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Improving the anti-toxin abilities of the CMG2-Fc fusion protein with the aid of computational design.

Xi Y, Wu X, Gao L, Shao Y, Peng H, Chen H, Chen H, Hu X, Yue J - PLoS ONE (2014)

Bottom Line: An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections.This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable.Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China.

ABSTRACT
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

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Toxin neutralization assay analyzing the CMG2-Fc mutants.The experiments were performed at different concentrations and dilutions due to the different abilities of the different mutants to neutralize LeTx. The data points represent the mean ± SD values of triplicate samples.
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pone-0104674-g004: Toxin neutralization assay analyzing the CMG2-Fc mutants.The experiments were performed at different concentrations and dilutions due to the different abilities of the different mutants to neutralize LeTx. The data points represent the mean ± SD values of triplicate samples.

Mentions: The ability of the CMG2-Fc mutants to neutralize LeTx toxicity in J774A.1 mouse macrophage cells was analyzed over a range of concentrations. The lethal doses in J774A.1 cells were 200 ng of LeTx. For the toxin neutralization assay, we designed different initial dilutions based on the respective EC50 calculated by the preliminary assay. Figure 4 shows a typical dose-response curve, from which an EC50 can be calculated. The average EC50 for CMG2-Fc is 49.2 ng/ml, whereas CMG2-Fc (E117Q) had an EC50 of 14.2 ng/ml. The ability of CMG2-Fc (E117Q) to protect J774A.1 cells against LeTx challenge is superior to CMG2-Fc (p<0.05). The neutralization ability of CMG2-Fc (Y158Q) is weaker than that of CMG2-Fc; the EC50 of CMG2-Fc is 131 ng/ml.


Improving the anti-toxin abilities of the CMG2-Fc fusion protein with the aid of computational design.

Xi Y, Wu X, Gao L, Shao Y, Peng H, Chen H, Chen H, Hu X, Yue J - PLoS ONE (2014)

Toxin neutralization assay analyzing the CMG2-Fc mutants.The experiments were performed at different concentrations and dilutions due to the different abilities of the different mutants to neutralize LeTx. The data points represent the mean ± SD values of triplicate samples.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125234&req=5

pone-0104674-g004: Toxin neutralization assay analyzing the CMG2-Fc mutants.The experiments were performed at different concentrations and dilutions due to the different abilities of the different mutants to neutralize LeTx. The data points represent the mean ± SD values of triplicate samples.
Mentions: The ability of the CMG2-Fc mutants to neutralize LeTx toxicity in J774A.1 mouse macrophage cells was analyzed over a range of concentrations. The lethal doses in J774A.1 cells were 200 ng of LeTx. For the toxin neutralization assay, we designed different initial dilutions based on the respective EC50 calculated by the preliminary assay. Figure 4 shows a typical dose-response curve, from which an EC50 can be calculated. The average EC50 for CMG2-Fc is 49.2 ng/ml, whereas CMG2-Fc (E117Q) had an EC50 of 14.2 ng/ml. The ability of CMG2-Fc (E117Q) to protect J774A.1 cells against LeTx challenge is superior to CMG2-Fc (p<0.05). The neutralization ability of CMG2-Fc (Y158Q) is weaker than that of CMG2-Fc; the EC50 of CMG2-Fc is 131 ng/ml.

Bottom Line: An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections.This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable.Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

View Article: PubMed Central - PubMed

Affiliation: Beijing Institute of Biotechnology, Beijing, China.

ABSTRACT
CMG2-Fc is a fusion protein composed of the extracellular domain of capillary morphogenesis protein 2 (CMG2) and the Fc region of human immunoglobulin G; CMG2-Fc neutralizes anthrax toxin and offers protection against Bacillus anthracis challenge. To enhance the efficacy of CMG2-Fc against anthrax toxin, we attempted to engineer a CMG2-Fc with an improved affinity for PA. Using the automatic design algorithm FoldX and visual inspection, we devised two CMG2-Fc variants that introduce mutations in the CMG2 binding interface and improve the computationally assessed binding affinity for PA. An experimental affinity assay revealed that the two variants showed increased binding affinity, and in vitro and in vivo toxin neutralization testing indicated that one of these mutants (CMG2-Fc(E117Q)) has superior activity against anthrax toxin and was suitable for further development as a therapeutic agent for anthrax infections. This study shows that the computational design of the PA binding interface of CMG2 to obtain CMG2-Fc variants with improving anti-toxin abilities is viable. Our results demonstrate that computational design can be further applied to generate other CMG2-Fc mutants with greatly improved therapeutic efficacy.

Show MeSH
Related in: MedlinePlus