Molecular characterization of digestive proteinases and sequence analysis of midgut cDNA transcripts of the yellow mealworm, Tenebrio molitor L. Insect Mol Biol

Department of Entomology, Kansas State University, Manhattan, KS, USA.
Insect Molecular Biology (Impact Factor: 2.59). 09/2007; 16(4):455-68. DOI: 10.1111/j.1365-2583.2007.00740.x
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Peptidase sequences were analysed in randomly picked clones from cDNA libraries of the anterior or posterior midgut or whole larvae of the yellow mealworm, Tenebrio molitor Linnaeus. Of a total of 1528 sequences, 92 encoded potential peptidases, from which 50 full-length cDNA sequences were obtained, including serine and cysteine proteinases and metallopeptidases. Serine proteinase transcripts were predominant in the posterior midgut, whereas transcripts encoding cysteine and metallopeptidases were mainly found in the anterior midgut. Alignments with other proteinases indicated that 40% of the serine proteinase sequences were serine proteinase homologues, and the remaining ones were identified as either trypsin, chymotrypsin or other serine proteinases. Cysteine proteinase sequences included cathepsin B- and L-like proteinases, and metallopeptidase transcripts were similar to carboxypeptidase A. Northern blot analysis of representative sequences demonstrated the differential expression profile of selected transcripts across five developmental stages of Te. molitor. These sequences provide insights into peptidases in coleopteran insects as a basis to study the response of coleopteran larvae to external stimuli and to evaluate regulatory features of the response.

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    • "One of the effects of Cry3Aa intoxication in T. molitor was a dramatic change in the expression of transcripts of serine (Ser) proteases in the larval gut (Oppert et al. 2012b). T. molitor larvae rely on a complex and highly compartmentalized digestive system, with mostly cysteine (Cys) proteases in the anterior and Serproteases in the posterior midgut (Vinokurov et al. 2006, Prabhakar et al. 2007). Of 48 Ser protease transcripts obtained from the gut of Cry3Aa-intoxicated T. molitor larvae, most were severely repressed, from 2-to 15-fold. "

    Insect Molecular Biology and Ecology, Edited by Klaus H. Hoffmann, 11/2014: pages 395; CRC press.
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    • "The occurrence of multiple isoforms may provide adaptive advantages for insects feeding on plants containing inhibitors. Therefore, it is not surprising that during the past decades, a large number of protease encoding genes were identified to be present in the digestive system of many different insects (Bown et al., 1997; Gatehouse et al., 1997; Ge et al., 2012; Marshall et al., 2008; Oliveira-Neto et al., 2004; Pedra et al., 2003; Prabhakar et al., 2007; Simpson et al., 2007). "
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    ABSTRACT: While technological advancements have recently led to a steep increase in genomic and transcriptomic data, and large numbers of protease sequences are being discovered in diverse insect species, little information is available about the expression of digestive enzymes in Orthoptera. Here we describe the identification of Locusta migratoria serine protease transcripts (cDNAs) involved in digestion, which might serve as possible targets for pest control management. A total of 5 putative trypsin and 15 putative chymotrypsin gene sequences were characterized. Phylogenetic analysis revealed that these are distributed among 3 evolutionary conserved clusters. In addition, we have determined the relative gene expression levels of representative members in the gut under different feeding conditions. This study demonstrated that the transcript levels for all measured serine proteases were strongly reduced after starvation. On the other hand, larvae of L. migratoria displayed compensatory effects to the presence of Soybean Bowman Birk (SBBI) and Soybean Trypsin (SBTI) inhibitors in their diet by differential upregulation of multiple proteases. A rapid initial upregulation was observed for all tested serine protease transcripts, while only for members belonging to class I, the transcript levels remained elevated after prolonged exposure. In full agreement with these results, we also observed an increase in proteolytic activity in midgut secretions of locusts that were accustomed to the presence of protease inhibitors in their diet, while no change in sensitivity to these inhibitors was observed. Taken together, this paper is the first comprehensive study on dietary dependent transcript levels of proteolytic enzymes in Orthoptera. Our data suggest that compensatory response mechanisms to protease inhibitor ingestion may have appeared early in insect evolution.
    Insect biochemistry and molecular biology 05/2014; DOI:10.1016/j.ibmb.2014.03.002 · 3.45 Impact Factor
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    • "Serine proteases play critical roles in an insect physiology. They serve as major insect gut enzymes in the digestion of dietary proteins, inactivate protein toxins, and show antimicrobial activity (Hegedus et al., 2003; Prabhakar et al., 2007; Chougule et al., 2008). Serine proteases also play critical roles in several insect biological processes such as blood coagulation, signal transduction, hormone activation, and development (Hedstrom, 2002; Clynen et al., 2005). "
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    ABSTRACT: A serine protease was isolated from midguts of the bumblebee male Bombus terrestris by a combination of precipitation procedures with column chromatography. The purified enzyme exhibited two bands with molecular masses of 25 and 26 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These bands showed a proteolytic activity in zymography assay. Midgut enzymes showed optimum proteolytic activity at pH 9 and 35°C using N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanine 4-nitroanilide as a substrate. The Michaelis constant (Km) and maximum reaction rate (Vmax) were 0.55 ± 0.042 mM and 0.714 ± 0.056 μmol p-nitroalanine produced min(-1) mg protein(-1) , respectively. Inhibition was affected by trypsin inhibitor, but not by phenylmethylsulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone, which indicated the trypsin-like but not chymotrypsin-like specificity. The identity of the serine protease was confirmed by nanoliquid-tandem mass spectrometry. Eleven unique peptides of the B. terrestris serine protease were found. It shows high homology to a previously reported B. ignitus serine protease covering more than 65% of the protein amino acid sequence.
    Archives of Insect Biochemistry and Physiology 03/2013; 82(3). DOI:10.1002/arch.21075 · 1.02 Impact Factor
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