Abstract
BackgroundThe major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.ResultsWe used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.ConclusionsThese data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.
Highlights
The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle
In the case of AaSPVI, the predicted zymogen form was primarily localized to the midgut epithelium, with only the mature form found in the food bolus, whereas both the zymogen and mature forms of AaSPVII and AaLT were found in the food bolus
By aligning the amino acid sequences of the four mosquito proteases with that of bovine trypsinogen using ClustalW [22], it can be seen that the most likely propeptide cleavage site is between the amino acids GRIV (AaET, AaSPVI) or GR-VV (AaSPVII, AaLT)
Summary
The major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. RNAi based studies using BApNA assays to analyze midgut extracts for trypsin activity from 0-48 hr post blood meal (PBM) showed that AaET is the only trypsin-like protease during the first 1-6 hr PBM, whereas the AaSPVI protease accounts for up to 75% of the BApNA cleaving activity during the late phase of digestion from 12-36 hr PBM [13] These same experiments showed that another midgut protease, AaSPVII, contributed significantly to overall fecundity in blood fed mosquitoes. RNAi knock down of AaLT expression, known as Late Trypsin, had no effect on BApNA activity in midgut extracts, even though it reduced fecundity by ~30% as did the AaSPVI RNAi knock downs [13] These data suggest that AaSPVI, and not AaLT, is the major late phase midgut trypsin in blood fed Ae. aegypti, and are consistent with bioinformatic analyses of protease encoding genes in the Ae. aegypti genome [15]
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