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In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus.

Borges-Veloso A, Saboia-Vahia L, Dias-Lopes G, Domont GB, Britto C, Cuervo P, De Jesus JB - Parasit Vectors (2015)

Bottom Line: These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds.Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. abv@ioc.fiocruz.br.

ABSTRACT

Background: Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins.

Methods: We used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features.

Results: Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.

Conclusion: We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.

No MeSH data available.


Related in: MedlinePlus

Representative zymographic profile and SDS-PAGE of total protein extracts of C. quinquefasciatus midgut extract. This figure shows two different electrophoretic systems used for characterize and identify, respectively, the trypsin-like serine peptidases: the zymography where proteins are resolved under non-reducing conditions and therefore their activity can be detected, and the denaturating SDS-PAGE ran under reducing conditions. SDS-PAGE slices were used for peptidase identification by mass spectrometry. The numbers on the left of each electrophoresis indicate the molecular mass of standards utilized in the gel (kDa). This figure also shows a representative image of midgut recorded by optical differential interference contrast microscopy (DIC)
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Fig4: Representative zymographic profile and SDS-PAGE of total protein extracts of C. quinquefasciatus midgut extract. This figure shows two different electrophoretic systems used for characterize and identify, respectively, the trypsin-like serine peptidases: the zymography where proteins are resolved under non-reducing conditions and therefore their activity can be detected, and the denaturating SDS-PAGE ran under reducing conditions. SDS-PAGE slices were used for peptidase identification by mass spectrometry. The numbers on the left of each electrophoresis indicate the molecular mass of standards utilized in the gel (kDa). This figure also shows a representative image of midgut recorded by optical differential interference contrast microscopy (DIC)

Mentions: The C. quinquefasciatus genome codes for 403 putative trypsin-like serine peptidase genes [14], but it is unknown which of them are expressed in the midgut tissue. Here, we identified seven trypsin-like serine peptidases constitutively expressed in the midgut of females fed a sugar diet using two independent search engines: Mascot (followed by Scaffold validation) and ProLuCID (Table 1). In addition, one trypsin-like serine peptidase was exclusively identified by MASCOT (followed by Scaffold validation) based on one peptide and one spectrum evidence (Table 1, B0WW44, gray filled). Interestingly, the SDS-PAGE bands where peptidases were identified by MS/MS coincide with the zymographic regions where peptidase activities were observed (Fig. 4). Although most of the peptidases were identified in electrophoretic bands migrating between 25 to 40 kDa (Fig. 4), the Trypsin5 and Trypsin7 were the only ones found in the high molecular mass region (Fig. 4). Conspicuously, these enzymes exhibited predicted sites for N-Glycosylation. Particularly, Trypsin5 also present predicted O-Glycosylation sites and transmembrane regions (Table 3). Such features could in fact alter the migration pattern of the mature protein. Nevertheless, as sample preparation for each electrophoresis is different, a comparison of peptidase mobility is difficult, but, in any case, both methodologies serve for mapping the identified peptidases. All identified trypsin proteins matched with C. quinquefasciatus protein sequences. The alignment of the full sequence of the peptidases identified by mass spectrometry showed several structural features typical of invertebrate digestive trypsin peptidases: (i) the conserved histidine, aspartic acid and serine residues forming the catalytic triad; (ii) six cysteine residues at conserved positions involved in the forming of disulfide bonds; (iii) the signal peptide sequence; (iv) the putative autocatalytic activation motifs immediately after an arginine or lysine residue (R/K- IVGG); (v) the motifs characteristic of active peptidases LTHAAC, DIAL, and GDSGGP (Fig. 5, Table 2) [56]. Interestingly, some trypsin peptidases identified here have distinct features. For example, we observed that the autocatalytic motif of Trypsin 4 has a His residue instead of R/K residues, which could suggest that this enzyme has a specific signal for activation. In addition, the activation motifs in Trypsin 5, IIGG, and cationic trypsin, VVGG, differ by one amino acid residue from the classical motif sequence (IVGG) [57, 58].Table 1


In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus.

