Expression of trypsin‐like serine peptidases in pre‐imaginal stages of Aedes aegypti (Diptera: Culicidae)

Abstract

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.

Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

“…Previous reports from our group have shown that preimaginal stages of Ae. aegypti also exhibit a complex profile of trypsin-like serine peptidases [33], similar to those observed here for Cx. quinquefasciatus .…”

“…Afterwards, samples were resolved as previously described [32]. Briefly, 10 μg of protein from each sample was mixed with SDS-PAGE sample buffer (125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 0.002% bromophenol blue) and loaded in 12% or 10% polyacrylamide gels co-polymerized with 0.1% porcine gelatin for separation at 4°C with a constant voltage of 110 V. Peptidase activity was detected as previously reported [33] with few modifications. The gels were incubated in the reaction buffer containing 100 mM sodium acetate (at pH 3.5 or 5.5) or 100 mM Tris–HCl (pH 7.5 or 10.0) at 37°C for 0.5, 1, 2 or 4 h for larvae; 0.5, 1, 2, 4, 6, 12 or 24 h for pupa; and 6, 12, 24 or 48 hours for egg homogenates.…”

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

“…aegypti trypsin was purified by reverse phase chromatography and presented a protein band of about 23 kDa by SDS-PAGE. It was recently shown by zymographic analysis that the serine proteases in larvae stadia present molecular masses ranging from 20 to 250 kDa (Mesquita-Rodrigues et al, 2011), which was in agreement the molecular mass calculated for other insect trypsin, 20,000 to 35,000 Da (Terra and Ferreira, 1994). The partial amino-terminal sequence of purified trypsin IVGGNEVSIA showed complete identity to the trypsin sequences AAEL005607, AAEL005614 and AAEL005609 in the Ae.…”

Temephos Resistance in Aedes aegypti in Colombia Compromises Dengue Vector Control

“…Serine proteases are a group of well-studied enzymes responsible for a variety of functions such as digestion, oogenesis, immune response, blood coagulation and metamorphosis [58], [59], [60]. In the present study, a chymotrypsin (AAEL011230-RA) was the most over transcribed gene in the temephos resistant population (30.2-fold difference between temephos survivors and non-exposed Cucuta larvae, and 87.1-fold difference between temephos survivors and NO).…”