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Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

Bottom Line: We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization.Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties.Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

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Related in: MedlinePlus

Urea-mediated unfolding of 2G12 variants. Change in intrinsic fluorescence upon addition of increasing concentrations of urea. Note that only the control IgM (IgM-617) could be completely unfolded. For the other mAbs, the upper plateau of the unfolding curve could not be determined. Hence, comparative estimates for the conformational stability of the antibodies could not be deduced.
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f0020: Urea-mediated unfolding of 2G12 variants. Change in intrinsic fluorescence upon addition of increasing concentrations of urea. Note that only the control IgM (IgM-617) could be completely unfolded. For the other mAbs, the upper plateau of the unfolding curve could not be determined. Hence, comparative estimates for the conformational stability of the antibodies could not be deduced.

Mentions: Finally, the conformational stability was probed by chemical denaturation of the four recombinant antibodies using increasing concentrations of urea (0–8 M). The change in tryptophan fluorescence intensity was monitored spectrofluorimetrically. Fig. 4 depicts the corresponding curves demonstrating that 2G12-IgG is stable up to 5 M urea. Unfolding starts at 5.5 M urea, but does not reach completion even at the highest urea concentration used (8 M). Its IgM counterpart IgM-012 already shows some minor unfolding between 1 and 4 M urea, but this is again followed by a steady increase in fluorescence without achieving an upper plateau. The presence of germline residues in IgM-012_GL only slightly increased its chemical stability, with the first (minor) transition being less pronounced than in the case of its wild-type counterpart. These data are comparable to natural IgM-617 that also follows a non-two-state transition with a first (minor) unfolding event below 3 M and a second (major) one within the range of 4–7 M urea (Fig. 4).


Introduction of germline residues improves the stability of anti-HIV mAb 2G12-IgM.

Chromikova V, Mader A, Hofbauer S, Göbl C, Madl T, Gach JS, Bauernfried S, Furtmüller PG, Forthal DN, Mach L, Obinger C, Kunert R - Biochim. Biophys. Acta (2015)

Urea-mediated unfolding of 2G12 variants. Change in intrinsic fluorescence upon addition of increasing concentrations of urea. Note that only the control IgM (IgM-617) could be completely unfolded. For the other mAbs, the upper plateau of the unfolding curve could not be determined. Hence, comparative estimates for the conformational stability of the antibodies could not be deduced.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582045&req=5

f0020: Urea-mediated unfolding of 2G12 variants. Change in intrinsic fluorescence upon addition of increasing concentrations of urea. Note that only the control IgM (IgM-617) could be completely unfolded. For the other mAbs, the upper plateau of the unfolding curve could not be determined. Hence, comparative estimates for the conformational stability of the antibodies could not be deduced.
Mentions: Finally, the conformational stability was probed by chemical denaturation of the four recombinant antibodies using increasing concentrations of urea (0–8 M). The change in tryptophan fluorescence intensity was monitored spectrofluorimetrically. Fig. 4 depicts the corresponding curves demonstrating that 2G12-IgG is stable up to 5 M urea. Unfolding starts at 5.5 M urea, but does not reach completion even at the highest urea concentration used (8 M). Its IgM counterpart IgM-012 already shows some minor unfolding between 1 and 4 M urea, but this is again followed by a steady increase in fluorescence without achieving an upper plateau. The presence of germline residues in IgM-012_GL only slightly increased its chemical stability, with the first (minor) transition being less pronounced than in the case of its wild-type counterpart. These data are comparable to natural IgM-617 that also follows a non-two-state transition with a first (minor) unfolding event below 3 M and a second (major) one within the range of 4–7 M urea (Fig. 4).

Bottom Line: We also investigated the effects of the class switch and germline substitutions on the ligand-binding properties of 2G12 and its capacity for HIV-1 neutralization.Our results demonstrate that the introduced germline residues improve the conformational and thermal stability of 2G12-IgM without altering its overall shape and ligand-binding properties.Interestingly, the engineered protein was found to exhibit much lower neutralization potency than its wild-type counterpart, indicating that potent antigen recognition is not solely responsible for IgM-mediated HIV-1 inactivation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Vienna Institute of BioTechnology at BOKU, University of Natural Resources and Life Sciences, Vienna, Austria.

Show MeSH
Related in: MedlinePlus