In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti.
Autor(es): Rascon Alberto A; Gearin Johnathon; Isoe Jun; Miesfeld Roger L
Resumo: The 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. We 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 (k(cat)/K(M)) 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. These 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.
Palavras-Chave: Trypsin ; Aedes aegypti; Hemoglobin; Serum albumin; Zymogen
Imprenta: BMC Biochemistry, v. 12, n. 43, 2011
Identificador do objeto digital: 10.1186/1471-2091-12-43.
Descritores: Aedes aegypti - Biochemistry ; Aedes aegypti - Molecular Structure ; Aedes aegypti - Pathogenesis ; Aedes aegypti - Proteins ; Aedes aegypti - Antibodies ; Aedes aegypti - Viral infections ; Aedes aegypti - Virus ; Aedes aegypti - Transmission ; Aedes aegypti - Dengue ; Aedes aegypti - Public health
Data de publicação: 2011