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

Persistent Link:
http://hdl.handle.net/10150/610098
Title:
In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti
Author:
Rascon, Alberto; Gearin, Johnathon; Isoe, Jun; Miesfeld, Roger
Affiliation:
Department of Chemistry & Biochemistry, and Center for Insect Science West Room 518, 1041 E. Lowell St., University of Arizona, Tucson, AZ, 85721, USA; Department of Chemistry & Biochemistry, BioSciences West Room 518, 1041 E. Lowell St., University of Arizona, Tucson, AZ, 85721, USA; Sandler Center for Drug Discovery, Byers Hall, 1700 4th Street N509, University of California at San Francisco, San Francisco, CA. 94143, USA
Issue Date:
2011
Publisher:
BioMed Central
Citation:
Rascón et al. BMC Biochemistry 2011, 12:43 http://www.biomedcentral.com/1471-2091/12/43
Journal:
BMC Biochemistry
Rights:
© 2011 Rascón et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)
Collection Information:
This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.
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.
EISSN:
1471-2091
DOI:
10.1186/1471-2091-12-43
Keywords:
trypsin; Aedes aegypti; hemoglobin; serum albumin; zymogen
Version:
Final published version
Additional Links:
http://www.biomedcentral.com/1471-2091/12/43

Full metadata record

DC FieldValue Language
dc.contributor.authorRascon, Albertoen
dc.contributor.authorGearin, Johnathonen
dc.contributor.authorIsoe, Junen
dc.contributor.authorMiesfeld, Rogeren
dc.date.accessioned2016-05-20T08:58:31Z-
dc.date.available2016-05-20T08:58:31Z-
dc.date.issued2011en
dc.identifier.citationRascón et al. BMC Biochemistry 2011, 12:43 http://www.biomedcentral.com/1471-2091/12/43en
dc.identifier.doi10.1186/1471-2091-12-43en
dc.identifier.urihttp://hdl.handle.net/10150/610098-
dc.description.abstractBACKGROUND: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.en
dc.language.isoenen
dc.publisherBioMed Centralen
dc.relation.urlhttp://www.biomedcentral.com/1471-2091/12/43en
dc.rights© 2011 Rascón et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0)en
dc.subjecttrypsinen
dc.subjectAedes aegyptien
dc.subjecthemoglobinen
dc.subjectserum albuminen
dc.subjectzymogenen
dc.titleIn vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegyptien
dc.typeArticleen
dc.identifier.eissn1471-2091en
dc.contributor.departmentDepartment of Chemistry & Biochemistry, and Center for Insect Science West Room 518, 1041 E. Lowell St., University of Arizona, Tucson, AZ, 85721, USAen
dc.contributor.departmentDepartment of Chemistry & Biochemistry, BioSciences West Room 518, 1041 E. Lowell St., University of Arizona, Tucson, AZ, 85721, USAen
dc.contributor.departmentSandler Center for Drug Discovery, Byers Hall, 1700 4th Street N509, University of California at San Francisco, San Francisco, CA. 94143, USAen
dc.identifier.journalBMC Biochemistryen
dc.description.collectioninformationThis item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
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