Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2003
Publication Date: 12/1/2003
Citation: ZHU, Y., ZENG, F., COHEN, A.C., OPPERT, B.S. MOLECULAR CLONING OF TRYPSIN-LIKE CDNAS AND COMPARISON OF PROTEINASE ACTIVITIES IN THE SALIVARY GLANDS AND GUT OF THE TARNISHED PLANT BUG LYGUS LINEOLARIS (HEMIPTERA: MIRIDAE). INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY. 2003. V33. pp. 889 - 899. Interpretive Summary: The tarnished plant bug has evolved sucking mouthparts to ingest plant juice. Extra-intestinal digestion is facilitated through injection of saliva containing variety of digestive enzymes into plant tissue. Proteins are critical nutrients for insect growth and development. This study was designed to understand and eventually manipulate protein digestion in the tarnished plant bug. Proteinases, such as trypsin, play an essential function for protein digestion and absorption in the tarnished plant bug. The introduction of proteinase inhibitors, especially those that target trypsin, into host plants may substantially suppress protein digestion, and subsequently achieve insect control through nutrient deficiency. We demonstrated that protein digestion in the tarnished plant bug depended primarily on proteinases from salivary glands, followed by gut proteinases for further digestion. Serine proteinases, especially trypsins, are the major proteinases in both salivary glands and guts. Protein digestion was affected by pH and was suppressible by many serine proteinase inhibitors.
Technical Abstract: Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseinase and 90% of BApNAse activities in the salivary glands were inhibited by aprotinin, benzamidine, and PMSF, whereas only 46% azocaseinase and 60% BApNAse activities in the gut were suppressed by benzamidine, leupeptin, and TLCK. The pH optima for azocaseinase activity in salivary glands ranged from 6.2 to 10.6, whereas the pH optima for gut proteinases was acidic for general and alkaline for tryptic proteinases. Zymogram analysis demonstrated that ~26-kDa proteinases from salivary glands were active against both gelatin and casein substrates. Three trypsin-like cDNAs, LlSgP2-4, and one trypsin-like cDNA, LlGtP1, were cloned from salivary glands and gut, respectively. Putative trypsin precursors from all cloned cDNAs contained a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included His, Asp, and Ser residues for the catalytic amino acid triad of serine proteinase active sites, residues for the binding pocket, and four pairs of cysteine residues for disulfide bridges. Deduced trypsin-like proteins from LlSgP2, LlSgP3, and LlGtP1 cDNAs shared 98-99% sequence identity with a previously reported trypsin-like precursor, whereas the trypsin-like protein of LlSgP4 shared only 44% sequence identity with all other trypsin-like proteins, indicating multi-trypsin forms are present in L. lineolaris.