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Proteinases in excretory-secretory products of Toxocara canis second-stage larvae: zymography and modeling insights.

González-Páez GE, Alba-Hurtado F, García-Tovar CG, Argüello-García R - Biomed Res Int (2014)

Bottom Line: Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0).By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases.These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ciencias Biológicas y Programa de Posgrado en Microbiología, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, 54714 Cuautitlán, MEX, Mexico.

ABSTRACT
Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32 kDa in TES were relatively similar over a broad pH range (5.5-9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

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Proteolytic activity towards different substrates by TES components from T. canis at different pH values. TES samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with the following substrates: 0.5 mg/gel laminin, 0.5 mg/gel fibronectin, 0.1% (w/v) bovine serum albumin, and 0.1% (w/v) goat IgG. After electrophoresis, proteinase activity was developed using acetate buffer (pH 5.5), phosphate buffer (pH 7.6), or glycine buffer (pH 9.0) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right of the panels indicated at the top. The pH values used are indicated at the bottom of each lane.
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fig3: Proteolytic activity towards different substrates by TES components from T. canis at different pH values. TES samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with the following substrates: 0.5 mg/gel laminin, 0.5 mg/gel fibronectin, 0.1% (w/v) bovine serum albumin, and 0.1% (w/v) goat IgG. After electrophoresis, proteinase activity was developed using acetate buffer (pH 5.5), phosphate buffer (pH 7.6), or glycine buffer (pH 9.0) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right of the panels indicated at the top. The pH values used are indicated at the bottom of each lane.

Mentions: The presence of proteinases in TES with similar activity at an ample pH range (5.5–9.0) on gelatin, an hydrolyzed form of collagen that is a very abundant protein of host's connective tissue, prompted us to determine if these enzymes could have the same activity on a series of physiologically important proteins (i.e., albumin, IgG, fibronectin, and laminin) that second-stage larvae eventually encounter while migrating across host tissues. In these substrate gel electrophoresis assays, the bands from TES with activity towards these different substrates and at different pH values are shown in Figure 3 and summarized in Table 1. The ~400 and 120 kDa bands were similarly active towards albumin and goat IgG at all the pH values tested, except for the case of goat IgG where these two bands displayed higher activity at pH 9. Of note, bands of 400 and 120 kDa displayed a significant difficulty to be separated in gels copolymerized with these two substrates. Otherwise some distinct bands with different pH-dependent activity were observed in gels copolymerized with laminin and fibronectin. The ~400 and 120 kDa bands appeared more separated to each other and displayed activity at all pH values while bands of 45 and 32 kDa displayed proteolysis only at basic pH (9.0). Moreover a 26 kDa proteinase was detected only with laminin at pH 9.0. Because a smear in the ~400–120 kDa range was observed using laminin and fibronectin as substrates, further densitometry analyses of these smears allowed defining reliable activity bands only by the ends of the aforementioned MW range (Figures 4(b) and 4(c)). As expected, the ~400- and 120 kDa sized proteinases could be detected separately from each other by densitometry when albumin and goat IgG were tested (Figure 4(d)). In additional assays in which proteinase inhibitors were used, as expected, only leupeptin abolished proteolysis of all these four protein substrates by TES components (not shown).


Proteinases in excretory-secretory products of Toxocara canis second-stage larvae: zymography and modeling insights.

González-Páez GE, Alba-Hurtado F, García-Tovar CG, Argüello-García R - Biomed Res Int (2014)

Proteolytic activity towards different substrates by TES components from T. canis at different pH values. TES samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with the following substrates: 0.5 mg/gel laminin, 0.5 mg/gel fibronectin, 0.1% (w/v) bovine serum albumin, and 0.1% (w/v) goat IgG. After electrophoresis, proteinase activity was developed using acetate buffer (pH 5.5), phosphate buffer (pH 7.6), or glycine buffer (pH 9.0) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right of the panels indicated at the top. The pH values used are indicated at the bottom of each lane.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150453&req=5

fig3: Proteolytic activity towards different substrates by TES components from T. canis at different pH values. TES samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with the following substrates: 0.5 mg/gel laminin, 0.5 mg/gel fibronectin, 0.1% (w/v) bovine serum albumin, and 0.1% (w/v) goat IgG. After electrophoresis, proteinase activity was developed using acetate buffer (pH 5.5), phosphate buffer (pH 7.6), or glycine buffer (pH 9.0) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right of the panels indicated at the top. The pH values used are indicated at the bottom of each lane.
Mentions: The presence of proteinases in TES with similar activity at an ample pH range (5.5–9.0) on gelatin, an hydrolyzed form of collagen that is a very abundant protein of host's connective tissue, prompted us to determine if these enzymes could have the same activity on a series of physiologically important proteins (i.e., albumin, IgG, fibronectin, and laminin) that second-stage larvae eventually encounter while migrating across host tissues. In these substrate gel electrophoresis assays, the bands from TES with activity towards these different substrates and at different pH values are shown in Figure 3 and summarized in Table 1. The ~400 and 120 kDa bands were similarly active towards albumin and goat IgG at all the pH values tested, except for the case of goat IgG where these two bands displayed higher activity at pH 9. Of note, bands of 400 and 120 kDa displayed a significant difficulty to be separated in gels copolymerized with these two substrates. Otherwise some distinct bands with different pH-dependent activity were observed in gels copolymerized with laminin and fibronectin. The ~400 and 120 kDa bands appeared more separated to each other and displayed activity at all pH values while bands of 45 and 32 kDa displayed proteolysis only at basic pH (9.0). Moreover a 26 kDa proteinase was detected only with laminin at pH 9.0. Because a smear in the ~400–120 kDa range was observed using laminin and fibronectin as substrates, further densitometry analyses of these smears allowed defining reliable activity bands only by the ends of the aforementioned MW range (Figures 4(b) and 4(c)). As expected, the ~400- and 120 kDa sized proteinases could be detected separately from each other by densitometry when albumin and goat IgG were tested (Figure 4(d)). In additional assays in which proteinase inhibitors were used, as expected, only leupeptin abolished proteolysis of all these four protein substrates by TES components (not shown).

Bottom Line: Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0).By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases.These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ciencias Biológicas y Programa de Posgrado en Microbiología, Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México, 54714 Cuautitlán, MEX, Mexico.

ABSTRACT
Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32 kDa in TES were relatively similar over a broad pH range (5.5-9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

Show MeSH