Limits...
Trypanosoma cruzi Binds to Cytokeratin through Conserved Peptide Motifs Found in the Laminin-G-Like Domain of the gp85/Trans-sialidase Proteins.

Teixeira AA, de Vasconcelos Vde C, Colli W, Alves MJ, Giordano RJ - PLoS Negl Trop Dis (2015)

Bottom Line: One peptide, named TS9, showed significant cell binding capacity and was selected for further studies.Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process.It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chemistry Institute, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT

Background: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the T. cruzi gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion.

Methodology/principal findings: An in silico analysis was utilized to identify peptide motifs shared by the gp85 T. cruzi proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents T. cruzi cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel β-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope.

Conclusions/significance: Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins.

Show MeSH

Related in: MedlinePlus

Selection of peptides common to all members of the gp85/trans-sialidase family.(A) General representation of gp85/TS proteins belonging to the group II. The signal peptides for endoplasmic reticulum (ER) localization and glycophosphatidylinositol (GPI) anchor (green boxes) are indicated; the sialidase ASP-box repeats (orange boxes) and the conserved peptides identified in this study (blue boxes), which include the VTVxNVxLYNRLN motif denominated FLY, are shown. (B) Moving average of nine amino acids for each position in the gp85/TS group II protein alignment. The signal peptide for ER localization (arrow) and the 10 most conserved peptides (*) are indicated. (C) Representation in sequence logo format of the gp85/TS derived peptides (shown within brackets). The letter size indicates amino acid conservation in each position and the color, whether amino acids are polar (green), hydrophobic (black), positively (blue) or negatively (red) charged.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4580646&req=5

pntd.0004099.g001: Selection of peptides common to all members of the gp85/trans-sialidase family.(A) General representation of gp85/TS proteins belonging to the group II. The signal peptides for endoplasmic reticulum (ER) localization and glycophosphatidylinositol (GPI) anchor (green boxes) are indicated; the sialidase ASP-box repeats (orange boxes) and the conserved peptides identified in this study (blue boxes), which include the VTVxNVxLYNRLN motif denominated FLY, are shown. (B) Moving average of nine amino acids for each position in the gp85/TS group II protein alignment. The signal peptide for ER localization (arrow) and the 10 most conserved peptides (*) are indicated. (C) Representation in sequence logo format of the gp85/TS derived peptides (shown within brackets). The letter size indicates amino acid conservation in each position and the color, whether amino acids are polar (green), hydrophobic (black), positively (blue) or negatively (red) charged.

Mentions: To identify conserved peptide motifs shared by members of the group II of the gp85/TS family, we aligned 117 protein sequences belonging to this family. Individual sequences were obtained from the TryTryp database [22] and considered as belonging to the group II of the gp85/TS family accordingly to a previously described classification [11]. As expected, analysis of the primary sequences showed that most proteins from group II have molecular mass ranging from 80 to 90 kDa and contain the ASP-box and the VTVxNVxLYNRPLN motifs common to all members of the family (Fig 1A). To visualize and quantify the results of our alignment, first we generated a grid profile to express amino acid frequencies for each position in the alignment (S1 Table). Next, the moving average for each position was calculated by averaging the frequencies for the initial position plus the frequency of the following eight amino acids. This was done to minimize fluctuations and to facilitate the visualization of conserved regions in the gp85/TS family members (Fig 1B). We chose a moving average of 9 amino acids because it approximates the peptide length that can be displayed by the bacteriophage (see below). Moreover, similar analysis performed with moving averages corresponding to peptides of 7 up to 12 amino acids produced similar profiles. Based on these results, a cut off of 85% identity was chosen to select the 10 most conserved peptides. The selected peptides ranged from 9 to 19 amino acids in length with average identities varying from 85.5 to 92.1% (Fig 1C). The selected peptides were denominated TS1 to TS10 (Table 1 and S2 Table). It is important to notice that almost all analyzed proteins have an amino-terminal signal peptide, which is not found in the mature protein, and therefore these regions were not considered for peptide selection (Fig 1B, arrow).


Trypanosoma cruzi Binds to Cytokeratin through Conserved Peptide Motifs Found in the Laminin-G-Like Domain of the gp85/Trans-sialidase Proteins.

