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Proteomic identification of dengue virus binding proteins in Aedes aegypti mosquitoes and Aedes albopictus cells.

Muñoz Mde L, Limón-Camacho G, Tovar R, Diaz-Badillo A, Mendoza-Hernández G, Black WC - Biomed Res Int (2013)

Bottom Line: Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa.In addition, viral particles bound high molecular weight proteins.Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, 07360 Mexico, DF, Mexico.

ABSTRACT
The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

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Related in: MedlinePlus

Amino acid sequence analysis of two different translation elongation factors EF-1 alpha/Tu. Alignment of these elongation factors (EJY57625 and ABF18239) identified in C6/36 cells, and MGs of Ae. aegypti mosquitoes of the IBO-11 and MORI strains is shown. Identified peptides are shown in red color.
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fig5: Amino acid sequence analysis of two different translation elongation factors EF-1 alpha/Tu. Alignment of these elongation factors (EJY57625 and ABF18239) identified in C6/36 cells, and MGs of Ae. aegypti mosquitoes of the IBO-11 and MORI strains is shown. Identified peptides are shown in red color.

Mentions: Proteomic analysis of proteins obtained from total protein extracts of Ae. aegypti mosquito DS3, IBO-11, and DMEB strains separated by SDS-PAGE that migrated as the purified proteins (57 and 67 kDa) with at least two peptide matches is shown in Table 1. The proteins identified were enolase, beta-ARK, translation elongation factor EF-1 alpha/Tu, and cadherin. Translation elongation factor EF-1 alpha/Tu and cadherin had been identified previously, thus ensuring that the procedure described in this work is suitable to the identified proteins bound to DENV and E protein domain III. Peptide sequence AKPGAEAHPPFRQHK has partial alignment with beta-ARK (ref/XP_001652291/) and with ATP-dependent RNA helicase (ref/XP_001648042.1/); however, the identification of beta-ARK was confirmed by the match of ESQELLGSMAKK peptide with beta-ARK identified in two mosquito strains (DS3 and DMEB). Although, cadherin is showing only one peptide, the peptide match to this protein has a very high score of 52 (16/17 amino acids). Proteins identified in C6/36 cells or mosquito MGs from DMEB, DS3, IBO-11, or Mori strains are also included in Table 1. Manual analysis was used to confirm peptide identity (Figure 4). Peptide sequence coverage was 35% for enolase, 2.6% for beta-ARK, and 20% for translation elongation factor EF-1 alpha/Tu. Because translation elongation factor EF-1 alpha/Tu matched two proteins, we manually verified mass spectra for presence of unique peptides for each homologous assignment. In Figure 5 we demonstrate the alignment for these two homologous proteins EJY57625 and ABF18239 and peptides identified in each of those two proteins. Peptides NNPPKQAA and K.GASDFTAQVIVLNHPGQIANGYTPVLDCHTAVIACK-FAEIQQK.V were specific for protein EJY57625 (Figure 5).


Proteomic identification of dengue virus binding proteins in Aedes aegypti mosquitoes and Aedes albopictus cells.

Muñoz Mde L, Limón-Camacho G, Tovar R, Diaz-Badillo A, Mendoza-Hernández G, Black WC - Biomed Res Int (2013)

Amino acid sequence analysis of two different translation elongation factors EF-1 alpha/Tu. Alignment of these elongation factors (EJY57625 and ABF18239) identified in C6/36 cells, and MGs of Ae. aegypti mosquitoes of the IBO-11 and MORI strains is shown. Identified peptides are shown in red color.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3842078&req=5

fig5: Amino acid sequence analysis of two different translation elongation factors EF-1 alpha/Tu. Alignment of these elongation factors (EJY57625 and ABF18239) identified in C6/36 cells, and MGs of Ae. aegypti mosquitoes of the IBO-11 and MORI strains is shown. Identified peptides are shown in red color.
Mentions: Proteomic analysis of proteins obtained from total protein extracts of Ae. aegypti mosquito DS3, IBO-11, and DMEB strains separated by SDS-PAGE that migrated as the purified proteins (57 and 67 kDa) with at least two peptide matches is shown in Table 1. The proteins identified were enolase, beta-ARK, translation elongation factor EF-1 alpha/Tu, and cadherin. Translation elongation factor EF-1 alpha/Tu and cadherin had been identified previously, thus ensuring that the procedure described in this work is suitable to the identified proteins bound to DENV and E protein domain III. Peptide sequence AKPGAEAHPPFRQHK has partial alignment with beta-ARK (ref/XP_001652291/) and with ATP-dependent RNA helicase (ref/XP_001648042.1/); however, the identification of beta-ARK was confirmed by the match of ESQELLGSMAKK peptide with beta-ARK identified in two mosquito strains (DS3 and DMEB). Although, cadherin is showing only one peptide, the peptide match to this protein has a very high score of 52 (16/17 amino acids). Proteins identified in C6/36 cells or mosquito MGs from DMEB, DS3, IBO-11, or Mori strains are also included in Table 1. Manual analysis was used to confirm peptide identity (Figure 4). Peptide sequence coverage was 35% for enolase, 2.6% for beta-ARK, and 20% for translation elongation factor EF-1 alpha/Tu. Because translation elongation factor EF-1 alpha/Tu matched two proteins, we manually verified mass spectra for presence of unique peptides for each homologous assignment. In Figure 5 we demonstrate the alignment for these two homologous proteins EJY57625 and ABF18239 and peptides identified in each of those two proteins. Peptides NNPPKQAA and K.GASDFTAQVIVLNHPGQIANGYTPVLDCHTAVIACK-FAEIQQK.V were specific for protein EJY57625 (Figure 5).

Bottom Line: Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa.In addition, viral particles bound high molecular weight proteins.Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Molecular Biology, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, 07360 Mexico, DF, Mexico.

ABSTRACT
The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin.

Show MeSH
Related in: MedlinePlus