombus ignitus. The B. ignitus serine protease (BiSP) gene spans 1702 bp and consists of four introns and five exons coding for 250 amino acid residues. Southern blot analysis of genomic DNA suggested that BiSP gene is a single copy gene. The cDNA encoding BiSP was expressed as a 28-kDa polypeptide in baculovirus-infected insect cells and the recombinant BiSP showed activity in a protease enzyme assay. BiSP was specifically expressed in the midgut of B. ignitus queens, males, and workers, suggesting that the BiSP is a gut enzyme involved in the digestion of dietary proteins.
Two glutathione S-transferase (GST) cDNAs, GSTD2 and GSTS2, were cloned from the silkworm Bombyx mori. The B. mori GSTD2 (BmGSTD2) gene spans 4371 bp and consists of four introns and five exons that encode 222 amino acid residues. The deduced amino acid sequence of BmGSTD2 showed 58% protein sequence identity to the Delta-class GST of Maduca sexta. The B. mori GSTS2 (BmGSTS2) gene spans 3470 bp and consists of three introns and four exons that encode 206 amino acid residues. The deduced amino acid sequence of BmGSTS2 revealed 67%, 63%, and 61% protein sequence identities to the Sigma-class GSTs from B. mori, Platynota idaeusalis, and M. sexta, respectively. The BmGSTD2 and BmGSTS2 cDNAs were expressed as 25 kDa and 23 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot and Western blot analyses showed that BmGSTD2 and BmGSTS2 were specifically expressed in three gut regions, indicating that the gut is the prime site for BmGSTD2 and BmGSTS2 synthesis in B. mori larvae.
By means of a tracer assay using a labeled synthetic angiotensin converting enzyme (ACE) substrate hippurylglycylglycine, we have detected high ACE activity in the testes of the African migratory locust, Locusta migratoria. Lower, but significant, ACE activity was observed in midgut and hemolymph. In a two-step purification procedure involving anion exchange and gel permeation chromatography, we have purified LomACE from the locust testes. The enzyme of approximately 80 kDa shows substantial amino-acid sequence homology with ACE from both vertebrate and invertebrate origin. The ACE identity of the purified enzyme was further confirmed by cDNA cloning of the Locusta ACE fragment, which, after in silico translation, revealed a mature protein of 623 amino acids with a large structural similarity to other known ACE proteins.