Teixeira AA, de Vasconcelos Vde C, Colli W, Alves MJ, Giordano RJ - PLoS Negl Trop Dis (2015)

Selection of peptides common to all members of the gp85/trans-sialidase family.(A) General representation of gp85/TS proteins belonging to the group II. The signal peptides for endoplasmic reticulum (ER) localization and glycophosphatidylinositol (GPI) anchor (green boxes) are indicated; the sialidase ASP-box repeats (orange boxes) and the conserved peptides identified in this study (blue boxes), which include the VTVxNVxLYNRLN motif denominated FLY, are shown. (B) Moving average of nine amino acids for each position in the gp85/TS group II protein alignment. The signal peptide for ER localization (arrow) and the 10 most conserved peptides (*) are indicated. (C) Representation in sequence logo format of the gp85/TS derived peptides (shown within brackets). The letter size indicates amino acid conservation in each position and the color, whether amino acids are polar (green), hydrophobic (black), positively (blue) or negatively (red) charged.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4580646&req=5

pntd.0004099.g001: Selection of peptides common to all members of the gp85/trans-sialidase family.(A) General representation of gp85/TS proteins belonging to the group II. The signal peptides for endoplasmic reticulum (ER) localization and glycophosphatidylinositol (GPI) anchor (green boxes) are indicated; the sialidase ASP-box repeats (orange boxes) and the conserved peptides identified in this study (blue boxes), which include the VTVxNVxLYNRLN motif denominated FLY, are shown. (B) Moving average of nine amino acids for each position in the gp85/TS group II protein alignment. The signal peptide for ER localization (arrow) and the 10 most conserved peptides (*) are indicated. (C) Representation in sequence logo format of the gp85/TS derived peptides (shown within brackets). The letter size indicates amino acid conservation in each position and the color, whether amino acids are polar (green), hydrophobic (black), positively (blue) or negatively (red) charged.
Mentions: To identify conserved peptide motifs shared by members of the group II of the gp85/TS family, we aligned 117 protein sequences belonging to this family. Individual sequences were obtained from the TryTryp database [22] and considered as belonging to the group II of the gp85/TS family accordingly to a previously described classification [11]. As expected, analysis of the primary sequences showed that most proteins from group II have molecular mass ranging from 80 to 90 kDa and contain the ASP-box and the VTVxNVxLYNRPLN motifs common to all members of the family (Fig 1A). To visualize and quantify the results of our alignment, first we generated a grid profile to express amino acid frequencies for each position in the alignment (S1 Table). Next, the moving average for each position was calculated by averaging the frequencies for the initial position plus the frequency of the following eight amino acids. This was done to minimize fluctuations and to facilitate the visualization of conserved regions in the gp85/TS family members (Fig 1B). We chose a moving average of 9 amino acids because it approximates the peptide length that can be displayed by the bacteriophage (see below). Moreover, similar analysis performed with moving averages corresponding to peptides of 7 up to 12 amino acids produced similar profiles. Based on these results, a cut off of 85% identity was chosen to select the 10 most conserved peptides. The selected peptides ranged from 9 to 19 amino acids in length with average identities varying from 85.5 to 92.1% (Fig 1C). The selected peptides were denominated TS1 to TS10 (Table 1 and S2 Table). It is important to notice that almost all analyzed proteins have an amino-terminal signal peptide, which is not found in the mature protein, and therefore these regions were not considered for peptide selection (Fig 1B, arrow).

Bottom Line: One peptide, named TS9, showed significant cell binding capacity and was selected for further studies.Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process.It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Chemistry Institute, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT

Background: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a disease that affects millions of people most of them living in South and Central Americas. There are few treatment options for individuals with Chagas' disease making it important to understand the molecular details of parasite infection, so novel therapeutic alternatives may be developed for these patients. Here, we investigate the interaction between host cell intermediate filament proteins and the T. cruzi gp85 glycoprotein superfamily with hundreds of members that have long been implicated in parasite cell invasion.

Methodology/principal findings: An in silico analysis was utilized to identify peptide motifs shared by the gp85 T. cruzi proteins and, using phage display, these selected peptide motifs were screened for their ability to bind to cells. One peptide, named TS9, showed significant cell binding capacity and was selected for further studies. Affinity chromatography, phage display and invasion assays revealed that peptide TS9 binds to cytokeratins and vimentin, and prevents T. cruzi cell infection. Interestingly, peptide TS9 and a previously identified binding site for intermediate filament proteins are disposed in an antiparallel β-sheet fold, present in a conserved laminin-G-like domain shared by all members of the family. Moreover, peptide TS9 overlaps with an immunodominant T cell epitope.

Conclusions/significance: Taken together, the present study reinforces previous results from our group implicating the gp85 superfamily of glycoproteins and the intermediate filament proteins cytokeratin and vimentin in the parasite infection process. It also suggests an important role in parasite biology for the conserved laminin-G-like domain, present in all members of this large family of cell surface proteins.

Show MeSH
Related in: MedlinePlus