Molecular characterization of genes encoding trypsin-like enzymes from Aedes aegypti larvae and identification of digestive enzymes

Abstract

Trypsin-like enzymes play an important role in the Aedes aegypti digestive process. The trypsin-like enzymes present in adults were characterized previously, but little is known about trypsins in larvae. In the present work, we identified one of the trypsin enzymes from Ae. aegypti larval midgut using a library of trypsin gene fragments, which was the sequence known as AAEL005607 from the Ae. aegypti genome. Quantitative PCR analysis showed that AAEL005607 was transcribed in all larval instars, but it was not present in adult midgut. In order to confirm transcription data, the trypsin-like enzymes from 4th instar larvae of Ae. aegypti midgut were purified and sequenced. Purified trypsin showed identity with the amino-terminal sequence of AAEL005607, AAEL005609 and AAEL005614. These three trypsins have high amino acids identity, and could all be used as a template for the design of inhibitors. In conclusion, for the first time, digestive enzymes of 4th larval instar of Ae. aegypti were purified and characterized. The knowledge of digestive enzymes present in Ae. aegypti larvae may be helpful in the development of a larvicide.

Trypsin-like serine peptidase profiles in the egg, larval, and pupal stages of Aedes albopictus

“…This result was corroborated by the quantitative in-solution assays using the fluorogenic substrate Z-Phe-Arg-AMC. Quantitative differential expression in larval instars has also been observed by other authors, specifically in the intestine, using alternative methods [21,39,56]. In the present study, the proteolytic classes of the enzymes were characterized using different inhibitors, revealing that peptidases from the four larval instars were inhibited by PMSF, an inhibitor of trypsin and chymotrypsin, and TLCK, an inhibitor of trypsin.…”

“…These results are in agreement with a recent transcriptome study showing that 12 serine peptidases-like genes were preferential expressed in the larvae of Ae. aegypti [39,59]. The broad spectrum of enzymatic activities detected in the larvae of Ae.…”

“…On the other hand, bands of proteolytic activities with different intensities within the same stage of development, among distinct stages and between the two species may indicate that some genes coding for trypsin are differentially expressed or that some isoforms with specific catalytic features are differentially regulated during the life cycle of the insect. Complex mechanisms regulating the expression of trypsin in insects have been previously described [39,53-56,60]. …”

First insights into insecticidal activity against Aedes aegypti and partial biochemical characterization of a novel low molecular mass chymotrypsin‐trypsin inhibitor purified from Lonchocarpus sericeus seeds

“…These enzymes have been reported to be involved in protein digestion for nutrition and development throughout the Ae. aegypti life cycle . After LsCTI had been demonstrated in vitro to inhibit the midgut enzymes of the larvae of this mosquito, in vivo assays were performed to determine how this inhibitor affects mosquito development.…”

A midgut‐specific serine protease, BmSP36, is involved in dietary protein digestion in the silkworm, Bombyx mori

“…The genomes of Anopheles gambiae , Drosophila melanogaster and Bombyx mori contain 305, 206 and 143 SP or SPH genes, respectively (Zdobnov et al ., ; Ross et al ., ; Zhao et al ., ). Many studies have shown that insect SPs play important roles in dietary protein digestion (Terra & Ferreira, ; Lehane et al ., ; Herrero et al ., ; Soares et al ., ), molting (Samuels et al ., ; Wei et al ., ; Liu et al ., ; He et al ., ), metamorphosis (Tsuji et al ., ; Danielli et al ., ; Kaji et al ., ) and the immune response (Jiang et al ., ; Gorman et al ., ; An et al ., ). Based on the residue composition of the substrate‐specific pocket, SPs are mainly categorized as trypsin (residues D189, G216 and G226), chymotrypsin (residues S189, G216 and G226) or elastase (residues V216 and T226) (Perona & Craik, , Hedstrom, ).…”