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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 of TES components of T. canis analyzed by acrylamide-gelatin gel electrophoresis. Effect of proteinase inhibitors. TES components were preincubated for 30 min at RT with one of the following compounds: control without inhibitor (lane 1); 2 μM trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (E64) to identify cysteine proteinases (lane 2); 0.1 μM pepstatin A for aspartic proteinases (lane 3); 1 mM ethylenediaminetetraacetic acid (EDTA) for metalloproteinases (lane 4); and 0.1 μM leupeptin for serine proteinases (lane 5). Samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with 0.1% (w/v) gelatin. Proteinase activity was developed using phosphate buffer (pH 7.6) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right.
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fig2: Proteolytic activity of TES components of T. canis analyzed by acrylamide-gelatin gel electrophoresis. Effect of proteinase inhibitors. TES components were preincubated for 30 min at RT with one of the following compounds: control without inhibitor (lane 1); 2 μM trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (E64) to identify cysteine proteinases (lane 2); 0.1 μM pepstatin A for aspartic proteinases (lane 3); 1 mM ethylenediaminetetraacetic acid (EDTA) for metalloproteinases (lane 4); and 0.1 μM leupeptin for serine proteinases (lane 5). Samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with 0.1% (w/v) gelatin. Proteinase activity was developed using phosphate buffer (pH 7.6) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right.

Mentions: TES samples collected from second-stage larva cultured in RPMI 1640 medium gave yields of protein production similar to that of 8 ng/day/larvae previously reported [30]. Initial electrophoretic analyses of TES were done in polyacrylamide gels without proteinase substrate. In these, up to eight main proteins with MW of ~400, 120 (doublet), 55, 45, 32, and 26 kDa were clearly visualized (Figure 1, lane 1). A band of 70 kDa was much less visible. When gels were copolymerized with gelatin, proteolytic (gelatinolytic) activities developed at the three different pH values employed (5.5, 7.6, and 9.0; Figure 1, lanes 2–4, resp.) and only the ~400 and 120 kDa bands displayed a significant activity that was similar at the different pH values evaluated. There was a very faint activity detected at Mr of 32 kDa. In order to determine the type of proteinases contained in TES, samples were subjected to electrophoretic separation using representative substrate (gelatin) and pH 5 (7.6) conditions and proteolytic activity was developed by previous treatment of gels with proteinase inhibitors with selectivity for cysteine, serine, aspartyl, and metalloproteinases. As shown in Figure 2, gelatinolytic activity in TES was virtually unaffected by E64 and EDTA (lanes 1 and 2), with pepstatin A exerting a partial inhibition on the ~400 and 120 kDa bands (lane 3). However leupeptin abolished all proteolytic activity in TES (lane 4) suggesting the presence of serine-type proteinases in these samples.


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 of TES components of T. canis analyzed by acrylamide-gelatin gel electrophoresis. Effect of proteinase inhibitors. TES components were preincubated for 30 min at RT with one of the following compounds: control without inhibitor (lane 1); 2 μM trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (E64) to identify cysteine proteinases (lane 2); 0.1 μM pepstatin A for aspartic proteinases (lane 3); 1 mM ethylenediaminetetraacetic acid (EDTA) for metalloproteinases (lane 4); and 0.1 μM leupeptin for serine proteinases (lane 5). Samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with 0.1% (w/v) gelatin. Proteinase activity was developed using phosphate buffer (pH 7.6) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right.
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Related In: Results  -  Collection

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

fig2: Proteolytic activity of TES components of T. canis analyzed by acrylamide-gelatin gel electrophoresis. Effect of proteinase inhibitors. TES components were preincubated for 30 min at RT with one of the following compounds: control without inhibitor (lane 1); 2 μM trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (E64) to identify cysteine proteinases (lane 2); 0.1 μM pepstatin A for aspartic proteinases (lane 3); 1 mM ethylenediaminetetraacetic acid (EDTA) for metalloproteinases (lane 4); and 0.1 μM leupeptin for serine proteinases (lane 5). Samples (10 μg/well) were electrophoretically separated in slab gels of 10% (w/v) acrylamide copolymerized with 0.1% (w/v) gelatin. Proteinase activity was developed using phosphate buffer (pH 7.6) and then gels were stained with Coomassie blue. Molecular weight of bands with activity is indicated at the right.
Mentions: TES samples collected from second-stage larva cultured in RPMI 1640 medium gave yields of protein production similar to that of 8 ng/day/larvae previously reported [30]. Initial electrophoretic analyses of TES were done in polyacrylamide gels without proteinase substrate. In these, up to eight main proteins with MW of ~400, 120 (doublet), 55, 45, 32, and 26 kDa were clearly visualized (Figure 1, lane 1). A band of 70 kDa was much less visible. When gels were copolymerized with gelatin, proteolytic (gelatinolytic) activities developed at the three different pH values employed (5.5, 7.6, and 9.0; Figure 1, lanes 2–4, resp.) and only the ~400 and 120 kDa bands displayed a significant activity that was similar at the different pH values evaluated. There was a very faint activity detected at Mr of 32 kDa. In order to determine the type of proteinases contained in TES, samples were subjected to electrophoretic separation using representative substrate (gelatin) and pH 5 (7.6) conditions and proteolytic activity was developed by previous treatment of gels with proteinase inhibitors with selectivity for cysteine, serine, aspartyl, and metalloproteinases. As shown in Figure 2, gelatinolytic activity in TES was virtually unaffected by E64 and EDTA (lanes 1 and 2), with pepstatin A exerting a partial inhibition on the ~400 and 120 kDa bands (lane 3). However leupeptin abolished all proteolytic activity in TES (lane 4) suggesting the presence of serine-type proteinases in these samples.

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