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Modulation of the immunogenicity of the Trypanosoma congolense cysteine protease, congopain, through complexation with alpha(2)-macroglobulin.

Huson LE, Authié E, Boulangé AF, Goldring JP, Coetzer TH - Vet. Res. (2009)

Bottom Line: These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation.Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-alpha(2)M complex.This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Genetics and Microbiology, University of KwaZulu-Natal (Pietermaritzburg campus), Private Bag X01, Scottsville, 3209, South Africa.

ABSTRACT
The protozoan parasite Trypanosoma congolense is the main causative agent of livestock trypanosomosis. Congopain, the major lysosomal cysteine proteinase of T. congolense, contributes to disease pathogenesis, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. The potential of different adjuvants to facilitate the production of antibodies that would inhibit congopain activity was evaluated in the present study. Rabbits were immunised with the recombinant catalytic domain of congopain (C2), either without adjuvant, with Freund's adjuvant or complexed with bovine or rabbit alpha(2)-macroglobulin (alpha(2)M). The antibodies were assessed for inhibition of congopain activity. Rabbits immunised with C2 alone produced barely detectable anti-C2 antibody levels and these antibodies had no effect on recombinant C2 or native congopain activity. Rabbits immunised with C2 and Freund's adjuvant produced the highest levels of anti-C2 antibodies. These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation. Rabbits receiving C2-alpha(2)M complexes produced moderate levels of anti-C2 antibodies and these antibodies consistently showed the best inhibition of C2 and native congopain activity of all the antibodies, with maximum inhibition of 65%. Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-alpha(2)M complex. This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.

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The interaction of the recombinant catalytic domain of congopain (C2) with bovine α2M. (A) The effect of increasing amounts of α2M on the activity of C2 against hide powder azure (▲) and Bz-Pro-Phe-Arg-pNA (□). Different concentrations of α2M were incubated with C2 (100 pmol for hide powder azure assay; 20 pmol for Bz-Pro-Phe-Arg-pNA assay) at molar ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 for 20 min at 37 °C. Proteolytic activity was determined by the extent of hydrolysis of substrate compared to that of a C2 control with no α2M (100% activity), and expressed as a percentage inhibition. Error bars represent the ± SEM (n = 3). (B) Non-denaturing PAGE (5% gel) analysis of the interaction between bovine α2M and activated C2. Lanes 1 and 9 500 ng α2M; lanes 2–8, α2M (500 ng) was incubated with increasing amounts of C2 (11–91 ng), corresponding to molar ratios of α2M:C2 of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 in the respective lanes. Proteins were silver stained. (C) Reducing SDS-PAGE (7.5% gel) after reaction of bovine α2M with activated C2. Lane 1, Bio-Rad molecular weight markers consisting of myosin (200 kDa), β-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa); lane 2, α2M (10 μg); lane 3, C2-α2M complex (10.372 μg). Proteins were stained with Coomassie blue R-250. Arrows represent bands at 95 and 85 kDa.
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Figure 1: The interaction of the recombinant catalytic domain of congopain (C2) with bovine α2M. (A) The effect of increasing amounts of α2M on the activity of C2 against hide powder azure (▲) and Bz-Pro-Phe-Arg-pNA (□). Different concentrations of α2M were incubated with C2 (100 pmol for hide powder azure assay; 20 pmol for Bz-Pro-Phe-Arg-pNA assay) at molar ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 for 20 min at 37 °C. Proteolytic activity was determined by the extent of hydrolysis of substrate compared to that of a C2 control with no α2M (100% activity), and expressed as a percentage inhibition. Error bars represent the ± SEM (n = 3). (B) Non-denaturing PAGE (5% gel) analysis of the interaction between bovine α2M and activated C2. Lanes 1 and 9 500 ng α2M; lanes 2–8, α2M (500 ng) was incubated with increasing amounts of C2 (11–91 ng), corresponding to molar ratios of α2M:C2 of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 in the respective lanes. Proteins were silver stained. (C) Reducing SDS-PAGE (7.5% gel) after reaction of bovine α2M with activated C2. Lane 1, Bio-Rad molecular weight markers consisting of myosin (200 kDa), β-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa); lane 2, α2M (10 μg); lane 3, C2-α2M complex (10.372 μg). Proteins were stained with Coomassie blue R-250. Arrows represent bands at 95 and 85 kDa.

Mentions: In order to study the interaction between congopain and the protease inhibitor, α2M, the extent of inhibition of C2 activity by α2M was assayed against both high (hide powder azure) and low (Bz-Pro-Phe-Arg-pNA) mol. wt. substrates. When proteases are trapped within the α2M tetramer, high mol. wt. substrates are excluded from interaction with the protease, resulting in complete inhibition, whereas low mol. wt. substrates are able to access the protease’s active site, resulting in partial inhibition [6]. Consistent with this observation bovine α2M inhibited 99% of the proteolytic activity of C2 towards hide powder azure and 45% of the peptidolytic activity against Bz-Pro-Phe-Arg-pNA (Fig. 1A). In contrast to cathepsin L and papain (result not shown), the peptide substrate experienced restricted access to the active site of congopain (15% inhibition at equimolar concentrations with α2M), and to a greater extent to that of C2, when in the α2M complex. A similar observation was made for cruzipain when assayed for inhibition with human α2M using the same peptide substrate [31]. From the inhibition assays it appeared that the enzyme and inhibitor interact in an equimolar ratio, since no further inhibition of C2 activity was observed above this ratio.Figure 1.


Modulation of the immunogenicity of the Trypanosoma congolense cysteine protease, congopain, through complexation with alpha(2)-macroglobulin.

