In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

Alberto A. Rascón, Johnathon Gearin, Jun Isoe, Roger Miesfeld

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Background: 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. Results: 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 (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. Conclusions: 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.

Original languageEnglish (US)
Article number43
JournalBMC Biochemistry
Volume12
Issue number1
DOIs
StatePublished - 2011

Fingerprint

Enzyme kinetics
Enzyme Activation
Dengue
Aedes
Serine Proteases
Chemical activation
Enteropeptidase
Trypsin
Culicidae
Blood
Peptide Hydrolases
Enzymes
Albumins
Hemoglobins
Substrates
Chromogenics
Enzyme Precursors
Proteins
Enzyme activity
Collagenases

Keywords

  • Aedes aegypti
  • hemoglobin
  • serum albumin
  • trypsin
  • zymogen

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti. / Rascón, Alberto A.; Gearin, Johnathon; Isoe, Jun; Miesfeld, Roger.

In: BMC Biochemistry, Vol. 12, No. 1, 43, 2011.

Research output: Contribution to journalArticle

@article{049433bd1a7649f49c4d63c4e87d9c61,
title = "In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti",
abstract = "Background: 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. Results: 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 (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. Conclusions: 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.",
keywords = "Aedes aegypti, hemoglobin, serum albumin, trypsin, zymogen",
author = "Rasc{\'o}n, {Alberto A.} and Johnathon Gearin and Jun Isoe and Roger Miesfeld",
year = "2011",
doi = "10.1186/1471-2091-12-43",
language = "English (US)",
volume = "12",
journal = "BMC Biochemistry",
issn = "1471-2091",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

AU - Rascón, Alberto A.

AU - Gearin, Johnathon

AU - Isoe, Jun

AU - Miesfeld, Roger

PY - 2011

Y1 - 2011

N2 - Background: 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. Results: 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 (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. Conclusions: 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.

AB - Background: 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. Results: 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 (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. Conclusions: 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.

KW - Aedes aegypti

KW - hemoglobin

KW - serum albumin

KW - trypsin

KW - zymogen

UR - http://www.scopus.com/inward/record.url?scp=79961130993&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79961130993&partnerID=8YFLogxK

U2 - 10.1186/1471-2091-12-43

DO - 10.1186/1471-2091-12-43

M3 - Article

C2 - 21827688

AN - SCOPUS:79961130993

VL - 12

JO - BMC Biochemistry

JF - BMC Biochemistry

SN - 1471-2091

IS - 1

M1 - 43

ER -