Limits...
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.

Show MeSH
Protein modeling of T. canis excretory-secretory components (TES) and superimposition with homologs from other organisms ((a)-(b)) and between TES proteins with high (c) and low (d) structure homology. The following structures were superimposed using the I-Tasser platform coupled to the TM-align tool: (a) TcMUC4 (cartoon) and lipase with serine proteinase triad from Rhizomucor miehei (PDB 1TGL, backbone trace in purple); (b) TcTES26 (cartoon) and carboxypeptidase Y from S. cerevisiae (PDB 1WPX, backbone trace in purple); (c) TcTES32 (cartoon in blue) and TcTES70 (cartoon in red); (d) TES120/MUC1 (cartoon in blue) and TcMUC4 (cartoon in red).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150453&req=5

fig5: Protein modeling of T. canis excretory-secretory components (TES) and superimposition with homologs from other organisms ((a)-(b)) and between TES proteins with high (c) and low (d) structure homology. The following structures were superimposed using the I-Tasser platform coupled to the TM-align tool: (a) TcMUC4 (cartoon) and lipase with serine proteinase triad from Rhizomucor miehei (PDB 1TGL, backbone trace in purple); (b) TcTES26 (cartoon) and carboxypeptidase Y from S. cerevisiae (PDB 1WPX, backbone trace in purple); (c) TcTES32 (cartoon in blue) and TcTES70 (cartoon in red); (d) TES120/MUC1 (cartoon in blue) and TcMUC4 (cartoon in red).

Mentions: In the case of serine proteinase activities observed at 400 and 45 kDa, none encoding sequence has been conclusively proposed. In the case of TES120/MUC1 there were no obvious homologs (TM score = 0.24, RMSD = 16.1, and Cscore = −4.52) and searches in PDB retrieved only a serine-type proteinase with low homology (human aminopeptidase A, PDB 4KxTH, Tm score = 0.443, RMSD = 5.12). For MUC2, 3, and 5 the homologs found were basically of the mucin family. Interestingly, the folding of MUC2 and MUC5 models was strikingly distinct from the remainder mucins as it was spindle-shaped while the others were globular-shaped. On the other hand, MUC4 had a reliable serine proteinase homolog which was the lipase with serine proteinase triad Ser/His/Asp from Rhizomucor miehei (PDB 1TGL, TM score = 0.554, RMSD = 4.90 with a sequence coverage of 0.90) (Figure 5(a)). In the primary amino acid sequence, the typical lipase serine active moiety VAVTGHSLGG is present as VVASQAA in TcMUC4. Besides MUC4, TES26 was the only other component with a serine proteinase homolog retrieved, which was the carboxypeptidase Y from Saccharomyces cerevisiae (PDB 1WPX, TM score = 0.557, RMSD = 2.47 with a sequence coverage of 0.63) (Figure 5(b)). At primary sequence, the archetypal serine active moiety FHIAGESYAG is present as FNLGSPYAG in TcTES26 while the histidine-containing active moiety FTYLRVFNGGHMVPFDVP is apparently a divergent PSTPAANTGVHRYVFLVY sequence in TcTES26. Regarding TES32 and TES70, both retrieved homologs bearing the typical C-type lectin domain mediating binding to oligosaccharides; nevertheless in pair-matching comparison of protein structures among these 8 TES components, TES32/TES70 had the highest structural homology (TM score = 0.605, RMSD = 3.0) (Figure 5(c)). As reference, superimposition of TES120/MUC1 to MUC4 did not give significant homology (TM score = 0.288, RMSD = 4.88) (Figure 5(d)).


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)

Protein modeling of T. canis excretory-secretory components (TES) and superimposition with homologs from other organisms ((a)-(b)) and between TES proteins with high (c) and low (d) structure homology. The following structures were superimposed using the I-Tasser platform coupled to the TM-align tool: (a) TcMUC4 (cartoon) and lipase with serine proteinase triad from Rhizomucor miehei (PDB 1TGL, backbone trace in purple); (b) TcTES26 (cartoon) and carboxypeptidase Y from S. cerevisiae (PDB 1WPX, backbone trace in purple); (c) TcTES32 (cartoon in blue) and TcTES70 (cartoon in red); (d) TES120/MUC1 (cartoon in blue) and TcMUC4 (cartoon in red).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Protein modeling of T. canis excretory-secretory components (TES) and superimposition with homologs from other organisms ((a)-(b)) and between TES proteins with high (c) and low (d) structure homology. The following structures were superimposed using the I-Tasser platform coupled to the TM-align tool: (a) TcMUC4 (cartoon) and lipase with serine proteinase triad from Rhizomucor miehei (PDB 1TGL, backbone trace in purple); (b) TcTES26 (cartoon) and carboxypeptidase Y from S. cerevisiae (PDB 1WPX, backbone trace in purple); (c) TcTES32 (cartoon in blue) and TcTES70 (cartoon in red); (d) TES120/MUC1 (cartoon in blue) and TcMUC4 (cartoon in red).
Mentions: In the case of serine proteinase activities observed at 400 and 45 kDa, none encoding sequence has been conclusively proposed. In the case of TES120/MUC1 there were no obvious homologs (TM score = 0.24, RMSD = 16.1, and Cscore = −4.52) and searches in PDB retrieved only a serine-type proteinase with low homology (human aminopeptidase A, PDB 4KxTH, Tm score = 0.443, RMSD = 5.12). For MUC2, 3, and 5 the homologs found were basically of the mucin family. Interestingly, the folding of MUC2 and MUC5 models was strikingly distinct from the remainder mucins as it was spindle-shaped while the others were globular-shaped. On the other hand, MUC4 had a reliable serine proteinase homolog which was the lipase with serine proteinase triad Ser/His/Asp from Rhizomucor miehei (PDB 1TGL, TM score = 0.554, RMSD = 4.90 with a sequence coverage of 0.90) (Figure 5(a)). In the primary amino acid sequence, the typical lipase serine active moiety VAVTGHSLGG is present as VVASQAA in TcMUC4. Besides MUC4, TES26 was the only other component with a serine proteinase homolog retrieved, which was the carboxypeptidase Y from Saccharomyces cerevisiae (PDB 1WPX, TM score = 0.557, RMSD = 2.47 with a sequence coverage of 0.63) (Figure 5(b)). At primary sequence, the archetypal serine active moiety FHIAGESYAG is present as FNLGSPYAG in TcTES26 while the histidine-containing active moiety FTYLRVFNGGHMVPFDVP is apparently a divergent PSTPAANTGVHRYVFLVY sequence in TcTES26. Regarding TES32 and TES70, both retrieved homologs bearing the typical C-type lectin domain mediating binding to oligosaccharides; nevertheless in pair-matching comparison of protein structures among these 8 TES components, TES32/TES70 had the highest structural homology (TM score = 0.605, RMSD = 3.0) (Figure 5(c)). As reference, superimposition of TES120/MUC1 to MUC4 did not give significant homology (TM score = 0.288, RMSD = 4.88) (Figure 5(d)).

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