Borges-Veloso A, Saboia-Vahia L, Dias-Lopes G, Domont GB, Britto C, Cuervo P, De Jesus JB - Parasit Vectors (2015)

Representative zymographic profile and SDS-PAGE of total protein extracts of C. quinquefasciatus midgut extract. This figure shows two different electrophoretic systems used for characterize and identify, respectively, the trypsin-like serine peptidases: the zymography where proteins are resolved under non-reducing conditions and therefore their activity can be detected, and the denaturating SDS-PAGE ran under reducing conditions. SDS-PAGE slices were used for peptidase identification by mass spectrometry. The numbers on the left of each electrophoresis indicate the molecular mass of standards utilized in the gel (kDa). This figure also shows a representative image of midgut recorded by optical differential interference contrast microscopy (DIC)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4502911&req=5

Fig4: Representative zymographic profile and SDS-PAGE of total protein extracts of C. quinquefasciatus midgut extract. This figure shows two different electrophoretic systems used for characterize and identify, respectively, the trypsin-like serine peptidases: the zymography where proteins are resolved under non-reducing conditions and therefore their activity can be detected, and the denaturating SDS-PAGE ran under reducing conditions. SDS-PAGE slices were used for peptidase identification by mass spectrometry. The numbers on the left of each electrophoresis indicate the molecular mass of standards utilized in the gel (kDa). This figure also shows a representative image of midgut recorded by optical differential interference contrast microscopy (DIC)
Mentions: The C. quinquefasciatus genome codes for 403 putative trypsin-like serine peptidase genes [14], but it is unknown which of them are expressed in the midgut tissue. Here, we identified seven trypsin-like serine peptidases constitutively expressed in the midgut of females fed a sugar diet using two independent search engines: Mascot (followed by Scaffold validation) and ProLuCID (Table 1). In addition, one trypsin-like serine peptidase was exclusively identified by MASCOT (followed by Scaffold validation) based on one peptide and one spectrum evidence (Table 1, B0WW44, gray filled). Interestingly, the SDS-PAGE bands where peptidases were identified by MS/MS coincide with the zymographic regions where peptidase activities were observed (Fig. 4). Although most of the peptidases were identified in electrophoretic bands migrating between 25 to 40 kDa (Fig. 4), the Trypsin5 and Trypsin7 were the only ones found in the high molecular mass region (Fig. 4). Conspicuously, these enzymes exhibited predicted sites for N-Glycosylation. Particularly, Trypsin5 also present predicted O-Glycosylation sites and transmembrane regions (Table 3). Such features could in fact alter the migration pattern of the mature protein. Nevertheless, as sample preparation for each electrophoresis is different, a comparison of peptidase mobility is difficult, but, in any case, both methodologies serve for mapping the identified peptidases. All identified trypsin proteins matched with C. quinquefasciatus protein sequences. The alignment of the full sequence of the peptidases identified by mass spectrometry showed several structural features typical of invertebrate digestive trypsin peptidases: (i) the conserved histidine, aspartic acid and serine residues forming the catalytic triad; (ii) six cysteine residues at conserved positions involved in the forming of disulfide bonds; (iii) the signal peptide sequence; (iv) the putative autocatalytic activation motifs immediately after an arginine or lysine residue (R/K- IVGG); (v) the motifs characteristic of active peptidases LTHAAC, DIAL, and GDSGGP (Fig. 5, Table 2) [56]. Interestingly, some trypsin peptidases identified here have distinct features. For example, we observed that the autocatalytic motif of Trypsin 4 has a His residue instead of R/K residues, which could suggest that this enzyme has a specific signal for activation. In addition, the activation motifs in Trypsin 5, IIGG, and cationic trypsin, VVGG, differ by one amino acid residue from the classical motif sequence (IVGG) [57, 58].Table 1

Bottom Line: These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds.Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia Molecular e Doenças Endêmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil. abv@ioc.fiocruz.br.

ABSTRACT

Background: Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins.

Methods: We used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features.

Results: Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far.

Conclusion: We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.

No MeSH data available.


Related in: MedlinePlus