Huson LE, Authié E, Boulangé AF, Goldring JP, Coetzer TH - Vet. Res. (2009)

The interaction of the recombinant catalytic domain of congopain (C2) with bovine α2M. (A) The effect of increasing amounts of α2M on the activity of C2 against hide powder azure (▲) and Bz-Pro-Phe-Arg-pNA (□). Different concentrations of α2M were incubated with C2 (100 pmol for hide powder azure assay; 20 pmol for Bz-Pro-Phe-Arg-pNA assay) at molar ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 for 20 min at 37 °C. Proteolytic activity was determined by the extent of hydrolysis of substrate compared to that of a C2 control with no α2M (100% activity), and expressed as a percentage inhibition. Error bars represent the ± SEM (n = 3). (B) Non-denaturing PAGE (5% gel) analysis of the interaction between bovine α2M and activated C2. Lanes 1 and 9 500 ng α2M; lanes 2–8, α2M (500 ng) was incubated with increasing amounts of C2 (11–91 ng), corresponding to molar ratios of α2M:C2 of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 in the respective lanes. Proteins were silver stained. (C) Reducing SDS-PAGE (7.5% gel) after reaction of bovine α2M with activated C2. Lane 1, Bio-Rad molecular weight markers consisting of myosin (200 kDa), β-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa); lane 2, α2M (10 μg); lane 3, C2-α2M complex (10.372 μg). Proteins were stained with Coomassie blue R-250. Arrows represent bands at 95 and 85 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: The interaction of the recombinant catalytic domain of congopain (C2) with bovine α2M. (A) The effect of increasing amounts of α2M on the activity of C2 against hide powder azure (▲) and Bz-Pro-Phe-Arg-pNA (□). Different concentrations of α2M were incubated with C2 (100 pmol for hide powder azure assay; 20 pmol for Bz-Pro-Phe-Arg-pNA assay) at molar ratios of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 for 20 min at 37 °C. Proteolytic activity was determined by the extent of hydrolysis of substrate compared to that of a C2 control with no α2M (100% activity), and expressed as a percentage inhibition. Error bars represent the ± SEM (n = 3). (B) Non-denaturing PAGE (5% gel) analysis of the interaction between bovine α2M and activated C2. Lanes 1 and 9 500 ng α2M; lanes 2–8, α2M (500 ng) was incubated with increasing amounts of C2 (11–91 ng), corresponding to molar ratios of α2M:C2 of 0.25:1, 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1 and 2:1 in the respective lanes. Proteins were silver stained. (C) Reducing SDS-PAGE (7.5% gel) after reaction of bovine α2M with activated C2. Lane 1, Bio-Rad molecular weight markers consisting of myosin (200 kDa), β-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), and ovalbumin (45 kDa); lane 2, α2M (10 μg); lane 3, C2-α2M complex (10.372 μg). Proteins were stained with Coomassie blue R-250. Arrows represent bands at 95 and 85 kDa.
Mentions: In order to study the interaction between congopain and the protease inhibitor, α2M, the extent of inhibition of C2 activity by α2M was assayed against both high (hide powder azure) and low (Bz-Pro-Phe-Arg-pNA) mol. wt. substrates. When proteases are trapped within the α2M tetramer, high mol. wt. substrates are excluded from interaction with the protease, resulting in complete inhibition, whereas low mol. wt. substrates are able to access the protease’s active site, resulting in partial inhibition [6]. Consistent with this observation bovine α2M inhibited 99% of the proteolytic activity of C2 towards hide powder azure and 45% of the peptidolytic activity against Bz-Pro-Phe-Arg-pNA (Fig. 1A). In contrast to cathepsin L and papain (result not shown), the peptide substrate experienced restricted access to the active site of congopain (15% inhibition at equimolar concentrations with α2M), and to a greater extent to that of C2, when in the α2M complex. A similar observation was made for cruzipain when assayed for inhibition with human α2M using the same peptide substrate [31]. From the inhibition assays it appeared that the enzyme and inhibitor interact in an equimolar ratio, since no further inhibition of C2 activity was observed above this ratio.Figure 1.

Bottom Line: These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation.Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-alpha(2)M complex.This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Genetics and Microbiology, University of KwaZulu-Natal (Pietermaritzburg campus), Private Bag X01, Scottsville, 3209, South Africa.

ABSTRACT
The protozoan parasite Trypanosoma congolense is the main causative agent of livestock trypanosomosis. Congopain, the major lysosomal cysteine proteinase of T. congolense, contributes to disease pathogenesis, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. The potential of different adjuvants to facilitate the production of antibodies that would inhibit congopain activity was evaluated in the present study. Rabbits were immunised with the recombinant catalytic domain of congopain (C2), either without adjuvant, with Freund's adjuvant or complexed with bovine or rabbit alpha(2)-macroglobulin (alpha(2)M). The antibodies were assessed for inhibition of congopain activity. Rabbits immunised with C2 alone produced barely detectable anti-C2 antibody levels and these antibodies had no effect on recombinant C2 or native congopain activity. Rabbits immunised with C2 and Freund's adjuvant produced the highest levels of anti-C2 antibodies. These antibodies either inhibited C2 and native congopain activity to a small degree, or enhanced their activity, depending on time of production after initial immunisation. Rabbits receiving C2-alpha(2)M complexes produced moderate levels of anti-C2 antibodies and these antibodies consistently showed the best inhibition of C2 and native congopain activity of all the antibodies, with maximum inhibition of 65%. Results of this study suggest that antibodies inhibiting congopain activity could be raised in livestock with a congopain catalytic domain-alpha(2)M complex. This approach improves the effectiveness of the antigen as an anti-disease vaccine candidate for African trypanosomosis.

Show MeSH
Related in: MedlinePlus